negative regulation of inflammation: implications …...nick dervisis tanya leroith liwu li kenneth...

Negative Regulation of Inflammation: Implications for Inflammatory Bowel Disease and Colitis Associated Cancer

Daniel E. Rothschild

Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

In Biomedical and Veterinary Sciences

Irving C. Allen, Chair Nick Dervisis

Tanya LeRoith Liwu Li

Kenneth Oestreich

September 21, 2018 Blacksburg, Virginia

Keywords: IRAK-M, inflammation, organoid, IBD, cancer



ABSTRACT

The ability to sense and respond to external environmental signals is closely regulated

by a plurality of cell signaling pathways, thereby maintaining homeostasis. In particular,

the inflammatory signaling cascade contributes to cellular homeostasis and regulates

responses prompted by external stimuli. Such responses are diverse and range from a

variety of processes, including tissue repair, cell fate decisions, and even immune-cell

signaling. As with any signaling cascade, strict regulation is required for proper

functioning, as abnormalities within the pathway are often associated with pathologic

outcomes. A hyperactive inflammatory response within the gastrointestinal tract, for

example, contributes to inflammatory bowel disease (IBD), presenting as Crohn’s

disease or ulcerative colitis. Furthermore, as a chronic condition, IBD is associated with

an increased risk for the development of colitis-associated cancer.

In order to resolve inflammation and thus restore homeostasis, negative regulation may

be utilized to mediate the activity of inflammatory molecules. The mechanistic action of

a specific negative regulator of interest, interleukin receptor associated kinase M (IRAK-

M), is explored in detail within the present dissertation. Investigation of IRAK-M in

mouse models of colitis, which mimics human IBD, and in mouse models of

inflammation-driven tumorigenesis, which models colitis associated cancer,

demonstrated that loss of this molecule contributes to host protection. Therefore, IRAK-

M may be a suitable target for inhibition in order to advance therapeutic options for

human patients afflicted with a GI-related inflammatory disease, such as IBD and colitis

associated cancer.

Furthermore, an ex vivo method that models the interaction of intestinal epithelial cells

with microbes present in the GI tract was optimized and is described in the present

dissertation. This method takes advantage of primary intestinal derived organoids, also

termed “mini-guts”, which display similar features corresponding to intestinal tissue in

vivo. For this reason, the use of “mini-guts” has several advantages, particularly for the

enhancement of personalized medicine. The method discussed herein aims to

normalize experimental conditions in order to enhance reproducibility, which can further

be used to uncover microbial-epithelial interactions that contribute to a pathological

state, such as IBD. Finally, this method of intestinal epithelial cell culture was utilized to

evaluate the role of a protein, termed NF-B inducing kinase (NIK), in intestinal

epithelial cell growth and proliferation. Ultimately, ex vivo organoid culture can serve as

an important model system to study the contribution of NIK in intestinal stem cell

renewal, cancer progression, as well as in maintenance of the integrity of the

gastrointestinal barrier.



GENERAL AUDIENCE ABSTRACT

Inflammation is a tightly regulated physiologic process that is employed by body

systems such as the gastrointestinal (GI) tract to handle pathogenic insult, aid in wound

healing, and help prevent infections. When abnormal inflammatory responses occur,

this can lead to the progression of severe diseases such as ulcerative colitis and

Crohn's disease. When inflammation persists in the GI tract, such as in inflammatory

bowel disease, this can predispose patients to the development of inflammation-

associated colorectal cancer. In order to improve the treatment options for patients

afflicted with these maladies, this dissertation is aimed at studying the signaling

pathways of the innate immune system that regulate such inflammatory responses.

Furthermore, this body of work encompasses a detailed method for isolating and

culturing intestinal stem cells, which can be applied in personalized medicine for

patients with intestinal diseases. This method was utilized in this dissertation to study

genetically modified intestinal stem cells, and can further be used to investigate the

interactions of intestinal epithelial cells with pyogenic bacteria that contribute to

inflammatory maladies in the GI tract.

Acknowledgements

First and foremost I would like to thank Dr. Irving Coy Allen for giving me the opportunity

to be a part of his lab. I am immensely grateful for the mentorship he has provided, and

for his open door policy and willingness to always discuss science. I have learned many

lessons, but most importantly how to think critically, and how to grow from failure. I will

take many of the lessons I learned as your protégé with me. Your mentorship has made

me a more critical and fundamentally better scientist.

I would like to thank my committee members for the helpful guidance, critiques, and

their time. Your support has helped me grow as a person and a scientist. For this, I am

immensely grateful.

I would also like to thank the other graduate students and the undergraduates in the

Allen lab; specifically, Dylan, Sheryl, Kristin, and Veronica. Your help, advice, company

and friendship has helped make the lab a positive and cohesive environment. I wish

everyone the best of luck in their future endeavors. I would also like to thank Becky

Jones for all her help and support.

I often question if I could have made it through this endeavor without the help of Dr.

Tara Srinivasan. I am forever indebted for your help throughout my career, and grateful

for your friendship. I will always cherish our conversations and it is safe to say that I

would not be where I am today without you.

I would also like to thank Dr. Renata Goncalves. Your critical suggestions always left

me in awe of your brilliance as a scientist; you are one of the most impressive I've come

across. You embody why we need more women in science.

Lastly, I would like to thank my extended family, my siblings and my parents. Staying

true to my personality, no words. I hope to see you out on the lake, cheers.

Attribution

Chapter 1 includes portions of previously published work that was co-first authored by

myself, Dylan McDaniel and Veronica Ringel-Scaia. The portions that have been

included in this dissertation from that publication were sections that I had written.

Footnotes that reference the original publication have been included in Chapter 1.

Chapter 2 includes work that was a collaborative effort. Figures 2.1, 2.2, 2.4-2.10

contains data that was generated in Dr. Irving Allen's lab by myself and Dr. Allen, while

figure 2.3 contains data contributed by Dr. Liwu Li's lab including: Yao Zang, Na Diao,

Christina K. Lee, and Keqiang Chen. I wrote the original draft of the manuscript,

including descriptions of the figures, with edits contributed by Dr. Liwu Li and Dr. Irving

C. Allen. Yao Zang provided the data and figure legend for figure 2.3. I solely generated

the data for figures: 2.7-2.10. Methodology as well as formal data analysis for the

remaining figures contained contributions from myself, Yao Zang, Na Dial, Christina K.

Lee, Keqiang Chen, Tanya LeRoith, Clayton C. Caswell, Daniel J. Slade, Richard Helm,

Liwu Li, and Irving C. Allen.

Chapter 3 includes a step-by-step method with contributions from myself and Tara

Srinivasan. I wrote the entire manuscript, and generated all of the figures. The

remaining authors provided described in the method. Tara Srinivasan provided technical

assistance, as well as scientific advice to help frame the method described.

Chapter 4 contains contributions from both myself and Kristin Eden. While working in

collaboration with Kristin Eden on this project, I conducted the experiments for and

generated figures 4.1 and 4.2, and wrote the entirety of chapter 4. Kristin Eden

contributed to the methodology as well as formal data analysis for figure 4.1 and 4.2.

The experiment that was conducted to generate Figure 4.3 contains equal contribution

from myself and Kristin Eden.

ix

Table of Contents

Acknowledgements v

Attribution vii

Table of Contents ix

List of Figures xi

Chapter 1: Introduction 1

Chapter 2: Enhanced Mucosal Defense and Reduced Tumor Burden in Mice with the

Compromised Negative Regulator IRAK-M

A. Abstract 67

B. Introduction 68

C. Materials and Methods 71

D. Results 79

E. Discussion 100

F. References 107

Chapter 3: The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of

Mouse Intestinal Organoids

A. Abstract 115

B. Introduction 116


D. Results 132

x

E. Discussion 138

F. References 139

Chapter 4: NF-B inducing kinase (NIK) is essential for adequate expression of the

stem cell marker LGR5 and results in enhanced proliferation of intestinal epithelial cells.

A. Abstract 142

B. Introduction 143


D. Results 147

E. Discussion 152

F. References 154

Chapter 5: Discussion and Future Directions 158

Appendix

A. Works Completed 181

B. Copyright Permissions 183

xi

Figures

Figure 1.1. Intestinal epithelial cells of only a single cell layer line the GI tract. 8

Figure 1.2. Three dimensional graphic of the small intestine depicting the lumen

lined with intestinal villi. 11

Figure 1.3. Wnt signaling drives intestinal stem cell niche. 12

Figure 1.4. MyD88 Dependent Signaling Pathway 19

Figure 2.1. IRAK-M Expression is Increased During Inflammatory Bowel

Disease Exacerbation and in Advanced Colorectal Cancer Patients. 81

Figure 2.2. Attenuated Experimental Colitis Pathogenesis in Irak-m-/- Mice. 84

Figure 2.3. Increased Neutrophil and T-cell Function in Irak-m-/- Mice 87

Figure 2.4. Increased Morbidity and Mortality in Antibiotic + DSS Treated Mice 89

Figure 2.5. Colitis Associated Tumorigenesis Progression is Significantly 91

Reduced in Irak-m-/- Mice

Figure 2.6. Irak-m-/- Mice Display Attenuated Polyp Formation in the Colitis

Associated Tumorigenesis Model. 93

Figure 2.7. Disruption of the Murine Irak-m Locus Results in the Formation 95

of an Irak-mrΔ9-11 Splice Variant.

Figure 2.8. Differences in TLR-2, TLR-7 and TLR-9 Stimulation in Irak-m 97

Mutant Mice.

Figure 2.9. Comparison of Male and Female Bone Marrow Derived Macrophages. 98

Figure 2.10. Western Blot Evaluation of Protein Expression of IRAK-M. 100

Figure 3.1. Small Intestinal Organoid Growth 133

Figure 3.2. mRNA Expression of Inflammatory Cytokines Following 24 hr 134

xii

PAMP Challenge

Figure 3.3. Relative Fluorescent Staining of Organoids for Normalization 135

Figure 3.4. Standard Curve generated from Caco-2 Cells 136

Figure 3.5. Graphic Representation of the Technique 137

Figure 4.1. Growth tracking of organoids from single cell suspensions 148

Figure 4.2. Relative expression of Lgr5 from Wild-type and Nik-/- colonic crypts 149

Figure 4.3. Organoid colonies and FACS analysis of single cell suspensions 151

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Chapter 1:

Introduction

The gastrointestinal (GI) system, similar to the integumentary system, has an important

immunological role, and acts as a physical barrier that helps to prevent the access of

microbes within the body. The cells of the GI system are renewed on a daily basis,

which maintains proper barrier and physiologic function. Several cells of the immune

system are located just beneath the epithelial cells of the GI system, mainly for

protection due to the high probability of encountering a foreign pathogen if this site is

breached. When a pathogen manages to invade host tissues, an immune response

ensues to handle the invading microbes. Many immune cells will then produce

inflammatory molecules that relay information to surrounding cells that directs a proper

cellular defense reaction in response to the foreign microbes. Resolution of these

inflammatory molecules must occur; otherwise continued production might lead to

catastrophic tissue damage. As such, several regulatory molecules are produced by the

cell to prevent hyperactive inflammation. The research in the present dissertation

focuses on these molecules, specifically on a positive and a negative regulator of NF-B

signaling.

As a primary focus of this dissertation, a molecule induced by the cell to limit the

inflammatory cascade, termed interleukin receptor associated kinase-M (IRAK-M), is

evaluated with respect to its function and contribution to inflammatory related diseases

in the GI system. Both epithelial and immune cells contribute to host defenses, but due

to complexities that exist with in vivo animal models, the elucidation of precise

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molecular mechanisms can be complicated. Therefore, in order to simplify and study the

epithelial contribution to disorders such as inflammatory bowel disease (IBD), a method

is described herein and is optimized utilizing intestinal derived organoids, or “mini-guts”,

aimed to simplify conditions through ex vivo techniques. Further, the method described

is integrated for experiments conducted in Chapter 4 and is used to help elucidate the

role of NF-B inducing kinase (NIK), a protein that participates in the induction of the

alternative NF-B pathway. As such, this dissertation collectively investigated the role of

a negative and positive regulator of the NF-B pathway, being IRAK-M and NIK,

respectively. Further, the method utilizing intestinal organoids was applied to study the

epithelial cell specific contribution of these molecules to intestinal epithelial cell biology.

1. Introduction to Innate immunity and adaptive immunity

From the perspective of a pathogenic microorganism, metazoans (i.e. multicellular

organisms) represent a well-suited environment to infect and reside. In particular,

mammalian physiology affords conditions that are favorable to foreign microbes, which

include a stable temperature, abundant energy source, and interaction with species

within a population to facilitate pathogenic transmission (Sund-Levander et al., 2002),

(Speakman, 2005), (Engering et al., 2013). Therefore, in order to prevent such

infections from occurring, evolution has selected elegant mechanisms for the host to

prevent and limit the spread of disease through the action of the immune system

(Schultz and Grieder, 1987). The mammalian immune system is a complex network of

cells that orchestrate diverse functions, most of which are attributed to protecting the

host from disease causing pathogens (Muller et al., 2008). This is accomplished by

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what evolution has afforded, and what immunologists have classified as the two main

branches of the immune system: the innate and adaptive immune system (Powers and

Dean, 2016). Simplistically, innate immunity is known for its ability to act as a barrier

from the external environment with internal tissues. Furthermore, innate immunity also

involves the recognition of broad components of pathogens, and can rapidly induce an

effector response once a pathogen is recognized (Akira et al., 2006). Adaptive immunity

is known for being acquired following pathogenic insult, is highly specific to previously

encountered pathogens through the action of T and B lymphocytes, and is long lasting

(Bonilla and Oettgen, 2010). Tight regulation of both branches of the immune system

are required for normal physiological process to occur, and pathologies ensue when

signaling aberrations deviate from homeostatic norms. Hyperactive aberrations in both

the innate and adaptive immune responses can result in severe tissue damage;

whereas hypoactive aberrations can result from improper recognition of a pathogen,

leading to opportunistic infection (Blach-Olszewska and Leszek, 2007), (Al Anazi,

2009).

1.1 Cells of the Innate Immune System

The importance of innate immunity cannot be understated. All metazoans have cells

that compose an innate immune system, which protects from pathogens; whereas, the

adaptive immune system is more specialized, and it is believed to have evolved

stemming from a common ancestor with an innate immune system (Kimbrell and

Beutler, 2001), (Beutler, 2004). The innate immune system is composed of a diverse

range of cell types, which are categorically assigned based on the criteria of being a

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foremost line of host defense (Alberts et al., 2008). It should be noted that clear cut

definitions of innate immune cells are not always possible. Recently, cell types with both

innate and adaptive immune cell characteristics have been discovered, and fittingly

termed innate lymphoid cells. Innate immune cells include cell types that compose and

maintain the anatomical barriers to the external environment; however, the main cell

types that are pertinent to the innate immune response usually refer to the white blood

cells including both mononuclear and polymorpho-nuclear phagocytes. Mononuclear

phagocytes include the monocytes, macrophages, and dendritic cells, whereas

polymorpho-nuclear phagocytes include neutrophils, basophils and eosinophils (Beutler,

2004). Monocytes are the circulating precursors to macrophages, and mature into

macrophages once they have migrated from the circulation into tissues. It should be

noted that not all macrophages are created equal. In particular, there are groups of

tissue resident macrophages that are present in all organs and they display alternate

epigenetic gene signatures. Currently, it is believed that the surrounding

microenvironment is in constant communication with, and contributes to the diverse

function of tissue resident macrophages (Lavin et al., 2014). Macrophages and dendritic

cells are well known for their ability to engulf foreign microbes, as well as cellular debris,

through a cellular process called phagocytosis, followed by presentation of foreign

antigens to the adaptive immune system on class II MHC molecules (Metchnikov,

1884), (Banchereau and Steinman, 1998). Neutrophils are polymorphonuclear cells that

are likely the first-responders to a pathogen. They undergo maturation in the bone

marrow and enter into the circulation following their differentiation (Bainton et al., 1971).

Ultimately, neutrophils have a limited lifespan following maturation, as they undergo

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apoptosis as shortly as 6 hours after their entry into circulation (Cronkite and Fliedner,

1964), (Brinkmann and Zychlinsky, 2007). Neutrophils have the ability, similar to

macrophages, to engulf foreign substances via phagocytosis (Cohn and Hirsch, 1960),

and have recently attracted attention for the finding pertaining to their ability to exude

their cellular contents, termed neutrophil extracellular traps (NETs), which combats

pathogenic bacteria (Brinkmann et al., 2004). Basophils and eosinophils compose a

small percentage of circulating leukocytes in the human body (Stone et al., 2010). There

are aspects of basophil function that are unknown; however, basophils are currently

understood to contribute to host defense against parasites (Min, 2008). They store

preformed granules that contain histamine, and thus contribute to the allergic responses

(Stone et al., 2010). Eosinophils are known for their response, mainly to helminth

infection and allergies, but can also play a role in rare diseases (Stone et al., 2010).

Once matured into fully differentiated eosinophils, they migrate into circulation, and will

increase in abundance and locality due to the presence of Th2 associated cytokines

(Rosenberg et al., 2007).

All of the cell types described above share a common feature, in that they all share a

common lineage ancestor. All of these cells described above differentiate from a

hematopoietic stem cell that normally resides in the bone marrow. These phenomena

are highly relevant, because depending on which type of cell immunologists wish to

investigate, isolated stem cells from the bone marrow can be differentiated ex vivo when

the proper signals are provided that drive differentiation to a specific lineage. This

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allows researchers to simplify complex conditions to solve specific questions, pertaining

to a specific cell.

1.2 Interplay of innate immune system with GI system

Barrier surfaces, such as the GI tract, are a highly susceptible point of entry for

pathogenic microorganisms when breached. Therefore, an adequate epithelial barrier,

and immune response is necessary to prevent and contain any pathogenic insult. The

GI system of humans, as well as other mammals, harbors a diverse array of microbes

that share a commensal relationship with the host (Human Microbiome Project, 2012).

This phenomenon poses a really fascinating question, one that currently perplexes

researchers, and is related to immune activation versus immune tolerance. How is the

body able to recognize and respond to a pathogen emanating from the GI system, while

also subverting an active immune response against the commensal microbes that can

share many similarities with the pathogen? The answer to this question is obviously not

a simple one, but the answer partially lies in the several molecular mechanisms that

regulate the interaction between the GI tract and immune system that allow

homeostasis with the microbiota. This collectively starts with the physical barrier of

epithelial cells that prevent translocation of bacteria from the intestinal lumen to the

lamina propria. This intestinal epithelium is composed of polarized columnar epithelial

cells that are remarkably only a single layer in thickness. (Abreu, 2010), (Mowat and

Agace, 2014), (Williams et al., 2015). The turnover rate of epithelial cells in the GI tract

is one of the fastest in the human body, occurring every 3-4 days, and is possibly

related to the exposure of this tissue to hostile substances (e.g. bacterial products,

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dietary components) (Creamer et al., 1961), (Cheng and Leblond, 1974). In the

unfortunate event that a pathogen breaches the epithelial barrier, immune cells of both

the innate and adaptive immune system strategically located beneath the layer of

epithelial cells within the lamina propria will recognize and engage the pathogen (Figure

1.1). The lamina propria of the GI tract contains the highest abundance of

macrophages, T-cells, and IgA secreting plasma cells; additionally, under normal

conditions, macrophages constitute the most abundant leukocyte in the lamina propria

of the GI tract (Mowat and Agace, 2014). Such a high abundance of phagocytic cells in

the lamina propria is likely due to the high occurrence, or probability of foreign microbial

encounter. Under homeostatic conditions, tissue resident macrophages found in the

lamina propria are under consistent turnover, renewed by circulating monocytes that

travel into the tissue from the blood stream (Bain et al., 2014). Once these cells migrate

into the lamina propria, they can be distinguished by three major factors. These include

the expression of the fractalkine receptor CX3CR1, the secretion of high amounts of IL-

10 and TNF, and by being relatively inert to the presence of LPS (Bain et al., 2013). The

low responsiveness of these macrophages to LPS suggests an important regulatory

function, one that likely includes phagocytosis of debris without inducing an immune

response. These cells contribute to several pleiotropic responses due to the secretion

of the cytokines IL-10 and TNF. IL-10 is important for the regulation of immune cell

function, and TNF can govern epithelial cell turnover (Shouval et al., 2014), Proper

tissue homeostasis is partially maintained by constant crosstalk between the epithelium,

immune cells, and the microbiota. It is fascinating that all the physiological processes

work, in most cases, without any insult or abnormalities. As can be expected, these

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interactions between tissue and commensal microbes, as well as the molecular

mechanisms that regulate immune tolerance versus immune activation are at the

forefront for many scientific investigators. Aberrations in any of these interactions can tip

Inte

stin

al lu

men

Lamina Propria

Intestinal bacteria

IgA

antibodies

Intestinal Epithelial cells

Intestinal Immune

cells

Figure 1.1. Intestinal epithelial cells of only a single cell layer line the GI tract, and physically separate intestinal microbes from entering the body. Beneath the epithelial cells are immune cells within the lamina propria, many of which are macrophages. Figure from: (Mowat, A. M. & Agace, W. W. 2014). See copyright permissions.

9

the homeostatic balance, and ultimately have the potential to contribute to tissue

pathologies.

2. Introduction to the GI system

The GI system collectively refers to the hollow tube that begins at the mouth and

terminates at the anus. The main functions of the GI system involve obtaining nutrients

via digestion and absorption followed by the excretion of waste, while also protecting

the host during these processes by forming a physical barrier with the external

environment (Cheng et al., 2010). As chemoorganoheterotrophs, humans require

energy sources from complex organic forms of carbon (i.e. carbohydrates, proteins and

lipids) and must break these macromolecules down to simpler subunits in order for

proper metabolism to occur. The GI system has evolved to mechanically and chemically

break down ingested food, beginning in the mouth and ending at the colon, while also

providing a large surface area of epithelial cells to maximize the absorption process,

occurring from small intestine to colon. The colon aids in absorption of nutrients and

water, and also harbors trillions of bacteria that form a commensal relationship with the

host, and is referred to as the microbiome.

2.1 Anatomy of Small intestine and Colon

The GI tract is a hollow tube that is derived from the endoderm during gastrulation

(Lewis and Tam, 2006). The small intestine is the segment of GI tissue that begins after

the stomach, and ends at the cecum, which is followed by the large bowel (i.e. the

colon). From its proximal to distal ends, the small intestine is further subdivided into

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three segments: duodenum, jejunum, and ileum, respectively. The function of the

duodenum pertains to acid neutralization, whereas the jejunum and ileum involve

nutrient absorption. The major functions of the small intestine are to aid in digestion of

ingested nutrients, and absorption of water, electrolytes and nutrients. The anatomical

structures of the small intestine are quite striking; the surface epithelium is lined with

fingerlike projections, termed villi (Figure 1.2 A, B). These fingerlike projections

maximize the intestinal surface area, which enhances the efficiency in which nutrients

can be absorbed into the blood stream. The intestinal epithelium is one of the most

rapidly dividing tissues in the human body, with epithelial turnover being every 3-5 days

(Cheng and Leblond, 1974), (Mayhew et al., 1999). Cell division is driven by intestinal

stem cells that reside in the base of the intestinal crypts, which in a conveyor belt

fashion, push the epithelial cells up towards the tip of the intestinal villus, where they

eventually undergo apoptosis (Williams et al., 2015) (Figure 1.2 C). This results in a

constant shedding of epithelial cells into the intestinal lumen to be excreted with the

feces (Schuijers and Clevers, 2012). The colon shares similarities with the small

intestine; however, there are distinct differences between the two tissues. Regarding

epithelial anatomy, the colon does not contain villi, but rather has a flat surface

epithelium (Colony, 1996), (Schuijers and Clevers, 2012). Similar to the small intestine,

the colon contains crypts that harbor stem cells, and the epithelial cells of the colon are

under constant renewal. The main epithelial cell types of the colon are columnar

epithelial cells, goblet cells, and enteroendocrine cells (Colony, 1996). One key feature

of the colon is that it is in constant contact with millions of bacteria, and thus contains

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several goblet cells to produce mucus, which provides another physical barrier to GI

bacteria.

2.2 Stem cells as contributors of epithelial cell fates and the Wnt signaling

pathway

Intestinal stem cells reside at the crypt base, and in the case of the small intestine,

reside next to the Paneth cells, which produce antimicrobial peptides and contribute to

stem cell maintenance (Cheng and Leblond, 1974), (Sato et al., 2011). Intestinal stem

cells give rise to all of the differentiated epithelial cells that compose the intestinal

epithelium (Cheng and Leblond, 1974), (Bjerknes and Cheng, 2006). The past two

decades of research has demonstrated the importance of the Wnt signaling pathway in

the maintenance of the intestinal epithelium (Figure 1.3). WNT is a ligand that binds to

Fizzled receptors that ultimately culminates in the stabilization and nuclear localization

Figure 1.2. A. Three dimensional graphic of the small intestine depicting the lumen lined with intestinal villi. B. Birdseye view of the fingerlike projections (villi). Intestinal crypts are at the base of the villi. C. Transverse section of intestinal villi. Intestinal crypts are the U-shaped structures at the base of the intestinal villi. Figure from: (Schuijers and Clevers, 2012). See copyright permissions.

A. B. C.

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of the transcription factor β-catenin. The importance of canonical Wnt signaling was first

demonstrated with transgenic mice lacking Tcf4, a key transcription factor that is

activated in response Wnt

ligands. Loss of Tcf4 in mice resulted in premature death, and interestingly, the

intestinal epithelium of neonatal mice was entirely differentiated (Korinek et al., 1998).

This provided the first clue that Wnt signaling contributed to the intestinal stem cell

niche. A major breakthrough came recently, when it was discovered that both small

intestine and colon intestinal stem cells express the cell surface receptor leucine-rich

repeat-containing G-protein coupled receptor 5 (LGR5) (Barker et al., 2007). LGR5 was

identified as a Wnt target gene, and further identified as the receptor for R-spondin. In

the presence of WNT ligands, R-spondin amplifies and sustains Wnt signaling that is

necessary to retain the stem cell niche (Kazanskaya et al., 2004), (Kim et al., 2008), (de

Lau et al., 2011). ). With this knowledge, isolated intestinal crypts or single Lgr5+/GFP

stem cells can be cultured ex vivo with the addition of several niche factors that

Figure 1.3. Wnt signaling drives intestinal stem cell niche. (left) intestinal stem cells divide and differentiate into all the epithelial cell types found in the GI system. (Right) Wnt signaling is amplified in intestinal stem cells by R-spondins that bind to LGR5. Figure from: (Koo and Clevers, 2014). See copyright permissions.

13

stimulate the Wnt signaling pathway (Sato et al., 2009). These “mini guts”, also termed

organoids possess the characteristics of intestinal epithelium, and are proving to be a

vital tool for GI research. Organoids are proving to be excellent tools to study GI

epithelial cell proliferation, because the confounding factors of the immune system have

been removed.

3. Pattern Recognition Receptors of the Innate Immune System

3.1 The Families of Pattern Recognition Receptors

Following pathogenic insult, the normal series of innate immune responses are to

recognize, respond, and resolve the insult from the foreign invader (Beutler, 2004), but

what mechanisms govern the ability of host cells to recognize a pathogen? It was

Charles Janeway that first proposed the existence of so called “pattern recognition

receptors” by innate immune cells, and rationalized that host innate immune cells likely

recognize conserved molecules (i.e. patterns) that are unique to foreign microbes

(Janeway, 1989). Time later proved that Janeway was ultimately correct in his

prediction. The major breakthrough came when it was demonstrated that Toll-like

receptor 4 (TLR4) was the bona fide receptor for the conserved bacterial molecule

lipopolysaccharide (LPS) (Poltorak et al., 1998). This discovery, along with work

conduced in the fruit fly by Jules Hoffman and works pertaining to dendritic cells linking

innate and adaptive immunity by Ralph Steinman, was ultimately awarded the Nobel

Prize in physiology and medicine in 2011. These findings were the first to demonstrate

the importance of pattern recognition receptors (PRRs) to innate immunity. Broadly, it is

now known that PRRs are located based on topology relative to the cell. Soluble

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extracellular PRRs include members of the complement system, as well as the

pentraxins, which bind to phosphocholine in a calcium dependent manner (Pepys and

Hirschfield, 2003); cell surface receptors include the Toll-like receptors (TLRs) and C-

type lectin receptors (CLRs); and finally, Nod-like receptors (NLRs), Rig-I-like helicases

(RLRs) and AIM2 receptors, which are located intracellularly in the cell cytosol.

PRRs recognize conserved molecules residing on or contained within foreign microbes

that are fundamentally distinct from healthy host cells. These foreign patterns (i.e.

molecules) can be of bacterial, viral, fungal, or protozoan origin; and certain PRRs can

even recognize self-damage patterns (Thompson et al., 2011). The molecules

recognized by PRRs are termed pathogen associated molecular patterns (PAMPs) and

self-patterns are termed damage/danger associated molecular patterns (DAMPs)

(Janeway, 1989), Following recognition of a PAMP/DAMP by a particular PRR leads to

a rapid cellular response. This ensues via multiple coordinated signal transduction

cascades that culminate in either the release of inflammatory molecules, an increase in

the transcription of genes involved in cellular migration of immune cells, and when the

signal is robust, activation of the adaptive immune system (Steinman and Witmer,

1978), (Medzhitov and Janeway, 1997), (Kawai et al., 2001), (Rahman et al., 2009).

Lastly, resolution of the immune response must ensue in order to prevent excessive

inflammation that can result in destructive tissue damage (Beutler, 2004). The resolution

phase occurs through negative feedback loops, which are a mechanism to restore

homeostasis, and are paramount to proper physiological function. This can occur

through a variety of means, for example, intracellular molecules that down-regulate the

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signaling cascades that culminate in the activation of the inflammatory response (PTEN,

SOCS-1, SHIP1, IRAK-M, CYLD, A20). Because both arms of the immune system are

highly involved in the production of inflammatory mediators, many inflammatory related

diseases can ensue when proper function is not maintained. Other mechanism to

restore homeostasis can include the production of anti-inflammatory cytokines, such as

interleukin-10 (IL-10), as well as the activation of regulatory cells that blunt the immune

system, i.e. T-regulatory cells (Tregs) and myeloid derived suppressor cells (MDSCs).

In summary, there are many ways the cell can restore balance once a response is

generated.

3.2 Cell types of both GI and innate immune systems that express PRRs

The expression of PRRs on both intestinal epithelial cells (IEC) and innate immune cells

has been shown to be crucial for proper intestinal and immune physiology (Vijay-Kumar

et al., 2007). The cells that line the GI tract are composed of polarized epithelial cells,

meaning they have an apical surface that borders the intestinal lumen, and a

basolateral surface that borders the lamina propria (Abreu, 2010). The families of PRRs

that have been most extensively studied pertaining to IEC are the TLRs, and their

expression varies regarding localization on either the apical or basolateral membrane of

the IEC. In the human colon, TLR5, which senses bacterial flagella, is expressed on the

basolateral membrane of IECs and not the apical membrane on IECs (Gewirtz et al.,

2001), (Rhee et al., 2005). This suggests that spatial separation of TLR5 from the

intestinal lumen is important in preventing constitutive interaction of this receptor with

the intestinal microbiota, and likely recognizes bacterial components once they have

16

breached beyond the apical membrane. Further, TLR1, TLR2, TLR4 and TLR9 are

expressed in human small intestinal epithelial cells (Otte et al., 2004). The expression of

these TLRs also display spatial polarization, with the highest expression on the

basolateral surface of IECs; however, their expression is not limited to the basolateral

membrane as they are found on the apical surface as well (Cario et al., 2002), (Lee et

al., 2006). This begets the question, if TLRs are expressed on the apical membrane of

IEC that are in contact with the luminal microbiota, why are these cells not constantly

driving inflammation? This is likely due to the importance of proper TLR expression, and

signaling for cellular maintenance of IECs. Indeed, TLR engagement of commensal

PAMPs results in the production of protective factors, and ablation of the GI microbiota

with antibiotics limits GI TLR activation, and results in profound susceptibility to dextran

sulfate sodium (DSS), which is a chemical that leads to colitis in mice (Rakoff-Nahoum

et al., 2004).

3.3 Inflammation and the NF-κB signaling pathway1 (Rothschild et al., 2018)

Inflammation, in certain contexts harbors a negative umbrella of pathologies; however,

under proper physiological conditions, it serves a very important purpose. Broadly,

inflammation functions to coordinate the repair of damaged tissue by informing the

immune system that tissue damage is taking place. Inflammation results in cellular

activation, increased blood flow to the affected area, and an influx of cells associated

with the immune system. This coordinated effort from cells of the host immune system

1 Published in: ROTHSCHILD, D. E., MCDANIEL, D. K., RINGEL-SCAIA, V. M. & ALLEN, I. C. 2018. Modulating

inflammation through the negative regulation of NF-kappaB signaling. J Leukoc Biol.

17

functions to clear the pathogenic insult, repair tissue damage and curb the inflammatory

response in order to restore tissue homeostasis. At the cellular level, this is coordinated

via an important regulator of transcription: the nuclear factor kappa light chain enhancer

binding protein (NF-κB) transcription factor.

NF-κB is an evolutionarily conserved transcription factor found in species ranging from

Drosophilia to humans, which underscores its critical role in the host immune response.

(Ghosh et al., 1998). The last 3 decades of research have contributed greatly to our

understanding of NF-κB. NF-κB functions as a prominent inducible transcription factor

that regulates the immune system Briefly, NF-κB it is known to regulate a vast array of

genes ranging from the development of the embryo, to cell fate decisions, and is well

known for its role as a prominent transcription factor that regulates the immune system

(Beg et al., 1995), (Alcamo et al., 2001), (Boersma et al., 2011), (Zhang et al., 2017).

Due to its diverse and broad biological functions, strict regulation of NF-κB signaling is

paramount to proper tissue allostasis. When NF-κB signaling is aberrant, several

maladies can ensue, such as susceptibility to infections, autoimmunity and cancer

(Oeckinghaus and Ghosh, 2009). (Sun et al., 2013), (Greten et al., 2004). Thus, as with

many major signal transduction pathways, several mechanisms tightly regulate NF-κB

signaling and maintain the proper balance of activation and repression.

NF-κB in mammals consists of a total of five proteins that are predominantly present in

an inactivated state in the cytosol as either homo- or hetero-dimers that include: RelA

(p65), RelB, c-Rel, p105 and p100. The p105 and p100 proteins are unique because

18

they must undergo post-translational processing through the proteasome to form the

active subunits, p50 and p52, respectively (Amir et al., 2004), (Ghosh et al., 1998). All

isoforms contain a common Rel homology domain, which is responsible for DNA

binding, as well as binding to the cytosolic inhibitory proteins, termed Inhibitor of –κB

(IκB) (Ghosh et al., 1998). Several families of cellular receptors signal through NF-κB to

initiate gene transcription and are known to coordinate signaling through either the

canonical NF-κB pathway or the non-canonical NF-κB pathway (which is also termed

the “alternative” NF-κB pathway). Molecules that signal via the canonical NF-κB

pathway include cytokines, such as tumor necrosis factor (TNF), interleukin-1 beta (IL-

1β), and the majority of PRRs (Ghosh et al., 1998). The non-canonical pathway is

initiated by a much smaller repertoire of molecules that are members of TNF family,

such as CD40, B-cell activating factor (BAFF), and lymphotoxin beta (LT-β) (Sun et al.,

2013).

3.4 Canonical NF-κB signaling pathway1

Convergence on the canonical NF-κB signaling pathway occurs through the activation

of several different families of receptors (i.e. TNFR, TLRs, NLRs, IL-1R) (Ghosh and

Hayden, 2012). As a classical example of canonical activation, we will focus on MyD88-

dependent TLR signaling (Figure 1.4), a process that occurs for the interleukin 1

receptor (IL-1R) and all TLRs with the exception of TLR3 (Yamamoto et al., 2003). IL-

1/TLR signaling commences via binding of their respective ligands. Engagement of a

PAMP to an extracellular TLR (i.e. TLR1, TLR2, TLR4, TLR5, TLR6) induces a

conformational change and hetero- or homo-dimerization of the TLR. Dimerization leads

19

to a change in conformation of the receptor, followed by recruitment of adaptor proteins

to the toll/interleukin receptor (TIR) domain of the TLR. The adaptor molecule MyD88 is

recruited to extracellular dimers composed of TLR5 receptors, whereas Mal followed by

MyD88 are recruited by TLR1/TLR2, TLR2/TLR6, TLR4 dimers (Kawai et al., 1999),

(Fitzgerald et al., 2001), (Yamamoto et al., 2002),(Horng et al., 2002),(Didierlaurent et

al., 2004), (Nishiya and DeFranco, 2004). MyD88 acts as a protein scaffold, as it

contains both a TIR domain and a death domain (DD) in its protein structure. The DD of

MyD88 recruits interleukin receptor associated kinase (IRAK) IRAK-4, followed by

IRAK-1, all in a helical assembly to form a Myddosome complex (Wesche et al., 1997),

(Lin et al., 2010). The close spatial proximity of IRAK-4 to IRAK-1 allows for IRAK-1 to

Lys63 Ubiquitination

Phosphorylation

Ubiquitin

Lys48 Ubiquitination

MyD88

IRAK-4

IRAK-1

TRAF6

TAK-1

IKK Complex

NF-! B

I! B

" TrCP

NF-! B Signaling (MyD88 Dependent )

26s Proteosome

Inflammation Cell Migration

Cell Survival

Negative Regulators

Figure 1.4. MyD88 Dependent Signaling Pathway. Engagement of a Toll-like receptor (TLR), with the exception of TLR3, induces a signal transduction cascade via the Myeloid Differentiation Factor 88 (MyD88) dependent pathway. MyD88 dependent signaling results in the activation of the canonical NF-κB pathway. Activation of NF-κB results in the transcription of multiple genes involved in inflammation, cell migration, cell survival, and negative feedback proteins to eventually restore homeostatic norms of the cell.

20

become rapidly phosphorylated by IRAK-4, and then undergo auto-phosphorylation

(Wesche et al., 1997), (Li et al., 2002). Once phosphorylated, IRAK-1 leaves the

receptor complex and interacts with TRAF-6. Upon activation, TRAF6 associates with

Uev1A and Ubc13 and results in TRAF6 lysine-63 (Lys-63)-mediated poly-ubiquitination

(Takaesu et al., 2000), (Deng et al., 2000). Lys63-mediated poly-ubiquitination of

TRAF6 acts as an important scaffold resulting in the recruitment and docking of TAB2/3

and TGF-β activated kinase-1 (TAK1) (Wang et al., 2001), (Kanayama et al., 2004).

Close association of the TAB2/3/TAK1 complex with poly-ubiquitinated TRAF-6 allows

TAK1 to undergo auto-phosphorylation, resulting in TAK1 activation (Wang et al., 2001).

Once TAK1 becomes phosphorylated, it can mediate downstream signaling by

phosphorylating the inhibitor of κB kinase (IKK) complex on IKKβ subunits (Ninomiya-

Tsuji et al., 1999), (Wang et al., 2001). IKKβ phosphorylation results in the IKK complex

activation, composed of NEMO/IKKα/ IKKβ subunits, to phosphorylate IκB (Mercurio et

al., 1997). Phosphorylation of IκB results in its subsequent recognition by the SCF-

βTrCP ubiquitin (Ub) ligase complex that covalently modifies IκB with Lys-48 poly-Ub

chains, which ultimately leads to the degradation of IκB by the 26s proteasome (Chen et

al., 1995). Once IκB is degraded, NF-κB dimers are liberated and predominantly shuttle

into the nucleus and bind to -κB promoter and enhancer elements involved in the

regulation of immunity, cell migration, cell adhesion, cell death and inflammation.

3.5 The Non-Canonical NF-κB Signaling Cascade1

The activation of the non-canonical or alternative NF-κB signaling cascade is tightly

regulated and has a much smaller group of ligands and receptors that can induce its

21

activation (Sun, 2012). Members of the TNF/TNFR superfamily, which include the

lymphotoxin-β receptor (LTβR), CD40, B-cell activating factor receptor (BAFFR),

receptor activator of NF-κB (RANK), TNFR2 and CD27 have all been shown to signal

through the non-canonical pathway (Sun, 2012). Upon ligation of TNF family members

to their receptors, the TRAF2-TRAF3-cIAP1/2 E3 complex is recruited to the receptor

(Sun, 2012). This recruitment leads to accumulation of TRAF2 molecules, allowing

TRAF2 to shift cIAP1/2’s Lys-48 Ub ligase activity towards TRAF3 via Lys-63 poly-

ubiquitination of cIAP1/2 (Sun, 2012), (Hostager et al., 2003). When TRAF3 is modified

via this mechanism, it ultimately leads to proteasomal degradation of TRAF3, allowing

for accumulation of MAP3K14 (also commonly known as NF-κB Inducing Kinase (NIK)),

resulting in increasing the levels of cytosolic NIK (Sun, 2012). Mechanistically, in an

unstimulated state, TRAF3 acts as a binding partner for NIK bringing it close to the

cIAP1/2 E3 ligase complex, which in this case leads to its degradation (Zarnegar et al.,

2008, Liao et al., 2004). NIK is essential for the processing of p100 into active p52(Xiao

et al., 2001), (Sun, 2012), which is a defining feature of the non-canonical NF-κB

signaling pathway. However, NIK has also been shown to phosphorylate IKKα, which

can act as a secondary kinase that mediates the activation of p100 (Senftleben et al.,

2001). For this to occur, NIK must be present in relatively high concentrations inside the

cell before interacting with IKKα. Thus, stabilization of NIK results in its accumulation

and interactions with IKKα, culminating in phosphorylation and activation (Sun,

2012),(Senftleben et al., 2001).

22

It is thought that IKKα-mediated phosphorylation of p100 occurs on Ser-872 in vitro and

Ser-866 and 870 in vivo(Sun, 2012),. While the cause of this discrepancy is currently

unclear, presumably, IKKα acts as a site-specific kinase for p100. This phosphorylation

step targets the C-terminal inhibitory domains (the PID and the ankyrin repeat domain)

of p100 for degradation via the proteasome(Sun, 2012),. Ankyrin repeat domains

(ARDs) have been shown to mask the nuclear localization sequence (NLS) of IκBα and

interestingly, p100’s phosphorylation site contains a sequence similar to IκBα (Sun,

2012),(Oeckinghaus and Ghosh, 2009). Therefore, disruption of the ARD of p100 via

proteasomal degradation may lead to unmasking of the NLS and subsequent

translocation of p52/RelB to the nucleus (Oeckinghaus and Ghosh, 2009). Once in the

nucleus, p52/RelB drives the transcription of a limited repertoire of genes, including

CCL19, CCL21, CXCL12, and CXCL13. In contrast, when the TNFR family members

are unstimulated, NIK is instead degraded via Lys-48 poly-ubiquitination(Sun, 2012).

Consequently, IKKα will not become activated and phosphorylate p100. Thus, in this

scenario, the NF-κB dimer cannot enter the nucleus and initiate transcription of target

genes. Due to its essential function, NIK represents a “bottleneck” in the non-canonical

NF-κB signaling cascade and is frequently targeted by mechanisms that have evolved

to negatively regulate this alternative pathway.

4 Negative Regulation of Inflammation

4.1 Inducible Negative regulators of Inflammation

Well-controlled mechanisms to resolve NF-κB activation are required to prevent its

potential destructive activities if this transcription factor if left unencumbered. These

23

include mechanisms that inhibit the action of NF-κB following its nuclear translocation.

There are over 785 different molecules that counteract the activity of NF-κB that are

either constitutively expressed in the cell, or induced by NF-κB following its activation;

there are even exogenous molecules that inhibit its activity (i.e. from plants) (Gilmore

and Herscovitch, 2006). The molecules (i.e. microRNA, proteins) that are induced are

often referred to as negative regulators, because they act to restore homeostasis via

negative feedback loops (Renner and Schmitz, 2009). Several, if not all of these

molecules serve a very important function that act as the “brakes” to halt NF-κB activity.

Certainly, these are not the only means utilized by the cell to reduce the activity of NF-

κB, but proteins expressed following NF-κB activation will be the focus here. Analogous

to an automobile losing its breaks, catastrophic outcomes can ensue if the cell loses

these abilities to halt the actions of NF-κB. These include diseases resulting from

abnormalities of the immune system, as well as certain types of cancers, mainly

lymphomas as well as GI cancers (Clevers, 2004), (Karin, 2009). One can anticipate

that loss of key components of a signal transduction cascade will result in severe

maladies, if not lethality. The same is true for cellular systems; in fact, mutations in

several of these proteins result in severe inflammatory disorders in both humans and

mice, if and when such mutations are actually viable.

4.2 Inhibitor of κB (IκB)1

There are 8 IκB proteins, known to negatively regulate NF-κB dimers, two of which are

the inhibitory regions on both p100 and p105 prior to proteosomal processing. The

mechanism of regulation functions, in the case of the canonical NF-κB pathway, by

24

binding to the Rel homology domain of NF-κB subunits via ankyrin repeats on the IκB

proteins. IκBα is induced by NF-kB, and this mechanism of induction demonstrates an

auto-regulatory feedback loop (Sun et al., 1993). The binding of IκBα to NF-κB results in

the concealment of the nuclear localization sequence on NF-κB dimers, thus keeping

NF-κB mainly contained in the cytosol under normal conditions (Ghosh and Hayden,

2012). This parallels the nuclear export signal on IκBα, which renders the flux of NF-κB

dimer mainly in the cytosol. Once IκBα is degraded by the proteasome, the nuclear

import signal on NF-κB is no longer blocked, and this allows for translocation to the

nucleus to regulate gene transcription (Sun et al., 1993). One question arises regarding

this feedback loop: if NF-κB upregulates IκBα, which contains a nuclear export signal,

and NF-κB is in the nucleus with a nuclear import signal, how does IκB-α access NF-κB

to halt its function? It turns out that both NF-κB and IκBα can shuttle into and out of the

nucleus; thus, they are in constant flux between these cellular compartments. When the

majority of IκBα is bound to NF-κB, the cytosolyic flux predominates and NF-κB is

limited to the nucleus. When NF-κB is free of IκBα, the nuclear flux predominates and

allows for NF-κB to bind enhancer regions on target DNA (Ghosh and Hayden, 2012).

4.3 Negative Regulation From The Interleukin Receptor Associated Kinase

Family1

There are four interleukin receptor associated kinases (IRAK) in mammals: three of

which are known as IRAK-1, IRAK-2 and IRAK-4. These three family members act as

positive regulators of NF-κB signal transduction (Thomas et al., 1999), (Muzio et al.,

1997), (Suzuki et al., 2002), (Kawagoe et al., 2008). One family member, IRAK-3 or

25

IRAK-M, is unique from the other family members, because it acts as an inducible

negative regulator of the canonical NF-κB signaling pathway (Wesche et al., 1999),

(Kobayashi et al., 2002). All four family members share a N-terminal death domain that

is important for homotypic protein-protein interactions with either MyD88, or with other

IRAK members for signal transduction. More central in the protein sequence for all

members is the kinase domain (Flannery and Bowie, 2010). IRAK-1 and IRAK-4 were

first described as being the only functional kinases in the family, with IRAK-2 and IRAK-

M being inactive or pseudo-kinases (Thomas et al., 1999), (Muzio et al., 1997), (Suzuki

et al., 2002), (Kobayashi et al., 2002). This was determined by both biochemical and

analytical methods. Analytically, the functional activity of a protein kinase can be

predicted by its primary amino acid sequence (Hanks and Hunter, 1995). There are

specific residues in the sequence of the kinase domain that are invariant for kinase

function, and these include residues in the kinase subdomain VIb known as the HRD

and DFG motifs (Hanks and Hunter, 1995), (Meylan and Tschopp, 2008). The invariant

aspartic acid residues, that are essential for proper function, are mutated in IRAK-2 and

IRAK-M to an asparagine and serine, respectively. This provides the rationale for the

nonfunctional activity of these IRAK family members (Meylan and Tschopp, 2008). All

IRAK proteins, apart from IRAK-4, contain a unique C-terminal domain (Flannery and

Bowie, 2010). This domain contains TRAF6 binding motifs that, as the name implies,

are important for interaction with TRAF6, and aid to propagate downstream signaling.

4.4 Molecular Mechanism of IRAK-M Function as a Negative Regulator1

26

Early functional studies suggested that IRAK-M shared a redundant function with IRAK-

1 and IRAK-2 (Wesche et al., 1999), despite the later prediction that IRAK-M was

inactive or a pseudo-kinase. In overexpression systems and luciferase reporter assays,

IRAK-1, IRAK-2, and IRAK-M were shown to function as positive regulators of NF-κB

signaling. However, with in vitro kinase assays, human IRAK-M was demonstrated to

have very weak intrinsic kinase activity, especially compared to IRAK-1 (Wesche et al.,

1999). Further, murine IRAK-M was shown to contain detectable kinase activity; albeit,

at a lower level when compared to the robust auto-phosphorylation of human IRAK-1

(Rosati and Martin, 2002). These data suggest that the auto-phosphorylation, and

therefore the kinase activity of IRAK-M is negligible for its function; however, it is

tempting to speculate that IRAK-M may indeed function as an active kinase on a

currently unknown cellular substrate following its induction. Despite these initial findings

and speculation, the prevailing literature suggests that IRAK-M functions as a negative

regulator of NF-κB signaling following TLR activation. These data are based on findings

utilizing genetically modified mice with targeted deletions of the Irak-m gene (Kobayashi

et al., 2002). The Irak-m gene contains 12 total exons and in order to define the role of

IRAK-M, exons 9-11 were targeted for deletion (Kobayashi et al., 2002). These exons

encode the amino acids predicted to constitute the putative kinase domain. Full length

IRAK-M was not detected when a western blot was performed with an antibody specific

for the C-terminus of IRAK-M (Kobayashi et al., 2002). Using these genetically modified

animals, Irak-m was found to be induced by NF-κB, along with IκB and A20 following

TLR stimulation, suggesting a negative feedback mechanism. Subsequent co-

immunoprecipitation experiments demonstrated that IRAK-M has the capacity to bind to

27

TRAF6, leading to the current model that IRAK-M functions to inhibit IRAK-1 and

TRAF6 interactions, which in turn inhibits downstream NF-κB activity (Kobayashi et al.,

2002). The expression of IRAK-M can be induced by other transcription factors, such as

C/EBP, Smad4, AP-1 and CREB (Lyroni et al., 2017), suggesting additional functions

beyond the feedback mechanism currently described. Phenotypically, Irak-m-/- mice do

not display any abnormal fetal or post-natal development, but do develop osteoporosis

later in life due to hyperactive osteoclast activity (Kobayashi et al., 2002), (Li et al.,

2005).

These Irak-m-/- mice were instrumental in defining IRAK-M as a negative regulator of

NF-κB signaling and have been widely utilized to define its function. For example,

consistent with increased NF-κB signaling, Irak-m-/- bone marrow derived macrophages

(BMDMs) display impaired endotoxin tolerance and hyper-production of inflammatory

cytokines (i.e. IL-6, TNF and IL-12p40) following TLR stimulation with specific PAMPS,

as well as, L. monocytogenes and S. typhimurium (Kobayashi et al., 2002). Consistent

with the ex vivo BMDM studies, Irak-m-/- mice were found to display enhanced small

intestinal inflammation following in vivo exposure to S. typhimurium (Kobayashi et al.,

2002). Interestingly, in these initial studies, Irak-m-/- mice do not display enhanced

morbidity or mortality following infection, despite the increased inflammation (Kobayashi

et al., 2002). These findings are consistent with a more recent study that showed the

Irak-m-/- mice are protected in models of experimental colitis and colitis associated

tumorigenesis (Rothschild et al., 2017). Mice lacking IRAK-M were found to have a

robust immune response to bacteria translocating from the lumen following chemical

28

induced damage to the intestinal epithelial cell barrier (Rothschild et al., 2017). The

attenuation in pathogenesis was associated with large expansions of gastrointestinal

associated lymphoid tissue (GALT), increased neutrophil function, and enhanced T-cell

recruitment (Rothschild et al., 2017). Complementary data revealed that the

gastrointestinal (GI) tract of the Irak-m-/- mice had a lower total colonic bacterial load

compared to wild type counterparts, which could also contribute to attenuation of

disease pathogenesis (Kesselring et al., 2016). Mechanistically, these data suggest the

immune system in Irak-m-/- animals is primed and more prone to a robust inflammatory

response. In the GI tract, this improves the efficiency of the host response to pathogenic

and commensal components of the host microbiome that drive disease processes.

It should be noted that while the consensus data identifies IRAK-M as a negative

regulator of NF-κB signaling, contrary data suggests a possible alternative mechanism.

Recently, it was demonstrated that IRAK-M participates in TAK-1 independent NF-κB

activation downstream of TLR7 through MEKK3 (Zhou et al., 2013). With the use of

IRAK-1/IRAK-2 double deficient mice and IRAK-1/-2/-M triple deficient mice, this study

demonstrated that under highly specific conditions, IRAK-M functions in the absence of

IRAK-1 and IRAK-2 and can actually activate NF-κB signaling (Zhou et al., 2013).

Mechanistically, IRAK-M interacts with IRAK-4 to form an IRAK-M myddosome complex

in the absence of both IRAK-1 and IRAK-2 that modulates NF-κB signaling through

TAK-1 independent mechanisms (Zhou et al., 2013). While these data are certainly

intriguing, the study ultimately concluded that IRAK-M exerts an inhibitory effect under

normal conditions by indirectly inducing inhibitory proteins (A20, SHIP-1, SOCS1 and

29

IκB-α) (Zhou et al., 2013). Further clouding mechanistic insight, it has recently been

revealed that the original Irak-m-/- mice commonly used to characterize this protein may

actually contain a truncated version of IRAK-M (Rothschild et al., 2017). Detailed

sequencing analysis of the Irak-m gene product was conducted following TLR

stimulation of BMDM from genotype confirmed Irak-m-/- mice (Rothschild et al., 2017).

Under these conditions, a splice variant of the Irak-m gene was identified that resulted

from the splicing of exon 8 with exon 12, in essence splicing around the neo cassette

(Rothschild et al., 2017). This type of splice variant is a common occurrence in

genetically modified animals where functional domains are targeted, as opposed to the

gene’s start site. Typically, these truncated proteins are dysfunctional and degraded by

the cell, which preserves the knockout status of the animals. It is also important to note

that the truncated IRAK-M protein has not yet been detected in situ and may not exist in

vivo (Rothschild et al., 2017). However, overexpression of Irak-mrΔ9-11 and functional

studies using the recombinant protein revealed that this truncation mutant is significantly

more potent at activating NF-κB signaling then the wild type version (Rothschild et al.,

2017).

Considering these data, we agree with the consensus findings that IRAK-M functions to

negatively regulate NF-κB signaling under normal conditions. However, the possibility

remains that IRAK-M may actually have a dual role under certain cell type or temporal

specific conditions to also activate NF-κB signaling. This could be directly correlated

with the cellular concentration of other IRAK family members or other critical cellular

substrates. Resolution of the crystal structure of IRAK-M would provide valuable insight

30

into these questions. It should also be pointed out that the history of IRAK-M is similar to

that of IRAK-2, which was first believed to function as an inactive pseudo-kinase

(Meylan and Tschopp, 2008). However, it was later determined that IRAK-2 function

was independent of IRAK-1 and plays a critical role in sustaining the late phase of NF-

κB signaling through potentially functioning as an active kinase (Kawagoe et al., 2008).

Future studies will indicate whether our interpretation into the function of IRAK-M will be

modified, as we have previously seen with IRAK-2.

4.5 IRAK-M and mucosal tissues

IRAK-M was appropriately named upon its discovery in mammals based on its

homology to the other IRAK family members and its tissue specific expression IRAK-M

mRNA expression was first found to be limited to cells of the myeloid lineage, hence the

“M” in IRAK-M. However, as scientific efforts intensified regarding the functional role of

IRAK-M, evidence emerged that suggested IRAK-M expression is not limited to cells of

myeloid origin. In fact, several reports have demonstrated the expression of IRAK-M in

a wide array of tissues comprising the bone, liver, and cells being osteoclasts, epithelial

cells, and neutrophils (Li et al., 2005), (Sumpter et al., 2011), (Kesselring et al., 2016),

(Rothschild et al., 2017). The Irak-m -/- mouse has greatly enhanced the current

understanding regarding the functional role of IRAK-M. Originally, Irak-m -/- mice

displayed enhanced inflammation in the small intestine when challenged with S.

typhimurium, and due to the revelation that these mice did not succumb to potentially

lethal effects due to endotoxic shock, it was concluded that these mice have enhanced

innate immunity compared to wild-type mice (Kobayashi et al., 2002). The lung and GI

31

tract are mucosal surfaces that are constantly interacting with foreign substances, often

described as luminal antigens, which include commensal microorganisms. Thus, these

mucosal tissues represent a first line defense against exposure to foreign substances,

and serve, therefore, as a strategic defense site for cells of the innate immune system.

For this reason, many studies have focused on the role of IRAK-M in the GI tract, as

well as other mucosal surfaces including the lung (Berglund et al., 2010), (Biswas et al.,

2011),(Deng et al., 2006). It is interesting to note that innate immune cells, particularly

monocytes isolated from septic patients display so called enhanced endotoxin

tolerance, and reduced response to antimicrobial peptides (Deng et al., 2006).

Interestingly, in peritonitis-induced sepsis models, Irak-m -/- mice display enhanced

bacterial clearance, and had higher survival rates compared to wild-type mice following

secondary challenge with the bacteria S. arengosa (Deng et al., 2006). This enhanced

bacterial clearance, and ultimate protection from endotoxin tolerance was attributed to

increased MIP-2 chemokine production, resulting in an influx of neutrophils to the lung

to handle secondary pulmonary bacterial challenge (Deng et al., 2006).

Several mouse models exist that are utilized to mimic GI inflammatory diseases, such

as ulcerative colitis and colitis-associated cancer (Okayasu et al., 1990), (Neufert et al.,

2007). These include both chemically induced models of colitis, as well as genetic

models utilizing mice that are predisposed to developing colitis (Wirtz et al., 2007),

(MacDonald, 1994). In one chemically induced model conducted with the agent dextran

sulfate sodium (DSS), Irak-m -/- mice displayed robust GI inflammation, with an

increase in plasma concentrations of inflammatory cytokines, namely IL-6 and TNF

32

(Berglund et al., 2010). Furthermore, Irak-m -/- mice displayed reduced splenic and

thymic weight, which normally increases in weight, and as these weights increase, a

correlation is observed with respect to increased disease severity of colitis (Berglund et

al., 2010). A genetic model of colitis is one that utilizes mice with a targeted disruption

for the gene that encodes the cytokine IL-10 (Kuhn et al., 1993). Regarding IRAK-M,

mice that are double deficient for IL-10/IRAK-M display increased inflammation in the

colon leading to exacerbated colitis, as well as increased pro-inflammatory cytokine

expression (Biswas et al., 2011). Evidence supports the claim that Irak-m-/- have an

enhanced inflammatory phenotype. This phenotype, however, appears uniquely titrated

at mucosal surfaces. Though these mice display robust inflammation following

pathogenic encounter, they also display enhanced innate immunity to these pathogenic

encounters, and mice are uniquely protected when monitoring their survival (Deng et al.,

2006), (Rothschild et al., 2017).

Regarding GI colitis-associated tumorigenesis, Irak-m -/- mice were first characterized

by robust tumorigenesis induction following completion of the AOM/DSS model

(Klimesova et al., 2013). Klimesova et al. attributed this finding to an altered microbiota

composition in Irak-m -/- mice, because wild-type mice were rescued from tumor

formation when supplemented with antibiotics in their drinking water. Conversely, Irak-m

-/- mice developed tumors both with and without antibiotics, and tumor formation in the

GI tract of Irak-m -/- mice was attributed to increased inflammatory cytokine production

(Klimesova et al., 2013). It is well recognized that inflammation is a growth promoting

condition that contributes to the progression of cancer (Greten et al., 2004); therefore,

33

the authors concluded that this was the main mechanism attributed to tumorigenesis in

the Irak-m -/- mice (Klimesova et al., 2013). It should be noted that microorganisms in

the GI tract are important contributing factors in models that use DSS as an inducing

agent for colitis. Laboratories often use different combinations of antibiotics to clear the

microbiota, which should minimize the catastrophic inflammatory effects of DSS;

however, this is not always the case. When a combination of ampicillin, vancomycin,

neomycin and mitronidizole are used in combination with DSS, mice often display

extreme morbidity and mortality, which is counter to what one would expect (Rakoff-

Nahoum et al., 2004),(Hernandez-Chirlaque et al., 2016). Results with a combination of

antibiotics should be interpreted with caution, because of heightened the variability of

due to fluctuations in the GI microbiota.

Reports from other groups have demonstrated Irak-m -/- mice with decreased tumor

progression in both xenograft models and AOM/DSS models (Xie et al., 2007),

(Standiford et al., 2011), (Kesselring et al., 2016), (Rothschild et al., 2017). This has

been attributed to enhanced innate immune functions in Irak-m -/- mice. Recently,

Kesselring et al. demonstrated reduced tumor progression in Irak-m -/- mice following

AOM/DSS colitis-associated tumorigensis (Kesselring et al., 2016). The authors of this

study reported increased GI inflammation in Irak-m -/- mice during early phase colitis

(i.e. acute colitis models); however, decreased inflammation driven tumorigenesis

following AOM/DSS. This decrease in tumor burden was attributed to multiple factors,

which include the absence of epithelial expressed IRAK-M, as demonstrated with bone

marrow chimeric mice, and substantiated by a reduced overall absolute bacterial load in

34

the colon of Irak-m -/- mice (Kesselring et al., 2016). Interestingly, co-housing studies,

which aim to transfer GI bacteria of mice, due to their coprophagic nature, did not

recapitulate tumor progression in wild-type mice with an inherited IRAK-M microbiome.

Therefore, the mechanism attributed to decreased carcinogenesis was the induced

expression of IRAK-M, leading to the stabilization of the transcription factor STAT-3,

which has a prominent oncogenic role in colon cancer (Kesselring et al., 2016),

(Jenkins, 2016), (Yu et al., 2014).

Evolution has provided unique and interesting mechanisms to tolerate commensal

microbes, while also maintaining an adequate immune response following pathogenic

encounter. There are multiple mechanisms, as well as negative regulatory proteins that

restore NF-κB transcriptional activity to homeostatic norms. It is clear that IRAK-M

participates in this process, and it is interesting that, at least in mice, tumor initiation and

progression appears to be reduced. This suggests that IRAK-M represents a novel

target for inhibition as a cancer therapeutic, with the intention of enhancing innate

immunity to limit the progression of cancer.

4.6 A20: An inducible negative regulator of inflammation that works via post-

translational modifications1

A20 or Tumor necrosis factor alpha-induced protein 3 (TNFAIP3) is a protein that is

rapidly induced by NF-κB, and functions through a feedback mechanism to halt the

canonical signaling cascade. A20 is one of the most studied negative regulatory

proteins targeting components of the NF-κB pathway and functions primarily by

35

modifying positive regulatory proteins in the cascade (Lee et al., 2000). The two

mechanisms in which A20 functions is by modifying the posttranslational status of target

proteins directly as a ubiquitin modifying enzyme (i.e. as both a ubiquitin ligase, and as

a protein deubiquitinase (Wertz et al., 2004). How can a single protein blunt NF-κB, and

other signaling pathways, by acting as both a ubiquitin ligase and de-ubiquitinase

(DUB)? The answer lies in the type of linkage of poly-ubiquitin chains to proteins.

Ubiquitin is a small 8.5 kDa peptide found in eukaryotic cells that can be covalently

attached to proteins to regulate their function post-translationally. This is accomplished

via the addition of poly-ubiquitin (poly-Ub) chains to amino acid residues on target

proteins. Ubiquitin itself contains seven different lysine residues, and the particular

lysine chain that is utilized to make the ubiquitin chain determines the regulatory status

of the protein. The seven-lysine residues found on ubiquitin are K6, K11, K27, K29,

K33, K48, and K63, with K48 and K63 linkages being the best studied, with currently

known regulatory function (Tenno et al., 2004), (Varadan et al., 2004). Generally, when

a target protein is modified by Lys-48-linked poly-ubiquitination, this serves as a

molecular tag on the protein for recognition, and further destruction by the 26S

proteasome (Wilkinson et al., 1980), (Voges et al., 1999). Additionally, Lys-63 linked

poly-Ub is a posttranslational modification that generally results in the activation of

tagged proteins (Tenno et al., 2004). In terms of A20, this describes the mechanism for

its ubiquitin ligase domain; removing Lys-63 linked poly-Ub chains from a protein results

in the attenuation of activity of the protein. By also functioning as a ubiquitin ligase, A20

has been shown to inhibit the canonical NF-κB pathway by the addition of Lys-48 linked

poly-Ub chains to receptor interacting protein 1 (RIP1) (Wertz et al., 2004), (Bertrand et

36

al., 2008). RIP1 is an essential molecule that activates canonical NF-κB pathway

downstream of the TNF receptor. Addition of Lys-48 linked poly-Ub chains to RIP1

targets this protein for degradation, thus removing a positive signal with the intent of

restoring cellular homeostasis.

The importance of A20 as a key negative regulator of inflammation was demonstrated

with the use of A20 knockout mice (Lee et al., 2000). In the absence of A20, mice

display a robust inflammatory phenotype in the liver, intestine, bone joints, skin and

kidney (Lee et al., 2000). Furthermore, severe cachexia occurs in several organs, and

mortality commences postnatally within a few weeks of age; this can be perpetuated

further when mice are given low doses of TNF or LPS (Lee et al., 2000). A20 does not

appear to be crucial for fetal development, because mice display an appropriate

Mendelian ratio when born. It was demonstrated in mouse embryonic fibroblasts (MEFs)

that in the absence of A20, NF-κB transcriptional activity is sustained, and IκB-α mRNA

is transcribed with similar, even possibly increased levels when compared to wild type

mice. However, IκB-α protein levels do not rebound following TNF treatment. This was

attributed to the continued activity of the IKK complex, because in the presence of a

proteasome inhibitor named MG-132, IκB-α levels were restored. Furthermore, it was

demonstrated that IκB-α was continually phosphorylated by the IKK complex following

translation, which resulted in its constant degradation in A20 null cells (Lee et al., 2000).

A20-Tnf and A20-Tnfr1 deficient mice also develop severe spontaneous inflammation,

indicating a role for A20 in modulating either alternative innate immune pathways, or

adaptive immune pathways (Boone et al., 2004). LPS stimulated A20-Tnf double

37

deficient BMDMs display a robust production of IL-6 and nitric oxide (NO) compared to

WT, which was slightly attenuated compared to A20 deficiency alone. This, along with

robust inflammation in A20-Rag1 double knockouts, suggested that A20 played a

prominent role in negatively regulating innate immune pathways beyond those regulated

by TNF (Boone et al., 2004).

Indeed, A20 was demonstrated to robustly regulate TLR signaling, as MyD88-A20

double deficient mice do not develop severe multi-organ inflammation and cachexia as

seen in A20 knockout mice alone Furthermore, components of the microbiota were

found to drive spontaneous inflammation, as chimeric mice reconstituted with A20-/-

bone marrow are rescued from severe spontaneous inflammation when treated orally

with an antibiotic cocktail that diminishes the intestinal microbiota (Turer et al., 2008).

4.7 CYLD1

CYLD is a constitutively expressed DUB that was originally described as a tumor

suppressor gene (Bignell et al., 2000). Loss of function of CYLD in humans is

associated with familial cylindromatosis, which are multiple benign skin cancers usually

localized to the face and neck (Bignell et al., 2000). Additional mechanistic studies

revealed that loss of CYLD contributes to an overall cellular resistance in apoptosis

(Brummelkamp et al., 2003), which likely contributes to cancer pathogenesis. CYLD is a

member of the Ub-specific protease family (USP), which represents the largest family of

DUBs in the human genome (Nijman et al., 2005). As a bona fide cysteine protease,

CYLD cleaves Lys-63 linked chains of poly-Ub on target proteins (Komander et al.,

38

2008). This serves as a repressive function because addition of poly-Ub on Lys-63 is

generally a post-translational modification that leads to increased activity of modified

proteins. Interestingly, in addition to Lys-63 mediated de-ubiquitination, CYLD has the

ability to de-ubiquitinate via linear Ub chains (Sato et al., 2015). It is interesting to note

that CYLD and A20 cleave Ub chains on several of the same substrates that participate

in the signal transduction cascade in the NF-κB pathway, including but not limited to

RIP1, IKKγ, TRAF2 and TRAF6 (Kovalenko et al., 2003, McDaniel et al., 2016,

Trompouki et al., 2003, Brummelkamp et al., 2003). This overlap in negative regulatory

function demonstrates the importance of signaling molecules, such as TRAF6, as

primary nodes in the induction of the inflammatory cascade. The post-translational

modification of several of the same molecular targets between A20 and CYLD could be

due to steady-state and inducible negative regulation. Further kinetics and substrate

avidity studies are necessary to explain the similar molecular targets between these two

molecules.

Studies utilizing independently generated Cyld-/- mice have yielded highly insightful

findings, albeit somewhat conflicting. In one study, the Cyld-/- animals demonstrated

significant defects in NF-κB and JNK signaling in macrophages following TLR and CD40

stimulation (Zhang et al., 2006). However, this effect appeared to be stimuli specific, as

TNF stimulation did not appear to be impacted by CYLD deficiency (Zhang et al., 2006).

Importantly, this study observed normal B and T-cell development and function (Zhang

et al., 2006). While there are several differences in the proposed mechanistic actions of

CYLD, these findings are in general agreement with other groups using these and other

39

independently generated Cyld-/- animals (Massoumi et al., 2006), (Lim et al., 2007).

However, at least one additional group has not observed these phenotypes using

another independently generated Clyd-/- mouse line (Reiley et al., 2004). These animals

did not recapitulate the defects in NF-κB, JNK, ERK, or p38 signaling (Reiley et al.,

2004). Rather, these animals showed significant attenuation of T cell receptor (TCR)

signaling in the absence of CYLD (Reiley et al., 2004).

5. Inflammatory maladies of the GI tract

5.1 Inflammatory Bowel Diseases

Inflammatory bowel disease (IBD) is an inclusive term for two similar, yet distinct

diseases, being ulcerative colitis (UC) and Crohn’s disease (CD); both diseases are

characterized by aberrant inflammation resulting in the pathogenesis of tissues localized

to the gastrointestinal (GI) system. The distinguishing features between these two

diseases are based on the localization of the GI inflammation, gross pathology, and

microscopic pathological characteristics (Ahmad et al., 2002), (Satsangi et al., 2006).

Based on locality, ulcerative colitis is generally limited to inflammation of the large

bowel, also termed the colon; whereas Crohn’s disease can cause inflammation-driven

pathologies, usually in an intermittent pattern, affecting any portion of the GI tract, even

the mouth. The most common GI area affected in Crohn’s disease is the colon and

terminal ileum of the small intestine (Abraham and Cho, 2009). Crohn’s disease patients

can have inflammatory lesions that affect all GI tissue layers, defined as transmural

inflammation; whereas, inflammation in ulcerative colitis usually only affects the

mucosal layer of the colon (Abraham and Cho, 2009). Ulcerative colitis, as the name

40

implies, display distinct ulcers present in the colon that may contribute to blood in the

stool. Crohn’s disease patients may have similar ulcerative lesions, but a diagnosis can

be distinguished over ulcerative colitis if these lesions are found in other interspersed

portions of the GI tract.

Gross pathologies are a method utilized to confirm a diagnosis between ulcerative

colitis and Crohn’s disease; however, because IBD is a multifactorial disease of differing

severities in the human population, diagnosis is not always simple (Yantiss and Odze,

2006). Gastroenterologists have built upon two models, aimed to guide clinicians to

determine if a patient suffers from ulcerative colitis or Crohn’s disease, which include

methods of identification termed the Vienna and Montreal models, which were

conveniently named after the cities where gastroenterologists convened when the

guidelines were established(Satsangi et al., 2006). The Vienna model, first proposed in

1998, described features to distinguish Crohn’s disease based on three predominant

elements. These include A, B and L classifications, which designated for age of onset

(A), disease behavior, and location of the disease (L), respectively (Satsangi et al.,

2006). In 2005, gastroenterologists reassessed the Vienna model and came up with

new guidelines that describe the Montreal model, which expanded on the guidelines to

identify Crohn’s disease, as well as the distinguishing features of ulcerative colitis

(Satsangi et al., 2006). Thus, the more comprehensive Montreal model is the preferred

system used among clinicians (Spekhorst et al., 2014).

5.2 Factors that influence IBD pathogenesis

41

There are several genetic mutations that may predispose individuals to IBD. Genome

wide association studies (GWAS) have demonstrated that NOD2 and IL23r mutations

are associated with the predisposition of individuals to Crohn’s disease; however, there

is currently no single gene mutation solely responsible for IBD pathogenesis (Ma et al.,

1999), (Hugot et al., 2001), (Satsangi et al., 1996), (Kanaan et al., 2012). Interestingly,

more genetic mutations have been found that are associated with predisposing

individuals to Crohn’s disease compared to ulcerative colitis, as demonstrated via

monozygotic twins, but different genetic alterations are seen in both diseases

(Thompson et al., 1996), (Orholm et al., 2000). A definitive cause of IBD onset and

pathogenesis is currently unknown, yet environmental and genetic components have

been described to play a significant role (Podolsky, 1991). Patients that have the

highest predisposition for IBD are Caucasians, usually of European decent, and disease

onset usually takes place in the fertile years of a patient’s life (Probert et al., 1993).

Interestingly, males and females are equally likely to develop IBD, thus, there are no

gender biases seen in human patients (Rosenblatt and Kane, 2015). Furthermore,

because there are several components that contribute to the development of IBD,

treatment regimens are generally aimed to improve quality of life for patients, as no cure

currently exists for both Crohn’s disease and ulcerative colitis (Braus and Elliott, 2009).

5.3 The Interrelationship Between Inflammation and Cancer

Chronic inflammation, which is common in IBD patients, is an enabling characteristic of

cancer (Hanahan and Weinberg, 2011). Notably, IBD patients, especially ulcerative

colitis patients, are at a heightened risk for the development of GI related cancers,

42

termed colitis-associated cancer (Thorsteinsdottir et al., 2011). Empirical evidence was

provided in mice that ultimately linked inflammation and cancer when components of the

NF-κB signaling cascade were mutated (Greten et al., 2004). Components of the

immune system are important players in this context, because they normally contribute

to the inflammatory milieu, especially in response to pathogens. As was seen earlier,

aberrations in NF-κB signaling can result in hyperactive inflammatory signaling, and

production of these cytokines can have a profound impact on the surrounding tissue. In

terms of chronic inflammation, this can result in a pro-growth environment that leads to

cancer progression. The revelation that inflammation is highly regulated by components

of the immune system, it is vital to understand the contribution of the immune system to

inflammatory related diseases, such as IBD.

Concluding Remarks

Several factors contribute to proper physiology and maintenance of the GI system.

Having a comprehensive understanding at the cellular level is a good starting point for

advancing therapeutic potential to treat maladies, particularly IBD and colon cancer.

For this reason, it is important to investigate specific components of cellular signaling

pathways, such as negative regulatory proteins, to determine their contribution

pertaining to these particular diseases. Further, broadening our perspectives to

encompass instances that alter the interactions of host cells and endogenous microbes

shows great potential to uncover unknown aspects that contribute to disease. There will

always be a need to understand these complex phenomena, and hopefully recent

43

advances, as well as ones to come, will not only impact our understanding, but also

strengthen treatment options for patients in the near future.

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Chapter 2:

Enhanced Mucosal Defense and Reduced Tumor Burden in Mice with the

Compromised Negative Regulator IRAK-M

Published as:

Rothschild, D. E., Zhang, Y., Diao, N., Lee, C. K., Chen, K., Caswell, C. C., Slade, D. J.,

Helm, R. F., Leroith, T., Li, L. & Allen, I. C. 2017. Enhanced Mucosal Defense and

Reduced Tumor Burden in Mice with the Compromised Negative Regulator IRAK-M.

EBioMedicine, 15, 36-47.

A. Abstract

Aberrant inflammation is a hallmark of Inflammatory Bowel Disease (IBD) and colorectal

cancer. As discussed in the previous chapter, the inflammatory signaling pathway has

many regulatory mechanisms, both through positive and negative regulation. IRAK-M is

a critical negative regulator of TLR signaling and overzealous inflammation. Here we

utilize data from human studies and Irak-m-/- mice to elucidate the role of IRAK-M in the

modulation of gastrointestinal immune system homeostasis. In human patients, IRAK-M

expression is up-regulated during IBD and colorectal cancer. Further functional studies

in mice revealed that Irak-m-/- animals are protected against colitis and colitis associated

tumorigenesis. Mechanistically, our data revealed that the gastrointestinal immune

system of Irak-m-/- mice is highly efficient at eliminating microbial translocation following

epithelial barrier damage. This attenuation of pathogenesis is associated with expanded

areas of gastrointestinal associated lymphoid tissue (GALT), increased neutrophil

migration, and enhanced T-cell recruitment. Further evaluation of Irak-m-/- mice revealed

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a splice variant that robustly activates NF-κB signaling. Together, these data identify

IRAK-M as a potential target for future therapeutic intervention.

B. Introduction

Crohn’s disease (CD) and ulcerative colitis (UC) represent the clinical manifestations of

inflammatory bowel disease (IBD). In humans, there is a strong association in patients

afflicted with IBD to further develop colitis associated cancer (CAC) (Greten et al.,

2004). Gastrointestinal (GI) immune homeostasis is, in part, maintained by pattern

recognition receptors (PRRs); including, but not limited to the Toll-like receptors (TLRs).

There are 10 known TLRs in humans that recognize pathogen associated molecular

patterns (PAMPs) associated with invading microbes. (Kawai and Akira, 2011). In the

gut, TLRs sense and respond to commensal flora translocation, resulting from damage

to the epithelial cell barrier during the course of disease progression (Saleh and Elson,

2011). Following TLR engagement to its pathogenic ligand, a highly coordinated

signaling cascade leads to the activation of specific transcription factors including

nuclear factor-κB (NF-κB), activating protein-1 (AP-1), or in the case of viral recognition,

interferon regulatory factor-3 (IRF-3). The dominant signaling pathway utilized depends

upon which TLR is stimulated in response to the pathogenic ligand; however, all TLRs,

with the exception of TLR-3, signal through the myeloid differentiation factor 88 (MyD88)

adaptor molecule (Kawai et al., 1999), (Takeuchi et al., 2000), (Jiang et al., 2006). A

MyD88 independent pathway exists for both TLR-3 and TLR-4 that has been shown to

signal through the adaptor molecule TIR-domain-containing-adapter-inducing interferon-

β (TRIF) (Yamamoto et al., 2003), (Hoebe et al., 2003), (Kawai et al., 2001). In healthy

69

individuals, these TLR signaling cascades are responsible for rapid activation of innate

host defenses and further activation of the adaptive immune response (Medzhitov and

Janeway, 1997). However, uncontrolled activation of TLR signaling promotes chronic

inflammation and is associated with a variety of autoimmune diseases, including IBD

(Cook et al., 2004).

The TLRs that utilize the MyD88 dependent pathway have been reported to signal

through a family of Interleukin Receptor Associated Kinases (IRAKs), which contain four

known family members being: IRAK-1, IRAK-2, IRAK-M, and IRAK-4 (Wesche et al.,

1997), (Muzio et al., 1997), (Wesche et al., 1999), (Li et al., 2002). IRAK-1, IRAK-2, and

IRAK-4 have been described to play a role in the transduction of downstream signaling

from MyD88; conversely, IRAK-M is rather unique and has been shown to be a negative

regulator of TLR signaling (Kobayashi et al., 2002). The initial reports pertaining to

IRAK-M described it as a member of the Pelle/IRAK family, and when overexpressed in

mammalian cells, had the capacity to activate NF-κB through interactions with TNF

receptor associated factor-6 (TRAF-6) (Wesche et al., 1999). Following the generation

of an Irak-m-/- mouse, IRAK-M was confirmed to function as a negative regulator of TLR

signaling (Kobayashi et al., 2002).

Here we report that IRAK-M is significantly up-regulated in human patients with IBD and

colitis associated neoplasia. We also show that IRAK-M is up-regulated in advanced

stages of colorectal cancer (CRC). Prior studies have evaluated IRAK-M in models of

experimental colitis and colitis associated tumorigenesis utilizing Irak-m-/- mice and

70

shown that these animals are highly sensitive to both inflammation and neoplasia.

However, contrary to these prior observations, more recent studies have revealed that

Irak-m-/- mice are actually protected from inflammation driven tumorigenesis in the colon

(Kesselring et al., 2016). IRAK-M was found to support colorectal cancer progression

through the reduction of antimicrobial defenses and the stabilization of STAT-3

(Kesselring et al., 2016). Due to these conflicting reports, we sought to better elucidate

the contribution of IRAK-M during IBD and colitis associated tumorigenesis. Our studies

are, in general, complementary to the more recent findings that support a role for IRAK-

M in disease pathogenesis. Here we report that Irak-m-/- mice were significantly

protected against dextran sulfate sodium (DSS)-mediated GI inflammation and

azoxymethane (AOM)/DSS mediated tumor formation. We further show that the

immune system in the GI tract of Irak-m-/- mice is primed and highly efficient at

eliminating components of the microbiome translocating from the GI lumen following

damage to the epithelial barrier. This priming and attenuation of disease appears to be

associated with expanded areas of gastrointestinal associated lymphoid tissue (GALT),

increased and efficient neutrophil migration, and enhanced T-cell recruitment.

Upon further evaluation of the Irak-m-/- mice, we discovered the formation of a Irak-m

splice variant. The splicing event joins exon 8 with exon 12, splicing around the

neomycin resistance cassette (Kobayashi et al., 2002), forming a Irak-mrΔ9-11 truncation

mutant. This truncation mutant functions to robustly induce NF-κB signaling when

overexpressed in HEK293T cells. Together, our data confirms previous findings that

71

IRAK-M functions as a critical mediator of inflammatory signaling pathways and is

essential in maintaining immune system homeostasis in the GI system.

C. Materials and Methods:

Human Metadata Analysis

IRAK-M expression was assessed using a publicly accessible microarray metadata

analysis search engine (http://www.nextbio.com/b/nextbioCorp.nb), as previously

described (Kupershmidt et al., 2010). The following array data series were analyzed to

generate the human patient and cell expression data: GSE10714; GSE59071;

GSE9686; GSE13367; GSE36807; GSE16879; GSE10191; GSE52746; GSE9452;

GSE38713; GSE4183; GSE37283; GSE37364; GSE10715.

Reagents

Ultra-pure LPS from E.coli, PGN-SA, Pam3CSK4, Poly (I:C) H.M.W, Flagellin-BS, ODN-

1668, Imiquimod were purchased from Invivogen. Mouse TNF-α was purchased from

Biolegend. Mouse IL-6, IL-10, TNF-α, IL-1β and IFNγ ELISA kits were purchased from

BD Biosciences. Rabbit polyclonal IRAK-M antibody was purchased from Millipore

(Millipore 07-1481). Rabbit monoclonal β-Actin (Cell Signaling 13E5), Rabbit

monoclonal-HA (Cell Signaling C29F4) and anti-rabbit-HRP were from Cell Signaling.

Antibodies for Ly6G, CD11b, CXCR2, CD14, Ly6C, IAE, and CD62L were from

Biolegend. PCR primers were generated against exons of murine Irak-m utilizing

Primer3 software and purchased from Integrated DNA Technologies. Dextran sulfate

sodium DSS salt (36,000-50,000 M.Wt.) was purchased from MP Biomedicals.

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Azoxymethane (AOM) was from Sigma. RNA isolation Miniprep kit was from Zymo

Research. Espresso mammalian expression CMV cloning kit was purchased from

Lucigen. Plasmid pGL4.32[luc2P/NF-ĸB-RE/Hygro] and pRL-null were from Promega.

Cell Culture

HEK293T cells (ATCC) were grown in Eagle’s Minimum Essential Medium (EMEM) +

10% Fetal bovine serum (FBS) (Atlanta Biologicals). THP-1 cells (ATCC) were grown in

RPMI + 10% FBS + 0.05 mM 2-mercaptoethanol.

Bone Marrow Derived Macrophage Assessments

Mouse bone marrow was harvested from both tibias and femurs for individual mice and

plated in non-tissue culture treated sterile petri dishes containing 1x DMEM

supplemented with 10% FBS + 20% L929 conditioned media for 7 days. Bone marrow

derived macrophages (BMDMs) were re-plated on day 7 by aspirating L929 growth

media and incubated with 10 ml ice cold sterile 1x PBS containing 5mM EDTA. Cells

were incubated at 4 oC for 30 minutes and gently scraped following incubation. Cell

containing supernatants were transferred to 50 ml conical tubes and centrifuged for 10

minutes at 300 x g. BMDMs were resuspended in 20 ml warm growth media and

replated in tissue culture treated sterile plastic ware at: 2x105 cells per well in triplicate

for 24 well plates; 2x106 cells per 60mm2 dish; 5x106 cells per 10mm2 dish. Wild-type

and Irak-m-/- BMDMs were re-plated in separate 24 well plates and allowed to adhere

overnight. Cell culture media was aspirated the following day and BMDMs were washed

with 1 ml 1x PBS. Following PBS aspiration the cells were treated with 500 μl total

73

volume per well of 1x DMEM +10% FBS containing specific pathogen associated

molecular patterns (PAMPs) for 24 hours at a concentration of: Poly I:C (6.25 μg/ml),

PGN-SA (10 μg/ml), LPS (10 ng/ml), Pam3CSK4 (300 ng/ml), TNF-α (75 ng/ml).

Colon organ culture supernatants

Following the acute colitis model or AOM/DSS model, colon sections were also

collected to establish organ cultures to determine local cytokine levels as previously

described (Greten et al., 2004), (Allen et al., 2010), (Williams et al., 2015). Briefly,

approximately 1-cm2 strip of the distal colon was removed and washed with 200 U/ml

Penicillin and 200 μg/ml streptomycin in 1x PBS. Following the wash, each respective

colon section had the total mass measured and recorded for downstream normalization.

Each individual distal colon section was then transferred to an individual well of a 24

well plate and supplemented with 1ml of DMEM media containing 200 U/ml Penicillin

and 200 μg/ml streptomycin with no additional supplements. Following overnight

incubation, the supernatants were collected and centrifuged at 300 x g for 10 minutes to

collect cell free supernatants. ELISAs were then conducted on the supernatants to

determine the respective cytokine level and each sample was normalized to their

respective colon section mass.

Bacterial Counts.

Bacterial counts were determined from mouse whole blood collected via cardiac

puncture. 25 μl of whole blood was plated, as well as two 10 fold serial dilutions were

74

prepared in sterile 1x PBS and 25 μl aliquots were plated on LB agar plates. Following

overnight incubation at 37 oC, bacterial colonies were counted.

Western Blot

Experiments utilizing WT and Irak-m-/- BMDMs were seeded in 60mm2 dishes and

allowed to adhere overnight. Experiments with HEK293T cells were seeded in 6 well

plates at 3x105 cells per well. BMDMs were treated with control media or stimulated with

10 ng/ml LPS, 10 μg/ml PGN-SA or 300 ng/ml Pam3CSK4 for 24 hr. Cells and BMDMs

were washed with ice cold 1x PBS and were subsequently lysed in cell lysis buffer

containing: 2% SDS (Fisher Sci), 100 mM NaCl (Fisher Sci), 10 mM Tris-HCl pH 7.4

(Fisher Sci), 1x Halt Protease inhibitor (Thermo). Whole cell lysates were sonicated to

shear endogenous nucleic acids. Protein concentration was quantified by BCA (Pierce)

and 15 - 20 μg total protein was boiled in 1x Blue Loading Buffer + 1x DTT (Cell

Signaling) at 95 oC for 5 min and electrophoresed with either Bolt 4-12% Bis-Tris gels

(Thermo) or Biorad 4-12% Criterion XT Bis-Tris Gel in 1x MOPs buffer (Thermo). Gels

were transferred onto 0.2 μm PVDF membranes (Millipore) in 1x Tris-glycine + 20%

methanol. Membranes were blocked for 30 minutes with 5% non-fat dry milk (Carnation

Brand) dissolved in TBS+0.05% Tween 20 (TBST). Primary antibodies were used

1:1000 and incubated at 4 oC overnight with gentle shaking. Primary antibodies were

either: anti-IRAK-M (Millipore 07-1481), anti-β-Actin (Cell Signaling 13E5) and anti-HA

(Cell Signaling C29F4). IRAK-M, β-Actin and HA were visualized using anti-rabbit IgG

linked to HRP, detected with ECL western blotting substrate Pico (Pierce) or Dura

(Pierce) by x-ray radiography. Densitometry was analyzed using ImageJ software.

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RT-PCR

BMDM were seeded in 10mm2 dishes and allowed to adhere overnight. Cells were then

treated with 1x DMEM +10% FBS without PAMP stimulation or 300 ng/ml Pam3CSK4

(Invivogen) for 24 hr. RNA was isolated using Zymo Research miniprep kit. Total RNA

was quantified and 2 μg was converted to cDNA with High Capacity cDNA reverse

transcription kit (Applied Biosystems) according to the manufacture’s instructions. 5-10

ng of cDNA was amplified for different exons of murine Irak-m by RT-PCR. The primer

sequences used for Irak-m exons are:

Irak-m Exon Primers

Exon: Forward Primer (5’3’) Reverse Primer (5’3’)

5-8 GGACATTCGAAACCAAGCAT TGTGCCATTTGTGCACTGTA

9-10 CCAGCTCCAACCCAAACTAA TGTTTCGGGTCATCCAGCAC

10-11 GGACCTCCTCATGGAACTGA CCAGAGAGGACAGGACTTCG

12 TCCTTCAGGTGTCCTTCTCCACTG CCTCTTCTCCATTGGCTTGCTC

5-12 GTGCAGAGAAAACGACCCTG TGGGAGGGTCTTCTGCAAAA

RT-PCR products were electrophoresed in 1x TBE and visualized on 1% agarose gel

stained with ethidium bromide.

Molecular Cloning, Overexpression and Dual Luciferase Assay.

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Molecular cloning of the Irak-m gene of both wild-type (Irak-m) and mutant (Irak-mrΔ9-11)

was performed from BMDMs stimulated for 24 hr with 300 ng/ml Pam3CSK4. RT-PCR

was conducted utilizing Phusion DNA high fidelity polymerase (Thermo) with primer

sequences generated with homology to either the WT Irak-m gene, or the Irak-mrΔ9-11

gene, and the espresso CMV cloning and expression system (Lucigen) according to the

manufacture’s instructions. Primer sequences are:

CMV-Irak-m-HA Cloning Primers

WT Irak-m-HA

(Forward) 5’3’

GAAGGAGATACCACCATGGCCGGCCGGTGCGGGGCCCGT

WT Irak-m-HA

(Reverse) 5’3’

GGGCACGTCATACGGATACTGCTTTTTGGACTGTTCATG

Irak-mrΔ9-11-HA

(Forward) 5’3’

GAAGGAGATACCACCATGGCCGGCCGGTGCGGGGCCCGT

Irak-mrΔ9-11-HA

(Reverse) 5’3’

GGGCACGTCATACGGATAAGGACGTGGGAGGGTCTT

6-well plates (Corning) were seeded with 3x105 HEK293T cells (ATCC) per well and

allowed to adhere overnight. Cells were transfected with a total of 2.7 μg of DNA using

Lipofectamine P3000 reagent (Thermo) according to the manufacturer’s instructions.

Transfection included: 100 ng pGL4.32[luc2P/NF-ĸB-RE/Hygro] (promega), 100 ng

pRL-null (promega), and decreasing amounts of either pME-CMV-Irak-m-HA tagged

(625ng-10ng); pME-CMV-Irak-mrΔ9-11-HA tagged (2500ng-40ng); 500 ng pME-CMV-β

77

galactosidase-HA; or 2500 ng pME-CMV-Empty Vector. The total amount of transfected

plasmid DNA remained constant for each treatment by co-transfecting with the proper

amounts pME-CMV-Empty Vector. 500 ng pME-CMV- -HA served as a

positive control for transfection efficiency. Following 18 hr of transfection, a pME-CMV-

galactosidase-HA transfected well was treated with 10 ng/mL human IL-

for 6 hr as a positive control for the dual luciferase assay. Following 24 hr transfection,

luminescence catalyzed by firefly luciferase and renilla luciferase of the cell lysates was

determined using Dual-Luciferase Reporter Assay System (promega) according to the

manufacturer’s instructions.

Experimental Animals

All mouse studies were approved by the Institute for Animal Care and Use Committee

(IACUC) at Virginia Tech and in accordance with the Federal NIH Guide for the Care

and Use of Laboratory Animals. The Irak-m-/- mice were generated as previously

described (Kobayashi et al., 2002) and purchased from The Jackson Laboratory. All

studies were controlled with either littermate and/or co-housed WT animals that were

maintained under specific pathogen-free conditions and received standard chow

(LabDiet) and water ad libitum. All experiments described utilized age matched male

mice.

Experimental Colitis and Colitis Associated Tumorigenesis

In order to assess acute experimental colitis, mice were given either 3 or 5% DSS

dissolved in drinking water available ad libitum for 5 days as previously described

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(Williams et al., 2015b), (Schneider, 2013). On day 5, mice were withdrawn from DSS

and given regular drinking water ad libitum until euthanasia was performed on day 7.

Cumulative semi quantitative clinical scores for acute experimental colitis were

assessed as previously described (Williams et al., 2015b), (Schneider, 2013).

Tumorigenesis was induced via a single intraperitoneal (i.p.) injection of AOM (10 mg/kg

of total body weight) and supplemented with three cycles of 2.5% DSS in drinking water

available ad libitum for 5 days with 2 weeks of recovery between cycles, as previously

described (Neufert et al., 2007), (Allen et al., 2010). While subjected to DSS, mice were

monitored for weight loss, physical body condition, stool consistency, and rectal

bleeding. Upon completion of each model, whole blood was collected by cardiac

puncture for bacterial counts, flow cytometry assessments of leukocyte populations, and

serum isolation. Colon sections were collected for H&E staining. Blinded to treatment

and mouse genotype, examination of histopathology was conducted by a board-certified

veterinary pathologist (T.L.). Colon H&E sections were evaluated and scored as

previously described (Williams et al., 2015b). Additional colon sections were further

prepared for immunohistochemistry and stained with anti-β-catenin and DAPI to

determine β-catenin levels in the AOM+DSS studies.

FACS

Leukocytes and lymphocytes were evaluated from either: bone marrow, spleen, or

whole blood utilizing flow cytometry. Cells were immunostained with antibodies for the

respective cell surface markers prior to FACS analysis. Sorted cells were further

evaluated for CXCR2, CD14, CD11b+, Ly6G+, CD4+, CD8+, Ly6C+, IAE, and CD62L.

79

ELISA

Cell culture supernatants or colon organ culture supernatants were collected from each

individual well in 1.7 ml tubes and centrifuged at 300 x g for 10 minutes to remove

residual cells. Cell-free supernatants were then assayed for mouse IL-6 and/ or IL-10

(BD Biosciences) according to the manufacturer’s instructions.

Statistical Analysis

Data are represented as mean ± standard error of mean (S.E.M.) unless otherwise

indicated. Graphs and statistical analysis were conducted via GraphPad PRISM

software. Complex data sets were analyzed by 1 way analysis of variance (ANOVA)

and followed by either Tukey-Kramer HSD or Newman-Keuls method. The Kaplan-

Meier test was conducted to determine group survival. A value of p<0.05 were

considered statistically significant.

D. Results

IRAK-M Expression is Significantly Increased in Human Patients with IBD and

CRC

Previous studies have shown that IRAK-M expression is increased in IBD patients

(Fernandes et al., 2016), (Gunaltay et al., 2014). Thus, we initially sought to evaluate

these findings and expand our analysis to further evaluate IRAK-M expression in the

context of colitis associated neoplasia and CRC using a retrospective metadata analysis

of publicly available gene expression data (Kupershmidt et al., 2010). Our analysis

revealed that the relative expression of IRAK-M is significantly increased in human

80

patients with active forms of IBD (Figure 2.1A). Patients that suffer from IBD have a

higher predisposition to CAC (Karlen et al., 1999); thus, we also included CAC patients

in our data analysis and found that IRAK-M is also significantly increased in patients

with active UC with inclusive areas of neoplasia (Figure 2.1A). Independent of CAC, we

were also interested in evaluating IRAK-M expression in the context of CRC. Thus, we

also analyzed expression levels of IRAK-M in patients diagnosed with both low and

high-grade CRC. CRC patients were stratified based on the Dukes’ staging system

(A/B) and (C/D). From these data, it is apparent that IRAK-M expression is significantly

increased in patients with more advanced CRC (grades C/D) compared to the patients

with less advanced CRC (grades A/B) and patients not diagnosed with CRC (Figure

2.1B). To gain greater insight into IRAK-M function, we next sought to evaluate IRAK-M

expression in different human cell types of relevance to IBD, CAC, and CRC, with

particular emphasis on specific immune cell populations and colon epithelial cells

Figure 2.1. IRAK-M Expression is Increased During Inflammatory Bowel Disease

Exacerbation and in Advanced Colorectal Cancer Patients.

81

Figure 1. IRAK-M Expression is Increased During Inflammatory Bowel Disease Exacerbation and in Advanced Colorectal Cancer Patients

A. B.

C.

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(Figure 2.1C). Prior literature documents the expression of IRAK-M in cells of the

myeloid linage (Wesche et al., 1999). This is supported by our metadata analysis

(Figure 2.1C). However, our findings further revealed that IRAK-M is also highly

expressed in eosinophils and neutrophils (Figure 2.1C). We also found IRAK-M

expressed in all of the other cell types assessed, albeit at lower levels than the

macrophages, myeloid progenitor cells and granulocytes (Figure 2.1C). Together, these

data suggest that IRAK-M plays a significant role in modulating the immune response in

the GI system and is up-regulated in the context of IBD and CRC. This up-regulation is

likely in response to increased TLR and interleukin 1 receptor (IL-1R1) signaling in

these disease states.

Irak-m-/- Mice Are Protected Against DSS Induced Colitis

Figure 2.1. IRAK-M Expression is Increased During Inflammatory Bowel Disease Exacerbation and in Advanced Colorectal Cancer Patients. A. Retrospective analysis of metadata from colonic biopsies revealed that IRAK-M expression was significantly increased in Crohn’s Disease (CD) and ulcerative colitis (UC) patients during exacerbation, compared to biopsies from currently diagnosed IBD patients during inactive disease (inactive) and patients not diagnosed with IBD (healthy). IRAK-M was also significantly up-regulated in UC patients with neoplasia (UC + Neo). The fold-change values were extracted and averaged from 7 separate datasets and reflect the change in IRAK-M expression between each patient population and the respective healthy controls. *, †, ‡, §, ¶, #p<0.05. B. Colorectal cancer (CRC) patients were stratified based on the Dukes’ staging system (A/B) and (C/D). IRAK-M expression was significantly increased in patients with more advanced disease (CRC C/D) compared to the less advanced (CRC A/B) patients and patients not diagnosed with CRC (healthy). The fold-change values were extracted and averaged from 2 separate datasets and reflect the change in IRAK-M expression between each patient population and the respective healthy controls. *, † p<0.05. C. IRAK-M is differentially expressed in multiple human cell types of relevance to IBD and CRC, with the highest levels of expression in cells of the myeloid lineage. The mean expression of IRAK-M in all human cell types is identified by the dotted line (218).

83

We next sought to better characterize the contribution of IRAK-M in the context of IBD.

Prior studies have evaluated Irak-m-/- mice in both dextran sulfate sodium (DSS) and

Il10-/- colitis models (Berglund et al., 2010), (Biswas et al., 2011). These prior studies

revealed that IRAK-M functions to attenuate the progression of experimental colitis

(Berglund et al., 2010), (Biswas et al., 2011). Here we utilized Irak-m-/- mice in an acute

colitis model induced with 5% DSS (Okayasu et al., 1990). This is a standard model

utilized previously by our laboratory in similar studies evaluating mediators of innate

immunity in IBD (Williams et al., 2015a), (Williams et al., 2015b), (Allen et al., 2012),

(Allen et al., 2010). We found that Irak-m-/- mice are protected in this experimental colitis

model (Figure 2.2). Clinically, the Irak-m-/- mice demonstrate significant improvements

in weight change and clinical parameters associated with disease progression

compared to the wild-type (WT) animals (Figure 2.2A and 2.2B). Consistent with our

clinical observations, Irak-m-/- mice also displayed significantly increased colon length

and less severe tissue damage as evident by histopathology evaluation of H&E stained

colon sections compared to the WT counterparts (Figure 2.2C and 2.2D). Interestingly,

when viewing the histopathology of the colon from Irak-m-/- mice we observed localized

and highly structured areas of lymphoid cells throughout the colon. These structures

were identified and confirmed to be expanded areas gut associated lymphoid tissue

(GALT) by a board certified veterinary pathologist (T.L.) (Figure 2.2E). We

hypothesized that the protective phenotype observed in the Irak-m-/- mice following

acute DSS exposure was due, in part, to the significant increase in GALT in the colon of

the Irak-m-/- mice, which were present irrespective of DSS treatment (Figure 2.2E).

Figure 2.2. Attenuated Experimental Colitis Pathogenesis in Irak-m-/- Mice.

84

0 2 4 6 8-15

-10

-5

0

5

Figure 2. Attenuated Experimental Colitis Pathogenesis in Irak-m-/- Mice

D. Mock Irak-m-/- +DSS Wild Type +DSS

*

B.

0

2

4

6

8

10

Mock

Irakm

r!9-

11

Wi ld

Type

*

Clin

ica

l Sc

ore

Cli

nic

al S

co

re

F.

0

100

200

300

Ba

cte

ria

l Co

un

ts in

Blo

od

*

Wi ld

Type

Irakm

r! 9

-11

Ba

cte

ria C

ou

nts

in

Blo

od

C.

0

2

4

6

8

10

Mock

Irakm

r!9-

11

Wi ld

Type

**

Co

lon

Le

ng

th (c

m)

Co

lon

Len

gth

(c

m)

G.

0

500

1000

1500

Co

lon

IL

-6 (p

g/m

l)

DSSMock

Wi ld

Type

Wi ld

Type

Irakm

r!9-

11

Irakm

r!9-

11

*

**

Co

lon

IL

-6 (

pg

/ml/

mg

)

Mock DSS

A.

*

†

* *

* †

† †

Day

% W

eig

ht

Ch

an

ge

Mock

Wild Type

Irak-m-/-

E. Irak-m-/- Mock GALT

* †

* †

DSS

85

To test this hypothesis, we measured the systemic bacteremia in whole blood of both

WT and Irak-m-/- mice following the acute DSS model. Our data lend support to this

hypothesis as bacterial counts were significantly reduced in Irak-m-/- mice (Figure 2.2F).

Further, we observed increased levels of IL-6 from Irak-m-/- colon organ culture

supernatant following DSS treatment, which is consistent with the increased GALT

(Figure 2.2G). While increased IL-6 is typically associated with detrimental

inflammation, our histopathology assessments revealed that the inflammation was

highly localized to these areas of GALT, with minimal damage to the epithelial cell

barrier (Figure 2.2D). Collectively, our data suggest that Irak-m-/- mice are protected

against DSS induced colitis by displaying increased resistance to bacterial translocation

and bacteremia.

Attenuation of Experimental Colitis in the Irak-m-/- Mice is Associated with

Enhanced Neutrophil and T-Cell Responses.

Figure 2.2. Attenuated Experimental Colitis Pathogenesis in Irak-m-/- Mice. A. Irak-m-/- mice demonstrated significantly improved weight change following DSS exposure compared to similarly treated WT mice. Irak-m-/- mock weight is not included for simplicity. B. Clinical features associated with disease progression were significantly attenuated in Irak-m-/- mice. C. Irak-m-/- mice had significantly increased colon length compared to WT animals. D. Representative H&E stained histopathology images of colon sections from WT and Irak-m-/- mice following the acute colitis model. Scale bars = 100 μm E. Irak-m-/- mice displayed large areas of Gut Associated Lymphoid Tissue (GALT), regardless of exposure to DSS. Scale bars = 100 μm. F. Bacteria count in 25μl of blood from WT (n=5) and Irak-m-/- mice (n=5) with DSS induced colitis. G. IL-6 levels were significantly increased in colon sections harvested from Irak-m-/- mice following DSS exposure. Data are represented as mean ± SEM. *p<0.05; †p<0.05 by 1 way ANOVA, Newman-Keuls post-test. WT mock, n=3; WT DSS, n=6; Irak-m-/- mock, n=3 (not shown); Irak-m-/-

DSS, n=5. Data are representative of 3 independent studies.

86

We hypothesized that the increased GALT observed in the Irak-m-/- mice was a

significant contributing factor in protecting these animals during experimental colitis. To

test whether Irak-m-/- protection from acute DSS was associated with increased

leukocyte recruitment and function, we evaluated the leukocyte composition in whole

blood using flow cytometry (Figure 2.3A). Under basal conditions, we found the

neutrophil count was significantly higher in the blood from naïve Irak-m-/- mice compared

to WT animals (Figure 2.3A). Interestingly, following exposure to DSS the number of

neutrophils significantly increased in WT mice; however, the number of neutrophils in

the Irak-m-/- mice maintained elevated levels with or without exposure to DSS (Figure

2.3A). We further measured the levels of CXCR2 and CD14 from both naïve animals

and mice in the experimental colitis model (Figure 2.3B and 2.3C). Under both naïve

and DSS treated conditions we observed increased levels of CXCR2 and CD14 from

Irak-m-/- Ly6G+/CD11b+ leukocytes, suggesting Irak-m-/- neutrophils are primed prior to

tissue insult, which likely improves the efficiency in recruitment to a site of infection,

such as the colon when bacterial translocation occurs. To further support this

hypothesis we performed a neutrophil chemotaxis assay in response to macrophage

inflammatory protein 2 (MIP-2). When bone marrow from WT and Irak-m-/- mice was

primed with doses of LPS and subjected to the chemotaxis assay, we observed

increases in neutrophils in a dose dependent manner for Irak-m-/- mice (Figure 2.3D).

Further, we also observed increased numbers of CD4+, CD8+ T-cells and monocytes

Figure 2.3. Increased Neutrophil and T-cell Function in Irak-m-/- Mice.

87

WT Irak-m-/- L

y6

G

CD11b

Na

ïve

D

SS

A.

C. D.

W T IR AK M - /-

2 0

4 0

6 0

8 0

Ly

6G

+C

D1

1b

+%

in

Blo

od

* *

Ly

6G

+C

D11b

+%

20

40

60

80

**

25

Ly

6G

+C

D1

1b

+%

in

Blo

od

W T IR A K M - /-

0

5

1 0

1 5

2 0

2 5 * *

0

5

10

15

20

Ly

6G

+C

D11b

+%

**

W T IR AK M - /-

0

2 0

4 0

6 0

8 0

1 0 0

CD

14

MF

I

* * *

CD

14

MF

I

20

0

40

60

80

100 ***

W T IR AK M - /-

4 0

6 0

8 0

1 0 0

1 2 0

CD

14

MF

I

* * *

CD

14

MF

I

40

60

80

100

120

***

CD

4+

%

W T IR AK M -/-

0

5

1 0

1 5

2 0

2 5

*

CD

8+

%

W T IR AK M -/-

0

5

1 0

1 5

*

Ly

6G

-Ly

6C

+C

D1

1b

+%

W T IR AK M -/-

0 .0

0 .5

1 .0

1 .5

2 .0

*

E.

0

5

10

15

20

25

CD

4+

%

0

5

10

15

CD

8+

%

0.0

0.5

1.0

1.5

Ly

6G

-LY

6C

+C

D11

b+

%

2.0 * *

*

WT

IRAKM-/-

IAE CD62L

WT Irak-m-/-

IAE

MF

I

W T IR AK M -/-

0

2 0

4 0

6 0

8 0

* *

CD

62

L M

FI

W T IR AK M -/-

0

2 0

4 0

6 0

8 0

1 0 0 * *

F.

0

20

40

60

IAE

MF

I

80

0

20

40

60

CD

62

L M

FI

80

100 ** **

WT Irak-m-/-

Naïve DSS

CD14 CD14

WT Irak-m-/- Irak-m-/-

WT

Figure 3. Increased Neutrophil and T-cell Function in Irak-m-/- Mice

B.

W T IR AK M -/-

2 0 0

2 5 0

3 0 0

3 5 0

4 0 0

CX

CR

2 M

FI

* * *

200

250

300

350

400

CX

CR

2 M

FI

***

W T IR AK M- /-

3 0 0

3 2 0

3 4 0

3 6 0

3 8 0

4 0 0

CX

CR

2 M

FI

*

CX

CR

2 M

FI

300

320

340

360

380

400 *

CXCR2

Naïve

CXCR2

DSS

WT Irak-m-/-

WT

Irak-m-/-

Irak

-m-/

-

Me

dia

M

IP-2

PBS 100pg/ml LPS

10ng/ml LPS

1µg/ml LPS

Wil

d T

yp

e

Me

dia

M

IP-2

Fo

ld I

ncre

ase

F

old

In

cre

ase

F

old

In

cre

as

e

0.5

1.0

1.5

2.0

0.5

1.0

1.5

2.0

*** **

* *

88

isolated from the spleen of Irak-m-/- mice following exposure to DSS (Figure 2.3E). This

finding is consistent with prior studies that indicated increased T-cell recruitment in Irak-

m-/- mice in the experimental colitis model (Berglund et al., 2010), (Klimesova et al.,

2013). Monocytes isolated from the spleen of Irak-m-/- mice following DSS exposure

displayed increased cell surface expression of IAE and decreased expression of CD62L

(Figure 2.3F). Further, we hypothesized that the differences in neutrophil and T-cell

recruitment in Irak-m-/- mice was the result of differences associated with the GI

microbiota composition. When mice were treated with antibiotics for two weeks prior to

DSS administration, similar susceptibilities to pathogenesis were observed between WT

and Irak-m-/- mice (Figure 2.4A-C). Collectively, our data suggests that Irak-m-/- mice

are protected from experimental colitis due to efficient recruitment of neutrophils and T-

cells following microbial translocation. These data provide a mechanism of acute colitis

protection and lend support to the phenotype that GALT in Irak-m-/- mice contributes to

the overall protection observed in the experimental colitis model.

Irak-m-/- Mice Are Protected Against Inflammation Driven Colon Tumorigenesis.

Figure 2.3. Increased Neutrophil and T-cell Function in Irak-m-/- Mice. A. Representative FACS plots of neutrophils and the percentage of neutrophils (Ly6G+CD11b+) in the blood of naïve mice and mice with DSS-induced ulcerative colitis. B. The expression of CXCR2 on the Ly6G+CD11b+ cells in the blood. C. The expression of CD14 on the Ly6G+CD11b+ cells in the blood. *p<0.05, **p<0.01, ***p<0.001, n=5. D. Chemotaxis of neutrophils to MIP-2. Neutrophils purified from the bone marrow were primed with different doses of LPS, then subjected to chemotaxis assay. PBS is used as control. *p<0.05, **p<0.01, ***p<0.001, n=5. Scale bars ≈ 10 μm. E. Percentages of CD4+, CD8+ T cells and monocytes in the spleen from mice with DSS-induced ulcerative colitis. F. The expression of IAE and CD62L on Ly6G-Ly6C+CD11b+ monocytes in the spleen from mice with DSS-induced ulcerative colitis. *p<0.05, **p<0.01, n=4.

89

Our retrospective metadata analysis revealed that IRAK-M likely modulates IBD, CAC,

and CRC in human patients (Figure 1A and 1B). Based on our findings in the IBD

model, we next sought to evaluate the Irak-m-/- mice in a model of colitis associated

tumorigenesis. Here we utilized the AOM+DSS model (Williams et al., 2015a),

Figure 2.4. Increased Morbidity and Mortality in Antibiotic + DSS Treated Mice

C

0 2 4 6 80

25

50

75

100

125

Irakm-/- + DSS + Antibiotic

Irakm-/- + Antibiotic

Wild Type+DSS+ Antibiotic

Wild Type + Antibiotic

Days

Pe

rce

nt s

urv

iva

l

A. B.

80

90

100

110

120

Irakm-/- + DSS + Antibiotic

Irakm-/- + Antibiotic

Wild Type + DSS + Antibiotic

Wild Type + Antibiotic

5% DSS

10 2 3 4 5 6 7

Days

Bo

dy

We

igh

t E

vo

lutio

n (

%)

4

5

6

7

8 N.S.

N.S. N.S.

N.S.

Co

lon

Le

ng

th (c

m)

90

(Allen et al., 2012), (Allen et al., 2010). Irak-m-/- mice displayed improved morbidity and

mortality throughout the course of the model compared to the WT animals, suggesting

attenuation of disease progression compared to the WT counterparts (Figure 2.5A-C).

When colon organ culture supernatants were assayed for IL-6 and IL-10, Irak-m-/- mice

displayed significant increases in both cytokines (Figure 2.5D-E). Beyond the significant

attenuation of clinical features and enhanced cytokine responses, the Irak-m-/- mice

displayed dramatic resistance to tumorigenesis (Figure 2.6). The overall gross polyp

formation was significantly higher in WT mice; conversely, no polyps were detected in

Irak-m-/- mice over the course of the study (Figure 2.6A-C). Decreased tumor burden

was further supported by histopathology assessments of H&E stained colon sections

(Figure 2.6D). As described for the experimental colitis model, GALT formation was

prominent in Irak-m-/- mice (Figure 2.6E). Consistent with the macroscopic colon

evaluation, histopathology scoring revealed significant reductions in areas of

hyperplasia and dysplasia in Irak-m-/- mice (Figure 2.6F-G). Likewise, we found that

Figure 2.4. Increased Morbidity and Mortality in Antibiotic + DSS Treated Mice Wild type and Irak-m mutant animals were subjected to two weeks of antibiotic (Ab) treatment prior to DSS administration available ad libitium consisting of 0.5mg/ml Ampicillin, 0.5mg/ml Metronidazole, 0.5mg/ml Neomycin, 0.25mg/ml Vancomycin and 12mg/ml splenda (Rutkowski et al., 2015), (Hernandez-Chirlaque et al., 2016). Wild type and Irak-m mutant animals were then given either Ab water or Ab + 5% DSS for 5 days, followed by Ab water alone for two days. A. Body weight evolution of mice once supplemented with DSS. B. Colon length of mice following completion of the study. C. Survival curve of mice throughout the study. n=5 Wild type + Ab; n=5 Irak-m-/- +Ab; n=5 Wild type Ab+DSS; n=5 Irak-m-/- + Ab+DSS.

91

Figure 2.5. Colitis Associated Tumorigenesis Progression is Significantly

Reduced in Irak-m-/- Mice

†

D a y s

Pe

rc

en

t s

urv

iva

l

0 2 0 4 0 6 00

2 0

4 0

6 0

8 0

1 0 0

Ira k m- /-

A O M + D S S

W ild T y p e A O M + D S S

M o c k + A O MMock AOM

Irak-m-/- AOM+DSS

WT AOM+DSS

0 20 40 60

Days

0

100

80

60

40

20

% S

urv

iva

l

*

*

†

A.

Figure 4. Colitis Associated Tumorigenesis Progression is Significantly Reduced in Irak-m-/- Mice

B.

D. E. C.

0 10 20 30 40 50 60

Days %

We

igh

t C

han

ge

-20

-15

-10

-5

0

5

10

15

20

25 Mock AOM

Irak-m-/- AOM+DSS

WT AOM+DSS

Clin

ica

l S

co

re

0.0

0.5

1.0

1.5

2.0

2.5

AOM+DSS

**

0

0.5

1.0

1.5

2.0

2.5

AOM + DSS AOM + DSS

0

100

200

300

400

500

AOM+DSS

**

IL-6

(p

g/m

l/mg

)

0

100

200

300

400

500

IL-6

(p

g/m

l/m

g)

0

10

20

30

40

50

IL-1

0 (

pg

/ml/m

g)

AOM+DSS

*

*

0

10

20

30

40

50

AOM + DSS

IL-1

0 (

pg

/ml/

mg

)

DSS DSS DSS

*

*

*

92

the levels of β-catenin are also markedly reduced in Irak-m-/- mice following AOM+DSS

(Figure 2.6H). Collectively, our data indicates that the Irak-m-/- mice are resistant to

both experimental colitis and inflammation driven tumorigenesis.

Identification of a Splice Variant of the Irak-m Gene in Irak-m-/- Mice

Expression levels of IRAK-M are highest in macrophages and previous studies have

utilized both human and mouse macrophages to study IRAK-M function (Wesche et al.,

1999), (Kobayashi et al., 2002). Thus, we were interested in determining the response

of IRAK-M in murine bone marrow derived macrophages (BMDM) when challenged with

diverse PAMPs. In our hands, BMDM from our Irak-m-/- mice display significantly

increased levels of IL-6 compared to WT BMDMs when stimulated for 24 hours with

different TLR ligands, specifically the TLR2 ligand Pam3CSK4 (Figure 2.7A). Further,

we assessed a broad panel of secreted cytokines following 24 hour treatment with TLR

1-9 agonists. Specifically, Irak-m-/- BMDMs display increased levels of TNF with TLR1/2

Figure 2.5. Colitis Associated Tumorigenesis Progression is Significantly Reduced in Irak-m-/- Mice. A. Kaplan-Meier plot of Irak-m-/- and WT survival. *p<0.05; †p<0.05. B. WT mice demonstrated significant weight loss throughout the AOM/DSS model, whereas weight loss in the Irak-m-/- mice was minimal. All mock and mock+AOM mice gained weight throughout the duration of the model, regardless of genotype. The WT mock+AOM are shown for comparison. C. Clinical features associated with disease progression were significantly improved in Irak-m-/-

mice. Data shown reflect the clinical scores on the final day of the model (Day 65). D-E. Both IL-6 and IL-10 levels were significantly increased in colons harvested from Irak-m-/- mice compared to mock, mock+AOM, and WT AOM+DSS treated mice. Data are represented as mean ± SEM. *p<0.05; †p<0.05; p<0.05 by 1 way ANOVA, Newman-Keuls post-test. Mock WT, n=5 (not shown); mock Irak-m-/-, n=5 (not shown); AOM+mock WT, n=5; AOM+mock Irak-m-/-, n=5 (not shown); AOM+DSS WT, n=18; AOM+DSS Irak-m-/-, n=5. Data are representative of 3 independent studies.

93

Figure 2.6. Irak-m-/- Mice Display Attenuated Polyp Formation in the Colitis

Associated Tumorigenesis Model.

Figure 5. Irak-m-/- Mice Display Attenuated Polyp Formation in the Colitis Associated Tumorigenesis Model

A. B.

F.

D. E.

Wild Type Irak-m-/-

Dis

tal

Pro

xim

al

C.

G.

Irak-m-/- AOM+DSS Wild Type AOM+DSS

GALT Irak-m-/-

Mock

*

*

ND ND

AOM + DSS

0

1

2

3

Nu

mb

er

of

Ma

cro

sc

op

ic P

oly

ps

/Co

lon

AOM+DSS

**

Nu

mb

er

of

Ma

cro

sc

op

ic P

oly

ps/C

olo

n

0

1

2

3

0

1

2

3

4

AOM+DSS

Po

lyp

Siz

e (m

m2)

**

ND ND

AOM + DSS

Po

lyp

Siz

e (

mm

2)

0

1

2

3

4

0

5

10

15

20

25

HA

I S

co

re

* *

AOM+DSSAOM + DSS

HA

I S

co

re

0

5

10

15

20

25

0

1

2

3

Hy

pe

rpla

sia

an

d D

ys

pla

sia

AOM+DSS

* *

AOM + DSS

Hyp

erp

lasia

& D

ys

pla

sia

0

1

2

3 DAPI Merge ! -catenin

Wil

d T

yp

e

Ira

k-m

-/-

H.

94

agonists and decreased IL-10 with TLR 1/2, 7, and 9 agonists (Figure 2.8A-E). This is

consistent with prior reports pertaining to IRAK-M being a negative regulator of TLR

signaling (Kobayashi et al., 2002). We also observed differences in IL-6 secretion

between male and female Irak-m-/- BMDMs when stimulated with Pam3CSK4 (Figure

2.9). However, during routine follow-up studies, we observed IRAK-M protein induction

in both the WT and Irak-m-/- strains when stimulated with various TLR ligands using an

antibody specific for the C-terminus of IRAK-M (Figure 2.10). This was unexpected, as

our Irak-m-/- mice were acquired from a commercial vendor, albeit reconstituted from

frozen embryos, and previously characterized (Kobayashi et al., 2002). It was previously

reported that generation of the Irak-m-/- mouse targeted exons 9-11 for deletion by

homologous recombination inserting the neomycin resistance cassette in place of these

Figure 2.6. Irak-m-/- Mice Display Attenuated Polyp Formation in the Colitis Associated Tumorigenesis Model. A. Representative image of macroscopic polyp formation in WT and Irak-m-/- mice. Black arrows indicate macroscopic polyps in WT mice, with none found in Irak-m-/- animals. Scale = mm. B. Average number of macroscopic polyps per colon of WT and Irak-m-/- mice following the AOM/DSS model. ND = none detected. C. Average macroscopic polyp size between WT and Irak-m-/- mice. D. Representative H&E stained histopathology images of colon sections from WT and Irak-m-/- mice following the AOM/DSS model. Scale bars = 100μm. E. Representative H&E staining of Gut Associated Lymphoid Tissue (GALT) from the colon of Irak-m-/-mice following the AOM/DSS model. Areas of expanded GALT were detected in the Irak-m-/- mice irrespective of exposure to AOM/DSS. Scale bar = 100μm F. Irak-m-/- mice demonstrated a significant attenuation in histopathological features associated with the AOM/DSS model, as indicated by the histopathological activity index (HAI) score. G. Histopathology assessments also revealed that Irak-m-/- mice had significantly attenuated development of hyperplasia and dysplasia compared to the WT animals. H. Representative images of immunofluorescence staining of the colon sections from WT and Irak-m-/- mice following the AOM/DSS treatment. The tissue sections were fixed and stained with anti–β-catenin (red) and DAPI (blue). Scale bars = 30 μm. Data are represented as mean ± SEM. *p<0.05; p<0.05 by 1 way ANOVA, Newman-Keuls post-test. Mock WT, n=5 (not shown); mock Irak-m-/-, n=5 (not shown); AOM+mock WT, n=5; AOM+mock Irak-m-/-, n=5 (not shown); AOM+DSS WT, n=18; AOM+DSS Irak-m-/-, n=5. Data are representative of 3 independent studies.

95

Figure 2.7. Disruption of the Murine Irak-m Locus Results in the Formation of an

Irak-mrΔ9-11 Splice Variant.

! "

#"

Figure 6. Disruption of the Murine Irak-m Locus Results in the Formation of an Irak-mr! 9-11 Splice variant

C.

800

400

600

1000

1500

Exon: 5-12

500/517

700

2000

(bp) 2531

! 400bp

WT Irak-m-/-

C Pam3 C Pam3 !

Neg.

A.

D.

Neo

WT Irak-m

Irak-mr! 9-11

1 2 3,4,5,6,7,8 9,10,11 12

1 2 34 5 6 7 8 9-11 12

1 2 34 5 6 7 8 12

1 2 3,4,5,6,7,8 12

B.

5-8, 9-10, 10-11, 12

!

Exon: 5-8 9-10 10-11 12

300

600

100

200

400 500/517

700

WT Irak-m-/- Neg. Control

C Pam3 C Pam3!

WT Irak-m-/- WT Irak-m-/- WT Irak-m-/-

C Pam3 C Pam3! C Pam3 C Pam3! C Pam3 C Pam3!

E.

102kDa

! actin (42 kDa)

HA (short exposure)

Em

pty

Ve

cto

r !

Ga

l!

Ga

l+IL

-1!

WT Irak-m Irak-mr! 9-11

150kDa

76kDa

52kDa

38kDa

HA (long exposure)

150kDa

102kDa

76kDa

52kDa

38kDa

Empty

Vecto

r

500n

g Beta

Gal

500n

g Beta

Gal +IL

-1beta

Wt 625

Wt 320

Wt 160

WT 80

Wt 40Wt 2

0Wt 1

0

Irakm

2500

Irakm

1250

Irakm

625

Irakm

320

Irakm

160

irakm

80

Irakm

40

0

20

40

60

80

*

*

†

†

WT Irak-m Irak-mr! 9-11

Re

lati

ve

Lu

min

es

cen

ce

Un

its

0

20

40

60

80

Mock

poly IC

PG

N-S

ALPS

Pam

3CSK4

TNFa

0

2000

4000

6000

80009000

14000

19000

24000

29000

WT

Irakm-/-

IL-6

Co

nc

en

tra

tio

n (

pg

/ml)

WT Irak-m-/-

*

96

three exons (Kobayashi et al., 2002). In order to test the hypothesis that IRAK-M was

present in our mutant mice, we proceeded by further investigating the phenomena at

the

Figure 2.7. Disruption of the Murine Irak-m Locus Results in the Formation of an Irak-mrΔ9-11 Splice Variant. A. Bone marrow derived macrophages (BMDM) from Irak-m-/- mice demonstrate increased production of IL-6 following 24 hr stimulation with specific PAMPs. Data shown are pooled from 3 separate and independent experiments. n=3 mice per genotype. B. Disruption of the Irak-m locus resulted in the loss of Exons 9-11. BMDM from WT and Irak-m-/- littermates were un-stimulated or stimulated for 24 hr with Pam3CSK4. After 24 hr, cells were harvested for RNA and RT-PCR was utilized to amplify Irak-m corresponding to exons: 5-8, 9-10, 10-11 and 12, respectively. C. A size difference was detected in the RT-PCR product corresponding to Exons 5-12 between WT and Irak-m-/- BMDMs treated for 24 hr with Pam3CSK4. The shift of approximately 400 nucleotides in the Irak-m-/- BMDMs corresponds to the deleted exons 9, 10 and 11. D. Sequencing of the exon 5-12 RT-PCR product revealed a splice immediately after exon 8 together with exon 12. Normal splicing occurs in the WT BMDM, whereas Irak-m-/- BMDM splice around the inserted Neo-cassette after exon 8 and generates a splice variant connecting exon 8 with exon 12. E. (Left) Dual-Luciferase assay of HEK293T cells transfected for 24 hr with equal amounts of NF-κB-firefly luciferase reporter plasmid and decreasing amounts of either CMV-WT Irak-m-HA plasmid (625ng-10ng) or CMV-Irak-mrΔ9-11-HA plasmid (2500ng-40ng) from three independent and pooled experiments. Empty Vector is shown as a negative control. β-Galactosidase-HA is shown as a positive control for transfection. A separate β-Galactosidase-HA transfection was treated with 10 ng/mL human IL-1β for 6hr following 18hr of transfection, and serves as a positive control for NF-kB-firefly luciferase activation. All treatment groups received a constant amount of plasmid DNA described in the Experimental Methods and are normalized to the Renilla luciferase activity. (Right) Representative western blot of WT IRAK-M and IRAK-MrΔ9-11 protein expression. Overexpression inserts are HA-tagged at the C-

-actin is shown as a loading control. The WT IRAK-M expresses readily as seen in the short exposure and migrates at ~ 68kDa. The IRAK-MrΔ9-11 protein is present in the long exposure migrating at ~ 38kDa. Data in panel A. is represented as mean ± SEM. *p<0.05 by 1 way ANOVA, Tukey-Kramer post-test. Data in panel E. is represented as mean ± SEM. *p<0.001, †p<0.01 by 1 way ANOVA, Tukey-Kramer post-test. Statistical significance between all treatment groups in E is not shown for simplicity. A minimum of 3 independent experiments was conducted for all assays.

97

2.8 Differences in TLR-2, TLR-7 and TLR-9 Stimulation in Irak-m Mutant Mice.

Con

torl

PolyIC

LPS

PGN-S

A

Pam

3CSK4

Flage

llin

CpG

DNA

Imiquim

od

IL-1

b

TNFa

0

1000

2000

3000WT

Irak-m-/-

***

**

TN

F C

oncentr

ation (

pg/m

l)

Con

torl

PolyIC

LPS

PGN-S

A

Pam

3CSK4

Flage

llin

CpG

DNA

Imiquim

od

IL-1

b

TNFa

0

WT

Irak-m-/-

No Detection

IFN

-g C

oncentr

ation (

pg/m

L)

Con

torl

PolyIC

LPS

PGN-S

A

Pam

3CSK4

Flage

llin

CpG

DNA

Imiquim

od

IL-1

b

TNFa

0

10

20

30

40

8000

10000

12000

14000

WT

Irak-m-/-

IL-1

b C

oncentr

ation (

pg/m

l)

Con

torl

PolyIC

LPS

PGN-S

A

Pam

3CSK4

Flage

llin

CpG

DNA

Imiquim

od

IL-1

b

TNFa

0

500

1000

1500

2000

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WT

Irak-m-/-

***

***

**

IL-1

0 C

oncentr

ation (

pg/m

L)

Con

trol

PolyIC

LPS

PGN-S

A

Pam

3CSK4

Flage

llin

CpG

DNA

Imiquim

od

IL-1

b

TNFa

0

5000

10000

15000

20000

25000

WT

Irak-m-/-

***

***

**

IL-6

Concentr

ation (

pg/m

l)

A.

B.

D.

E.

C.

98

Figure 2.9. Comparison of Male and Female Bone Marrow Derived Macrophages

Figure 2.8. Differences in TLR-2, TLR-7 and TLR-9 Stimulation in Irak-m Mutant Mice. Bone marrow was harvested from 8-week age matched males. BMDMs were derived and seeded in 24 well plates as described in the Materials and Methods. Cells were stimulated with either: Control media, 6.25μg/ml Poly(I:C), 10 ng/ml LPS, 10 μg/ml PGN-SA, 300 ng/ml Pam3CSK4, 50 ng/ml standard Flagellin-BS, 5 μM CpG DNA ODN1668, 1 μg/ml Imiquimod (R837), 20 ng/ml m-IL-1β, 75 ng/ml m-TNFα. Cell free supernatants were collected and assayed by ELISA for extracellular: A. IL-6. B. IL-10. C. TNF (75ng/ml TNFα treatment indicates the upper limit/saturation range of the ELISA). D. IL-1β (Data plotted is extrapolated, as the values were below the detection limit of the ELISA). E. IFN-γ. Data indicates the mean from n=3 WT, n=3 Irak-m-/- mice. Error bars indicate SEM. Data was analyzed by 1-way ANOVA followed by Tukey’s HSD. *p<0.05, **p<0.01,***p<0.001.

99

mRNA level. When BMDMs were stimulated for 24 hours with the TLR1/2 ligand

Pam3CSK4 to induce Irak-m transcription, we observed an amplification band

corresponding specifically to exon 12 of Irak-m by RT-PCR (Figure 2.7B). PCR primers

were further designed to amplify the region spanning the length of exons 5 and 12

(Figure 2.7C). Interestingly, a shift of approximately 400bp was observed in the BMDMs

from our Irak-m-/- animals, which suggested a splice variant had been generated. This

was indeed confirmed by Sanger sequencing, which revealed a splice occurred after

exon 8 and connecting it with exon 12 in the amplification band (Figure 2.7D). Loss of

the neo cassette only occurred at the mRNA level, likely after exon splicing, and not at

the DNA level. This is evident because the genotyping primers are targeted against a

portion of the neo cassette, which is present in the genotyping for the Irak-m-/- mice.

Based on this revelation, we have defined this splice variant as Irak-mrΔ9-11. In order to

test the functionality of this truncated transcript, we cloned both the WT and mutant Irak-

mrΔ9-11 transcripts. Overexpression in HEK293T cells of both the WT and truncated Irak-

mrΔ9-11 transcripts both had the capacity to activate an NF-

reporter. However, NF-κB activation was robustly increased following overexpression of

Irak-mrΔ9-11 compared to the WT (Figure 2.7E), suggesting a functional and potent role

for this mutant protein. We further validated the specificity of the commercially available

Figure 2.9. Comparison of Male and Female Bone Marrow Derived Macrophages. IL-6 ELISA of BMDM stimulated for 24 hrs with respective Pamps. Concentrations of Pamps are described in the Materials and Methods. n=3 WT males, n=3 Irak-m-/- males, n=3 WT females, n=3 Irak-m-/- females. Data was analyzed by 1-way ANOVA followed by Tukey’s HSD. ***p<0.001.

100

Figure 2.10. Western Blot Evaluation of Protein Expression of IRAK-M.

IRAK-M antibody used in Figure S1, which displayed no specificity for the WT-IRAK-M

or Irak-mrΔ9-11 overexpression constructs (data not shown).

E. Discussion

Overzealous inflammation associated with dysregulated innate immune signaling is a

significant component of IBD pathogenesis. This hyper-inflammation is often associated

with aberrant TLR signaling. There is significant interest in better characterizing the

contribution of proteins, like IRAK-M, that regulate PRR signaling in IBD, CAC, and

CRC. This interest is due to the revelation that these regulatory proteins significantly

IRAK-M

(68 kDa)

β-actin

(42 kDa)

(4.2) (0.08) (0.4) (0.9) (0.7) (0.08) (0.58) (0.9) (0.6)

Figure 2.10. Western Blot Evaluation of Protein Expression of IRAK-M. Western blot evaluation revealed that IRAK-M protein is present in both the WT and Irak-m-/- treatment groups following 24hr stimulation with specific PAMPs. Western blot is normalized to β-actin and human THP-1 cells are used as a positive control. Densitometry is quantified with numerical values below β-actin using ImageJ. Further, independent validation of the IRAK-M antibody used displayed no specificity for the murine WT-IRAK-M-HA or Irak-mrΔ9-11-HA overexpression constructs (data not shown).

101

attenuate disease pathogenesis in IBD mouse models. For example, prior studies by

our group and others have shown that a diverse group of negative regulatory proteins,

including NLRP12, NLRX1, TOLLIP, A20, and GIT2, also function to maintain immune

system homeostasis in the gut through the attenuation of hyper-responsive

inflammation (Mukherjee and Biswas, 2014), (Vereecke et al., 2014), (Hammer et al.,

2011), (Boj et al., 2015), (Allen et al., 2012), (Zaki et al., 2011), (Soares et al., 2014),

(Singh et al., 2015). Many of these negative regulatory proteins have been found

dysregulated in IBD and CRC patients. For example, previous reports have indicated

that IRAK-M expression is significantly increased in IBD patients with both UC and CD

(Fernandes et al., 2016), (Gunaltay et al., 2014). These findings are consistent with our

analysis of retrospective metadata that revealed increased IRAK-M expression, not only

in active UC and CD patients, but also in the context of colitis associated neoplasia

(Figure 2.1). Our retrospective metadata analysis revealed that IRAK-M expression

increases with severity of CRC progression (Figure 2.1). Together these data likely

reflect an increase in the innate immune response during both IBD and CRC that is

driven by PRR activation. These data are consistent with recent findings that revealed

increased IRAK-M induction in colon tumor cells associated with the combined effects of

Wnt and TLR activation (Kesselring et al., 2016). The increase in IRAK-M expression,

as well as the expression of other genes that encode negative regulators of PRRs, is

likely an attempt to reign in overzealous inflammation and maintain some level of

immune system homeostasis during IBD and CRC.

102

The Irak-m-/- mouse model has been an essential tool to determine the negative

regulatory mechanisms underlying TLR signaling. Irak-m-/- mice have proven to be more

sensitive to TLR stimulation and display impaired endotoxin tolerance, likely due to the

hyper-activation of NF-κB signaling (Kobayashi et al., 2002). In addition to negatively

regulating canonical NF-κB signaling, IRAK-M inhibits the non-canonical NF-κB

cascade, in part, through modulating the degradation of NF-ĸB inducing kinase (NIK)

(Su et al., 2009). In prior tumor injection models, loss of Irak-m-/- has been shown to

result in enhanced innate immune responses and the attenuation of tumor growth (Xie

et al., 2007), (Standiford et al., 2011). These studies were based on tumor injection

models with Irak-m-/- mice. However, the overall conclusions of each study are

consistent with the observations reported here. In both prior studies, the Irak-m-/-animals

demonstrated significant resistance to tumor growth and pathogenesis following

inoculation. Mechanistically, the attenuation of tumorigenesis was associated with

increased activation and proliferation of B cells and T cells, specifically CD4+ and CD8+

T cells (Xie et al., 2007). Our group has previously shown that Irak-m-/- macrophages

display enhanced uptake of acLDL, as well as increased percentages of macrophages

that uptake apoptotic thymocytes (Xie et al., 2007). Reports have demonstrated IL-10R

signaling plays a dramatic role in macrophage function (Shouval et al., 2014);

additionally, IL-10 is produced from macrophages following the uptake of apoptotic cells

(Chung et al., 2007), (Zhang et al., 2010). Therefore, though we have not explicitly

tested, we speculate that increased phagocytosis of apoptotic cells by Irak-m mutant

macrophages results in increased IL-10 leading towards a tolerant, and potentially

protective phenotype.

103

Our findings in the experimental colitis and colitis associated tumorigenesis models

were initially surprising, as Irak-m-/- mice appear to have increased inflammation.

However, upon further investigation we discovered that the inflammation is actually

highly compartmentalized and isolated to regions of enhanced GALT, with little damage

or effect to the epithelial cell barrier. As we detail in the current manuscript, we believe

that these expanded areas of GALT are actually beneficial to these mice and function to

improve the efficiency of the immune response to translocating microbes from the GI

lumen. This is further supported as (Kesselring et al., 2016) described increased GI

epithelial barrier permeability in Irak-m-/- mice using FITC labeled dextran.

IRAK-M modulation of experimental colitis and colitis associated tumorigenesis has

been previously evaluated in Irak-m-/- mice (Berglund et al., 2010), (Biswas et al., 2011,

(Klimesova et al., 2013). In the initial study that evaluated IRAK-M function during DSS-

induced colitis, Irak-m-/- mice were treated with 3% DSS for 5 days and allowed 2 days

to recover prior to harvest (Berglund et al., 2010). In this model, Irak-m-/- mice were

found to be sensitive to DSS and presented with significantly increased clinical and

histopathological features associated with disease progression (Berglund et al., 2010).

This study also found elevated cytokine, chemokine, and T-cell transcription factor

mRNA expression in Irak-m-/- colon tissue and increased systemic IL-6 and TNF levels

in the plasma following DSS administration (Berglund et al., 2010). Subsequent studies

by a different group utilizing the AOM+DSS colitis associated tumorigenesis model in

both conventional and germfree conditions also reported increased sensitivity and

104

tumorigenesis in the Irak-m-/- mice (Klimesova et al., 2013). Similar to the study by

Berglund et al, the Irak-m-/- mice were shown to have enhanced pro-inflammatory

responses and increased T-cell accumulation in the tumor tissue and local lymph nodes

(Klimesova et al., 2013). The mechanism associated with the increased sensitivity was

correlated with altered commensal microbe metabolic activity in Irak-m-/- mice,

suggesting a difference in the microbiome between the WT and Irak-m-/- animals

(Klimesova et al., 2013). Beyond DSS based models, IRAK-M has also been evaluated

in the Il-10-/- model of spontaneous colitis (Biswas et al., 2011). Similar to the findings

from the DSS models, loss of IRAK-M resulted in increased TLR signaling, resulting in

increased inflammation and expression of pro-inflammatory signaling pathways (Biswas

et al., 2011). The Il-10-/- model is highly dependent on the intestinal commensal flora

and subsequent germfree studies evaluating Irak-m expression further suggest that

Irak-m-/- sensitivity in this experimental colitis model is dependent on the composition of

the resident GI microbiota (Biswas et al., 2011). The microbiota composition cannot be

underestimated for DSS based models as the severity to DSS colitis can drastically

change based on the microbiota composition (Hernandez-Chirlaque et al., 2016). We

postulate that differences observed between labs utilizing Irak-m-/- mice in DSS models

could be due to altered microbiome compositions or differing percentages and lots of

DSS used among labs.

Though our data provides contradictory evidence compared to the previous findings

pertaining to IRAK-M, (Berglund et al., 2010), (Biswas et al., 2011), (Klimesova et al.,

2013) our data is consistent with a more recent study that also showed Irak-m-/- mice

105

display reduced tumor burden compared to their WT counterparts in the AOM/DSS

model (Kesselring et al., 2016). This was attributed to enhanced epithelial cell barrier

function, specifically localized to tumor sites in the GI tract of Irak-m-/- mice (Kesselring

et al., 2016). This was shown to be associated with reduced activity of the oncogene

STAT-3. Our findings described in the current manuscript lend support to this

conclusion pertaining to Irak-m-/- mice challenged in the AOM/DSS model (Figure 2.5

and Figure 2.6). We further confirm the findings pertaining to decreased Wnt signaling

(Kesselring et al., 2016) as demonstrated by the reduced intracellular β-catenin in our

study (Figure 2.6H). Finally, the analysis of human biopsies from this prior study

revealed that human patients with increased IRAK-M expression have worse cancer

survival (Kesselring et al., 2016), which corroborates our metadata analysis described

(Figure 2.1). Collectively, our data pertaining to IRAK-M is complementary to the major

findings by Kesserlring et al. Together, these studies extend the mechanistic insight

associated with IRAK-M modulation of IBD and colitis associated tumorigenesis.

IRAK-M functions as a negative regulator of TLR and IL-1R1 signaling by either

attenuating the IRAK-1/IRAK-4 phosphorylation event or stabilizing the

TLR/MyD88/IRAK-4 complex (Kobayashi et al., 2002). Originally, we sought to better

define the mechanisms associated with IRAK-M attenuation of inflammation in the

context of experimental colitis and colitis associated tumorigenesis. Through the course

of our experiments, our in vitro data suggested that BMDMs from Irak-m-/- mice

displayed an augmented inflammatory response (Figure 2.7A), which is consistent with

the initial reports characterizing IRAK-M and the Irak-m-/- mice (Kobayashi et al., 2002).

106

However, we were intrigued after discovering a protein band pertaining to IRAK-M in

BMDMs from the Irak-m-/- mice after treating with specific pathogenic ligands (Figure

2.4). As previously described in the original manuscript reporting the generation of Irak-

m-/- mice, exons 9-11 were targeted for deletion by homologous recombination and the

insertion of a neo cassette (Kobayashi et al., 2002). Our genotyping confirms the

successful targeting of Irak-m. However, as we show in our current studies, a splice

variant of the Irak-m gene is formed following BMDM stimulation in the targeted Irak-m-/-

animals. The splicing event circumvents the neo cassette and joins exon 8 with exon 12,

defined here as Irak-mrΔ9-11. Together, our data suggests that the inclusion of exon 12 in

the mRNA effectively stabilizes the transcript. Further functional studies using

overexpression systems revealed that this truncation has the potential to robustly

activate NF-κB signaling (Figure 2.7E). If this splice variant is present in the Irak-m-/-

mice, then it is possible that the Irak-mrΔ9-11 variant could result in potential phenotypes

not characteristic of a true knockout mouse. These could include reduced or hyperactive

activity for IRAK-M functioning as either a dominant negative or potentially even a

dominant positive.

In conclusion, our data strongly suggests that IRAK-M functions to modulate

inflammatory signaling pathways and is critical in maintaining immune system

homeostasis in the gut. However, increased IRAK-M is associated with increased

disease pathogenesis and increased cancer severity in human patients. Our findings in

mice revealed that the immune system in Irak-m-/- animals is primed and highly efficient

at eliminating microbes translocating from the GI lumen. This increased microbial

107

clearance is associated with reduced experimental colitis and colitis associated

tumorigenesis. Together, our data identify IRAK-M as an essential regulator of

inflammation and is critical in the maintenance of mucosal immune system homeostasis

in health and disease.

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Chapter 3:

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of

Mouse Intestinal Organoids

Published as:

Rothschild, D. E., Srinivasan, T., Aponte-Santiago, L. A., Shen, X. & Allen, I. C. 2016.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal

Organoids. J Vis Exp. May 18; (111).

A. Abstract:

While the previous chapter employed conventional in vivo and in vitro techniques to

study the role of IRAK-M, the present chapter discusses the utility and potential of

optimizing primary intestinal organoids as a model system in mucosal immunology.

The complexities of the organoid growth characteristics carry significant caveats for

the investigator. Specifically, the growth patterns of each individual organoid are

highly variable and create a heterogeneous population of epithelial cells in culture.

With such caveats, common tissue culture practices cannot be simply applied to the

organoid system due to the complexity of the cellular structure. Counting and plating

based solely on cell number, which is common for individually separated cells, such

as cell lines, is not a reliable method for organoids unless some normalization

technique is applied. Normalizing to total protein content is made complex due to the

resident protein matrix. These characteristics in terms of cell number, shape and cell

type should be taken into consideration when evaluating secreted contents from the

organoid mass. This protocol has been generated to outline a simple procedure to

culture and treat small intestinal organoids with microbial pathogens and pathogen

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associated molecular patterns (PAMPs). It also emphasizes the normalization

techniques that should be applied when gene expression and protein analysis are

conducted after such a challenge.

B. Introduction:

The ability to harvest and culture primary organoids have been described for small

intestine, colon, pancreas, liver and brain and are exciting advances germane to

understanding a more physiologically representative phenomena for tissue biology(Sato

et al., 2009)-(Lancaster et al., 2013). The first methods describing the culture and

maintenance of small intestinal organoids was reported by Sato et al. out of the lab of

Hans Clevers(Sato et al., 2009). Prior to this method, harvesting and culture of primary

intestinal epithelial cells proved to be limited and ineffective in sustaining epithelial cell

growth. Methods included dissociation of tissue via incubation with enzymes, such as

collagenase and dispase, which would ultimately lead to the outgrowth of intermixed

primary fibroblast cells(Freshney and Freshney, 2002). These conditions would also be

time restricted in sustaining the epithelial cell culture. Minimal to no epithelial cell niche

would form, as the epithelial cells would enter apoptosis due to the lack of appropriate

growth factors or loss of contact integrity, termed anokis(Vachon et al., 2002). The

advent of the 3D-organoid culture system has provided a method to culture primary

intestinal cells containing a spectrum of intestinal cell types in sustained culture(Sato et

al., 2009). These epithelial organoids have advantages over cell lines being that they

are composed of several differentiated cells, and better mimic the organ they are

derived from in vivo(Hynds and Giangreco, 2013). The process to ultimately “grow a

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mini gut in a dish” has proven to be a valuable tool for assessing the response of

intestinal epithelium under different stimuli. Investigating the interaction of primary

intestinal cells with microbial PAMPs is relevant to the field of immunology as these

molecular patterns can regulate diverse responses from both host and microbe(Kaiko

and Stappenbeck, 2014). Not only can investigators now explore these interactions with

mouse organoids, but they can be cultured from humans as well(Sato et al., 2011). This

technology has the potential to dramatically alter personalized medicine and it is

tempting to speculate about advances that this technique will make possible in the near

future.

The overall goal of this method is to provide a protocol for the culture, expansion,

and treatment of intestinal organoids with a variety of stimuli. Such stimuli can

ultimately range from vaccines, bacterial pathogen associated molecular patterns

(PAMPs), live pathogens, gastrointestinal (GI) and cancer therapeutics. This

method is focused on describing an adequate technique for proper normalization

when working with non-homogenous cell structures, which must be taken into

consideration when conducting an assay based on cell number.

C. Materials and Methods:

All research was approved and conducted under Virginia Tech IACUC guidelines

1. Prepare R-Spondin1 Conditioned Media From HEK293T-Rspo1 Cell Line

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1.1) Seed HEK293T-Rspondin1 secreting cells at 5-10% confluency in a T-175 flask

with 40 ml of growth media and incubate at 37 oC + 5% CO2. R-spondin1 can be

alternatively purchased as a recombinant growth factor.

1.2) Grow the HEK293T-Rspondin1 cells for seven days or until reaching 95%

confluency determined by bright-field microscopy. Harvest the 40 ml of conditioned

media and transfer to a 50 ml conical tube. The T-175 flask of HEK293T-Rspondin1

secreting cells can be discarded.

1.3) Centrifuge at 300 x g for 10 min at 4 oC to pellet cellular debris. Collect the

supernatant, termed R-spondin1 conditioned media, and aliquot into 15 ml tubes. Store

aliquots at -20 oC for short term or -80 oC for long term storage.

2. Preparation of Organoid Growth Media and Reagents for Harvesting Small

Intestinal Crypts

2.1) One-day prior to harvesting, place the frozen protein matrix on ice and thaw

overnight at 4 oC. Autoclave dissecting scissors, forceps and glass slides that will be

used for crypt harvest.

2.2) The following day, begin by preparing 40 ml of organoid growth media containing

supplements and growth factors by adding stock concentrations to 40 ml of 1x

Advanced DMEM/F-12. Media supplements contain: 10 mM HEPES - , 1x

glutamine supplement - , 1x Vitamin B27 without vitamin A - , 1x N2 - 200

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, 1 mM N-acetyl-cysteine - , 100 ng/ml m-Noggin - , 50 ng/ml m-EGF -

and place in a 37 oC water bath until usage. Warm a 15 ml aliquot of R-spondin1 to 37

oC in a water bath.

2.3) Warm three sterile 24 well plates to 37 oC by placing in an incubator. Prepare 50 ml

per mouse 1x PBS + 2 mM EDTA and cool to 4 oC. Prepare 100 ml per mouse 1x PBS

containing 10% FBS and cool to 4 oC.

3. Harvesting Mus musculus Small Intestinal Crypts for Organoid Culture(Sato et

al., 2009)

3.1) Sacrifice a male C57B6/J mouse age 6-12 weeks of age raised on standard rodent

chow and water available ad libitum according to institutional guidelines. Euthanize via

CO2 asphyxiation followed by cervical dislocation. Note: Keep carcass on ice until

tissue harvest and perform the tissue harvest in a biological safety cabinet to minimize

the risk of contamination.

3.2) Note: It is optional shave the mouse to remove the fur before submersing in 70%

ethanol.

Submerse euthanized mouse in 70% ethanol for 2 min and pin limbs to pinning board

with the dorsal side of the mouse touching the board.

3.3) Use pre-sterilized dissecting scissors and forceps to make a mid-line incision on

the ventral portion of the mouse. Make the incision at the genitalia proceeding cranial to

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the base of the neck. Open the skin incision laterally with tweezers and pin the skin to

the pinning board to expose the peritoneum.

3.4) Make a mid-line incision in the peritoneum of the abdomen with dissecting scissors

and fold the peritoneum with forceps laterally and pin to the pinning board in order to

expose the abdominal organs. Note: take care to avoid cutting abdominal organs.

3.5) Remove the small intestine from the abdominal cavity with dissecting forceps and

scissors by cutting the proximal junction of the small intestine connected to the stomach

and the distal junction connected to the caecum. Place the small intestine a sterile petri

dish containing 10 ml of ice cold 1x PBS.

3.6) Flush the contents contained within the lumen of the small intestine with ice cold 1x

PBS using a 1 ml pipette. Repeat this step until the debris within the lumen of the small

intestine is removed.

3.7) Cut the small intestine with dissecting scissors into 3-4 approximately equal length

strips and lay the strips longitudinally on a new sterile petri dish. For each strip, insert

the cutting edge of the dissecting scissors inside the lumen of the small intestine to

make an incision the entire length of the strip. Use forceps to laterally fold open the

incision in order to expose the lumen of the small intestine.

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3.8) Use a sterile glass slide to gently scrape away the villi on the luminal surface of the

small intestine. Cut the small intestine into 1-2 cm length strips with dissecting scissors

and transfer these strips to a 50 ml conical tube containing 10 ml ice cold 1x PBS.

3.9) Mix the contents gently and allow the tissue contents to settle to the bottom of the

50 ml conical tube. Aspirate the 1x PBS and repeat this three times by washing the

tissue with 10 ml of ice cold 1x PBS. On the final wash leave the conical tube on ice for

10 min containing 10 ml 1x PBS and the small intestine tissue segments.

3.10) Aspirate the 1x PBS and add 25 ml of 1x PBS + 2 mM EDTA. Place on a

rocking platform at 4 oC or in an ice bucket for 45 min. While the tissue is incubating,

label six 15 ml conical tubes “fraction #1-6.”

3.11) After incubation, allow the tissue contents to settle to the bottom of the tube

and aspirate 1x PBS + 2 mM EDTA. Add 10 ml of 1x PBS +10% FBS and shake the

tube vigorously by hand 10 times.

3.12) Allow the tissue contents to settle to the bottom of the tube. Remove and

transfer the supernatant to the tube labeled “fraction 1”. Repeat the 1x PBS + 10%

FBS shaking step five times and transfer the supernatant contents in order to the

appropriately labeled fraction tube.

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3.13) Centrifuge at 125 x g for 5 min. Aspirate the supernatant and resuspend

pellets in 5 ml pre-warmed 1x Advanced DMEM/F-12, with no growth factors added.

3.14) Centrifuge at 78 x g for 2 min. Aspirate 4 ml of the supernatant and resuspend

each pellet in 1 ml of remaining media.

3.15) Remove 0 μl from each fraction and add to a glass slide. Visualize crypts

and debris under a light microscope and determine which fractions to combine and

pool accordingly to achieve the greatest percentage of crypts to debris ratio. The

authors find that fractions 2-6 yield the greatest percentage of crypts to be plated.

The fractions to pool are most often fraction 3 with fraction 4, and fraction 5 with

fraction 6.

3.16) Centrifuge fractions to be plated at 125 x g for 5 min. Aspirate supernatant

leaving 50-100 remaining above the pellet.

3.17) Keep tubes on ice and add 1 ml of protein matrix to the pooled fractions. Pipet

up and down slowly to prevent addition of air bubbles.

3.18) Remove pre-warmed 24 well plates from 37 oC incubator and add 50 μl of

protein matrix/crypt suspension in the middle of each well. Transfer the seeded plate

to a 37 oC incubator for 10 min to allow the protein matrix drop to solidify.

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3.19) Add 450 μl of previously prepared organoid growth media + 50 μl of R-

spondin1 conditioned media per well. Incubate plates at 37 oC + 5% CO2 overnight.

3.20) Add 50-100 μl of R-spondin1 conditioned media per well daily. Every 3rd day

replace entire organoid growth media. The protein matrix drop maintains its integrity

for one week, and after 7 days proceed to passaging organoids.

4. Passaging Organoids Every 7th Day

4.1) Thaw the protein matrix on ice overnight at 4 oC the day before passaging.

Warm one sterile 24 well plate to 37 oC for at least 30 min. Prepare organoid growth

media described in step 2.2 and keep at 37 oC until usage.

4.2) Remove organoid plate from incubator and commence passaging of plate on

ice.

Aspirate growth media with a 10 ml pipette from 3-4 wells to start.

4.3) In the aspirated wells, pipette up and down in order to dislodge the protein

matrix drop from the plate. Gentle scraping with the tip of a 10 ml pipette is helpful in

dislodging the protein matrix drop. Transfer the contents to a 15 ml conical tube.

4.4) Incubate the 15 ml conical tubes on ice for 10 min and centrifuge at 125 x g for

5 min at 4 oC. Observe a white pellet at the bottom of the conical tube. Gently

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aspirate the majority of the supernatant above the organoid pellet leaving 100-200 μl

above the pellet.

4.5) Break up the organoid crypts by using a 23 gauge needle attached to a 1 ml

syringe. Aspirate and eject 10-15 times in order to dissociate the crypts. Minimize air

bubble formation due to excessive pipetting.

4.6) Resuspend the organoids in 500 μl of protein matrix and add 50 μl per well in a

new pre-warmed 24 well plate. Add 450 of previously prepared organoid growth

media + 50 μl of R-spondin1 conditioned media per well. Incubate plates at 37 oC

overnight.

5. Plating Organoids on Day 14 for Pattern Recognition Receptor Stimulation

with PAMPs and Listeria monocytogenes for Gene Expression Analysis

5.1) Two days prior to challenge, streak out L. monocytogenes on BHI agar plate.

5.2) The following day pick a L. monocytogenes colony and grow in 10 ml of BHI

broth at 37 oC overnight shaking at 200 rpm.

5.3) Thaw the protein matrix on ice overnight at 4 oC the day before organoid

challenge. Warm one sterile 24 well plate to 37 oC for at least 30 min. Prepare

organoid growth media described in step 2.2 and keep at 37 oC until usage.

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5.4) Remove organoid culture from incubator and commence passaging of

organoids.

5.5) Aspirate media from 3-4 wells and pipette up and down in order to dislodge the

protein matrix drop from the plate. Gentle scraping with the tip of a 10 ml pipette is

helpful in dislodging the protein matrix drop. Transfer the contents to a 15 ml conical

tube.

5.6) Incubate the 15 ml conical tubes on ice for 10 min. Centrifuge at 125 x g for 10

min at 4 oC. Observe a white pellet at the bottom of the conical tube. Gently aspirate

the supernatant leaving approximately 100 μl of residual supernatant behind.

5.7) Resuspend the organoid pellet in 5 ml of ice cold 1x PBS and remove a 10 μl

aliquot for counting. Add the 10 μl aliquot to the middle of a hemocytometer without

the glass cover slip. Gently overlay the glass coverslip on top of the drop. The

authors find that the hemocytometer will clog with organoids if the glass slide is not

first removed.

5.8) Count the total number of organoids via a light microscope in every grid/ the

entire chamber of the hemocytometer and determine the organoid concentration:

number of total organoids counted/10 μl.

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5.9) Remove a appropriate volume from the 15 ml conical tube that will allow

adequate numbers of organoids to be seeded for the assay and centrifuge this

suspension at 125 x g for 10 min. Note: The authors find that a range between

40-100 organoids per well of a 24 well plate is sufficient for RNA analysis.

5.10) Aspirate the supernatant and resuspend the pellet in 500 μl of protein matrix

without generating air bubbles. Add 50 μl of organoid/protein matrix suspension per

well in a 24 well plate. Note: The amount of protein matrix used to resuspend

organoids will vary for the number of treatment conditions and technical

replicates.

5.11) Add 500 μl of organoid growth media (without N-acetyl-cysteine) to each well

and incubate at 37 oC + 5% CO2 for 1 hr.

5.12) Prepare 2x concentrations of PAMPs and live L. monocytogenes. Measure the

OD of live L. monocytogenes and steak on a BHI plate for counting. Treat organoids

at 1 x 106 CFU/ml. For example, the authors find that an OD of 0.6 ≈ 1 x 109 CFU/ml

under typical conditions in their laboratory. The OD to CFU determination will vary

by bacteria type and should be assessed prior to organoid stimulation.

5.13) Streak out a serial dilution from the treatment stock of L. monocytogenes to

accurately determine the treatment CFU/ml. The authors find that a 1 x 108 dilution

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of 100 μl on a BHI agar plate is sufficient to count colonies to determine an accurate

CFU/ml.

5.14) Prepare a heat killed solution of L. monocytogenes by taking a 1 ml aliquot of

the live L. monocytogenes solution and centrifuge at 5,000 x g for 10 min. Aspirate

the supernatant and wash the bacterial pellet with 1 ml of 1x PBS.

5.15) Centrifuge the L. monocytogenes at 5,000 x g for 10 min and resuspend the

pellet in 1 ml of 1x PBS. Heat kill the L. monocytogenes in a 1.7 ml polypropylene

tube by heating at 80-90 oC for 1hr. Culture a 100 μl aliquot of the heat killed L.

monocytogenes on a BHI agar plate to confirm no live bacteria remain.

5.16) Add 500 μl of the appropriate 2x PAMP and/or microbe treatment to 500 μl

resident media in designated wells determined by the user. Adding a 500 μl aliquot

of a 2x PAMP/microbe treatment to 500 μl of resident media will bring the

treatment concentration to 1x. Incubate at 37 oC + 5% CO2 for 24 hr.

5.17) The following day count L. monocytogenes colonies for an accurate

determination of treatment CFU/ml.

5.18) Aspirate media from the plate of treated organoids and wash wells three times

with 1x PBS.

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5.19) Proceed to RNA extraction via phenol/chloroform(Chomczynski and Sacchi,

2006) or a RNA extraction kit according to manufacturers instructions. Evaluate

gene expression using standard qRT-PCR(Allen et al., 2011).

6. Plating Organoids on day 14 for PAMP and L. monocytogenes Challenge for

Protein Analysis in Supernatant

6.1) Two days prior to challenge start a cell culture of Caco-2 cells in a T-75 flask

with 1x DMEM + 10% FBS and incubate the Caco-2 cells at 37 oC + 5% CO2. Begin

a bacterial culture of L. monocytogenes on BHI plate and incubate plate in a

bacterial incubator at 37 oC.

6.2) The following day pick a L. monocytogenes colony and grow in 10 ml of BHI

broth at 37 oC overnight. Thaw the protein matrix on ice overnight at 4 oC the day

before organoid challenge.

6.3) The following day warm one sterile 96 well black-walled plate to 37 oC for at

least 30 min. Prepare organoid growth media without N-acetyl-cysteine described in

step 2.2 and keep at 37 oC until usage. Remove organoid culture from incubator and

commence passaging of organoids.

6.4) Aspirate media from 3-4 wells and resuspend with a pipette in order to dislodge

the protein matrix drop from plate. Gentle scraping with a 10 ml pipette is helpful in

dislodging the protein matrix drop. Transfer the contents to a 15 ml conical tube.

129

6.5) Incubate the 15 ml conical tubes on ice for 10 min. Centrifuge at 125 x g for 10

min at 4 oC. Observe a white pellet at the bottom of the conical tube. Gently aspirate

the supernatant leaving 100 μl of residual supernatant behind.

6.6) Resuspend the organoid pellet in 5 ml of ice cold 1x PBS and remove a 10 μl

aliquot for counting. Add the 10 μl aliquot to the middle of a hemocytometer and

gently overlay the glass coverslip. The authors find that the hemocytometer will clog

with organoids if the glass slide is not first removed.

6.7) Count the total number of organoids via a light microscope in every grid of the

hemocytometer/ the entire chamber and determine the organoid concentration:

number of total organoids counted/10 μl.

6.8) Remove a appropriate volume from the 15 ml conical tube that will allow

adequate numbers of organoids to be seeded for the assay and centrifuge this

suspension at 125 x g for 10 min. Note: The amount of protein matrix used to

resuspend organoids will vary for the amount of treatment conditions and

technical replicates. The authors find that 40 - 100 organoids is sufficient for

secreted protein analysis.

6.9) Aspirate the supernatant and resuspend the pellet in 500 μl of protein matrix

/well without generating air bubbles. The amount of protein matrix used to

130

resuspend organoids will vary for the amount of treatment conditions and

technical replicates.

6.10) Leave at least two columns empty on the 96 well black-walled tissue culture

plate as these will serve as wells seeded with Caco-2 cells for the generation of a

standard curve. Add 50 μl of protein matrix + organoid suspension per well to a pre-

warmed 96 well black-walled tissue culture plate. Store the plate at 37 oC for 10 min

to allow protein matrix to solidify.

6.11) Add 100 μl of organoid growth media (without N-acetyl-cysteine) to each well

and incubate at 37 oC + 5% CO2 for 1 hr.

6.12) Prepare 2x concentrations of PAMPs and live L. monocytogenes. Add 100 μl of

each given treatment condition per well and incubate for 24 hr. Incubation and

Treatment times to be determined by the investigator.

6.13) The following day plate Caco-2 cells in the standard curve wells. Begin by

warming sterile 1x PBS, 0.25% trypsin and 1x DMEM + 10% FBS to 37 oC.

6.14) Remove the T-75 flask containing Caco-2 cells and aspirate the cell culture

media. Wash the cells with 10 ml 1x PBS. Aspirate the 1x PBS, add 2 ml 0.25%

Trypsin and place T-75 flask in 37 oC incubator for 15 min.

131

6.15) Visualize the flask under a light microscope to ensure the cells have detached

then add 8 ml of 1x DMEM + 10% FBS to the detached Caco-2 cells and transfer

into a 15 ml conical tube. Remove a 10 μl aliquot and count the cells by light

microscopy using a hemocytometer. Note: The authors find that a upper cell

number of 60,000 cells/well work well and dilutions can be conducted for generation

of the standard curve down to 2500 cells/well.

6.16) Resuspend the Caco-2 cells in protein matrix and add 50 μl per well to

appropriate wells. Allow the protein matrix to solidify at 37 oC for Caco-2 cells.

6.17) Following the treatment time for the organoid assay, aspirate and save the

organoid cellular supernatant media and keep on ice. Wash the wells three times

with 1x PBS. Add 100 µl of 100% methanol and incubate at 4 oC or on ice for 20-30

min to fix the organoids.

6.18) Following the methanol incubation, wash the wells three times with ice cold 1x

PBS. The plate can now be stored at 4 oC for 1-2 weeks.

6.19) Add nuclear staining dye at a concentration of 1 µg/ml per well, cover the plate

with aluminum foil and incubate at 4 oC overnight.

132

6.20) Use a 96 well plate reader to measure nuclear staining dye excitation/emission

(350nm/461nm respectively) and normalize each organoid well to the standard

curve of Caco-2 cells.

6.21) Proceed to downstream assays, such as ELISA(Allen et al., 2011), of cell

culture supernatant normalizing to cell number.

D. Results:

When following this protocol to cultivate intestinal organoids, characteristic sphere

shaped organoids will be present after harvesting. The addition of R-spondin1

conditioned media daily will initiate the growth and budding of the organoids. The

growth of organoids is shown in Figure 1 (A-F), and is representative of intestinal

organoids on days 1, 2, 4, 5, 6 and day 14. Figure 1F represents the non-

homogeneous growth characteristics of organoids on day 14 of PAMP challenge.

Once the organoids are grown to an adequate number they can be replated and

challenged with various PAMPs and/or microbes. Expression analysis can be

performed via standard techniques. This is represented in Figure 2 (A-C) which

shows the mRNA expression of inflammatory cytokines IL-18, IL-6, and

a 24 hr challenge of organoids with heat

killed L. monocytogenes and the PAMP flagellin. Figure 3 (A-C) demonstrates the

relative nuclear staining of intestinal organoids with nuclear staining dye following

fixation with minimal background staining of debris in the resident protein matrix.

133

This method of fixation and staining can be applied to normalize assays, which will

account for differing cell numbers in each well. This is apparent in Figure 4 when

generating a standard curve of serial dilutions of Caco-2 cells plated in protein

matrix, then fixed and stained with nuclear staining dye. The R2 value of 0.89

indicates a linear relationship between cell number and mean fluorescent intensity,

and the linear equation can be used to normalize organoids to cell number.

Figure 3.1

134

Figure 3.1. Small Intestinal Organoid Growth

Time course of growth for murine small intestine derived organoids following isolation.

A) Day 1. B) Day 2. C) Day 4. D) Day 5. E) Day 6. F) Day 14. Images are

representative of organoid growth for each given day.

Figure 3.2

135

Figure 3.2. mRNA Expression of Inflammatory Cytokines Following 24 hr PAMP

Challenge

Relative mRNA expression of wild-type intestinal organoids challenged for 24 hr with

PAMPs and heat killed Listeria monocytogenes. A-C) represent fold change of the

inflammatory cytokines IL-18, IL-

Figure 3.3

136

Figure 3.3. Relative Fluorescent Staining of Organoids for Normalization

Nuclear staining of organoids with following fixation. A) Bright field of organoids. B)

Fluorescent staining of organoids with nuclear staining dye. C) Merged image of

bright field and fluorescent staining.

Figure 3.4

Figure 3.4. Standard Curve generated from Caco-2 Cells

Standard curve generated from triplicate wells. Cell seeding had a range starting at

60,000 cells per well with dilutions down to 2,500 cells per well.

137

Figure 3.5

Figure 3.5. Graphic Representation of the Technique

1. Harvesting R-spondin conditioned media. 2. Harvesting small intestinal crypts,

and growing organoids. 3. Plating organoids, followed by PAMP treatment, and

normalization.

Cell Number

Em

issi

on

Stomach {

Small intestine {

Cecum/ Large intestine {

} Villus

} Crypts

2 mM EDTA

Fractionate and Plate

2

3 4 5 6

14 Days

A. B. C.

D.

Caco-2

6 5 4 3 2 1

1

2

3

4

5

6

Figure 5.

1.

2.

3.

138

E. Discussion:

The culture and maintenance of intestinal organoids is a procedure that can be

mastered by any individual with adequate tissue culture technique. There are subtleties

in passaging when compared to growing cells in a more conventional monolayer, but

these subtleties are not difficult to overcome. The critical steps of this method involve

being able to grow the organoids to a high enough density for optimal seeding.

Experiments must be scaled down with organoids as large seeding densities that can

commonly be achieved with cell lines are not practical. This becomes especially

apparent when there are multiple treatment groups.

This protocol is intended to provide a step-by-step method to study host-pathogen

interactions of the intestinal epithelium with a variety of different bacterial, viral, and

fungal pathogens, as well as address difficulties using this system with respect to

normalization. Reports are available that describe the interactions of intestinal

organoids with the bacterial pathogen Salmonella, yet do not address normalization

methods when measuring secreted cytokines(Zhang et al., 2014). There are several

difficulties that are encountered when normalizing organoid cultures by different

methods. Normalizing secreted protein via BCA is an option; however, the growth

components required for organoid culture (N2 and/or Vitamin B27) interfere with the

BCA assay (data not shown). Normalizing via cellular viability, such as a modified MTT

assay has been described(Grabinger et al., 2014); however, a treatment that will alter

the mitochondrial metabolic activity of the organoids will introduce an inaccurate method

for normalization via this technique as MTT is based on reduction by the action of

139

mitochondrial dehydrogenases(Slater et al., 1963). It is also necessary to remove N-

acetyl-cysteine from the media if normalization via the MTT technique is desired.

The authors find that fixing with methanol and staining nuclei with nuclear staining dye

is an effective normalization technique against a standard curve of Caco-2 cells.

Although the growth characteristics of this colon cancer cell line do not exactly mimic

the growth characteristics of intestinal organoids, using a nuclear stain and measuring

mean fluorescence intensity is a good strategy to normalize against total cell number.

Taken together, the technique described here provides a good starting point to mimic

host-pathogen interactions with adequate normalization that is essential for making

accurate interpretations using this model system. The significance of this technique with

respect to alternative methods is that proper normalization must be performed when

conducting any evaluation of secreted protein, such as ELISA based assays.

F. References: ALLEN, I. C., MOORE, C. B., SCHNEIDER, M., LEI, Y., DAVIS, B. K., SCULL, M. A.,

GRIS, D., RONEY, K. E., ZIMMERMANN, A. G., BOWZARD, J. B., RANJAN, P.,

MONROE, K. M., PICKLES, R. J., SAMBHARA, S. & TING, J. P. 2011. NLRX1

protein attenuates inflammatory responses to infection by interfering with the

RIG-I-MAVS and TRAF6-NF-kappaB signaling pathways. Immunity, 34, 854-65.

CHOMCZYNSKI, P. & SACCHI, N. 2006. The single-step method of RNA isolation by

acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something

years on. Nat Protoc, 1, 581-5.

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FRESHNEY, R. I. & FRESHNEY, M. G. 2002. Culture of epithelial cells, New York,

Wiley-Liss.

GRABINGER, T., LUKS, L., KOSTADINOVA, F., ZIMBERLIN, C., MEDEMA, J. P.,

LEIST, M. & BRUNNER, T. 2014. Ex vivo culture of intestinal crypt organoids as

a model system for assessing cell death induction in intestinal epithelial cells and

enteropathy. Cell Death Dis, 5, e1228.

HYNDS, R. E. & GIANGRECO, A. 2013. Concise review: the relevance of human stem

cell-derived organoid models for epithelial translational medicine. Stem Cells, 31,

417-22.

KAIKO, G. E. & STAPPENBECK, T. S. 2014. Host-microbe interactions shaping the

gastrointestinal environment. Trends Immunol, 35, 538-48.

LANCASTER, M. A., RENNER, M., MARTIN, C. A., WENZEL, D., BICKNELL, L. S.,

HURLES, M. E., HOMFRAY, T., PENNINGER, J. M., JACKSON, A. P. &

KNOBLICH, J. A. 2013. Cerebral organoids model human brain development

and microcephaly. Nature, 501, 373-9.

SATO, T., STANGE, D. E., FERRANTE, M., VRIES, R. G., VAN ES, J. H., VAN DEN

BRINK, S., VAN HOUDT, W. J., PRONK, A., VAN GORP, J., SIERSEMA, P. D.

& CLEVERS, H. 2011. Long-term expansion of epithelial organoids from human

colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology,

141, 1762-72.



141



SLATER, T. F., SAWYER, B. & STRAEULI, U. 1963. Studies on Succinate-Tetrazolium

Reductase Systems. Iii. Points of Coupling of Four Different Tetrazolium Salts.

Biochim Biophys Acta, 77, 383-93.

VACHON, P. H., HARNOIS, C., GRENIER, A., DUFOUR, G., BOUCHARD, V., HAN, J.,

LANDRY, J., BEAULIEU, J. F., VEZINA, A., DYDENSBORG, A. B., GAUTHIER,

R., COTE, A., DROLET, J. F. & LAREAU, F. 2002. Differentiation state-selective

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ZHANG, Y. G., WU, S., XIA, Y. & SUN, J. 2014. Salmonella-infected crypt-derived

intestinal organoid culture system for host-bacterial interactions. Physiol Rep, 2.

142

Chapter 4:

NF-B inducing kinase (NIK) is essential for adequate expression of the stem cell

marker LGR5 and results in enhanced proliferation of intestinal epithelial cells.

A. Abstract

In the previous chapter, we discussed an optimized method for the culture and

stimulation of intestinal organoids. Here, we utilized this method further to study

intestinal epithelial cell proliferation. Intestinal epithelial cell (IEC) proliferation is

required to maintain homeostasis throughout the gastrointestinal (GI) tract. IECs act as

a protective barrier from the external environment within the GI lumen, exhibiting rapid

turnover and thus maintaining the integrity of the barrier. Cellular renewal relies on

several signal transduction pathways working in synchronization; and aberrancies in this

regenerative process contribute to cancer initiation within the intestine. The non-

canonical NF-B signaling pathway is emerging as an important pathway that aids in

adequate proliferation of intestinal epithelial cells. Here, we investigated the contribution

of the non-canonical NF-B pathway to IEC proliferation in a mouse model deficient for

NF-B inducing kinase (NIK). In the absence of NIK, IEC organoids demonstrated

defects in proliferation, consistent with limited expression of the intestinal stem cell

marker, LGR5. Our results demonstrate the essential role of NIK to proper IEC

proliferation, and ultimately GI maintenance.

143

B. Introduction

Epithelial cells lining the small intestine and colon within the GI tract undergo rapid

turnover compared to those residing in other tissues. For example, epithelial cells

composing the skin turnover on a 47-48 day basis while, in contrast, intestinal epithelial

cells turnover every 4-5 days (Iizuka, 1994), (Creamer et al., 1961). This rapid renewal

process is associated with protection from various diseases, such as colon cancer,

since increased turnover on a bi-weekly basis prevents cells from accumulating

mutations due to toxic substances encountered within the GI tract. Intestinal epithelial

cells differentiate from intestinal stem cells (ISC) at the base of the intestinal crypt and

migrate up the villus in single-file manner. Upon reaching the apex of the villus, IECs

undergo apoptosis and are subsequently shed into the intestinal lumen to be excreted.

This consistent and rapid renewal is important in protecting the GI tract from the hostile

environment within the lumen of the intestine. In the absence of constant IEC renewal,

any breach in the intestinal epithelial barrier may lead to deleterious effects. Such

consequences may include endotoxic shock due to the plethora of bacterial pathogen

associated molecular patterns (PAMPS) that stimulate surrounding immune cells.

Furthermore, neutrophils responding to local invaders produce vast quantities of

reactive oxygen species (ROS), which can be genotoxic to surrounding cells.

The non-canonical, or alternative NF-B signaling pathway has been shown to be vital

for the proper development of secondary lymphoid organs, as well as proper B-cell

maturation (Claudio et al., 2002). This alternative NF-B pathway is activated

144

downstream of TNF-family receptors, including BAFF, LTR, and CD40; thus, absence

of this pathway results in immunodeficiency of the adaptive immune system. One crucial

molecule that propagates downstream signaling when this pathway is stimulated is NF-

B inducing kinase (NIK). In quiescent cells, NIK is constantly poly-ubiquinated and

degraded, thus maintaining the level of NIK within the cell at low levels. Following a

stimulus, NIK is stabilized, and levels rise within this cell, allowing NIK to act as a

functional kinase on its substrate IKK, which ultimately leads to NF-B activation,

primarily through RelB-p52 heterodimers. Recently, it has been demonstrated that the

silencing of NIK in the surrounding stroma of the intestine results in increased

metastasis of colon cancer cells to distant sites in the body (Maracle et al., 2018). The

purpose of this study was to investigate the role of NIK in relation to the GI system. In

vivo, NIK maintains GI homeostasis by preventing aberrant cellular hyperplasia and

inflammation. When cultured ex vivo, NIK is required for proper IEC proliferation. Our

results demonstrate that loss of Nik results in decreased IEC turnover due to reduced

expression of the stem cell receptor LGR5.

C. Materials and Methods

Reagents

Advanced DMEM F12 media, N2 supplement, B27 without Vitamin A, HEPES,

Glutamax supplement were purchased from ThermoFisher Scientific. HEK-293T-

Rspondin1 cells were cultured as previously described (Kim et al., 2005), and utilized to

generated R-spondin 1 conditioned media previously described (Rothschild et al.,

2016). L- cells were purchased from ATCC and utilized to generate Wnt3a conditioned

145

media as described by ATCC. qRT-PCR primers for mouse LGR5 and KRT20 were

purchased from TheromFisher Scientific. Antibodies for EphB2, EpCAM4, anti-mouse

APC, anti-donkey Alexaflur488 were purchased from ThermoFisher.

Colonic Organoid Isolation and Culture.

Colons from age matched 6-12 week old C57BL6J and Nik-/- mice were dissected

separately and colonic crypts were isolated as previously described (Sato et al., 2011).

For each genotype, approximately 1000 crypts were plated in each well of a 6-well

tissue culture (TC)-treated plate, containing in 100ul matrigel per well. Matrigel was

allowed to solidify for 10 minutes in a 37oC TC incubator. Organoids were cultured

900ul basal media containing: 1x N2, 1x B27without vitamin A, 10mM HEPES, 1x

Glutamax supplement, 100ng/ml m-Noggin, 50ng/ml m-EGF, 1mM N-acetyl cysteine,

25nM Y27632, and 1x Penicillin/Streptomycin. An additional 100 ul R-spondin1

conditioned media and 100 ul Wnt-3a media was added to each well during seeding

(day 0) for initial culturing. After 24 hours, the culture media was aspirated, each well

was washed with warm 1x PBS without Calcium/Magnesium; fresh medium without

Y27632 was then added to each well. Following day 2, 100ul R-spondin 1 and 100ul

Wnt-3a media was added to each well daily. The medium was completely replaced

every 3 days.

Growth tracking of organoids

Following 7 days of culture, organoids for each respective genotype were passaged and

single cell suspensions were made. This was accomplished by scraping and

146

transferring matrigel drops to a 50ml conical tube. Tubes were allowed to incubate on

ice for 15 min, and were subsequently centrifuged at 300g for 10 min. PBS was

aspirated and matrigel pellets were manually disrupted using a syringe with a 22G

needle. Organoids were centrifuged again and pellets were incubated for 10 min at

37oC with 1x TrypLE, centrifuged and TrypLE was aspirated. Cells were then passed

through a 70 um cell strainer to make single cell suspensions. Cells were counted,

assessed for viability with Trypan blue, and plated at 20,000 cells per well in a 6 well

dish. Single cells were tracked for growth over the next 6 days. Media was changed

each day, and each genotype received the same media, media volume, and

concentration of organoid growth factors.

qRT-PCR

Colon crypts were isolated as previously described (Sato et al., 2011). Crypts were

pelleted by centrifugation and harvested for RNA using Zymogen quick RNA isolation

kit. 2g RNA was converted to cDNA using high-capacity cDNA reverse transcription kit

according to the manufacturer’s instructions. 50ng cDNA was plated in each well as a

template, and amplified using TaqMan gene expression master mix according to the

manufacturer’s instructions.

FACS

Single cell suspensions of colonic crypts were made by first isolating colonic crypts.

Crypts were incubated for 20 min at 37oC in TrypLE (ThermoFisher). Crypt digests were

centrifuged and resuspended in 1x DMEM + 10 % FBS and passed through a 70 m

147

cell strainer. Single cell suspensions were stained with respective antibodies for 30 min

at 4oC, and washed with 1x PBS before FACS sorting.

Statistical Analysis

Graphs are represented as the mean ± standard error of mean (S.E.M.). Graphs and

statistical analysis were conducted via GraphPad PRISM software. Data sets were

analyzed by Mann-Whitney U test, and statistical significance was determined using a P

value < 0.01 unless otherwise indicated.

D. Results

NIK is needed for proper proliferation of colonic organoids.

Previous studies have demonstrated that the Wnt signaling pathway is critical when

culturing intestinal organoids ex vivo (Barker et al., 2007). Furthermore, a correlation

has been observed between inflammation driven by the classical NF-B pathway and

colon cancer (Greten et al., 2004). Here we tested the alternative NF-B pathway in

relation to colonic organoid proliferation by utilizing Nik-/- mice. In order to assess

whether NIK influences colonic proliferation, we cultured colonic crypts from Nik-/- mice

ex vivo for one week using both wild-type and ApcMin crypts as negative and positive

controls, respectively (Figure 4.1A). As anticipated, ApcMin crypts proliferated at an

accelerated rate compared to those derived from wild-type mice. To our surprise, Nik-/-

crypts proliferated, however the proliferation rate was drastically reduced when

compared to both wild-type and ApcMin crypts. This was observed both qualitatively

148

using microscopy (Figure 4.1A), and quantitatively when organoid diameter was

measured after one week (Figure 4.1B).

Colonic organoids from Nik-/- mice have impaired proliferation due to reduced

Lgr5 expression and enhance Krt20 expression.

In order to determine the cause of the reduced proliferation of Nik-/- colonic organoids,

stem cells derived from each of the different strains were characterized. This was

accomplished by evaluating the expression of Lgr5, a crucial receptor and marker of

intestinal stem cells (ISC), from freshly isolated colonic crypts, and evaluating gene

Figure 4.1 A. Growth tracking of organoids from single cell suspensions of wild-type,

ApcMin, and Nik-/-. Images are representative for day 1-6. Scale bar = 100m B. Quantification of organoid diameter following one week of growth. 30 organoids were measured in a similar manner for both wild-type and Nik-/- from randomly selected tissue culture wells. ApcMin quantification was omitted to not obscure the graph. Experiments were repeated a minimum of two times with similar results.

149

expression by qRT-PCR. Interestingly, we found Lgr5 levels of Nik-/- crypts to be

significantly reduced compared to the wild-type crypts (Figure 4.2A).

A reduction in the expression of Lgr5 suggests either that the number of stems cells

from Nik-/- is lower compared to the wild-type, or the amount of receptor on a per stem

cell basis is drastically reduced. We also analyzed the expression of Krt20, which has

been shown to increase in expression to correlate with differentiated IEC, as opposed to

ISC (Merlos-Suarez et al., 2011). A greater expression of Krt20 was observed from

Nik-/- crypts (Figure 4.2B). Collectively, these expression results suggest that in the

absence of NIK, not only are stem cell pools reduced, but the differentiated intestinal

Figure 4.2 A. Relative expression of Lgr5 from wild-type and Nik-/- colonic crypts. B. Relative expression of Krt20 from wild-type and Nik-/- colonic crypts. For both figures: wild-type n=3 mice, Nik-/- n=3 mice. Gapdh was used as the housekeeping gene and experiments were repeated a minimum of two times with similar results.

150

epithelial cell lineages are greatly increased. These data provide a possible mechanistic

explanation for the reduced proliferation of the Nik-/- colonic organoids.

Single cell suspensions of individual ISC from Nik-/- crypts yields reduced

organoid colonies

In order to determine whether NIK plays a role in not only stem cell number, but in

proliferation as well, we tested single cell suspensions from harvested colonic crypts. As

was consistent with our pervious findings, this experiment yielded a reduction in the

total number of colonic organoid colonies from Nik-/- crypts (Figure 4.3A). This

suggests that the stem cell pool from Nik-/- crypts are reduced, thus leading to a

reduction in the number of individual organoid colonies that were derived from single

stem cells. To further evaluate the difference in stem cell number between wild-type and

Nik-/- stem cells, we performed FACS analysis on the ISC surface marker EphB2 and

the IEC marker EpCAM1 as shown by Merlos-Suárez et al. (Figure 4.3B). The results,

though subtle, do display altered populations in the EphB2-high pool and EpCAM1-high

pool between wild-type and Nik-/- crypts. Separate gating strategies are indicated to

assess the differences between these two groups.

151

Figure 4.3 A. Total organoid colonies from single cell suspension made after one week of culturing colonic organoids. B. FACS analysis of single cell suspensions made from both wild-type and Nik-/- colonic crypts. Cells were stained for EphB2 and EpCAM. A separate gating strategy is depicted as previously described by Merlos-Suárez et al. to emphasize the ISC pools between the genotypes. Experiments were performed once.

152

E. Discussion

One major role of the NF-B pathway is to enhance cellular survival by driving anti-

apoptotic mechanisms (Karin, 2009). The ability of the NF-B pathway to act as an

oncogene is possible, yet rare, and limited to certain types of leukemia (Karin, 2009).

Evidence suggests that the classical and alternative NF-B pathways are capable of

crosstalk between each other, with NIK having the ability to stimulate the classical

pathway (O'Mahony et al., 2000), (Ramakrishnan et al., 2004). Based on our ex vivo

culture of Nik-deficient colonic organoids, reduced growth and proliferation may result

from globally reduced stimulation of the classical NF-B pathway as a direct

consequence of the loss of NIK. Reduced cellular survival, and increased apoptosis

would be expected with reduced classical NF-B activation, similar to the phenotype

observed in Nik-deficient organoids. However, reduced Lgr5 expression in Nik-deficient

organoids suggests a mechanism that may involve the collaboration between the

alternative NF-B pathway and the Wnt signaling pathway, or some component of the

alternative pathway that directly contributes to the reduced expression of LGR5.

The discovery of LGR5 as the receptor responsible for contributing to ISC maintenance

has greatly advanced our understanding of intestinal stem cell biology (Barker et al.,

2007). Not only is Lgr5 a Wnt responsive gene, but changes in Lgr5 expression suggest

the Wnt signaling pathway has been altered in the intestine (Huels and Sansom, 2017).

It was recently shown that both R-spondins and Wnt ligands are both necessary to

maintain ISCs and allow them to differentiate into IECs that compose the crypt-villus

153

architecture (Yan et al., 2017). Yan et al. demonstrated that when Wnt ligands are

absent, R-spondins are unable compensate for the loss of Wnt signaling, resulting in

crypt death. Conversely, if R-spondins are lost, Wnt signaling is temporarily sufficient for

crypt maintenance; however, the LGR5 stem cell pool is lost, and the crypt eventually

dies. A similar phenomenon may explain the results observed in Nik deficient organoids;

one possible explanation is that there may be alternative sources of secreted R-

spondins other than epithelial cells in vivo, thereby promoting crypt survival.

Previous studies have shown NIK to be an essential protein kinase that participates in

the activation of NF-B downstream of TNF, CD95, and IL-1 receptors in the alternative

NF-B pathway, (Malinin et al., 1997). Furthermore, Nik deficiency in mice leads to

reduced B-cells numbers in the lymph nodes and spleen, which also contributes to

impaired IgA production (Brightbill et al., 2015). Since IgA is the predominant class of

antibody to be produced in mucosal tissues, one possibility for the alternative responses

observed in vivo and ex vivo could be explained by reduced IgA production in NIK

deficient mice. An impaired IgA response may ultimately contribute to a disrupted

intestinal microbiome, which may perpetuate an enhanced inflammatory state within the

intestinal system. Since IgA coating has been shown to predict the bacterial populations

that contribute to colitis in mice (Palm et al., 2014), it is possible that loss of IgA coating,

and ultimately immune defenses, may perpetuate a hypersensitive state in the GI tract.

Our studies demonstrate NIK to be a pleiotropic molecule that is of critical importance to

IEC homeostasis; therapeutic targeting of NIK may be eventually used for treatment of

GI-related maladies.

154

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M., YUAN, J., LI, X., BOLEN, C. R., WILHELMY, J., DAVIES, P. S., UENO, H.,

VON FURSTENBERG, R. J., BELGRADER, P., ZIRALDO, S. B., ORDONEZ, H.,

HENNING, S. J., WONG, M. H., SNYDER, M. P., WEISSMAN, I. L., HSUEH, A.

J., MIKKELSEN, T. S., GARCIA, K. C. & KUO, C. J. 2017. Non-equivalence of

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Wnt and R-spondin ligands during Lgr5(+) intestinal stem-cell self-renewal.

Nature, 545, 238-242.

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Chapter 5

Discussion and Future Directions

5.1 The Contribution of IRAK-M to GI Immunology

Our findings pertaining to the contribution of IRAK-M were slightly unexpected. One

would anticipate that absence of a negative regulatory protein of the inflammatory

cascade would lead to a runaway inflammatory effect in the host, as well as contribute

to increased carcinogenesis, because increased inflammation can lead to increased

cancer, especially in the colon (Greten et al., 2004). However, in DSS-induced colitis

and AOM/DSS-induced colitis-associated tumorigenesis studies, it was apparent that

absence of IRAK-M was actually protective in both of these respective models. In our

hands, Irak-m-/- mice display decreased morbidity and mortality when challenged in the

acute DSS colitis model. Our mechanistic data explaining this discrepancy suggests

that Irak-m-/- mice have increased inflammation following DSS challenge, but also

enhanced innate immune functions, particularly with neutrophils. Furthermore, the

innate immune function of these neutrophils greatly contributes to and counteracts the

destructive tissue damage that occurs from bacteria, which breach the intestinal barrier

following DSS challenge. We propose that this enhanced state of immunity present in

Irak-m-/- mice also signals the adaptive immune system, mainly the T-lymphocytes, to

be in a heightened activation state. Following this logic, it is likely that cells of the

adaptive immune system from Irak-m-/- mice contribute to the anti-tumorigenic state

displayed in these animals following AOM/DSS challenge, as T cells are known to

destroy cancerous cells (Martinez-Lostao et al., 2015). An alternative explanation is that

it is likely that difference in the microbiome in the GI tract between Irak-m-/- mice and

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the wild-type mice plays a role in the differences observed in the acute colitis model. In

fact, when an antibiotic cocktail of ampicillin, vancomycin, neomycin, and metronidazole

were given to mice for two weeks prior to DSS challenge, both wild-type and Irak-m-/-

mice displayed similar morbidity and mortality following DSS administration. This

suggests that specific components of the microbiota contribute to the protective

phenotype displayed in Irak-m-/- animals. Metabolomics of specific bacterial products

produced in the GI tract could help explain the discrepancies observed between our

laboratory and others that have utilized these mice.

5.1.1 The Significance of the Irak-mrΔ9-11 Truncation Mutant

The central dogma of biology is that cells contain DNA, and the genes that are

expressed from DNA are transcribed into mRNA; further, mRNA is then translated into

proteins that carry out the functional roles in the cell. At the transcription level,

specifically in eukaryotes, mRNA is processed in a “cut and paste” fashion in the

nucleus by a small nuclear RNA (snRNA)-protein complex called the spliceosome

(Berget et al., 1977). The major function of the spliceosome is to process newly

transcribed mRNA into fully mature mRNA that then leaves the nucleus and encodes for

proteins in the cytosol. In eukaryotes, not all of the nascent mRNA that is transcribed in

the nucleus is encoded into proteins. Exons of the mRNA template, which only make up

a small fraction of the encoded mRNA, are spliced together to form much smaller

mature mRNA, while introns are removed from the mRNA template and degraded

(Berget et al., 1977). As such, a single gene can encode for many altered versions of a

protein based upon which exons are spliced together, and are termed splice variants.

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One unexpected and interesting finding was that the commercially available Irak-m-/-

animals form a spliced Irak-m mRNA that joins exons 8 with exon 12. Murine Irak-m is

located on chromosome 10, and contains 12 total exons that form the coding region for

the Irak-m gene. There are 5 naturally occurring Irak-m splice variants, and only two

form fully encoded proteins, using 1-12 and exons 2-12 to encode for splice variant

proteins, respectively (ensemble.org). The truncation mutant that is present in Irak-m

mutant animals is strictly a result of the knockout strategy used by the scientists that

generated these animals, and is not a normal splice variant that is known to occur in

wild-type mice. This was unexpected, because the first reports describing this knockout

mouse did not describe exon 12 being present in the mRNA transcript that was seen via

northern blot (Kobayashi et al., 2002). The relevance to having exon 12 present in this

transcript lies in the potential to encode a possibly stable truncated IRAK-M protein.

There are several factors that contribute to the stability of mRNA inside the cell, and one

factor for stability that contributes to mRNA translation is the addition of a

polyadenylation (poly-A) tail to the 3’ end of the mRNA. Since exon 12 is the last

naturally occurring exon for the Irak-m gene, the poly-A tail is likely contained in the

mRNA transcript, and it is therefore equally likely that the addition of this exon

contributes to the stability of this spliced transcript for protein translation. Therein lies

the problem for a truncated protein to be encoded from this transcript, which may

function in an unknown mechanism inside the cells. As it happens, the exons that were

targeted for deletion, being exons 9 through 11, encode a portion of the kinase domain

of IRAK-M. Interestingly, the current understanding for the function of IRAK-M describes

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this molecule as a non-functional kinase, so a mutation was targeted in this gene in an

already non-functional domain. Further, even more relevance is apparent because the

mechanism for IRAK-M has been described that demonstrates this protein’s function is

mediated through protein-protein interactions with other molecules, namely IRAK-1 and

IRAK-4 (Wesche et al., 1999), (Zhou et al., 2013). This mainly occurs through death

domain- death domain interactions with other proteins, and the death domain for IRAK-

M is encoded at the 5’end (N-terminal) region of this protein, which would be present in

a protein encoded from the resulting Irak-mrΔ9-11 splice variant.

Though this finding is novel and poses interesting questions into the functional role for

IRAK-M, it still remains highly speculative as to whether a protein is present and

functional. The fact that these Irak-m mutant mice display an alternative phenotype

compared to wild-type animals demonstrates the successful mutation of this gene. It is

highly likely that the mutation generated to make this mouse was successful in creating

a null Irak-m allele; however, research is still required to fully validate whether or not this

is the case. A strategy that could be used to validate whether a truncated IRAK-M

protein is present in these animals, would be to generate an antibody that is specific for

the N-terminal domain of IRAK-M, as most of the current commercially available

antibodies target the C-terminal region of this protein. Then, a simple western blot could

be conducted to validate if a protein is or is not present in these mice. Of the five

commercially available antibodies specific for murine IRAK-M, only one of these

antibodies were successfully validated in our laboratory. This was conducted by cloning

wild-type IRAK-M, along with a C-terminal HA tag, into a plasmid containing the strong

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mammalian CMV promoter. In this fashion, wild-type IRAK-M will be highly expressed

when transfected into HEK293T cells. Unfortunately, the antibody that was successfully

validated will not provide answers as to whether a IRAK-MrΔ9-11 protein is present in the

mutant mouse strain, because the epitope that this antibody recognizes is encoded in

exons 9 and 10.

5.1.2 Studies to Overcome the Truncation Mutant Conundrum

An alternative strategy, without the use of antibodies and western blot would be a

genetic approach. An option would be to test whether mice that are heterozygous for

this mutation have similar inflammatory phenotype as wild-type mice or the Irak-m-/-

mice. Assuming a truncated protein is present, the heterozygous mouse will display

similar responses to the knockout mouse if the mutation created is in fact a dominant

mutation. Alternatively, if the mutation generated is a null mutation, then the resulting

wild-type allele from the heterozygous mice will function in a similar manner to the

homozygous wild-type mouse.

Overall, our results, along with other laboratories demonstrate that IRAK-M is an

important intracellular signaling molecule with complex functions. Our research appears

to demonstrate that the function of IRAK-M following TLR ligation is TLR specific. Our

results suggest that some, but not all of the murine TLRs, when stimulated with

respective ligands displayed significant differences in IL-6, TNF, and IL-10. These

included TLR-2, TLR-7 and TLR-9, and all of these respective TLRs utilize the MyD88

dependent pathway exclusively. One possibility is that that these particular TLRs signal

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through similar NF- κB dimers, recall that NF-κB can form up to 15 combinations of

dimers, and each dimer can have slightly alternating functions (Zhang et al., 2017). This

also could be due to the concentration and treatment time of the particular PAMPs

utilized to stimulate the cells. This poses an interesting question related to dose

response and receptor threshold limits. The complexity of cellular signaling is

highlighted in many aspects of dosage, timing and kinetics, because stimulation of a cell

can result in early activation, late activation, and repressive responses at the gene level

(Wang et al., 2012). An interesting avenue to potentially pursue would be to determine

the precise mechanistic role for IRAK-M when specific TLRs are activated. This has

already been done in the case of TLR-7(Zhou et al., 2013); however, investigation of

TLR-9 and TLR-2 would provide additional interesting insight into the mechanistic

functions of IRAK-M.

5.1.3 Therapeutic Targeting of IRAK-M and the Clinical Implications

Our findings implicate IRAK-M to be an candidate molecular target for patients afflicted

with inflammatory maladies, such as IBD and inflammation driven colorectal cancer.

IRAK-M appears to be a molecule that shifts the balance toward host protection in both

acute colitis and inflammation driven tumorigenesis models. Though more mechanistic

research is required to fully elucidate the functions of IRAK-M, it is tempting to envision

a therapeutic strategy targeting IRAK-M in patients that currently suffer from, or are

predisposed to IBD and inflammation driven colorectal cancer. Based on our findings,

acute inhibition of IRAK-M would be an optimal therapeutic approach to treat patients

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with such maladies. Though no molecular inhibitors for IRAK-M currently exist,

compounds that inhibit the interaction of IRAK-M with other death domain containing

proteins would be a potential strategy. Another interesting approach could be to inhibit

IRAK-M and the mRNA level with a DNAzyme approach. DNAzymes are catalytically

active antisense strands of DNA that will bind to sense mRNA targets, and cleave them

for degradation (Burgess, 2012). Designing a DNAzyme specific for IRAK-M mRNA

could be utilized as a strategy to block the function of IRAK-M with minimal off target

effects, due to the rationale that IRAK-M is expressed in a cell type specific manner,

and is induced following cellular activation by transcription factors like NF-κB and AP-1,

as opposed to being constitutively transcribed (Lyroni et al., 2017). From our results,

one would anticipate that utilizing this approach would lead to a reduction in colitis, and

even prevention of colitis associated tumorigenesis in patients with active tumors.

IRAK-M is an interesting target for modulating the immune system, specifically via

inhibition, because it does not appear to be as essential of an inhibitory molecule when

compared to other negative regulatory molecules of inflammation like A20. Absence of

IRAK-M, at least acutely, does not result in severe multi-organ inflammation and tissue

damage, as is the case in mice with the loss of A20 (Lee et al., 2000). Therefore, if this

molecule is nonfunctional in mutant Irak-m-/- animals, complete inhibition of IRAK-M

may result in similar therapeutic benefits, as our results have demonstrated in the Irak-

m mutant mice. Further, IRAK-M may be a key molecular target for patients suffering

from life-threating infections. One can envision a therapeutic strategy that would aim to

inhibit the function of IRAK-M, which would result in heightened immunological functions

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of the immune system. This could be a novel strategy to treat patients with a

suppressed immune system, as is common under conditions of sepsis, as well as

elderly patients with infectious pneumonia. IRAK-M appears to be a novel molecule for

this type of approach, because its inhibition results in enhanced bacterial clearance,

without negative consequences, such as destructive tissue damage.

5.1.4 IRAK-M Targeted Therapies

Recent technologies have emerged that are very promising for gene therapy;

specifically, with enzymes that can be manipulated to efficiently edit DNA, via the use of

CRISPR-Cas9 technology (Brouns et al., 2008). Utilizing a gene editing approach

would be an option for creating a similar IRAK-M mutant in humans. One would

anticipate that targeting exons 9 through 11 of human IRAK-M for deletion, with

CRISPR-Cas9, would result in a similar outcome that has been seen in the mutant Irak-

m-/- mice. Further, it would be expected that this type of edit of IRAK-M would lead to

diminished colitis, and resistance to colorectal cancer in patients that carried a mutation

in the kinase domains of IRAK-M. This type of application could be conducted on the

precursor cells to macrophages, many of which are the hematopoietic stem cells found

in the bone marrow. In a manner that similar to generating chimeric antigen receptor

(CAR) T-cells, bone marrow could be extracted from human patients, hematopoietic

stem cells could then be individually isolated and transfected in the laboratory with the

specific CRISPR-Cas9 guide RNAs targeting exons 9-11 of IRAK-M for deletion. The

successfully transfected stem cells could then be returned to the patient that carry the

desired IRAK-M mutation, and would ideally result in a permanent therapeutic benefit

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for the patient. One of the promising options of this technique is that the therapy is

essentially permanent; therefore, the need for additional medications in the future

should be reduced. Additionally, one can envision an option in which expression or

repression of a gene, following the CRISPR-Cas9 strategy, is induced with a chemical

compound; analogous to the inducible mouse models that utilize estrogen receptor-

CRE-lox system. In this way, a mutation could be induced, or a gene repressed when

an exogenous molecule, such as tamoxifen is administered.

5.1.5 Future Directions with IRAK-M

In terms of future research regarding IRAK-M, it is necessary to develop and validate a

functional antibody to the murine protein, preferably a monoclonal antibody that

recognizes the N-terminus of IRAK-M. This would help solve the question of what is

occurring in the knockout mouse. In addition to this, one would anticipate the actual

mechanistic role of human IRAK-M to be reexamined. Though this molecule is currently

believed to act as a non-functional kinase, intensive biochemical analysis would help

validate whether IRAK-M is completely devoid of kinase activity. First and foremost,

currently IRAK-M has not been crystalized, and it would be highly relevant to determine

the full 3-D structure of this molecule. In addition, when IRAK-M was first discovered, it

was shown to have weak autophosphorylation capacity (Wesche et al., 1999), yet it is

believed that this molecule is completely devoid of kinase activity. One hypothesis is

that this molecule actually behaves as a functional kinase, but doesn’t have as strong

phosphorylation capacity when compared to other IRAK family members. It would be

very interesting to determine if IRAK-M acts on an unknown substrate that it modulates

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via post-translational modification to affect cellular signaling. An approach to solve this

perplexing question would be to make an overactive kinase mutant in kinase subdomain

VIb. Would changing the endogenous serine in the HRD motif to an aspartic acid

restore kinase function, and if so, what would be the substrate that IRAK-M

phosphorylates? If indeed IRAK-M acts as a functional kinase on a currently unknown

substrate, it is essential to determine what this substrate would be, as it is difficult to

decipher the exact cellular signaling events that IRAK-M would modulate without fully

understanding the kinase-substrate relationship (Berwick and Tavare, 2004).

If indeed IRAK-M phosphorylates a particular substrate, a “chemical genetic” approach

could be used experimentally to answer this question (Shah and Shokat, 2003). This

would involve making a mutation in the ATP binding pocket of the kinase of interest, in

this case IRAK-M, and converting a hydrophobic amino acid to one with a small side

chain. In this manner, the kinase would still be catalytically active, but has now gained

the capacity to use a form of ATP that can be labeled at the γ-phosphate with a

covalently attached traceable substituent, like biotin (Shah and Shokat, 2003), (Berwick

and Tavare, 2004). Even cell permeable ATP analogs can be used, so this technique

would not be limited to use with only cell lysates (Fouda and Pflum, 2015). Furthermore,

because this mutated kinase is the only kinase able to use this analog of ATP as a

substrate, the phosphorylated species, i.e. the substrate of the kinase, will become

uniquely labeled. Regarding IRAK-M, this could entail making a mutation that converts

the tyrosine gatekeeper of the ATP binding pocket of IRAK-M to a glycine or alanine.

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Then, this type of assay could be conducted to determine whether IRAK-M functions as

an active kinase on a particular substrate.

Another interesting method would be to use a Reverse In Gel Kinase Assay (RIKA) (Li

et al., 2007). This method is optimal because of its ingenuity and the relative ease in its

practical application; it requires common sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) reagents to perform this experimental procedure. To

summarize, a catalytically active kinase of interest is mixed with the reagents to make a

SDS-PAGE gel, and in this fashion, the kinase will be embedded into the gel matrix

once the gel has polymerized. Then, cell lysates can be run by electrophoresis, and

following its completion, a series of buffer exchange steps can be performed that cause

the proteins within the gel to refold back into their native conformations. Further,

radiolabeled ATP at the γ-phosphate can be incubated with this gel, and because of the

close proximity of the embedded kinase with potential substrates, the kinase will add

radioactive or “hot” ATP to the potential substrates. Now, any radioactive molecules can

be visualized with radiographic methods, and excised from the gel to be processed via

mass spectrometry. Thus, this provides a relatively inexpensive method to determine

previously unidentified substrates of the protein kinase.

4.2 Impact of Recent Methodologies to Study GI Immunology.

4.2.1 Elucidation of the GI Microbiota

Significant progress within the last decade has been made with respect to GI research.

The use of transgenic technology, particularly the use of genetically modified mice, has

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been at the forefront of many of these advances (Thomas and Capecchi, 1987), and

additional creativity from the scientific community continues to fuel progress within the

field. With respect to GI immunology and pathology, momentum has been building with

research pertaining to the symbiotic interactions between the GI systems and the

intestinal microbiota, and how the composition of the microbiota can influence health

and disease of the host. Studying these interactions are complex due to several factors,

but are partially due to the overwhelming diversity of bacterial genera present in the GI

tract, particularly in the colon (Human Microbiome Project, 2012). In fact, the

reproducibility between laboratories with respect to DSS colitis studies can be

confounded, and attributed in part, to the variability in the microbiota composition within

mice from lab to lab (Hufeldt et al., 2010). In order to minimize confounding factors

associated with the GI microbiota, researchers should make every effort to understand

and validate the composition of the microbiome present in the GI tract of mice, which

will help limit discrepancies among investigators. In addition, many laboratories have

adopted the practice of utilizing germ-free animal facilities, in which animals are derived

under sterile conditions, and are thus unable to be colonized by the microorganisms that

constitute the microbiome. This provides an excellent tool for researchers as specific

bacterial species can be added back on an individual microbe basis to these sterile

mice, allowing their microbiome to be colonized by a single microbial species. This

allows the interaction between host and microbe to be studied without other

confounding microbial communities. Of course, this system is not perfect: because

humans have many diverse types of microbes in the GI tract, it is likely that a shift in

composition of the whole microbial community contributes to health and disease.

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Though germ-free animals are a very useful and powerful scientific tool, interpretations

should be made with this caveat in mind.

4.2.2 Bioinformatics and the Impact of Intestinal Organoids on Future Research

A strategy to accomplish such a thorough undertaking for determining the microbiome

associated between laboratories would be to obtain microbiome profiles of the mouse

colony; specifically, with 16s ribosomal DNA sequencing technology, as well as

conducting metabolomics of bacterial products produced in the GI tract. This would

greatly advance our understanding of specific bacterial communities’ contribution

regarding colitis and cancer models, as well as how the composition may vary between

differing genotypes of mice. Additionally, ex vivo tissue culture, specifically with

intestinal organoids derived from the intestinal stem cells is an excellent strategy to

reduce complex confounding effects that occur when using live animal models. This is

one significant advantage of culturing ex vivo organoids from specific tissues, such as

the gut. Specifically, gastric, small intestinal and colonic organoids can be cultured ex

vivo and screened with substances such as specific drugs, microbial species, and

PAMPs to assess, for example, proliferative and anti-proliferative effects on epithelial

cells. A great example that has demonstrated the usefulness of this strategy was

conducted by Kaiko et. al.,2016. With the use of a Cdc25a-luciferase reporter mouse,

Kaiko et. al. screened colonic organoids with a broad panel of microbiome derived

metabolites with the aim of determining if any metabolites enhanced or inhibited cellular

proliferation. Kaiko et. al. determined that the microbial metabolite butyrate inhibited

stem cell proliferation under physiologic concentrations that are normally present in the

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GI tract. Further, convincing evidence supported their hypothesis that the production of

butyrate contributed to the invaginations that form the intestinal crypt (Kaiko et al.,

2016). The rationale was that differentiated colonic epithelial cells toward the top of the

crypt metabolize butyrate rapidly, which prevents its availability and interaction with the

colonic stem cells at the base of the crypt. Therefore, it is likely that the relationship of

microbial metabolites, namely butyrate, contributes to the U-shaped invaginations that

form the colonic crypt. The work by Kaiko et. al. highlight the powerful importance of the

ex vivo organoid culture, along with screening technology to the scientific method to

advance our current understanding of intestinal anatomy and physiology.

4.2.3 Research Applications Utilizing Intestinal Organoids

One advantage of the organoid system over traditional immortalized cell lines is due to

the inclusion of many intestinal cell types in the organoid (Sato et al., 2009). Mimicking

the intestinal stem cell niche allows for maintenance of the intestinal stem cells that give

rise to the fully differentiated epithelial cells present in the GI system; thus, this closely

models the physiology of the GI tract without the complex confounding interactions with

other organs that are present in a live animal, such as the immune system and others.

The importance of this methodology is demonstrated by the ability to culture intestinal

organoids from human patients with a minimally invasive biopsy taken from a patient.

For example, ex vivo organoids derived from patients with genetic mutations that cause

cystic fibrosis have been useful for devising and evaluating therapeutic strategies.

Patients with cystic fibrosis have a mutated gene that encodes for a chloride ion

channel, which results in the inability to make surfactant that moistens the lungs

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(Dekkers et al., 2013). Because this global mutation occurs throughout the genome,

intestinal organoids derived from cystic fibrosis patients can be cultured and monitored

over time, screened for compounds that restore proper activity of the intestinal organoid,

and advance chemotherapeutic options for patients with genetic diseases (Dekkers et

al., 2013).

An exciting application with ex vivo organoids is the concept of microinjection of

individual bacterial strains directly within the organoid lumen. This application is a novel

concept, and one of relevance to help simplify complex variables that occur between the

intestinal microbiota with the intestinal epithelial cells. A method has been described

that utilizes this very concept with gastric organoids, and the bacterium H. pylori

(Bartfeld et al., 2015), but can also be applied to colon derived organoids. Specific

species of bacteria have been associated with the induction of colorectal cancer

(Castellarin et al., 2012), (Abed et al., 2016), and introduction of these pro-carcinogenic

bacterial strains with intestinal organoids may have utility as a primary model to

determine the full contribution of these carcinogenic bacteria. Additionally, organoids

from genetically modified mice can be used as a model to study growth and proliferation

of intestinal stem cells ex vivo.

5.2.4 Future Directions with Organoids: a model system to study both genetic

and host-microbiome interactions

The alternative NF-kB pathway and principally NF-kB inducing kinase (NIK) is known for

its role in immune cell function and B-cell maturation (Eden et al., 2017), (Claudio et al.,

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2002), yet its role in intestinal epithelial cells is currently under investigation. The results

in the present dissertation suggest NIK to be critical for proper proliferation of intestinal

organoids and maintenance of epithelial cells. Since NIK has been shown to contribute

to cellular proliferation in the adaptive immune system (Claudio et al., 2002), (Li et al.,

2016), the results shown herein are consistent with these prior studies. Importantly,

however, our research findings are novel, as we have demonstrated NIK to be important

in intestinal stem cell (ISC) proliferation. Moreover, it was recently shown that Nik-/-

mice develop spontaneous eosinophilic esophagitis (Eden et al., 2017); this association

in combination with the results of the present dissertation further bridges the importance

of NIK to epithelial cell barrier function. It will be an interesting line of further

investigation to determine whether the alternative NF-kB pathway and the Wnt signaling

pathway collaborate in regulating the expression of LGR5. Indeed, future mechanistic

studies will be critical in uncovering the role of NIK with proper stem cell proliferation.

Intestinal organoids are a great model system to further study host-pathogen

interactions, or host-commensal interactions between GI epithelial cells with respective

microorganisms. One particular bacterium that our group has worked with, in

collaboration with Dr. Dan Slade (Virginia Tech, Biochemistry Department), is

Fusobacterium nucleatum. F. nucleatum has received attention in recent years because

of its association and presence in tumors of human patients with colon cancer. Thus,

this bacterium is implicated with contributing to the oncogenic transformation of

intestinal epithelial cells. Our group has been interested in determining the functional

role that F. nucleatum plays in colorectal cancer with the use of mouse colonic

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organoids. The interesting phenomenon of F. nucleatum is that it is a normal portion of

the microflora found in the human mouth, and is not normally found in the GI tract;

which provokes several intriguing questions: how is it that this bacterium is localized to

colonic tumors? Does it actually cause colon cancer or does it localize to a colon tumor

after a tumor has been initiated by alternative mechanisms? One hypothesis is that F.

nucleatum is able to migrate through the bloodstream, entering from the gums and can

localize to the colon through this route, ultimately resulting in its tumorigenic potential. A

likely reason is that F. nucleatum levels increase in the blood after the brushing of one’s

teeth, thus, it is possible that humans receive a substantial septic F. nucleatum dose

twice a day, depending on hygienic habits. The use of colonic organoids is a strategic

model to test this hypothesis, because it is relatively straightforward to treat colonic

organoids in vitro with this bacterium. One practical use of this technique is that colonic

epithelial cells that compose the organoid demonstrate cellular polarity when grown in

matrigel, similar to that of the actual organ under in vivo conditions (Sato et al., 2009).

Thus, the basolateral portion of the epithelial organoid would be the point of entry the

bacteria would exploit when traveling from the bloodstream to the tissue.

5.2.5 Gene Therapy Targeting Multi-potent Stem Cells

One can envision stem cell based therapeutics, in combination with gene therapy, to

advance treatments for human patients that suffer from genetic mutations, specifically

point mutations. One major hurdle with gene therapy is the ability to correct a mutation

that becomes retained throughout the genome of future daughter cells. This can be

overcome if the mutation is corrected in the multi-potent stem cells themselves,

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because it is these cells that are retained in adult tissue, and further give rise to fully

differentiated cell types that populate the organ. In this respect, the corrected mutation

will be retained in future daughter cells, and essentially cure that aberrant genetic

disease. This is one of the very promising aspects of CRISPR-Cas9 based gene

therapy; however, if this strategy is to be used in adult humans, not all multipotent stem

cells have been able to be isolated, so currently, only a limited number of diseases

could be addressed with this approach.

5.3 Conclusion

It will be exciting to view what the future holds regarding research advances in IBD,

mucosal immunology, and cancer fields. With the use of intestinal organoids, many

novel compounds may be discovered that improve therapeutic options for patients with

inflammatory related diseases. Furthermore, stem cell based approaches in

combination with gene therapy offer a promising methodology to treat or potentially cure

diseases caused by point mutations. Though the cost of this type of treatment may be

expensive, this is the type of personalized medical approach that could be utilized to

treat an individual’s particular type of malady.

There remain many unanswered question regarding cellular signaling and how

modulation of one single molecule can affect so many aspects related to the cell. In

conjunction with our current findings, continued research efforts focused on IRAK-M will

be essential and contribute to the global understanding of how this particular molecule

modulates aspects of the immune system. In the larger clinical context, the elucidation

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of the mechanistic role of IRAK-M offers significant promise towards the development of

novel therapeutic strategies for the treatment of GI pathologies, such as IBD and

colorectal cancer.

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Appendix

A. Works Completed

1. Rothschild, D. E., McDaniel, D. K., Ringel-Scaia, V. M., and Allen, I. C. (2018)

Modulating inflammation through the negative regulation of NF-kappaB signaling.

Journal of leukocyte biology

2. Rothschild, D. E., Zhang, Y., Diao, N., Lee, C. K., Chen, K., Caswell, C. C., Slade,

D. J., Helm, R. F., LeRoith, T., Li, L., and Allen, I. C. (2017) Enhanced Mucosal

Defense and Reduced Tumor Burden in Mice with the Compromised Negative

Regulator IRAK-M. EBioMedicine 15, 36-47

3. Eden, K., Rothschild, D. E., McDaniel, D. K., Heid, B., and Allen, I. C. (2017)

Noncanonical NF-kappaB signaling and the essential kinase NIK modulate crucial

features associated with eosinophilic esophagitis pathogenesis. Dis Model Mech 10,

1517-1527

4. Scott, G. K., Chu, D., Kaur, R., Malato, J., Rothschild, D. E., Frazier, K.,

Eppenberger-Castori, S., Hann, B., Park, B. H., and Benz, C. C. (2016) ERpS294 is a

biomarker of ligand or mutational ERalpha activation and a breast cancer target for

CDK2 inhibition. Oncotarget

5. Rothschild, D. E., Srinivasan, T., Aponte-Santiago, L. A., Shen, X., and Allen, I. C.

(2016) The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse

Intestinal Organoids. Journal of visualized experiments : JoVE

6. McDaniel, D. K., Jo, A., Ringel-Scaia, V. M., Coutermarsh-Ott, S., Rothschild, D. E.,

Powell, M., Zhang, R., Long, T. E., Oestreich, K., Riffle, J. S., Davis, R. M., and Allen,

I. C. (2016) TIPS pentacene loaded PEO-PDLLA core- shell nanoparticles have

similar cellular uptake dynamics in M1 and M2 macrophages and in corresponding in

vivo microenvironments. Nanomedicine

7. Brickler, T., Gresham, K., Meza, A., Coutermarsh-Ott, S., Williams, T. M.,

Rothschild, D. E., Allen, I. C., and Theus, M. H. (2016) Nonessential Role for the

NLRP1 Inflammasome Complex in a Murine Model of Traumatic Brain Injury.

182

Mediators Inflamm 2016, 6373506

8. Williams, T. M., Leeth, R. A., Rothschild, D. E., McDaniel, D. K., Coutermarsh-Ott,

S. L., Simmons, A. E., Kable, K. H., Heid, B., and Allen, I. C. (2015) Caspase-11

attenuates gastrointestinal inflammation and experimental colitis pathogenesis.

American journal of physiology. Gastrointestinal and liver physiology 308, G139-150

9. Williams, T. M., Leeth, R. A., Rothschild, D. E., Coutermarsh-Ott, S. L., McDaniel,

D. K., Simmons, A. E., Heid, B., Cecere, T. E., and Allen, I. C. (2015) The NLRP1

inflammasome attenuates colitis and colitis-associated tumorigenesis. Journal of

immunology 194, 3369-3380

183

B. Copyright Permissions

1. Rothschild, D.E., Zhang Y., Diao N., et al. “Enhanced Mucosal Defense and Reduced Tumor Burden in Mice with the Compromised Negative Regulator IRAK-M.” EBioMedicine. 2017 Feb;15:36-47.

Please note that, as the author of this Elsevier article, you retain the right to include it in a thesis or dissertation, provided it is not published commercially. Permission is not required, but please ensure that you reference the journal as the original source. For more information on this and on your other retained rights, please visit: https://www.elsevier.com/about/our-business/policies/copyright#Author-rights.

2. Rothschild D.E., Srinivasan T., Aponte-Santiago L.A., et al. “The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids.” J Vis Exp. 2016 May 18;(111), e54033.

In general, authors own the copyright to the written article, but grant JoVE the exclusive, royalty-free, perpetual use of the article, regardless of access type. Video copyrights are owned by JoVE unless the author selects open access, where it is then published under the creative commons license(CRC 3.0, by,nc,nd). The author may use the video and article for non-commercial purposes. Full copyright information is available in our Author License Agreement, or for UK-funded authors our Author License Agreement UK.

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3. Rothschild D.E., McDaniel D.K., Ringel-Scaia V.M., and Allen I.C. “Modulating inflammation through the negative regulation of NF-κB signaling.” J Leukocyte Bio. 2018 Jun;103(6):1131-1150.

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4. FIgure 1.1 (MOWAT, A. M. & AGACE, W. W. 2014. Regional specialization within the

intestinal immune system. Nat Rev Immunol, 14, 667-85.)

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