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Graduate Theses and Dissertations Graduate School

January 2012

Maternal Immune Dysregulation in thePathogenesis of Neurodevelopmental Disorders:Interleukin-6 as a Central Mechanism andTherapeutic Target of FlavonoidsEllisa Carla Parker-AthillUniversity of South Florida, eparkera@health.usf.edu

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Scholar Commons CitationParker-Athill, Ellisa Carla, "Maternal Immune Dysregulation in the Pathogenesis of Neurodevelopmental Disorders: Interleukin-6 as aCentral Mechanism and Therapeutic Target of Flavonoids" (2012). Graduate Theses and Dissertations.http://scholarcommons.usf.edu/etd/4195

Maternal Immune Dysregulation in the Pathogenesis of Neurodevelopmental Disorders:

Interleukin-6 as a Central Mechanism and Therapeutic Target of Flavonoids

by

Ellisa Carla Parker-Athill

A dissertation submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy Department of Molecular Pharmacology and Physiology

College of Medicine University of South Florida

Co-Major Professor: Jun Tan, M.D., Ph.D. Co-Major Professor: Javier Cuevas, Ph.D.

Craig Doupnik, Ph.D. R. Douglas Shytle, Ph.D.

Edwin Weeber, Ph.D.

Date of Approval: March 8, 2012

Keywords: Prenatal infection, Signal transducer and activator of transcription-3 (Stat3),

Luteolin, Autism Spectrum Disorder (ASD), Inflammation, Astroglia

Copyright © 2012, Ellisa Carla Parker-Athill

DEDICATION

I dedicate this work to my grandmother, Viola Athill, who has been a constant

source of strength and encouragement throughout my life. Your unfailing love and

unwavering faith has helped make me the person I am today. You have taught me to be

compassionate and strong, to appreciate all life’s blessings, to persevere through life’s

struggles and to always trust in God. I thank you for all these lessons; I could not have

made it through this journey without them. Although you are not here to see me end this

chapter and begin a new one, your encouragement and love has stayed with me and will

continue to guide me through all of life’s journeys. Just as I knew you were always

praying for me always loving and supporting me, I know your love and strength will

always be with me and for that I am eternally grateful and truly blessed.

ACKNOWLEDGEMENTS

Graduate school has been an unforgettable experience filled moments of

gratification, frustration and the gamut of emotions in between. I thank God for the

people he has placed in my life throughout this journey. Without their constant support

and encouragement I would not have made it through the low points and would never

have experienced the highs. To my family and friends, my grandmother, whose prayers

and love has been a constant source of strength, my mother, Maureen Athill, and

brother, Karim Parker, for their encouragement and my friends Shara Pantry and

Yvonne Davis who set out on this journey with me and have been constant sources of

encouragement, patient listeners, and welcomed escape partners, I am forever grateful

to you. To my major professor Dr. Jun Tan, my co-major Dr. Javier Cuevas, and my

committee members Dr. Douglas Shytle, Dr. Craig Doupnik and Dr. Edwin Weeber,

thank you for your mentorship and guidance. My committee chairman, Dr. Hossein

Fatemi, thank you for your time and invaluable feedback. I also have to thank the

members of my lab, both past and present specifically Dr. Zhu, who was a patient

teacher and mentor. Last of all and most importantly I would like to thank my cousin

Nichole ‘Nicky’ Murray, who encouraged me to do my first lab rotation, and set me on

the course I am on now. She realized my passion and love for research at a time when I

could not see it for myself. Without her I would not have had the courage to set out on

this journey and I am forever grateful for her encouragement, support, and unwavering

faith in me and my abilities. You have all made the completion of this dissertation

possible.

i

TABLE OF CONTENTS

LIST OF TABLES iii LIST OF FIGURES iv ABSTRACT vi CHAPTER 1 INTRODUCTION 1

1.1 Autism Spectrum Disorder (ASD) 1 1.2 Prenatal Infection, Cytokines and Pregnancy 3 1.3 Cytokines and Neurodevelopment 5 1.4 Prenatal infection and Neurodevelopmental Disorders:

Evidence from Fetal Inflammatory Response Syndrome (FIRS) 6 1.5 Prenatal infection and Neurodevelopmental Disorders:

IL6 as a Central Mechanism and Therapeutic Target 7 1.6 Therapeutic potential of flavonoids – ‘Flavones’ 8

CHAPTER2 MATERIALS AND METHODS 10

2.1 Reagents 10 2.2 Cell culture 10

2.2.1 Murine derived primary neural stem cells 10 2.2.2 Murine immortalized neuroblastoma cells 11 2.2.3 Murine derived primary splenocytes 11 2.2.4 Treatments 12

2.3 Mice 12 2.3.1 Housing and maintenance 12 2.3.2 Treatments 12

2.4 Lysate and Tissue Preparation 13 2.5 Western Blot Analysis 13 2.6 ELISA Cytokine Analysis 14 2.7 Cyquant Proliferation assay 14 2.8 Tissue Section Preparation 15 2.9 Immunofluorescence Analysis 15 2.10 Immunohistochemistry 16 2.11 Behavioral Analysis 16

2.11.1 Open Field 16 2.11.2 Social Interaction 17

2.12 Statistical analysis 17

CHAPTER 3 LUTEOLIN ATTENUATES IL6 MEDIATED PATHOLOGIES: IMPLICATIONS FOR PRENATAL INFECTION AND ADVERSE NEUROLOGICAL OUTCOMES 18

3.1 Regulation of CNS development 18

ii

3.1.1 Role of Gp130 cytokines and Stat3 18 3.1.2 Stat3 in Neurodevelopmental Disorders 19

3.2 Results 21 3.2.1 Luteolin attenuates IL-6 mediated neuronal Jak2/Stat3

phosphorylation 21 3.2.2 Luteolin attenuates IL-6 mediated effects in neural stem

cells through attenuation of Jak2/Stat3 phosphorylation 25 3.2.3 Luteollin attenuates IL-6 mediated activation of Protein Kinase

B (Akt) and Mitogen Activated Protein Kinase (Erk1/Erk2) pathways in neural stem cells 28

3.2.4 Luteolin attenuates IL-6 induced proliferation in Neuro-2a cells 31 3.2.5 Effects of IL-6 in neural stem cells may reflect in vivo pathological

consequences of prenatal infection 33 3.2.6 Luteolin attenuates IL-6 induced morphological changes in neural

stem cells 39 3.2.7 Luteolin attenuates IL-6 mediated expression of GFAP positive

cells 41 3.2.8 IL6 and Luteolin activity dependent on extrinsic factors

in the cellular microenvironment 44 3.3 Discussion 47

CHAPTER 4 FLAVONOIDS, A PRENATAL PROPHYLAXIS FOR

ADVERSE PERINATAL OUTCOMES AFTER PRENATAL INFECTION 50

4.1 Prenatal infection, Cytokines and the Placental Barrier 50 4.1.1 Current animal models of adverse perinatal outcomes

following prenatal infection 51 4.1.2 Luteolin as a therapeutic intervention – potential mechanism 52

4.2 Results 53 4.2.1 Luteolin attenuates maternal immune response following prenatal

infection 53 4.2.2 Luteolin attenuates adverse fetal outcomes following prenatal

infection 60 4.2.3 Structural analog diosmin reduce pro-inflammatory cytokine

production and Jak2/Stat3 phosphorylation in the brain tissue of IL-6 newborn mice 62

4.2.4 Maternally blocking the Stat3 signaling pathway reduces abnormal behaviors in prenatally exposed animals 65

4.2.5 Diosmin reduced pro-inflammatory cytokine and Stat3 phosphorylation in prenatally exposed animals 68

4.3 Discussion 71 CHAPTER 5 CONCLUSION 73 REFERENCES 78 APPENDIX 1 PUBLICATION CONTRIBUTING TO THE

DISSERTATION 105 APPENDIX 2 COPYRIGHT PERMISSION 106

iii

LIST OF TABLES

4.1 Luteolin attenuates adverse fetal outcomes following prenatal infection 61

iv

LIST OF FIGURES

3.1 Luteolin attenuated IL-6 mediated Jak2 phosphorylation in neuroblastoma cells 22 3.2 Luteolin attenuated IL6 mediated Stat3 phosphorylation in neuroblastoma cells 24 3.3 Luteolin attenuated IL-6 mediated phosphorylation of Jak2/Stat3

in neural stem cells 26

3.4 Luteolin attenuated IL-6 mediated phosphorylation of Protein kinase B (Akt) in neural stem cells 29

3.5 Luteolin attenuated IL-6 mediated phosphorylation of Migtogen

activated protein kinase (Erk1/Erk2) in neural stem cells 30

3.6 Luteolin attenuates IL6 induced proliferation in neuroblastoma cells 32 3.7 Effects of IL-6 in neural stem cells may reflect in vivo pathological consequences of prenatal infection 34

3.8 The ability of luteolin to attenuate IL-6 mediated pathologies modulated by the presence of growth factors (EGF and FGF) 36

3.9 IL-6 and Luteolin does not alter neurosphere size or number 38 3.10 Luteolin attenuates IL-6 mediated morphological changes in neural

stem cells 40

3.11 Luteolin attenuates IL-6 induced GFAP expression in neural stem cells 42 3.12 IL6 and Luteolin activity dependent on extrinsic factors in the cellular

microenvironment 45

4.1 Luteolin attenuates maternal immune responses following prenatal infection: LPS and conA challenged splenocyte cultures 54

4.2 Luteolin attenuates pro-inflammatory cytokine expression from LPS or

conA challenged splenocytes 57

4.3 Luteolin attenuates Stat3 phosphorylation in maternal spleens following prenatal infection 59

v

4.4 Luteolin attenuates adverse fetal outcomes following prenatal infection 61 4.5 Diosmin reduced pro-inflammatory cytokine expression in the brain homogenates of prenatally exposed newborn mice 63

4.6 Diosmin reduced Jak2/Stat3 phosphorylation in the brain homogenates

of prenatally exposed newborn mice 64

4.7 Maternally blocking Stat3 activity reduces abnormal behaviors in prenatally exposed animals 67

4.8 Diosmin reduces pro-inflammatory cytokine expression in the brain

homogenates in prenatally exposed animals 69 4.9 Diosmin attenuates Stat3 phosphorylation in the brain homogenates of

prenatally exposed animals 70

vi

ABSTRACT

Activation of the maternal immune system and resultant maternal cytokine

expression due to prenatal infection has been implicated as a significant contributor to

the pathology of neuropsychiatric and neurodevelopmental disorders such as

schizophrenia and Autism Spectrum Disorder (ASD). Increased maternal interleukin-6

(IL-6) expression, observed clinically and in animal models of prenatal infection, and

resultant activation of key signaling pathways, has been shown to be a biological

indicator of pathology, and a central component of the pathological mechanism. In

animal models of prenatal infection and clinically in pregnancy disorders hallmarked by

immunological irregularities and increased IL-6 expression, inhibition of IL-6 has been

shown to reduce pathological symptoms both maternally and in the exposed offspring.

This study aims to demonstrate the ability of IL-6 expression, resulting from prenatal

infection, to induce neuropathological and behavioral outcomes that mirror clinical

observations seen in disorders such as ASD. More importantly, it shows how flavones

luteolin and diosmin, a subclass of the flavonoid family, through inhibition of IL-6

mediated activation of Signal Transducer and Activator of Transcription-3 (Stat3) can

reduce these pathologies both in vitro and in vivo.

Evidence suggests that flavonoids, a polyphenolic class of naturally occurring

plant secondary metabolites, are potent anti-inflammatory agents that can attenuate the

expression of cytokines such as IL-6, possibly through the modulation of tyrosine kinase

activity. They have been shown to have significant therapeutic potential in disorders

hallmarked by increased inflammation or disruptions in immune regulation, such as

vii

neurodegenerative disorders and certain cancers. Members such as diosmin have also

been shown to be safe during pregnancy, and are currently utilized in the treatment of

certain vascular disorders associated with pregnancy.

In vitro work undertaken in this study showed that co-administration of luteolin

with IL-6 in neural stem cells (NSC) was able to attenuate pathological outcomes

induced by IL-6 including aberrant proliferation, over expression of astroglial marker,

glial fibrillary acidic protein (GFAP) and changes in cellular morphology. In vivo studies

involving luteolin and diosmin further confirmed the therapeutic efficacy of these

compounds as similar attenuation of IL-6 mediated maternal and fetal pro-inflammatory

cytokine expression and abnormal behaviors in prenatally exposed offspring was

observed. Mechanistically, these effects were mediated through inhibition of Stat3

activation although other pathways activated by IL-6 were modulated by flavone co-

treatment. Flavonoid treatment during periods of prenatal infection may prove to be a

therapeutic intervention for the resultant pathological outcomes seen in offspring through

attenuation of the maternal and fetal immune response to infection as well as modulation

of signaling pathways in the fetal brain. These compounds may prove therapeutically

efficacious for the application in perinatal conditions hallmarked by increased

inflammation during pregnancy.

1

CHAPTER 1

INTRODUCTION

1.1 Autism Spectrum Disorder (ASD)

It is estimated that approximately 13% of children in the U.S. ages 3 to 17 suffer

from at least one developmental disability that affects their behavior, memory, or

learning capability (Boulet et al., 2009, Boyle et al., 2011). The resulting impact on the

social, economic, educational, and health care delivery systems are significant, as the

economic cost of neurodevelopmental disorders in the US have been estimated to be

$81.5 to $167 billion per year (Boyle et al., 1994). Recently, Autism Spectrum Disorder

or autism (ASD) has gained significant focus as there has been an unprecedented

increase in the number of children diagnosed with the disorder, with an average increase

of 57% between 2002 and 2006 (Autism and Developmental Disabilities Monitoring

Network Surveillance Year 2006 Principal Investigators; Centers for Disease Control and

Prevention, 2009). Although this increased prevalence has been attributed to

improvements in diagnostic criterion, it has nonetheless underscored the necessity for

increased research as an exact etiology for the disorder is still unknown. This increased

prevalence, coupled with the lack of a definitive etiological factor and therapeutic options

for patients and their families has fueled the urgency felt by parents, clinicians and

researchers to find a definitive etiology and therapeutic strategy for this disorder which

often carries a significant economic and emotional burden with lifetime care cost

estimated at $3.2 million per individual (Ganz, 2007)

2

The term autism was originally used by Eugen Bleuler to describe a cluster of

symptoms in schizophrenic individuals that included withdrawal or ‘self-isolation’. The

term would later be used by Leo Kanner in 1943 and Hans Asperger in 1944, to describe

a similar group of symptoms in children. Today however, the term autism is often used

synonymously for Autism Spectrum Disorder (ASD) to describe the cluster of symptoms

manifested in children displaying withdrawn or ‘isolated’ social behaviors (American

Psychiatric Association, 2000). The definition has now evolved to describe the group or

spectrum of behaviors characterized by impairments in reciprocal social interaction and

verbal and nonverbal communication skills compounded by symptoms of restrictive and

repetitive behaviors and/or stereotyped patterns of interest that are abnormal in intensity

or focus (American Psychiatric Association, 2000, Koenig and Scahill, 2001,

Zwaigenbaum et al., 2005). Included in this spectrum is ‘classical’ autism, which usually

involves social isolation, impaired verbal communication and stereotypical and repetitive

behaviors; Asperger syndrome, often described as a milder form of autism, lacking the

components of impaired verbal communication skills while retaining the intense

repetitive behaviors and difficulties in social interaction. Also in the spectrum is

pervasive developmental disorder that usually encompasses disorders that cannot

otherwise be classified (American Psychiatric Association, 2000, Allen et al., 2001,

Koenig and Scahill, 2001, Zwaigenbaum et al., 2005).

Despite the advances in diagnostic criterion, the exact etiology of ASD has not

been identified, and although there have been significant advancements made in the

identification of key genes that are abnormally expressed within the autistic population a

single gene candidate has not been found (Andres, 2002, Belmonte and Bourgeron,

2006, Bernardet and Crusio, 2006, Chen et al., 2006, Campbell et al., 2008, Li et al.,

2008, Tetreault et al., 2009, Virkud et al., 2009, Kim et al., 2011). The genetic variation

and phenotypic heterogeneity within this population has led researchers to suggest

3

alternative hypotheses including the collective contributions of multiple genetic

disruptions as well as the synergistic interaction of environmental factors with genetic

polymorphisms or instabilities (Hornig et al., 1999, Fatemi et al., 2002, Fatemi et al.,

2005, Campbell et al., 2008, Fatemi et al., 2008b).

Prenatal infection has been one such ‘environmental factor’ with observations

from murine studies confirming the potential for prenatal infection, and more importantly

the resulting activation of the maternal immune system, to induce aberrant neurological

and behavioral outcomes in offspring reminiscent of the phenotypes observed clinically

in disorders such as schizophrenia and ASD (Fatemi et al., 2002, Shi et al., 2003,

Zuckerman et al., 2003, Fatemi et al., 2005, Smith et al., 2007, Fatemi et al., 2008b,

Winter et al., 2008, Ibi et al., 2009, Kirsten et al., 2010, Ghiani et al., 2011). Similarly,

epidemiological data and clinical cases of prenatal infection have supported the potential

for this event to precipitate adverse neurological outcomes with an increased incidence

of preterm births and cerebral palsy, a neurological disorder involving impairments in

motor and cognitive function, visual and auditory perception observed in prenatally

exposed infants. Fetal Inflammatory Response Syndrome (FIRS), a disorder

characterized by systemic inflammation and uncharacteristically high levels of plasma IL-

6 in the affected newborn as well as increased circulating IL-6 in the placental and

umbilical cord plasma is also seen in infants exposed prenatally to intra-amniotic

bacterial infection or viral infection and often results in preterm births, and adverse

neurological outcomes such as cerebral palsy (Gomez et al., 1998, Chaiworapongsa et

al., 2002, Gotsch et al., 2007).

1.2 Prenatal infection, Cytokines and Pregnancy

Although the exact mechanism may not be fully understood, the activation of the

maternal immune system following prenatal infection, and specifically the increases in

4

pro-inflammatory cytokine expression has been shown to be integral in precipitating the

adverse neurological and behavioral outcomes observed in prenatally exposed offspring

(Ashdown et al., 2006, Fortier et al., 2007, Jonakait, 2007, Ghiani et al., 2011).

Cytokines, small signaling molecules secreted by cells to mediate intercellular

communication, have long been shown to play a vital role in immune regulation,

particularly in the progression of inflammatory responses to stimuli including pathogenic

insults (Bromberg and Wang, 2009, Sweeney, 2011, Liu et al., 2012). Their role extends

beyond their pathological activities however as they have been shown to be equally

important in physiological events particularly the development and function of various

cellular populations. In the immune system, cytokines regulate the differentiation and

mobilization of T-cell populations, an important event in response to pathogenic stimuli

as well as during pregnancy (Makhseed et al., 2000, Schluns and Lefrancois, 2003,

Norton et al., 2010, Robertson et al., 2010, Zhu and Paul, 2010, Zwirner and Domaica,

2010, Morrison et al., 2011, Tower et al., 2011). It is this regulatory role that may begin

to explain the implications of maternal immune activation during prenatal infection in

precipitating adverse perinatal outcomes as disruptions in cytokine expression have

been observed in adverse perinatal outcomes (Tosun et al., 2010, Vitoratos et al., 2010,

Hsiao and Patterson, 2011, Wang et al., 2011).

During pregnancy, cytokines serve as regulators at the maternal-fetal immune

interface, preventing maternal rejection of the fetus by maintaining the balance between

immune-suppressive regulatory T cells (T-regs) and cytotoxic T cells (Th1, Th17)

(Piccinni and Romagnani, 1996, Makhseed et al., 2000, Piccinni et al., 2000, Piccinni,

2002, Kanellopoulos-Langevin et al., 2003, Aluvihare et al., 2004, Poole and Claman,

2004, Aluvihare et al., 2005, Petroff, 2005, Piccinni, 2005, 2010, Saito et al., 2010,

Shima et al., 2010). Murine models and human clinical studies have confirmed this

interplay, with observations of changes in cytokine expression and a shift in the

5

differentiation of naïve T cells toward a T-regulatory phenotype during the peri-

implantation period (Zenclussen, 2006b, Zenclussen et al., 2006, Guerin et al., 2009,

Ernerudh et al., 2011, Leigh R. Guerin, 2011). Additionally, adverse pregnancy

outcomes such as recurrent miscarriages, preeclampsia, and instances of restricted fetal

growth have been attributed to placental dysfunction, specifically the inability of the

placenta to fully maintain an immunological balance as increased cytotoxic T-cells,

particularly Th17 cells, have been observed in these disorders along with increased

levels of pro-inflammatory cytokines particularly IL6 (Makhseed et al., 2000, Zenclussen,

2006b, a, Piccinni, 2007, Lai et al., 2010, Laresgoiti-Servitje et al., 2010, Saito et al.,

2010, Tosun et al., 2010, Jin et al., 2011, Liu et al., 2011a, Liu et al., 2011b, Saini et al.,

2011, Raghupathy et al., 2012).

1.3 Cytokines in neurodevelopment

The activity of cytokines, both physiologically and pathologically have also been

extensively described beyond their role in the peripheral immune system as they

participate in the central nervous system (CNS) as key mediators in neurodegenerative

disorders and other instances characterized by neuroinflammation and also in normal

CNS development and function (Ajmone-Cat et al., Burns et al., 1993, Pousset, 1994a,

Zhao and Schwartz, 1998, Balschun et al., 2004, Bauer et al., 2007, Deverman and

Patterson, 2009, Jellinger, 2010, Appel et al., 2011, Matousek et al., 2011). During CNS

development, cytokines are present and functional in the fetal brain, with receptors

identified on both neuronal and glial cells (Pousset, 1994b, Rothwell et al., 1996, Dame

and Juul, 2000). They exert their regulatory effects propagating permissive or inhibitory

signals to coordinate the birth and terminal differentiation of neuronal and glial cell

populations, their migration and positioning in the brain and the formation and

maintenance of functional synapses (Bauer et al., 2007, Deverman and Patterson,

6

2009). This regulatory role may explain the ability for pathological expression of

cytokines such as IL-6, during prenatal infection to produce disruptions in cellular events

including changes in migratory patterns and synaptic function that may manifest in the

adverse neurological symptoms observed clinically in disorders such as ASD.

1.4 Prenatal infection and implications for neurod evelopmental disorders:

Evidence from fetal inflammatory response syndrome (FIRS)

The idea of an ‘environmental insult’ precipitating neurodevelopmental disorders

is not a novel hypothesis as several compounds, including toxins and heavy metals,

have been shown to cause neurological and congenital abnormalities in children

exposed prenatally (Silbergeld and Patrick, 2005, Wigle et al., 2007, Sutton et al., 2010,

Schoeters et al., 2011). The link between prenatal infection and disruptions in fetal

development is also not a novel idea. Several researchers have observed correlations

between maternal infections, pre-term births and neurological disorders such as cerebral

palsy, ASD and schizophrenia in these prenatally exposed infants (Barak et al., 1999,

Jacobsson and Hagberg, 2004, Atladottir et al., 2010, Kinney et al., 2010, Monji et al.,

2011). FIRS is one example of how prenatal infection can lead to adverse pregnancy

outcomes and compromise fetal development. FIRS is observed in fetuses and

newborn infants exposed to infections in utero and is characterized by systemic

inflammation including abnormally high levels of IL-6 and has been correlated with an

increased occurrence of neurological disorders such as cerebral palsy. Although

described in the context of fetal pathology, FIRS is precipitated by maternal infection,

specifically, bacterial infections of the amniotic fluid, placenta, uterus or fetal

membranes, although viral infections can induce similar pathologies (Fahey, 2008).

Known as intra-amniotic infections, they elicit maternal and fetal immunological

responses including cytokine release, leukocyte migration and rupture of the fetal

7

membranes. It is this inflammatory response that is thought to induce maternal and fetal

pathological outcomes including preterm birth and neurological disruptions (Gomez et

al., 1998, Romero et al., 2000, Romero et al., 2003, Jacobsson and Hagberg, 2004,

Gotsch et al., 2007, Fahey, 2008, Fuksman and Mazzitelli, 2009). Additionally, murine

models of prenatal infection have also mirrored clinical findings in FIRS including

observations of increased maternal cytokines such as IL-6 which appear to precipitate

the adverse behaviors and neuropathologies observed in exposed offspring.

1.5 Prenatal infection and neurodevelopmental disor ders– IL-6 as a central

mechanism and therapeutic target

Indeed findings from FIRS, murine models of prenatal infection and preeclampsia

have supported a role maternal and potentially fetal inflammatory response and cytokine

expression, particularly IL-6, in the adverse perinatal outcomes precipitated by prenatal

infection including the behavioral abnormalities in exposed offspring (Boksa, 2004,

Zenclussen, 2006a). These models have shown that IL-6 is a key component of the

pathological mechanism, with its inhibition attenuating pathological symptoms (Campbell

et al., 1993, Smith et al., 2007, Tosun et al., 2010, Covey et al., 2011, Wang et al.,

2011). Although the exact mechanism by which IL-6 contributes to these pathologies

may not be fully understood, the physiological role of cytokines and in this instance IL-6,

during CNS development and in immune regulation may help elucidate how its

disruption following prenatal infection can lead to pathological outcomes.

A member of the gp130 family of cytokines, IL-6 and its counterparts propagate

their regulatory and pathological effects through the activation of a common signaling

component, Signal Transducers and Activators of Transcription (Stat3) (Heinrich et al.,

1998, Deverman and Patterson, 2009, Islam et al., 2009). It is this common pathway

that may allow pathological expression of IL-6 during instances of prenatal infection to

8

disrupt placental function and developmental events mediated by Stat3 activity. The

differentiation of cytotoxic T-cell populations for example has been shown to be a Stat3

dependent highly influenced by IL-6 expression. T-cells play a critical role during

pregnancy and clinical studies have suggested that T-cell disturbances may play an

important role in pregnancy disorders (Kimura and Kishimoto, 2010, Saini et al., 2011).

Similarly, the differentiation of astroglial cells from neural stem cell populations has also

been shown to be Stat3 dependent and disruptions in this population have been

observed in neurological disorders associated with prenatal infection such as ASD

(Brunello et al., 2000, He et al., 2005, Islam et al., 2009, Bilbo, 2011, Sicca et al., 2011).

Observations that the inhibition of IL-6 may attenuate pathological symptoms

arising from prenatal infection have provided a potential mechanism for understanding

the role of IL-6 in these pathologies as well as a new strategy that can be utilized to

develop therapeutic alternatives. In preeclampsia for example, a disorder in pregnancy

characterized by the sudden onset of hypertension and protein in the urine after the 20th

week, increased levels of IL6 has been identified as a key indicator of disease pathology

as well as a therapeutic target with its inhibition attenuating hypertensive symptoms and

other associated pathologies (Lockwood et al., 2008, Lamarca et al., 2011, Zhou et al.,

2011). Similarly in murine models of prenatal infection, increased levels of IL-6 have

been observed following infection and have been shown to play a major role in the

neuropathologies and abnormal behaviors seen in the exposed offspring while the

inhibition of IL-6, and Stat3 activity have been shown to counteract these pathologies

(Smith et al., 2007).

1.6 Therapeutic potential of Flavonoids – ‘Flavones ’

Recently flavonoids, a class of polyphenolic plant metabolites have gained

significant attention in cancer and inflammation research because of their therapeutic

9

potential, acting as immune modulators in various pathological disorders (Hirano et al.,

2004, Kawai et al., 2007, Seelinger et al., 2008, Geraets et al., 2009). Grouped on the

basis of their structural attributes, flavonoids have properties ranging from anti-

inflammatory to anti-carcinogenic attenuating immune reactions in inflammatory cells

and aberrant proliferation in cancer cells largely through the modulation of tyrosine

kinase activity. Luteolin, a member of the flavone class of flavonoids, and found in

chamomile and celery among other plants, is known to have significant anti-inflammatory

and anti-tumor potential attenuating cytokine production in immune cell populations,

while displaying pro-apoptotic properties in tumor cell lines and murine models of cancer

(Rezai-Zadeh et al., 2008, Seelinger et al., 2008, Kang et al., 2011). Luteolin, like many

of the other flavonoids has also been shown to be blood-brain barrier permeable, a

characteristic that makes it an appealing therapeutic strategy for the neurological

disorders precipitated by prenatal infection, when coupled with its anti-inflammatory

potential (Jang et al., 2008). Diosmin, a semisynthetic flavonoid derivative, has also

been shown to have similar therapeutic potential being utilized for the treatment of

vascular disorders. This research outlines how flavonoids luteolin, and diosmin, can

inhibit the adverse effects of IL6, including the disruptions in maternal immunity following

prenatal infection and the neuropathological and behavioral abnormalities observed in

the exposed offspring, potentially serving as a useful therapeutic intervention during

instances of prenatal infection and immune dysfunction during pregnancy.

10

CHAPTER 2

MATERIALS AND METHODS

2.1 Reagents

Luteolin (>95% purity by HPLC) was generously donated by Pharma Science

Nutrients (Ocala, FL, USA). Diosmin (>90% purity by HPLC) was purchased from Axxor

(San Diego, CA, USA). Antibodies against Jak2, phospho-Jak2, Stat3, phospho-Stat3,

phospho-PI3K, phospho-Akt, Akt, phosphor-ERK1/2 (p44/42), vimentin and nestin were

obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against

GFAP, Doublecortin (DCX), and Beta-tubulin were obtained from Abcam (Cambridge,

MA, USA). Murine recombinant IL-6 (mIL-6 or IL-6) and cytokine ELISA kits were

purchased from eBioscience (San Diego, CA, USA). BCA protein assay kit was

purchased from Pierce Biotechnology (Rockford, IL, USA) and Cyquant Cell Proliferation

assay kit from Invitrogen (Grand Island, NY).

2.2 Cell culture

2.2.1 Murine derived Primary neural stem cells (NSC )

Cerebral cortices isolated from C57BL/6 mouse embryos, 14 days in utero were

obtained from Stem Cell Technologies. In preparation for culture, neurospheres were

mechanically dissociated, centrifuged at 90 x g for 5 minutes, and resuspended and

maintained in 1:1 Dulbecco’s Modified Eagle Medium: Nutrient Mixture F12 (DMEM/F12)

supplemented with 2% B27 serum free supplement, 5µg/µl fibroblast growth factor (FGF,

Sigma-Aldrich, St. Louis, MO, USA) and 10µg/ml epidermal growth factor (EGF, Sigma-

11

Aldrich, St. Louis, MO, USA). Prior to treatment, cells were plated in 96 well collagen-

coated culture plates at a density of 2x104 cells per well, 8 well collagen coated, poly-d-

lysine treated culture plates at a density of 2.5 x 105 cells per well, or 6 well collagen-

coated culture plates at a density of 1 x 106. All cultures were maintained in the

previously described media formulation prior to treatment unless otherwise indicated and

incubated at 37°C in 10% CO2 before and during treat ment.

2.2.2 Murine immortalized neuroblastoma cells (N2a)

N2a (murine neuroblastoma) cells, purchased from the American Type Culture

Collection (ATCC, Manassas, VA) were maintained in complete DMEM supplemented

with 10% fetal calf serum. Cells were plated in 96 well collagen-coated culture plates at

a density of 2x104 cells per well, or 6 well collagen-coated culture plates at a density of

1x106 cells per well. All cultures were maintained in the previously described media

formulation prior to treatment and incubated at 37°C in 10% CO2 before and during

treatment.

2.2.3 Murine derived primary splenocytes

Spleens isolated from female C57BL/6 mice were dissected and mechanically

dissociated to obtain a single cell suspension. Cells were re-suspended and washed in

complete Roswell Park Memorial Institute 1640 (RPMI 1640) containing 10% serum,

centrifuged at 400 x g for 5 minutes, then washed in PBS, followed by centrifugation at

400 x g for 5 minutes. To remove red blood cells, splenocytes were resuspended in ACK

lysis buffer and incubated per manufacturer’s instructions. Cells were washed 3 times in

PBS, resuspended in RPMI 1640 containing 10% serum, and incubated at 37ºC, 5%

CO2 prior to treatment.

12

2.2.4 Treatments

For proliferation experiments, after overnight incubation cells incubated in 96 well

plates were treated with various concentrations of murine recombinant IL-6 (0, 3.125,

6.25, 12.5, 25, 50ng/mL) or cotreated with 50ng/mL murine recombinant IL-6 and

various concentrations of luteolin (0, 1.25, 2.5, 5, 10, 20 µM) for 24 hours. For western

blot analysis, cells were treated with 50 ng/mL murine recombinant IL-6 for a range of

time points (0, 15, 30, 45, 60 or 75 minutes) or 50 ng/mL murine recombinant IL-6 in the

presence of various concentrations of luteolin (0, 1.25, 2.5, 5, 10, 20 µM) for 30 minutes.

For immunocytochemistry, cells were treated with 50 ng/mL murine recombinant IL-6 in

the presence or absence of 20uM luteolin.

For assays performed under differentiated conditions, neural stem cells were

incubated in neurobasal media supplemented with 2% B27 while neuroblastoma cells

were incubated in neurobasal media supplemented with 3 mM dibuturyl cAMP. Reagent

concentrations and length of treatment maintained. All experiments were performed in

triplicate.

2.3 Mice

2.3.1 Housing and maintenance

C57BL/6 mice were obtained from Jackson Laboratory (Bar Harbor, MA) and

maintained in an animal facility of the University of South Florida (USF). All animals

were given ad libitum access to food and water and were maintained on a 12-hour

light/dark schedule. All subsequent experiments were performed in compliance with

protocols approved by the USF Institutional Animal Care and Use Committee.

2.3.2 Treatments

Pregnant mice were injected intraperitoneally with IL6 (5ug/mouse),

13

lipopolysaccharide (LPS) (50ug/kg), or phosphate buffered saline (PBS) as control on

embryonic day 12.5 (E12.5) and divided equally among three separate groups. The first

group was co-injected with Luteolin (20mg/kg), the second group was placed on diosmin

supplemented diets (10 mg/kg/day, 0.005% in NIH31 chow) and the third group co-

injected with the STAT3 inhibitor S31-201. A minimum of four pregnant females per

treatment were utilized for all experiments.

2.4 Lysate and homogenate preparation

Cultured cells were lysed in ice-cold lysis buffer (20 mM Tris, pH 7.5, 150 mM

NaCl, 1 mM EDTA, 1 mM EGTA, 1% v/v Triton X-100, 2.5 mM sodium pyrophosphate, 1

mM β-glycerolphosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, 1 mM PMSF) for 5 minutes

followed by scraping. Lysates were collected, briefly vortexed and centrifuged at 14,000

x g for 15 minutes at 4º C.

Mouse brains and spleens were isolated under sterile conditions on ice and

placed in ice-cold lysis buffer. Tissues were sonicated on ice for approximately 3

minutes, allowed to stand for 15 minutes at 4°C, and then centrifuged at 15,000 x g for

15 minutes at 4°C. Total protein content in cell lysates a nd tissue homogenates were

estimated using the bicinchoninic acid assay (BCA protein assay, Pierce Biotechnology)

per manufactures instructions and normalized.

2.5 Western Blot Analysis

Aliquots corresponding to 100ug of total protein were electrophoretically

separated using 12% SDS gels. Electrophoresed proteins were then transferred to

nitrocellulose membranes (Bio-Rad, Richmond, CA), washed in Tris buffered saline with

0.1% Tween-20 (TBS/T), and blocked for one hour at ambient temperature in TBS/T

containing 5% (w/v) non-fat dry milk. After blocking, membranes were hybridized

14

overnight at 4°C with various primary antibodies. Me mbranes were then washed three

times for five minutes each in TBS/T and incubated for one hour at ambient temperature

with the appropriate HRP-conjugated secondary antibody (1:1,000, Pierce

Biotechnology). Primary antibodies were diluted in TBS/T containing 5% BSA and

secondary antibodies in TBS containing 5% (w/v) of non-fat dry milk. Blots were

developed using the luminol reagent (Pierce Biotechnology). Densitometric analysis

was conducted using a FluorS Multiimager with Quantity One™ software (BioRad,

Hercules, CA). For phospho-Stat3 detection, membranes were probed with a phospho-

Ser727 and phosphor-Tyr705 Stat3 antibody (1:1,000) and stripped with stripping

solution and then re-probed with antibody that recognizes total Stat3 (1:1,000). Similarly

for phospho-Jak2 detection, phosphor-Akt, and phosphor-Erk(1/2) membranes were

probed with appropriated phosphor antibodies (1:1,000) and stripped and re-probed for

corresponding total antibody (1:1,000).

2.6 ELISA Cytokine Analysis

Mouse brain and spleen homogenates were prepared as described above and

used at a dilution of 1:10 in cell lysis buffer for these assays. Tissue-solublized

cytokines were quantified using commercially available ELISAs that allow for detection of

IL-1β, IL-6 and TNF-α. Cytokine detection was carried out according to the

manufacturer’s instruction. Data was represented as pg of cytokine/mg total cellular

protein for each cytokine.

2.7 Proliferation Assay

Cells plated in 96 well plates were treated as described in cell culture treatments

subsection. Following treatment proliferation was quantified using the Cyquant

proliferation assay per manufactures instructions.

15

2.8 Tissue Section Preparation

Postnatal day 0 mice were anesthetized with in an ice bath. Brains being utilized

for immunofluorescence and immunohistochemistry were immediately isolated and

incubated in paraformaldehyde at 4ºC for 24hrs. Brains were then sequentially

transferred to increasing concentrations of sucrose for 72hrs up to a final concentration

of 30% sucrose. Brains were then cyrosectioned at 30um and placed in freezing media

until staining.

2.9 Immunofluoresence Analysis

Cells cultured in eight well culture slides were treated as described outlined in

chapter 3. After treatment, cultured cells were washed, and then fixed with 4%

paraformaldehyde in phosphate-buffered saline (PBS; 0.1 M, pH 7.4) for 30 min at room

temperature. Cells were then washed in PBS three times for five minutes each then

permeabilized with 0.05% Triton X-100 (Sigma), washed with PBS three times for five

minutes each, then blocked with 5% horse serum in dilution buffer (0.05% Triton X-100)

for one hour. Cells were then incubated overnight at 4ºC with various primary antibodies

diluted in blocking buffer. After overnight incubation, cells were washed three times for

five minutes each in PBS and incubated for 45 minutes at room temperature with the

appropriate HRP-conjugated secondary antibody. After incubation, cells were washed

three times for five minutes in PBS, mounted using vector shield with DAPI, and

analyzed using confocal microscopy.

Brain sections were washed three times for five minutes then blocked in 5%

horse serum for one hour. Sections were then washed and incubated in primary

antibody overnight at 4ºC. After incubation, sections were washed and incubated in

secondary antibody, for 45 minutes. Cells were then washed three times for five

minutes, mounted onto slides and cover slipped with DAPI mounting media.

16

2.10 Immunohistochemistry

Brains being utilized for immunohistochemistry studies were prepared as

mentioned in immunofluorescence procedure. After incubation in secondary antibody,

sections were incubated in VectaStain Elite ABC reagent (Vector Laboratories,

Burlingame, CA) coupled with diaminobenzidine (DAB) substrate. Stained sections were

mounted onto coverslips, air dried, and washed in distilled deionized water two times for

five minutes. Sections were then rinsed through three rapid changes of ethanol, cleared

through three xylene washes and cover slipped with permount. Sections were visualized

using bright field Olympus BX-51 Sections microscope.

2.11 Behavioral analysis

2.11.1 Open Field

General locomotor activity of mice was assessed using the open field test. Mice

were placed in a 50 x 50cm white Plexiglas box brightly lit by fluorescent room lighting

and six 60 W incandescent bulbs approximately 1.5 m above the box. Activity was

recorded by a ceiling-mounted video camera and analyzed from digital video files either

by the automated tracking capabilities of Ethovision or counted using the behavior

tracker (version 1.5, www.behaviortracker.com), a software-based event-recorder. The

total distance moved and numbers of entries into the center of the arena (central 17 cm

square) were determined in a 10 minute session. General activity levels were evaluated

by measurements of horizontal activity and vertical activity. Anxiety was also assessed

by examining the ratio of center distance traveled to total distance traveled. As the

natural tendency of the mice is to avoid open areas, animals that show greater tendency

to explore in the center of the field show a lower tendency for anxiety, or the reverse

situation can indicate a higher anxiety level.

17

2.11.2 Social Interaction

To assess social interaction, treatment mice were allowed to interact with a

stranger mouse and the level of interaction, including sniffing and other investigative

behaviors was measured. The testing apparatus consisted of a 60 x 40 cm Plexiglas

box divided into three chambers. Mice were be able to move between chambers

through a small opening (6 x 6 cm) in the dividers. Plastic cylinders in each of the two

side chambers contained the probe mice, and numerous 1cm holes in the cylinders

enabled test and probe mice to contact each other. Test mice were placed in the center

chamber, with an overhead camera recording their movements. Mice were allowed five

minutes of exploration time in the box, after which an unfamiliar, same-sex probe mouse

from the same experimental group was placed in one of two restraining cylinders

(Radyushkin et al., 2009). The Ethovision software (Noldus, Leesburg, VA) program

measured time spent in each of the three chambers, and social preference was defined

as follows: (% time spent in the social chamber) - (% time spent in the opposite

chamber).

2.12 Statistical analysis

All data were normally distributed; therefore, in instances of single mean

comparisons, Levene's test for equality of variances followed by t-test for independent

samples was used to assess significance. In instances of multiple mean comparisons,

analysis of variance (ANOVA) was used, followed by post-hoc comparison using

Bonferroni's method. Alpha levels were set at 0.05 for all analyses unless otherwise

indicated. The STATistical package for the social sciences release 10.0.5 (SPSS Inc.,

Chicago, IL, USA) was used for all data analysis.

18

CHAPTER 3

LUTEOLIN ATTENUATES IL-6 MEDIATED CHANGES IN NEURAL STEM CELLS VIA

INHIBITION OF STAT3 ACTIVITY: IMPLICATIONS FOR PREN ATAL INFECTION

3.1 Regulation of central nervous system (CNS) deve lopment

3.1.1 Role of gp130 cytokines and Stat3

It has been established that CNS development is an inherently vulnerable period

with subtle changes in the immediate cellular environment leading to disruptions in the

morphology and function of neural populations that can precipitate adverse neurological

outcomes. This vulnerability has been attributed to the high degree of regulation

necessary during this period to coordinate the actions of the milieu of morphogenic

factors, including cytokines. Cytokines are important in the development of the CNS as

they play a key role in the fate specification and terminal differentiation of neuronal and

glial populations, the migration of neurons to the final position in the CNS, the formation

and maintenance of functional synapses, the sequential manner in which these

cytokines are expressed and the temporal nature in which these events occur

(Pekkanen-Mattila et al., 2010).

During early embryogenesis for example, the emergence of the neuroectoderm

from embryonic stem cells signals the formation of the CNS and coincides with the

expression of transcription factors such as sex determining region Y – box1 (Sox1).

Sox1 is implicated in directing the fate specification and expansion of this multipotent

cellular population as well as ensuring its maintenance through active proliferation and

19

the inhibition of further differentiation (Suter et al., 2009, Elkouris et al., 2011). By

contrast, paired box 6 (Pax6), expressed after Sox1, induces neuroectoderm to

differentiate into radial glial and subsequently neuronal cells (Warren et al., 1999, Mo

and Zecevic, 2008, Sansom et al., 2009). Similarly, the transition from neurogenesis to

gliogenesis is regulated by the sequential expression of pro-neuronal and pro-astroglial

cytokines and morphogenic factors in addition to cell intrinsic mechanisms that act on

multi-potent radial glial populations. At the onset of neurogenesis, precursor cell pools

differentiate preferentially into neuronal cells under the availability of pro-neuronal

cytokines such as neurogenin, while gliogenic genes are repressed through methylation,

and transcription factors inhibited through protein modification (Deverman and

Patterson, 2009). These inhibitory mechanisms are coupled with the reduced availability

of pro-glial cytokines, which altogether support the differentiation of neuronal cells while

restricting the astroglial differentiation. As neurogenesis progresses, the accumulation

of neuronal proteins, such as cardiotrophin-1 (CT-1), a gp130 cytokine expressed by

newly differentiated neurons, induces astroglial differentiation through activation of the

Stat3 (Sauvageot and Stiles, 2002, Barnabe-Heider et al., 2005).

3.1.2 Stat 3 in neurodevelopmental disorders

Maternal immune dysregulation, following prenatal infection and resultant

expression of cytokines such as IL6 provide a unique opportunity for neuropathological

consequences due to the disruption of the Stat3 pathway, and the developmental events

it regulates. Murine models of Stat3 mutations have highlighted the role of Stat3 in

neuropathological outcomes as these models have yielded observations ranging from

disruptions in synaptic transmission, neuronal migration, and abnormal neuronal and

glial morphologies to embryonic lethality depending on the extent of the mutation

(Campbell et al., 1993, Brunello et al., 2000). Particularly, disruptions in synaptic

20

transmission, potentially due to neuronal overgrowth or astroglial abnormalities have

been thought to precipitate the behavioral pathologies in animals possessing these

mutations. Similar disruptions in neuronal populations have also been implicated in the

behavioral abnormalities observed clinically in disorders such as ASD (Campbell et al.,

1993, Brunello et al., 2000).

The ability of IL6 mediated Stat3 disruptions to alter astroglial differentiation and

function may particularly suggest an indirect mechanism of precipitating these

pathologies. During development, expression and subsequent binding of gp130

cytokines such as CT-1 to the gp130-Janus tyrosine kinase-2/Signal Transducer and

Activator of Transcription-3 (Jak2/Stat3) receptor complex triggers a series of

phosphorylation including the activation of Stat3, its entry into the nucleus and

subsequent transcription of astroglial genes such as GFAP (Akira, 1997, Heinrich et al.,

1998, Deverman and Patterson, 2009). Astrocytes are important constituents of the

CNS providing migratory scaffolding for neurons during development and the later

regulating synaptic transmission through the release of neuroactive compounds,

clearance of neurotransmitters from the synaptic cleft, or direct communication with

neurons (Brunello et al., 2000). Disruption of astroglial function have been shown to

have deleterious effects, and has been implicated in disorders such as epilepsy, a

condition found to be a common comorbidity with ASD in a subset of this population

(Dutuit et al., 2000, Brenner et al., 2001, Laurence and Fatemi, 2005, Vargas et al.,

2005, Jabs et al., 2008). Astrocyte disruptions have also been directly implicated in the

pathology of ASD with disruptions in several astroglial markers including GFAP elevated

in the brains of autistic individuals, a potential fetal inflammatory response to prenatal

infection (Fatemi et al., 2008a). Stat3 mediated regulation of astrocyte development and

function is therefore important in regulating CNS function.

21

3.2 Results

3.2.1 Luteolin attenuates IL-6 mediated neuronal Ja k2/Stat3 phosphorylation

The effects of IL-6 has been shown to be a largely Stat3 dependent event.

During CNS development, the maintenance of stem cell populations through active

proliferation and the initiation of gliogenesis in neural stem cells have also been shown

to be mediated by activation of Stat3. To determine the efficacy of luteolin in attenuating

IL-6/Stat3 mediated activation and resultant pathologies, Jak2/Stat3 phosphorylation

was measured in murine neuroblastoma (Neuro-2a) cells. IL-6, like other gp130

cytokines bind to their respective gp130 receptor complex to activate Stat3 through a

series of phosphorylation events beginning with the phosphorylation of Jak2, followed by

the phosphorylation, dimerization and nuclear entry of Stat3. Once in the nucleus, Stat3

binds target DNA sequences to initiate the transcription of Stat3 dependent genes such

as glial fibrillary acidic protein (GFAP) at the onset of gliogenesis. To examine IL-6

mediated activation of the Jak2/Stat3 pathway, Neuro-2a cells were treated with 50

ng/mL of murine recombinant IL-6 (mIL-6) in a time dependent manner. Alternatively,

cells were also co-treated with mIL-6 and increasing concentration of luteolin (0µM -

20µM) for 30 minutes. Western blot analysis of cell lysates showed that IL-6 treatment

induced a time dependent increase in Jak2 phosphorylation beginning at 30 minutes and

continuing until 75 minutes, when the timed analysis was concluded (fig. 3.1a). When

cell lysates of luteolin co-treated cells were analyzed, there was significant inhibition of

Jak2 phosphorylation observed beginning at 10µM.

22

Figure 3.1a-b

Luteolin attenuated IL-6 mediated Jak2 phosphorylation in neuroblastoma cells. Densitometric analysis of the ratio of phospho Jak2 to total Jak2, and one way ANOVA showed mIL-6 treatment significantly induced Jak2 phosphorylation in a time dependent manner beginning at 30 minutes (**p<0.005) (a). Luteolin co-treatment significantly inhibited the effects of IL-6, attenuating IL-6 mediated phosphorylation beginning at 10µM (**p<0.01) (b).

**

**

a

b

23

Stat3 phosphorylation in Neuro-2a cells was analyzed next. IL-6 treatment

resulted in a significant increase in Stat3 phosphorylation at residue Ser727 beginning at

30 minutes, and remaining significant until the end of treatment at 75 minutes. Luteolin

co-treatment significantly inhibited IL-6 mediated Stat3 phosphorylation beginning at

10µM.

24

Figure 3.2a-b

Luteolin attenuated IL-6 mediated Stat3 phosphorylation in neuroblastoma cells. Densitometric analysis of the ratio of phosphor Stat3 to total Stat3, and one way ANOVA showed mIL-6 treatment significantly induced Stat3 phosphorylation in a time dependent manner beginning at 30 minutes (**p<0.005) (a). Luteolin co-treatment significantly attenuated this phosphorylation beginning at 10 µM (*p<0.01, **p<0.001) (b). Similar results were obtained using antibody specifically against phospho-Star3 (Tyr705).

**

**

** *

**

**

b

a

25

3.2.2 Luteolin attenuates IL-6 mediated effects in neural stem cells through

inhibition of the Jak2/Stat3 phosphorylation

Although neuroblastoma cells are often utilized as an in vitro model to examine

CNS pathologies the effects of IL-6 and the therapeutic efficacy of luteolin was further

examined in neural stem cells isolated from embryonic mouse cortices, to closer

approximate the in vivo environment during CNS development. Phosphorylation of Jak2

was examined following mIL-6 treatment or luteolin and mIL-6 co-treatment. In cultures

treated with mIL-6 Jak2 phosphorylation was significantly increased at 15 minutes post

treatment, reaching a maximal intensity at 45 minutes (fig. 3.3a). When Stat3

phosphorylation was examined, there was a similar phosphorylation pattern observed

with a significant increase in phosphorylation in response to IL-6 treatment at residue

Tyr705. Phosphorylation at residue Ser727 was significant at 45 minutes (fig. 3.3c).

Luteolin co-treatment significantly inhibited IL-6 mediated phosphorylation of Jak2 and

Stat3 at both residues at 20µM (fig. 3.3b&d).

26

Figure 3.3a-b

Luteolin attenuated IL-6 mediated phosphorylation of Jak2/Stat3 in neural stem cells. Densitometric analysis showed mIL-6 treatment significantly induced Jak2 phosphorylation in a time dependent manner beginning at 15 minutes, remaining significant until 45 minutes (a). This phosphorylation was attenuated by luteolin co-treatment (b). (*=p<0.01)

a

b

27

Figure 3.3c-d

Densitometric analysis showed mIL-6 treatment significantly induced Stat3 phosphorylation in a time dependent manner beginning at 15 minutes, remaining significant until 30 minutes (c). This phosphorylation was attenuated by luteolin co-treatment (d). (*=p<0.01)

* *

*

c

d

*

28

3.2.3 Luteolin attenuates IL-6 mediated activation of Protein kinase B (Akt) and

Migtogen activated protein kinase (Erk1/Erk2) pathw ays in neural stem

cells

Although Stat3 has been shown to be a key transcription factor and regulatory

agent during neurodevelopment, other pathways, such as protein kinase B, also known

as Akt, and mitogen activated protein kinase or Erk1/Erk2 have been shown to play

important roles. These pathways have also been shown to interact and regulate the

activity of Stat3 and are responsive to IL-6 treatment. Erk1/Erk2 for example in addition

to regulating neuronal differentiation has been shown to regulate Stat3 activity at Ser727,

through phosphorylation. Although the exact purpose of this event is not fully

understood, it has been hypothesized that Erk1/Erk2 mediated serine phosphorylation

regulates Stat3 homodimerization, while other groups have hypothesized a role in

regulating transcriptional activity and DNA binding (Wen et al., 1995, Zhang et al., 1995,

Chung et al., 1997). The effects of luteolin on these pathways were examined as well.

In neural stem cells cultures treated with IL-6 there were significant increases in Akt

phosphorylation and Erk1/Erk2 phosphorylation at which was inhibited by luteolin co-

treatment. Although it is not clear how activation of these pathways, or inhibition by

luteolin for that matter impacts the pathological effects of mIL-6, or the ability of luteolin

to attenuate these events, their interactions with Stat3, and role in development suggest

that they may play some role in regulation.

29

Figure 3.4 Luteolin attenuated IL-6 mediated phosphorylation of Protein kinase B (Akt) in neural stem cells. Densitometric analysis showed mIL-6 treatment significantly induced Akt phosphorylation in a time dependent manner beginning at 15 minutes, remaining significant until 30 minutes (a). This phosphorylation was attenuated by luteolin co-treatment (b). (*=p<0.01)

*

*

a

b

30

Figure 3.5a-b

Luteolin attenuated IL-6 mediated phosphorylation of Mitogen activated protein kinase (Erk1/Erk2) in neural stem cells. Densitometric analysis showed mIL-6 treatment significantly induced Stat3 phosphorylation in a time dependent manner beginning at 15 minutes, remaining significant until 30 minutes (a). This phosphorylation was attenuated by luteolin cotreatment (b). (*=p<0.01)

*

* *

a

b

31

3.2.4 Luteolin attenuates IL-6 induced proliferatio n in neuroblastoma cells

Increases in the number of neuronal cells in certain regions of the brain have

been one pathological feature described in ASD, and may be attributed to disruptions in

the proliferative or apoptotic mechanisms during key phases of neurodevelopment.

Activation of the Stat3 pathway by gp130 cytokines has been shown to be a potent

inducer of proliferation, with dysregulation of Stat3 being an important mechanism in

certain cancers. The potential of IL-6 to induce proliferation in neuronal cells was

examined in Neuro-2a cells. Undifferentiated and differentiated Neuro-2a were treated

with murine recombinant IL-6 (mIL-6) for 24hours and proliferation measured as a

function of fluorescent intensity of labeled nucleic acid residues. IL-6 treatment

significantly increased proliferation in undifferentiated Neuro-2a cells at even minimal

concentrations of 1.625ng/mL, with proliferation peaking at 12.5ng/mL. Although this

proliferative effect declined with increasing concentration, 25 – 100ng/mL, it remained

significant in comparison to control levels (fig. 3.6a). By contrast, in differentiated N2a

cells, the effects of IL6 on proliferation were minimal, peaking at 3.125ng/mL and

declining significantly thereafter although remaining marginally significant (fig. 3.6b).

32

Figure 3.6a-b

Luteolin attenuates IL-6 induced proliferation in neuroblastoma cells. Measurement of fluorescent intensity, from labeled nucleic acid residues indicated that mIL6 treatment significantly increased proliferation in undifferentiated n2a cells, with fluorescent intensity doubling at 1.625ng/mL (p0.0002), and peaking at 12.5ng/mL (p0.0003) (a). In differentiated cells, the increase in proliferation was much lower albeit significant peaking at 3.125ng/mL (p0.009) and drastically declining thereafter. Proliferation at 100ng/mL was not significant (b). (* p<0.05, **p<0.01, ***p<0.001)

a

b

33

3.2.5 Effects of IL-6 in neural stem cells may refl ect in vivo pathological

consequences of prenatal infection.

The development of the CNS has been shown to be largely dependent on a

series of tightly regulated events including periods of proliferation and targeted

apoptosis. Although neuroblastoma cells are often utilized as an in vitro model to

examine CNS pathologies the proliferative capabilities of IL-6 and the therapeutic

efficacy of luteolin was further examined in neural stem cells isolated from embryonic

mouse cortices, to closer approximate the in vivo environment. Interestingly, the

proliferative effects of mIL-6 observed in undifferentiated and differentiated Neuro-2a

cells, were not replicated in neural stem cell (NSC) cultures although significant

proliferation was still observed (fig. 3.7a). This difference may be attributable to the

tumorigenic nature of Neuro-2a cells which has been shown to exhibit significant

proliferative potential and their ability to proliferate independent of exogenous growth

factors primarily through the autocrine expression of transforming growth factors (van

Zoelen et al., 1984). When cells were co-treated with luteolin there was no significant

inhibition of proliferation observed, as levels were comparable and potentially slightly

increased to that observed in IL-6 and even control cells (fig. 3.7b).

34

Figure 3.7a-b

Effects of IL-6 in neural stem cells may reflect in vivo pathological consequences of prenatal infection. In NSC cultures, (a) mIL6 treatment resulted in a significant increase in proliferation at concentrations 12.5ng/mL – 50ng/mL. (b) Luteolin treatment did not significant attenuate IL6 induced proliferation in NSC. In fact there was a trend toward an increase in proliferation with all concentrations except 3.125uM and 50uM, which showed a trend toward a decrease. (*=p<0.05)

b

35

To further investigate this observation, the effects of luteolin were compared in

the presence and absence of IL-6 treatment. As initially eluded to in the experiment

represented in figure 3.8b, the effects of luteolin were influenced by the presence of IL-6.

In the absence of IL-6, luteolin co-treatment attenuated proliferation at higher

concentrations. When IL-6 was present, the effects of luteolin were highly variable. This

effect may be attributed by the presence of growth factors such as epidermal growth

factor (EGF), which maintains stem cells in a proliferative state, and signals through

activation of Stat3. Stat3 has also been shown to regulated by the interactions of

multiple signaling pathways that converge at Stat3 such as the Erk pathway. Although

luteolin has been shown to modulate Erk, the combined actions of IL-6 and EGF may

impact the therapeutic effects of luteolin on inhibiting IL-6 mediated pathologies.

36

Figure 3.8

The ability of luteolin to attenuate IL-6 mediated pathologies modulated by the presence of growth factors (EGF and FGF). The proliferative effects of IL-6 in neural stem cells are also affected by the presence of growth factors.

37

As the proliferative effects of IL-6 and the modulatory effects of luteolin in NSC

cultures were somewhat unexpected, proliferation was next assessed optically, using

fluorescent microscopy. Characteristically, neural stem cells form spherical structures in

suspension known as neurospheres when maintained in proliferative media,

supplemented with the appropriate growth factors, described previously in chapter 2,

section 2.1. The effects of IL6 and luteolin co-treatment on neurosphere morphology

and number were examined using FITC labeling of cellular nucleic acid, to confirm

previous results. Interestingly there again appeared to be no significant proliferative

effects as there was no significant difference in the total amount of neurospheres or the

size of neurospheres under the different treatment conditions. There was however a

trend towards a difference in neurosphere size following mIL-6 treatment, with an

increase in medium and small neurosphere populations and a corresponding decrease

in the large neurosphere population. Although not significant, these effects were

reversed by luteolin co-treatment (fig3.9).

38

Figure 3.9

IL-6 and Luteolin does not alter neurosphere size or number. Neurospheres were classified as small (less than 100um), medium (101-200um) or large (greater than 201um). There were no significant changes in the total population of neurospheres induced by treatment. There were however changes in neurosphere size following mIL6 treatment, with medium sized (101 – 200um) neurosphere populations increasing. Similarly, luteolin co-treatment did not show significant modulation of IL6 activity although the increase in the number of medium neurosphere population was reversed.

39

3.2.6 Luteolin attenuates IL-6 mediated morphologic al changes in neural stem

cells

The morphological effects of IL6 and luteolin co-treatment were examined next.

IL6 treatment induced observable changes in neurosphere morphology; specifically

there was a shift from a stem cell phenotype to a commitment to terminal differentiation

evidenced by increased branching and attachment to the culture surface, which was

significantly inhibited by luteolin co-treatment (fig3.10). This effect was not evident in all

neurosphere clones as IL6 seemed to maintain a consistent population of neurospheres

while simultaneously inducing differentiation in others. To further examine this effect

cultures were treated with IL6 in the absence of growth factors. In these cultures,

neurosphere morphology was maintained at levels comparable to that of control

suggesting that IL6 can maintain neural stem cells in a self-renewal phase characterized

by continuous proliferation or shift them toward lineage commitment and differentiation.

These effects appear to be highly dependent on the immediate microenvironment as can

be seen from the outcomes of this experiment and the observations of proliferation in

differentiated and undifferentiated N2a cells (fig. 3.6).

40

Figure 3.10

Luteolin attenuates IL-6 mediated morphological changes in neural stem cells. IL6 treatment (GF/IL6) resulted in increased attachment of cells to the culture surface and increased branching and dissociation of neurospheres. This effect was attenuated by luteolin co-treatment (GF/IL6-Lt). In the absence of growth factors, cells fail to maintain active proliferation, resulting in significant decreases in neurosphere numbers (Control). IL6 treatment, in the absence of growth factors was able to maintain neurosphere morphology and active proliferation (IL6), an effect also attenuated by luteolin co-treatment.

41

3.2.7 Luteolin attenuates IL-6 mediated differentia tion of GFAP positive cells

To confirm that mIL-6 had induced differentiation and lineage commitment, cells

were fluorescently labeled with neuronal and glial specific cellular markers. When IL-6

treated cultures were examined there was in fact increased differentiation and lineage

commitment evidenced by increased expression of neuronal and glial markers Beta-

tubulin and GFAP respectively. Surprisingly, there was no corresponding decrease in

vimentin expression, a marker of neural stem cells, further suggesting the ability of IL6 to

maintain some cells in an uncommitted fate (fig3.11). When cells were co-treated with

luteolin there was a significant decrease in GFAP expression however, this effect was

not seen with Beta-tubulin although there was a trend toward a decrease.

Since GFAP expression has been shown to be a Stat3 dependent event, the

selective attenuation of GFAP expression suggests luteolin effects may be mediated

through modulation of Stat3 signaling. Additionally, the limited effects on neuronal

populations further confirm, a Stat3 dependent mechanism as well as suggest that the

effects on neuronal populations may be secondary. This secondary effect shows an

indirect mechanism by which the function of neuronal populations are altered by the

actions of astroglial cell populations, as these cells have been shown to be supportive to

neuronal cells cooperating in synaptic transmission as well as providing metabolic

support through the supply of neuroactive molecules.

42

43

Figure 3.11

Luteolin attenuates IL-6 induced GFAP expression in neural stem cells. Following treatment, cells were triple labeled with anti-GFAP, anti-btub and anti-vimentin and lineage determined and quantified using confocal microscopy and analytical software. IL6 treatment (GF/IL6) resulted in a significant increase GFAP (***p<0.0002) and btub (***p<0.0001) expression although vimentin expression remained constant. GFAP expression was attenuated by luteolin co-treatment (GF/IL6-Lt) (***p<0.0005) however there was no significant effect observed with btub expression although there was a trend toward a decrease.

44

3.2.8 IL6 and luteolin activity influenced by extri nsic conditions in the cellular

microenvironment

To further examine the effects of IL-6 and luteolin on lineage specification, IL6

and luteolin co-treatment was examined in differentiated cells already committed to a

specific cellular lineage. When NSC cells, three days post differentiation were treated

with IL6, there were no significant changes observed in GFAP and Beta-tubulin

expression although both populations showed a trend toward an increase. Interestingly,

nestin expression, a marker of uncommitted neural stem cells, was also increased

suggesting an increase in this population of cells. There were also observable changes

in neuronal morphology, specifically increased and less ordered axonal branching. This

effect did not appear to be significantly attenuated by luteolin co-treatment.

45

46

Figure 3.12

IL-6 and Luteolin activity dependent on extrinsic factors in the cellular microenvironment. Neural stem cells 3 days post differentiation did not show significant changes following treatment although there was a trend toward an increase in Beta-tubulin and GFAP expression. This trend was not attenuated by luteolin, as co-treated cultures appeared to show slight increases although not significant.

47

3.3 Conclusions

Prenatal infection and the ensuing increase in maternal cytokines, has been

regarded as a key etiological factor in the pathogenesis of neurodevelopmental

disorders such as ASD. IL6, already described as a key mediator in inflammatory

disorders, and an important immune regulator, has been cited as being critical to the

adverse outcomes following prenatal infection, as increases in this cytokine have been

strongly correlated to disease pathology, pathologies which have been ameliorated by

following IL-6 inhibition. Additionally, inhibition of this cytokine during prenatal infection

has been shown to attenuate pathological outcomes. In this chapter, the efficacy of

flavonoids such as luteolin in reducing IL-6 mediated pathologies was examined in NSC,

an in vitro model of the effects of prenatal infection on the fetal CNS. Luteolin co-

administration was able to attenuate the pathological effects of IL6 particularly increases

in proliferation and excess glial cell expression. Targeted luteolin attenuation of glial cell

commitment, a Stat3 dependent event, and its attenuation of Stat3 phosphorylation

suggest that the neuropathologies were mediated through IL6-Stat3 activation and that

the inhibition of this pathway may be therapeutically beneficial.

A somewhat unexpected but noteworthy observation was the dependence of

mIL-6 activity on cell extrinsic and intrinsic factors, as the pathologies observed in

undifferentiated cells were significantly different than those seen in differentiated cells.

These observations support epidemiological findings that suggest that there is an ‘ideal

window’ where the effects of prenatal infection are more deleterious. In the in vitro

model utilized in this chapter, the effects of IL-6 seemed most significant in

undifferentiated stem cells which suggest that early infections may be most detrimental

to CNS development. Several studies have emphasized these findings in vivo

suggesting earlier infections provide an ideal opportunity for disruption of key processes

such as cellular proliferation and differentiation. These disruptions precipitate further

48

pathologies including alterations in cellular migration and synapse development which

manifest clinically in the neurological and behavioral abnormalities observed in disorders

such as ASD and schizophrenia which have been correlated with prenatal infection

(Meyer et al., 2007). Dopaminergic neurons for example have been shown to emerge

during the early phases of neurodevelopment from precursor populations both in murine

animals and in humans (Bayer et al., 1995, Brana et al., 1996, Marti et al., 2002).

Prenatal infection and resultant maternal and/or fetal cytokine expression during this

period can lead to disruptions in the proliferation, differentiation, migration of these cells

and subsequent disruptions in dopaminergic cellular populations (Brown et al., 2004). It

is plausible that these neuropathologies induced by prenatal infection may account for

the behavioral and neurological abnormalities observed in disorders such as

schizophrenia where disruptions in the dopaminergic system have been a consistent

finding. These findings however do not preclude the ability of prenatal infection during

late gestation to induce adverse pathological outcomes. Indeed infections during this

developmental window have been shown to induce disruptions in cellular migration,

neuronal organization, and synaptogenesis (Buka et al., 2001, Gilmore et al., 2004).

It is evident that infections during pregnancy have to potential to lead to adverse

neurological outcomes in exposed offspring and the timing of infection may impact the

nature of pathologies observed clinically. Early infections allow for opportunities to

disrupt proliferation, differentiation and migration of cellular populations that can lead to

multiple morphological, structural and functional abnormalities due to the potential to

impact multiple cellular populations. By contrast, later infections may have more

targeted effects, producing disruptions to the positioning and interactions of specific

cellular populations that may be vulnerable due to migration, synapse formation (Buka et

al., 2001, Meyer et al., 2007). The implications of these divergent outcomes can be

seen in the nature of the pathologies attributed to earlier infections, where

49

symptomology is often more heterogeneous as can be seen in disorder such as ASD

and schizophrenia. By contrast, later infections may give rise to certain cognitive deficits

and similar neurological abnormalities.

50

CHAPTER 4

FLAVONOIDS, A PRENATAL PROPHYLAXIS FOR ADVERSE PERI NATAL

OUTCOMES AFTER PRENATAL INFECTION

4.1 Prenatal infection, Cytokines and the Placental Barrier

The pleiotropic nature of cytokines, particularly their involvement in both

pathological and physiological events and their ubiquitous expression throughout the

body and at various developmental periods make them prime targets as mediators of the

adverse outcomes resulting from prenatal infection. Despite evidence of their function in

this capacity however, there is still disagreement as to their role in neurodevelopmental

and/or neuropsychiatric disorders such as ASD (Dassa et al., 1995, Chen et al., 2004).

Adding to this disagreement has been a lack of consensus as to whether or not maternal

cytokines, produced during prenatal infection, can traverse the placental barrier and

disrupt fetal development. Research focusing on the placental migration of cytokines,

following prenatal infection has yielded contradictory results with some studies reporting

a positive results while others have reported no migration (Bell et al., 2004, Aaltonen et

al., 2005, Ashdown et al., 2006). These contradictions may be attributed to variations in

experimental design as comparative studies have shown that the ability of maternal

cytokines to traverse the placental barrier may be highly dependent on the time of

immune challenge. IL-6 for example has been shown to traverse the rat placenta during

mid-gestation, approximately embryonic days 11–13, while this observation is not seen

during embryonic days 17–19 (Bell et al., 2004, Dahlgren et al., 2006).

51

It is worthwhile to note, that even with no observable cytokine migration during

late gestation, prenatal infection during this period has still been shown to increase

cytokine levels in the fetal CNS, and precipitate adverse neurological outcomes.

Additionally, murine models of prenatal infection utilizing LPS, which does not traverse

the placental barrier, have induced both maternal and fetal inflammatory responses

following prenatal administration (Bell et al., 2004, Elovitz et al., 2011). These findings

suggest that instances of prenatal infection not only activate the maternal immune

system, but can propagate a cascade of inflammatory responses inducing inflammation

in the placenta as well as the fetal CNS. This can in turn lead to adverse changes in

placental function and the development and function of the fetal CNS.

4.1.1 Current animal models of adverse perinatal ou tcomes following prenatal

infection

Although the mechanism by which maternal cytokines disrupt fetal CNS

development is not fully understood, findings from animal models have highlighted their

role in the pathological changes that may precipitate later neuroanatomical,

immunological and behavioral abnormalities in prenatally exposed offspring. In murine

models of prenatal influenza exposure, neuropathologies including changes in cell

density, cell atrophy and neurochemical abnormalities such as disruptions in serotonin

levels were observed. These pathologies mirror clinical findings in disorders such as

schizophrenia and ASD, and may explain the alterations in the levels of

neurotransmitters such as serotonin and its metabolites observed in these disorders

(Fatemi et al., 2002, Meyer et al., 2008, Winter et al., 2008). Behavioral correlates

among prenatally exposed murine offspring have also been observed. Particularly

deficits in prepulse inhibition in adult offspring following prenatal exposure to bacterial

endotoxin LPS or synthetic viral analog polyinosinic:polycytidylic acid (poly(I:C)) have

52

been observed, a pathology representative of deficits in sensorimotor gating, an inability

to filter out extraneous information, and a well-known pathological feature in disorders

such as ASD and schizophrenia (Fortier et al., 2007).

In addition to neurodevelopmental abnormalities, these models have also shown

instances of pregnancy disorders including preeclampsia and spontaneous recurrent

miscarriages that seem to result from disruptions in maternal immunity (Makhseed et al.,

2000, Laresgoiti-Servitje et al., 2010, Tosun et al., 2010, Holder et al., 2011, Jin et al.,

2011). These disorders have described observations of aberrant T cell differentiation

with uncharacteristic increases in cytotoxic T cell populations, an IL-6 influenced event,

and reductions in immune suppressant T cell populations (Kimura and Kishimoto, 2010,

Fujimoto et al., 2011). This scenario contrast observations in normal pregnancies where

increases in the number of immunosuppressive T-regs preceding and after implantation

of the fetus, prevent the proliferation and activation of cytotoxic Th17 cells ensuring the

ability to maintain a viable pregnancy (Aluvihare et al., 2004, Heikkinen et al., 2004,

McCracken et al., 2004, Somerset et al., 2004, Hunt et al., 2005, Zenclussen et al.,

2006, Kallikourdis et al., 2007, Kallikourdis and Betz, 2007, Kahn and Baltimore, 2010,

Dimova et al., 2011). IL6 has therefore been shown to be a recurrent pathological

feature and potential site of intervention in disorders precipitated by prenatal infection.

4.1.3 Luteolin as a therapeutic intervention – pote ntial mechanism

As the inhibition of IL-6 and its mediated activation of Stat3 are known to reduce

pathological outcomes following prenatal infection, prenatal flavonoid treatment may

prove to be of therapeutic value for both maternal and fetal pathologies. Several studies

have shown that flavonoids are potent anti-inflammatory agents that act through

modulation of tyrosine kinase activity to inhibit cytokine production, and the expression

of other pro-inflammatory mediators. This has been confirmed in studies involving

53

stimulation of immune cells with flavonoids such as luteolin resulting in a reduction in the

expression of cytokines such as IL6 and Tumor necrosis factor-alpha (TNF-α), with

inhibition of Stat3, nuclear factor kappa light chain enhancer of activated B cells (NF-κB)

and Protein Kinase B (Akt) being implicated as potential mechanisms (Hirano et al.,

2006, Jang et al., 2008, Zhu et al., 2011).

4.2 Results

4.2.1 Luteolin attenuates maternal immune response following prenatal infection

Activation of the maternal immune system following prenatal infection,

particularly the release of maternal cytokines has been cited as the major contributor to

adverse perinatal outcomes. Particularly, disruptions in cytokine expression and T-cell

differentiation have been cited as key mediators in adverse perinatal events through

their disruption of placental function. The spleen is an important immune organ and

source of pro-inflammatory cytokines. During prenatal infection, the spleen has been

shown to be an important mediator of the maternal inflammatory response and cytokine

expression (Athanassakis and Iconomidou, 1996). Here, splenocytes isolated from

female C57BL/6 mice were challenged with lipopolysaccharide (LPS) or concavalin A

(conA) and co-treated with luteolin to examine attenuation of inflammatory responses

and splenic cytokine expression. LPS is a bacterial endotoxin utilized in animal models

to elicit immune reactions including pro-inflammatory cytokine production, while. conA is

often utilized as a T-cell mitogen. When LPS challenged splenocyte cultures were co-

treated with luteolin, there was a significant inhibition of IL-6 and TNF-α expression.

Similarly, in cultures challenged with conA, luteolin co-treatment also significantly

inhibited IL-6 and TNF- α expression.

54

Figure 4.1a-b

Luteolin attenuates pro-inflammatory cytokine expression from LPS or conA challenged splenocytes. Elisa cytokine analysis showed luteolin co-treatment significantly inhibits LPS mediated IL6 (a) and TNF-α (b) expression from splenocyte cultures in a concentration dependent manner (***p<0.001). These results represent five independent experiments.

a

b

55

Figure 4.1c-d

Elisa cytokine analysis showed luteolin co-treatment significantly inhibits LPS mediated IL6 (c) and TNF-α (d) expression from splenocyte cultures in a concentration dependent manner (***p<0.001). These results represent five independent experiments.

a

b

***

56

To further confirm the efficacy of flavonoids such as luteolin in attenuating

maternal immune activation during prenatal infection, cytokine production was further

examined in the spleens of recently delivered prenatally infected mice. As IL-6 mediated

activation of Stat3 have been shown to play a critical role in immune regulation,

particularly in the propagation of inflammatory responses, including the differentiation of

cytotoxic or pro-inflammatory T-cell populations, and pro-inflammatory cytokine

expression following infection, the role of Stat3 in mediating this inflammation, and the

ability of luteolin to attenuate Stat3 activation was also examined. Pregnant mice were

treated with 5 µg/mL mIL-6, 50ug/kg LPS, or co-treated with these compounds in the

presence of 10mg/kg luteolin at embryonic day 12 (E12). Mice injected prenatally with

LPS and IL6 showed significant increases in TNF-α and interleukin1- β (IL1-β)

expression which was attenuated by luteolin co-treatment.

57

Figure 4.2a-b

Luteolin attenuates maternal immune responses following prenatal infection. Luteolin co-treatment significantly inhibited cytokine production in spleens from mice prenatally injected with LPS or IL-6. Luteolin alone also significantly reduced cytokine expression below that of control levels (***p<0.001). Stat3 has also been shown to play a major role in the progression of

b

a

58

inflammatory processes following infection. Additionally, IL-6 mediated activation of

Stat3 has been implicated as key in the differentiation of cytotoxic T-cell populations

from naïve T-cells. Stat3 activity was analyzed and the effect of luteolin co-treatment

was assessed in spleens isolated from prenatally treated mice. In mice treated

prenatally with LPS, there was a significant increase in Stat3 phosphorylation confirming,

its involvement in potentiating inflammatory responses. When luteolin was co-

administered with LPS, Stat3 phosphorylation was significantly reduced, to levels

comparable to that of control animals.

59

Figure 4.3

Luteolin attenuates Stat3 phosphorylation in maternal spleens following prenatally infection. Luteolin co-treatment significantly inhibited Stat3 phosphorylation in spleens from mice prenatally injected with LPS or IL6 (**p<0.05).

60

4.2.2 Luteolin attenuates adverse fetal outcomes fo llowing prenatal infection

Several adverse perinatal events including premature delivery, in utero growth

restriction, and often more serious outcomes such as neurological disorders, recurrent

miscarriages and preeclampsia have been attributed to prenatal infection. Attenuation of

the maternal immune response to infection may serve as a therapeutic alternative not

only for developmental outcomes but also for adverse pregnancy outcomes. In mice

exposed prenatally to LPS or IL-6 at E12, there was a significant decrease in the birth

weight of offspring suggestive of in utero growth restriction. This effect was attenuated

by luteolin co-treatment. An unexpected result of luteolin treatment was an apparent

increase in birth weight. There were no visible abnormalities observed after treatments.

61

Table 4.1

Luteolin attenuates adverse fetal outcomes following prenatal infection.

Treatment Ctl/PBS LPS IL6 LPS/Lt IL6/Lt

Average litter weight 1.33±0.10 1.27±0.13* 1.25±0.09* 1.35±0.13* 1.36±0.13*

Figure 4.4

Luteolin attenuates adverse fetal outcomes following prenatal infection. Offspring exposed prenatally to IL6 showed an average decrease in birth weight when compared to their non-exposed counterparts (*p<0.05). This was attenuated by luteolin, although there was an increase in birth weight associated with luteolin treatment (*p<0.05). There were no observable physical abnormalities observed.

62

4.2.3 Diosmin reduced pro-inflammatory cytokine pro duction and Jak2/Stat3

phosphorylation in the brain tissues of IL-6 newbor n mice

To further expand on the efficacy of flavonoid treatment during instances of

prenatal infection, an in vivo model of dietary intervention was utilized. For the following

experiments, pregnant mice were treated with 5ug/mL mIL-6 in the presence or absence

of diosmin (10 mg/kg/day diosmin) administered orally in their chow from E12 until birth.

As a positive control, a subgroup of mice were co-treated with IL-6 and Stat3 inhibitor

S31-201 (4ug/mL) at embryonic day 12 (E12). Cytokine production and Jak2/Stat3

phosphorylation was examined in the brain homogenates of new born mice. In mice

prenatally exposed to IL-6, there was a significant increase in pro-inflammatory

cytokines TNF-α and IL-1β levels compared to controls. These increases were

significantly reduced by almost 50% in the presence of S31-201 or diosmin. Western

blot analysis of brain homogenates show that both S31-201 and diosmin significantly

reduce IL-6 induced Jak2 and Stat3 phosphorylation.

63

Figure 4.5

Diosmin reduced pro-inflammatory cytokine production in the brain homogenates of prenatally exposed newborn mice. Pro-inflammatory cytokine analysis by ELISA was conducted on these newborn mouse brain homogenates. Analysis of results revealed a significant reduction of TNF-α and IL-1β cytokines in brain homogenates from IL-6/S31-201 and IL-6/Diosmin/newborn mice when compared to IL-6 only (MIA model) newborn mice (**P < 0.01).

64

Figure 4.6 Diosmin reduced Jak2/Stat3 phosphorylation in the brain homogenates of prenatally exposed newborn mice. Brain homogenates were prepared from newborn mice from mothers injected with IL-6, IL-6/S31-201, IL-6/diosmin or PBS (control). Treatment of S31-201 or diosmin significantly inhibited IL-6-induced Jak2/Stat3 phosphorylation in brain tissues from newborn mice. Densitometry analysis shows the ratio of phospho-Jak2/Stat3 to total Jak2/Stat3 as indicate below the figures. One-way ANOVA showed that both significantly inhibit Jak2/Stat3 signaling (P <0.005).

65

4.2.4 Prenatal diosmin co-treatment reduces abnorma l behaviors in offspring

following prenatal infection through inhibition of Stat3 pathway

Abnormal social behaviors, and in some instances anxiety are prominent

features in disorders such as ASD and schizophrenia. To further examine the efficacy of

flavonoid dietary intervention in attenuating the pathological effects of prenatal infection,

the behavior of adult offspring exposed prenatally to IL-6 and flavonoid intervention was

examined. The open field test was used to assess general locomotor activity and

anxiety while the social interaction test was used to assess social behaviors. Pregnant

mice were treated as described in the previous section and their adult offspring

examined for behavioral outcomes. In mice exposed prenatally to IL-6, there were

significant behavioral disruptions observed. In the open field test, these mice spent less

time in the inner section compared to control, S31-201 or diosmin treated mice.

Offspring of mice co-treated with either S31-201 or diosmin showed behaviors

comparable to that of control mice. The open field test measures anxiety as a function

of entries the test animal makes into an arbitrarily defined ‘center’. Since this measure is

highly dependent on the total distance travelled within the apparatus, changes in

locomotor activity in one group could significantly skew the outcome measures. To

account for this, ANOVA on distance traveled in the inner section was performed and did

not indicate a main effect. Analyses of distance traveled in the outer section did not

reveal a significant difference or a statistical trend towards a significant difference among

groups. There was not a significant difference between PBS-treated mice and S31-

treated control (non-IL-6 injected) mice.

In the social interaction test, the adult offspring of control mice showed a strong

preference for the social chamber, almost double that of the adult offspring of IL-6

treated mice confirming impairments in social behaviors in mice exposed prenatally to

IL-6. This social impairment was significantly reduced by maternal co- treatment with

66

S31-201 or diosmin as these mice showed a preference for the social chamber

comparable to that of control adult offspring. To quantify social interaction, exploration

demonstrated as sniffing time was analyzed via ANOVA. There was a main effect of

group; the S31-201 or diosmin mice exhibited an increase in sniffing compared to IL-

6/MIA mice. There were no observations of social grooming, chasing, dominant mounts,

pinning, boxing, or biting. There were no significant differences in total move time and

total distance traveled.

67

Fig. 4.7

Maternally blocking Stat3 activity reduces abnormal behaviors in prenatally exposed animals. Offspring of mice (n = 8, 4 female/4 male) intraperitoneally (i.p.) treated with IL-6 (5 µg/mouse) in the absence or presence of STAT3 inhibitor (S31-201; 4 µg/mouse; i.p.) (Siddiquee et al., 2007) or with diosmin [oral administration; (10 mg/kg/day)]. (a) In the open-field test, offspring of mice treated with either S31-201 or diosmin enter the center more often than IL-6 (**P < 0.01) and are nearly similar to control mice. (b) In the social interaction test, as previously reported, the social chamber was defined as (percentage of time in social chamber) – (percentage of time in opposite chamber). Most notably, control mice reveal a strong preference for the social chamber. Interestingly, the social impairment of offspring is significantly improved by maternal administration of STAT3 inhibitor, S31-201 or diosmin (**P < 0.005).

68

4.2.5 Diosmin reduces pro-inflammatory cytokines an d Stat3 phosphorylation in

prenatally exposed animals

After behavioral testing, adult offspring were sacrificed to confirm that inhibition of

Stat3 phosphorylation by diosmin attenuates IL-6/Stat3 induced behavioral

abnormalities. At sacrifice, brain homogenates were prepared from control mice and

mice exposed prenatally to IL-6, IL-6/S31-201, and IL-6/Diosmin. Pro-inflammatory

cytokine ELISA showed significant increases in TNF-α and IL-1β levels in the

homogenates of IL-6 adult offspring. Maternal co-treatment with S31-201 or diosmin

significantly reduced TNF-α and IL-1β cytokine levels, with diosmin showing slightly

more significant reductions as shown in Fig. 4.8.

Western blot analysis of phospho- and total Stat3 showed that prenatal IL-6

treatment increased Stat3 phosphorylation in the brain homogenates of adult offspring

while co-treatment with either S31-201 or diosmin lead to a significant reduction of

STAT3 phosphorylation. This data suggest that activation of the Stat3 pathway following

prenatal infection is a plays a major role in the resultant pathologies including behavioral

deficits. Inhibition of Stat3 activation, by diosmin reduces these behavioral deficits and

may be a key therapeutic intervention strategy.

69

Fig. 4.8

Diosmin reduces pro-inflammatory cytokine expression in the brain homogenates of prenatally exposed animals. Pro-inflammatory cytokine analysis by ELISA revealed a significant reduction of TNFα and IL-1β cytokines in brain homogenates from IL-6/31-201 and IL-6/Diosmin adult offspring when compared to offspring prenatally exposed to IL-6 only.(**P < 0.05).

70

Fig. 4.9

Diosmin attenuates Stat3 phosphorylation in the brain homogenates of prenatally exposed animals. Western blot analysis showed a significant reduction of STAT3 phosphorylation in brain homogenates from IL-6/S31-201 and IL-6/Diosmin/adult offspring when compared to offspring of IL-6 offspring. These reductions were further showed by densitometric analysis (**P < 0.005).

71

4.3 Discussion

Epidemiological data, clinical findings from pregnancy disorders and

observations from murine models of prenatal infection have supported a role for

activation of the maternal immune system, and subsequent maternal cytokine

expression as a key component of their pathological mechanism (Hwang and Chen,

Brown et al., 2000, Brown and Susser, 2002, Li et al., 2009, Atladottir et al., 2010).

Furthermore, IL-6 has been shown to be an important mediator of these pathological

events through its dysregulation of the Stat3 pathway. This chapter shows how

flavonoids luteolin and diosmin not only attenuate IL-6 mediated Stat3 activity but more

importantly the adverse maternal and fetal outcomes stemming from prenatal infection.

To confirm the ability of luteolin to attenuate maternal immune responses during

prenatal infection, cytokine production from the spleen was examined following an

immunological challenge. The spleen is an important mediator during immune activation

and a reservoir of pro-inflammatory cytokines. In splenocytes challenged with LPS or

conA luteolin co-treatment significantly inhibited IL-6 and TNF-α expression.

Immunological disruption including increased IL-6 expression has been shown to be a

consistent feature in adverse perinatal outcomes precipitated by prenatal bacterial or

viral infections or instances of chronic immunological dysregulation. Increased plasma

levels of IL-6 for example has been observed in FIRS precipitated by bacterial infections

as well as in preeclampsia, thought to be connected to immunological disruptions

(Gomez et al., 1998, Chaiworapongsa et al., 2002, Tosun et al., 2010, Zhou et al.,

2011). The ability of luteolin to inhibit inflammatory response to both LPS and conA

suggest it may be a key therapeutic alternative in a wide range of pregnancy disorders

as these two mitogens elicit immune reactions through very different mechanisms.

These findings were also confirmed by in vivo analysis. When spleens isolated

from recently delivered mice challenged with LPS or IL-6 were analyzed, TNF-α and IL-

72

1β, were reduced by luteolin in co-treatment. TNF-α and IL-1β have both been shown to

indirectly induce IL-6 expression and Stat3 activity (Mori et al., 2011). As increased

Stat3 activity is a known consequence of IL-6 expression, this was also examined. Stat3

phosphorylation was significantly reduction by luteolin co-treatment in prenatally

challenged animals. The attenuation of maternal immunological disruptions and

cytokine expression by luteolin also reduced fetal pathological outcomes, particularly

growth restrictions, as mice exposed prenatally to LPS or IL-6 had significantly lower

birth weights than control offspring, a pathology not seen in the offspring of mice that

received luteolin co-treatment.

It is clear that maternal immunological disruptions following prenatal infection

play an important role in adverse perinatal outcomes, however fetal immunological

responses have also been observed to play an equally important role in pathologies,

particularly the neurological abnormalities observed in prenatally exposed offspring

(Dammann and Leviton, 2000, Chaiworapongsa et al., 2002). To examine the ability of

flavonoids to attenuate fetal immune responses, fetal cytokine expression and Stat3

activity were examined in newborn and adult offspring, while abnormal behaviors were

analyzed in the adult offspring of prenatally exposed animals. A more chronic

administration route of flavonoid treatment was also examined. Diosmin dietary

intervention was utilized to examine prenatal supplementation of flavonoids and their

effects on fetal outcomes. Similar to maternal outcomes, there was an increase in

inflammatory cytokines and Stat3 phosphorylation in the brains of prenatally exposed

animals which was attenuated by diosmin co-treatment. Additionally, behavioral deficits

observed in these animals were also attenuated following diosmin co-treatment. It is

therefore evident that flavonoid co-treatment is a beneficial therapeutic intervention in

prenatal infection, as it can reduce the neuropathological and abnormal behavioral

outcomes seen in prenatally exposed offspring.

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CHAPTER 5

CONCLUSION

Clinical observations in disorders such as preeclampsia, schizophrenia and ASD

have highlighted the role of maternal immune activation and subsequent IL-6

expression, often a consequence of prenatal infection, in the pathological features

observed in these disorders. Similarly, murine models replicating these disorders have

identified this immune reaction and IL-6 expression as a central component of the

pathological mechanism. Studies have also shown that IL-6 may be a measurable

indicator of pathology and target of therapeutic intervention as inhibition of IL6 has been

shown to reduce associated disease pathology (Smith et al., 2007, Zhou et al., 2011). In

disorders such as preeclampsia for example, elevations in IL-6 have been implicated as

an observable and measurable mediator of hypertension through its regulation of

vasoconstrictive proteins and its inhibition has been shown to reduce this pathology

(Zhou et al., 2011). Similarly, in models of prenatal infection meant to recapitulate

neurodevelopmental disorders such as ASD, IL-6 has been shown to induce not only the

neuropathologies such as abnormal cellular morphology, but also abnormal behaviors

such as impaired social interactions, outcomes all attenuated by inhibition of IL-6 (Smith

et al., 2007).

This study further explores how prenatal infection can lead to adverse perinatal

outcomes, particularly changes in neuroanatomy that seem to precipitate the abnormal

behavioral outcomes observed in prenatally exposed offspring and clinically in disorders

such as ASD. More specifically, it focuses on role of IL-6 mediated activation of the

74

Stat3 pathway in these pathologies and how attenuation of this pathway, by flavonoids

can reduce the pathological outcomes associated with prenatal infection. Chapter three

explores this relationship by utilizing an in vitro model of pathological IL-6 expression to

examine the neuropathologies that may occur following prenatal infection and

subsequent exposure to IL-6. Treatment of neural stem cells with IL-6 revealed

significant pathologies including increases in proliferation, and aberrant patterns of

differentiation. Most notably, and possibly most relevant to clinical observations in

disorders such as ASD were findings of increased astroglial differentiation following IL-6

treatment, an effect inhibited by luteolin co-treatment. Astrocyte pathologies have been

implicated in neurological disorders such as epilepsy due largely to their role in

regulating synaptic transmission, and their supportive role in maintaining optimal

neuronal function and integrity in the CNS (Brunello et al., 2000, Dutuit et al., 2000,

Brenner et al., 2001). These types of pathologies may be particularly relevant in

disorder such as ASD as increased expression of astroglial markers such as GFAP and

aquaporin 4 have been observed in post mortem autistic brain samples (Fatemi et al.,

2004, Laurence and Fatemi, 2005, Fatemi et al., 2008a). When NSC cultures were co-

treated with IL-6 and luteolin, astroglial differentiation, a Stat3 mediated event was

significantly reduced. An examination of Stat3 phosphorylation showed that this was

also significantly reduced following luteolin co-treatment suggesting prenatal luteolin

treatment has the potential to attenuate IL-6/Stat3 mediated pathologies.

To expand on these findings, luteolin’s effects were investigated in vivo. The

effects of chronic prenatal administration were also examined using diosmin. The

experiments outlined in chapter four looked at the ability of luteolin and diosmin to inhibit

activation of the maternal immune system as well as adverse fetal outcomes such as low

birth weight, indicative of in utero growth restriction and abnormal behaviors. To look at

maternal immune reactions, luteolin activity was examined in splenocyte cultures as well

75

as the spleens of recently delivered prenatally exposed animals. Luteolin treatment was

able to attenuate inflammatory response following prenatal infection, an event mediated

by attenuation of Stat3 phosphorylation, which was also inhibited by luteolin co-

treatment. Specifically, luteolin inhibited expression of IL-6 and TNF-α in splenocytes

challenged with LPS or conA, confirming the ability of luteolin to attenuate maternal

immune responses independent of the pathogenic stimuli. In spleen tissue, luteolin

inhibited TNF-α and IL-1β expression in the spleens of mice challenged with IL-6 or LPS.

Like IL-6, these cytokines have been shown to be elevated following prenatal infection

and during other instances of immune dysregulation and can indirectly induce IL-6

expression and resultant activation of Stat3 (Mori et al., 2011).

The inhibition of maternal immune activation following prenatal infection by

luteolin suggests fetal pathologies resulting from prenatal infection can also be

attenuated by prenatal flavonoid treatment. To confirm this, several fetal outcomes were

examined, including birth weight, as decreased birth weight is a known consequence of

prenatal infection. As expected, prenatal infection not only activated the maternal

immune system but also led to decreased birth weight in exposed offspring a feature

often correlated to subsequent neurological deficits. These effects were significantly

inhibited by luteolin co-treatment. The neurological consequences of prenatal infection

were examined next. Although maternal cytokine expression has been noted as central

to the pathologies attributed to prenatal infection, fetal immune activation has also been

observed and are considered a key mediator of the neuropathologies and resultant

neurological deficits observed in exposed offspring. Chronic flavonoid administration as

a potential prophylactic intervention was examined next using diosmin dietary

supplementation. Examination of the brain homogenates of newborn mice exposed

prenatally to IL-6 showed significant increases in TNF-α and IL-1β expression as well as

increased Stat3 phosphorylation. In the offspring of animals maintained on a diosmin

76

enriched diet from the time of prenatal challenge until birth, these pathologies were

inhibited. To confirm the role of Stat3 activity in the neurological deficits following prental

infection, behavioral outcomes were examined in prenatally exposed adult offspring.

The efficacy of flavonoids and inhibition of IL-6/Stat3 activity in reducing these abnormal

behavioral outcomes were also examined. In mice prenatally exposed to IL-6, there

were significant behavioral abnormalities observed including deficits in social interaction,

a prominent feature in ASD and schizophrenia. In animals co-treated with diosmin, this

deficit was significantly attenuated. More importantly, analysis of brain homogenates

following behavioral assessment showed an attenuation of pro-inflammatory cytokine

production and Stat3 phosphorylation.

Currently there is no cure for ASD and therapeutic interventions focus primarily

on the management of symptoms, utilizing behavioral therapy to control the abnormal

behaviors and pharmacological intervention to manage comorbid conditions. The role of

prenatal infection in precipitating adverse pregnancy and fetal outcomes has not only

provided a potential mechanism but a focus for therapeutic intervention as targeted

inhibition of IL6 and Stat3 activation has been shown to attenuate, and in many cases

completely ameliorate pathological outcomes. In addition to neurological disorders

attributed to prenatal infection, disorders of pregnancy such as preeclampsia and

recurrent spontaneous abortions share a similar pathology to those precipitated by

prenatal infection due to the involvement of maternal immune dysregulation. This

overlap in pathological features suggests that modulation of the maternal immune

system may also be beneficial in these disorders and may be a potential therapeutic

alternative. While some of these disorders such as recurrent miscarriages seem to be

characterized by a persistent dysregulation of the maternal immune system and may

require more aggressive and chronic treatments, others such as preeclampsia often

appear more transient with a sudden onset of pathological symptoms and may benefit

77

from short term interventions. The results of this study show the potential for both

chronic and one time flavonoid intervention to yield significant therapeutic effects.

Additionally, the relative safety of many of these flavonoid compounds make their

application during pregnancy for chronic or transient treatment feasible as compounds

such as diosmin have already been shown to be safe when administered during

pregnancy (Buckshee et al., 1997). Although there is room for more research in this

area, the potential application of flavonoid compounds, particularly diosmin and luteolin,

is evident in light of their therapeutic efficacy in attenuating both maternal and fetal

adverse outcomes during periods of maternal immune activation, or dysregulation.

78

REFERENCES

Aaltonen R, Heikkinen T, Hakala K, Laine K, Alanen A (2005) Transfer of

proinflammatory cytokines across term placenta. Obstetrics and gynecology

106:802-807.

Ajmone-Cat MA, Cacci E, Ragazzoni Y, Minghetti L, Biagioni S Pro-gliogenic effect of IL-

1alpha in the differentiation of embryonic neural precursor cells in vitro. Journal

of neurochemistry 113:1060-1072.

Akira S (1997) IL-6-regulated transcription factors. The international journal of

biochemistry & cell biology 29:1401-1418.

Allen DA, Steinberg M, Dunn M, Fein D, Feinstein C, Waterhouse L, Rapin I (2001)

Autistic disorder versus other pervasive developmental disorders in young

children: same or different? Eur Child Adolesc Psychiatry 10:67-78.

Aluvihare VR, Kallikourdis M, Betz AG (2004) Regulatory T cells mediate maternal

tolerance to the fetus. Nat Immunol 5:266-271.

Aluvihare VR, Kallikourdis M, Betz AG (2005) Tolerance, suppression and the fetal

allograft. J Mol Med (Berl) 83:88-96.

79

American Psychiatric Association A (2000) Diagnostic and statistical manual of mental

disorders. 4th ed. American Psychiatric Association.

Andres C (2002) Molecular genetics and animal models in autistic disorder. Brain Res

Bull 57:109-119.

Appel SH, Zhao W, Beers DR, Henkel JS (2011) The microglial-motoneuron dialogue in

ALS. Acta myologica : myopathies and cardiomyopathies : official journal of the

Mediterranean Society of Myology / edited by the Gaetano Conte Academy for

the study of striated muscle diseases 30:4-8.

Ashdown H, Dumont Y, Ng M, Poole S, Boksa P, Luheshi GN (2006) The role of

cytokines in mediating effects of prenatal infection on the fetus: implications for

schizophrenia. Molecular psychiatry 11:47-55.

Athanassakis I, Iconomidou B (1996) Cytokine production in the serum and spleen of

mice from day 6 to 14 of gestation: cytokines/placenta/spleen/serum. Dev

Immunol 4:247-255.

Atladottir HO, Thorsen P, Ostergaard L, Schendel DE, Lemcke S, Abdallah M, Parner

ET (2010) Maternal infection requiring hospitalization during pregnancy and

autism spectrum disorders. J Autism Dev Disord 40:1423-1430.

Autism and Developmental Disabilities Monitoring Network Surveillance Year 2006

Principal Investigators; Centers for Disease Control and Prevention C (2009)

Prevalence of autism spectrum disorders - Autism and Developmental

80

Disabilities Monitoring Network, United States, 2006. MMWR Surveillance

summaries : Morbidity and mortality weekly report Surveillance summaries / CDC

58:1-20.

Balschun D, Wetzel W, Del Rey A, Pitossi F, Schneider H, Zuschratter W, Besedovsky

HO (2004) Interleukin-6: a cytokine to forget. Faseb J 18:1788-1790.

Barak Y, Kimhi R, Stein D, Gutman J, Weizman A (1999) Autistic subjects with comorbid

epilepsy: a possible association with viral infections. Child Psychiatry Hum Dev

29:245-251.

Barnabe-Heider F, Wasylnka JA, Fernandes KJ, Porsche C, Sendtner M, Kaplan DR,

Miller FD (2005) Evidence that embryonic neurons regulate the onset of cortical

gliogenesis via cardiotrophin-1. Neuron 48:253-265.

Bauer S, Kerr BJ, Patterson PH (2007) The neuropoietic cytokine family in development,

plasticity, disease and injury. Nature reviews 8:221-232.

Bayer SA, Wills KV, Triarhou LC, Ghetti B (1995) Time of neuron origin and gradients of

neurogenesis in midbrain dopaminergic neurons in the mouse. Experimental

brain research Experimentelle Hirnforschung Experimentation cerebrale 105:191-

199.

Bell MJ, Hallenbeck JM, Gallo V (2004) Determining the fetal inflammatory response in

an experimental model of intrauterine inflammation in rats. Pediatric research

56:541-546.

81

Belmonte MK, Bourgeron T (2006) Fragile X syndrome and autism at the intersection of

genetic and neural networks. Nature neuroscience 9:1221-1225.

Bernardet M, Crusio WE (2006) Fmr1 KO mice as a possible model of autistic features.

ScientificWorldJournal 6:1164-1176.

Bilbo SD (2011) How cytokines leave their mark: the role of the placenta in

developmental programming of brain and behavior. Brain, behavior, and

immunity 25:602-603.

Boksa P (2004) Animal models of obstetric complications in relation to schizophrenia.

Brain research Brain research reviews 45:1-17.

Boulet SL, Boyle CA, Schieve LA (2009) Health care use and health and functional

impact of developmental disabilities among US children, 1997-2005. Arch Pediatr

Adolesc Med 163:19-26.

Boyle CA, Boulet S, Schieve LA, Cohen RA, Blumberg SJ, Yeargin-Allsopp M, Visser S,

Kogan MD (2011) Trends in the prevalence of developmental disabilities in US

children, 1997-2008. Pediatrics 127:1034-1042.

Boyle CA, Decoufle P, Yeargin-Allsopp M (1994) Prevalence and health impact of

developmental disabilities in US children. Pediatrics 93:399-403.

Brana C, Caille I, Pellevoisin C, Charron G, Aubert I, Caron MG, Carles D, Vital C, Bloch

B (1996) Ontogeny of the striatal neurons expressing the D1 dopamine receptor

82

in humans. The Journal of comparative neurology 370:23-34.

Brenner M, Johnson AB, Boespflug-Tanguy O, Rodriguez D, Goldman JE, Messing A

(2001) Mutations in GFAP, encoding glial fibrillary acidic protein, are associated

with Alexander disease. Nat Genet 27:117-120.

Bromberg J, Wang TC (2009) Inflammation and cancer: IL-6 and STAT3 complete the

link. Cancer Cell 15:79-80.

Brown AS, Begg MD, Gravenstein S, Schaefer CA, Wyatt RJ, Bresnahan M, Babulas

VP, Susser ES (2004) Serologic evidence of prenatal influenza in the etiology of

schizophrenia. Archives of general psychiatry 61:774-780.

Brown AS, Schaefer CA, Wyatt RJ, Goetz R, Begg MD, Gorman JM, Susser ES (2000)

Maternal exposure to respiratory infections and adult schizophrenia spectrum

disorders: a prospective birth cohort study. Schizophr Bull 26:287-295.

Brown AS, Susser ES (2002) In utero infection and adult schizophrenia. Mental

retardation and developmental disabilities research reviews 8:51-57.

Brunello AG, Weissenberger J, Kappeler A, Vallan C, Peters M, Rose-John S, Weis J

(2000) Astrocytic alterations in interleukin-6/Soluble interleukin-6 receptor alpha

double-transgenic mice. Am J Pathol 157:1485-1493.

Buckshee K, Takkar D, Aggarwal N (1997) Micronized flavonoid therapy in internal

hemorrhoids of pregnancy. International journal of gynaecology and obstetrics:

83

the official organ of the International Federation of Gynaecology and Obstetrics

57:145-151.

Buka SL, Tsuang MT, Torrey EF, Klebanoff MA, Wagner RL, Yolken RH (2001) Maternal

cytokine levels during pregnancy and adult psychosis. Brain, behavior, and

immunity 15:411-420.

Burns TM, Clough JA, Klein RM, Wood GW, Berman NE (1993) Developmental

regulation of cytokine expression in the mouse brain. Growth Factors 9:253-258.

Campbell DB, Li C, Sutcliffe JS, Persico AM, Levitt P (2008) Genetic evidence

implicating multiple genes in the MET receptor tyrosine kinase pathway in autism

spectrum disorder. Autism Res 1:159-168.

Campbell IL, Abraham CR, Masliah E, Kemper P, Inglis JD, Oldstone MB, Mucke L

(1993) Neurologic disease induced in transgenic mice by cerebral

overexpression of interleukin 6. Proceedings of the National Academy of

Sciences of the United States of America 90:10061-10065.

Chaiworapongsa T, Romero R, Kim JC, Kim YM, Blackwell SC, Yoon BH, Gomez R

(2002) Evidence for fetal involvement in the pathologic process of clinical

chorioamnionitis. Am J Obstet Gynecol 186:1178-1182.

Chen GK, Kono N, Geschwind DH, Cantor RM (2006) Quantitative trait locus analysis of

nonverbal communication in autism spectrum disorder. Molecular psychiatry

11:214-220.

84

Chen W, Landau S, Sham P, Fombonne E (2004) No evidence for links between autism,

MMR and measles virus. Psychological medicine 34:543-553.

Chung J, Uchida E, Grammer TC, Blenis J (1997) STAT3 serine phosphorylation by

ERK-dependent and -independent pathways negatively modulates its tyrosine

phosphorylation. Mol Cell Biol 17:6508-6516.

Covey MV, Loporchio D, Buono KD, Levison SW (2011) Opposite effect of inflammation

on subventricular zone versus hippocampal precursors in brain injury. Ann

Neurol 70:616-626.

Dahlgren J, Samuelsson AM, Jansson T, Holmang A (2006) Interleukin-6 in the maternal

circulation reaches the rat fetus in mid-gestation. Pediatric research 60:147-151.

Dame JB, Juul SE (2000) The distribution of receptors for the pro-inflammatory

cytokines interleukin (IL)-6 and IL-8 in the developing human fetus. Early human

development 58:25-39.

Dammann O, Leviton A (2000) Role of the fetus in perinatal infection and neonatal brain

damage. Curr Opin Pediatr 12:99-104.

Dassa D, Takei N, Sham PC, Murray RM (1995) No association between prenatal

exposure to influenza and autism. Acta Psychiatr Scand 92:145-149.

Deverman BE, Patterson PH (2009) Cytokines and CNS development. Neuron 64:61-78.

85

Dimova T, Nagaeva O, Stenqvist AC, Hedlund M, Kjellberg L, Strand M, Dehlin E,

Mincheva-Nilsson L (2011) Maternal Foxp3 expressing CD4+ CD25+ and CD4+

CD25- regulatory T-cell populations are enriched in human early normal

pregnancy decidua: a phenotypic study of paired decidual and peripheral blood

samples. Am J Reprod Immunol 66 Suppl 1:44-56.

Dutuit M, Didier-Bazes M, Vergnes M, Mutin M, Conjard A, Akaoka H, Belin MF, Touret

M (2000) Specific alteration in the expression of glial fibrillary acidic protein,

glutamate dehydrogenase, and glutamine synthetase in rats with genetic

absence epilepsy. Glia 32:15-24.

Elkouris M, Balaskas N, Poulou M, Politis PK, Panayiotou E, Malas S, Thomaidou D,

Remboutsika E (2011) Sox1 maintains the undifferentiated state of cortical neural

progenitor cells via the suppression of Prox1-mediated cell cycle exit and

neurogenesis. Stem Cells 29:89-98.

Elovitz MA, Brown AG, Breen K, Anton L, Maubert M, Burd I (2011) Intrauterine

inflammation, insufficient to induce parturition, still evokes fetal and neonatal

brain injury. Int J Dev Neurosci 29:663-671.

Ernerudh J, Berg G, Mjosberg J (2011) Regulatory T helper cells in pregnancy and their

roles in systemic versus local immune tolerance. Am J Reprod Immunol 66 Suppl

1:31-43.

Fahey JO (2008) Clinical management of intra-amniotic infection and chorioamnionitis: a

review of the literature. Journal of midwifery & women's health 53:227-235.

86

Fatemi SH, Araghi-Niknam M, Laurence JA, Stary JM, Sidwell RW, Lee S (2004) Glial

fibrillary acidic protein and glutamic acid decarboxylase 65 and 67 kDa proteins

are increased in brains of neonatal BALB/c mice following viral infection in utero.

Schizophrenia research 69:121-123.

Fatemi SH, Earle J, Kanodia R, Kist D, Emamian ES, Patterson PH, Shi L, Sidwell R

(2002) Prenatal viral infection leads to pyramidal cell atrophy and macrocephaly

in adulthood: implications for genesis of autism and schizophrenia. Cellular and

molecular neurobiology 22:25-33.

Fatemi SH, Folsom TD, Reutiman TJ, Lee S (2008a) Expression of astrocytic markers

aquaporin 4 and connexin 43 is altered in brains of subjects with autism.

Synapse (New York, NY 62:501-507.

Fatemi SH, Pearce DA, Brooks AI, Sidwell RW (2005) Prenatal viral infection in mouse

causes differential expression of genes in brains of mouse progeny: a potential

animal model for schizophrenia and autism. Synapse (New York, NY 57:91-99.

Fatemi SH, Reutiman TJ, Folsom TD, Huang H, Oishi K, Mori S, Smee DF, Pearce DA,

Winter C, Sohr R, Juckel G (2008b) Maternal infection leads to abnormal gene

regulation and brain atrophy in mouse offspring: implications for genesis of

neurodevelopmental disorders. Schizophrenia research 99:56-70.

Fortier ME, Luheshi GN, Boksa P (2007) Effects of prenatal infection on prepulse

inhibition in the rat depend on the nature of the infectious agent and the stage of

pregnancy. Behavioural brain research 181:270-277.

87

Fujimoto M, Nakano M, Terabe F, Kawahata H, Ohkawara T, Han Y, Ripley B, Serada

S, Nishikawa T, Kimura A, Nomura S, Kishimoto T, Naka T (2011) The influence

of excessive IL-6 production in vivo on the development and function of Foxp3+

regulatory T cells. J Immunol 186:32-40.

Fuksman RB, Mazzitelli NG (2009) Second-trimester histopathological placental findings

in maternal-fetal inflammatory response syndrome. Pediatr Dev Pathol 12:42-46.

Ganz ML (2007) The lifetime distribution of the incremental societal costs of autism. Arch

Pediatr Adolesc Med 161:343-349.

Geraets L, Haegens A, Brauers K, Haydock JA, Vernooy JH, Wouters EF, Bast A,

Hageman GJ (2009) Inhibition of LPS-induced pulmonary inflammation by

specific flavonoids. Biochemical and biophysical research communications

382:598-603.

Ghiani CA, Mattan NS, Nobuta H, Malvar JS, Boles J, Ross MG, Waschek JA,

Carpenter EM, Fisher RS, de Vellis J (2011) Early effects of lipopolysaccharide-

induced inflammation on foetal brain development in rat. ASN neuro 3.

Gilmore JH, Fredrik Jarskog L, Vadlamudi S, Lauder JM (2004) Prenatal infection and

risk for schizophrenia: IL-1beta, IL-6, and TNFalpha inhibit cortical neuron

dendrite development. Neuropsychopharmacology 29:1221-1229.

Gomez R, Romero R, Ghezzi F, Yoon BH, Mazor M, Berry SM (1998) The fetal

inflammatory response syndrome. Am J Obstet Gynecol 179:194-202.

88

Gotsch F, Romero R, Kusanovic JP, Mazaki-Tovi S, Pineles BL, Erez O, Espinoza J,

Hassan SS (2007) The fetal inflammatory response syndrome. Clin Obstet

Gynecol 50:652-683.

Guerin LR, Prins JR, Robertson SA (2009) Regulatory T-cells and immune tolerance in

pregnancy: a new target for infertility treatment? Hum Reprod Update 15:517-

535.

He F, Ge W, Martinowich K, Becker-Catania S, Coskun V, Zhu W, Wu H, Castro D,

Guillemot F, Fan G, de Vellis J, Sun YE (2005) A positive autoregulatory loop of

Jak-STAT signaling controls the onset of astrogliogenesis. Nature neuroscience

8:616-625.

Heikkinen J, Mottonen M, Alanen A, Lassila O (2004) Phenotypic characterization of

regulatory T cells in the human decidua. Clin Exp Immunol 136:373-378.

Heinrich PC, Behrmann I, Muller-Newen G, Schaper F, Graeve L (1998) Interleukin-6-

type cytokine signalling through the gp130/Jak/STAT pathway. The Biochemical

journal 334 ( Pt 2):297-314.

Hirano T, Higa S, Arimitsu J, Naka T, Ogata A, Shima Y, Fujimoto M, Yamadori T,

Ohkawara T, Kuwabara Y, Kawai M, Matsuda H, Yoshikawa M, Maezaki N,

Tanaka T, Kawase I (2006) Luteolin, a flavonoid, inhibits AP-1 activation by

basophils. Biochemical and biophysical research communications 340:1-7.

Hirano T, Higa S, Arimitsu J, Naka T, Shima Y, Ohshima S, Fujimoto M, Yamadori T,

89

Kawase I, Tanaka T (2004) Flavonoids such as luteolin, fisetin and apigenin are

inhibitors of interleukin-4 and interleukin-13 production by activated human

basophils. Int Arch Allergy Immunol 134:135-140.

Holder BS, Tower CL, Jones CJ, Aplin JD, Abrahams VM (2011) Heightened Pro-

Inflammatory Effect of Preeclamptic Placental Microvesicles on Peripheral Blood

Immune Cells in Humans. Biology of reproduction.

Hornig M, Weissenbock H, Horscroft N, Lipkin WI (1999) An infection-based model of

neurodevelopmental damage. Proceedings of the National Academy of Sciences

of the United States of America 96:12102-12107.

Hsiao EY, Patterson PH (2011) Activation of the maternal immune system induces

endocrine changes in the placenta via IL-6. Brain, behavior, and immunity

25:604-615.

Hunt JS, Petroff MG, McIntire RH, Ober C (2005) HLA-G and immune tolerance in

pregnancy. Faseb J 19:681-693.

Hwang SJ, Chen YS Congenital rubella syndrome with autistic disorder. J Chin Med

Assoc 73:104-107.

Ibi D, Nagai T, Kitahara Y, Mizoguchi H, Koike H, Shiraki A, Takuma K, Kamei H, Noda

Y, Nitta A, Nabeshima T, Yoneda Y, Yamada K (2009) Neonatal polyI:C

treatment in mice results in schizophrenia-like behavioral and neurochemical

abnormalities in adulthood. Neuroscience research 64:297-305.

90

Islam O, Gong X, Rose-John S, Heese K (2009) Interleukin-6 and neural stem cells:

more than gliogenesis. Mol Biol Cell 20:188-199.

Jabs R, Seifert G, Steinhauser C (2008) Astrocytic function and its alteration in the

epileptic brain. Epilepsia 49 Suppl 2:3-12.

Jacobsson B, Hagberg G (2004) Antenatal risk factors for cerebral palsy. Best Pract Res

Clin Obstet Gynaecol 18:425-436.

Jang S, Kelley KW, Johnson RW (2008) Luteolin reduces IL-6 production in microglia by

inhibiting JNK phosphorylation and activation of AP-1. Proceedings of the

National Academy of Sciences of the United States of America 105:7534-7539.

Jellinger KA (2010) Intrathecal levels of IL-6 in Alzheimer's disease. J Neurol 257:142.

Jin LP, Fan DX, Zhang T, Guo PF, Li DJ (2011) The costimulatory signal upregulation is

associated with Th1 bias at the maternal-fetal interface in human miscarriage.

Am J Reprod Immunol 66:270-278.

Jonakait GM (2007) The effects of maternal inflammation on neuronal development:

possible mechanisms. Int J Dev Neurosci 25:415-425.

Kahn DA, Baltimore D (2010) Pregnancy induces a fetal antigen-specific maternal T

regulatory cell response that contributes to tolerance. Proceedings of the

National Academy of Sciences of the United States of America 107:9299-9304.

91

Kallikourdis M, Andersen KG, Welch KA, Betz AG (2007) Alloantigen-enhanced

accumulation of CCR5+ 'effector' regulatory T cells in the gravid uterus.

Proceedings of the National Academy of Sciences of the United States of

America 104:594-599.

Kallikourdis M, Betz AG (2007) Periodic accumulation of regulatory T cells in the uterus:

preparation for the implantation of a semi-allogeneic fetus? PLoS One 2:e382.

Kanellopoulos-Langevin C, Caucheteux SM, Verbeke P, Ojcius DM (2003) Tolerance of

the fetus by the maternal immune system: role of inflammatory mediators at the

feto-maternal interface. Reprod Biol Endocrinol 1:121.

Kang KP, Park SK, Kim DH, Sung MJ, Jung YJ, Lee AS, Lee JE, Ramkumar KM, Lee S,

Park MH, Roh SG, Kim W (2011) Luteolin ameliorates cisplatin-induced acute

kidney injury in mice by regulation of p53-dependent renal tubular apoptosis.

Nephrology, dialysis, transplantation : official publication of the European Dialysis

and Transplant Association - European Renal Association 26:814-822.

Kawai M, Hirano T, Higa S, Arimitsu J, Maruta M, Kuwahara Y, Ohkawara T, Hagihara

K, Yamadori T, Shima Y, Ogata A, Kawase I, Tanaka T (2007) Flavonoids and

related compounds as anti-allergic substances. Allergol Int 56:113-123.

Kim SJ, Silva RM, Flores CG, Jacob S, Guter S, Valcante G, Zaytoun AM, Cook EH,

Badner JA (2011) A quantitative association study of SLC25A12 and restricted

repetitive behavior traits in autism spectrum disorders. Molecular autism 2:8.

92

Kimura A, Kishimoto T (2010) IL-6: regulator of Treg/Th17 balance. Eur J Immunol

40:1830-1835.

Kinney DK, Hintz K, Shearer EM, Barch DH, Riffin C, Whitley K, Butler R (2010) A

unifying hypothesis of schizophrenia: abnormal immune system development

may help explain roles of prenatal hazards, post-pubertal onset, stress, genes,

climate, infections, and brain dysfunction. Med Hypotheses 74:555-563.

Kirsten TB, Taricano M, Maiorka PC, Palermo-Neto J, Bernardi MM (2010) Prenatal

lipopolysaccharide reduces social behavior in male offspring.

Neuroimmunomodulation 17:240-251.

Koenig K, Scahill L (2001) Assessment of children with pervasive developmental

disorders. J Child Adolesc Psychiatr Nurs 14:159-166.

Lai M, You Z, Ma H, Lei L, Lu F, He D, Liu H, Yin S (2010) [Effects of shoutai pills on

expression of Th1/Th2 cytokine in maternal-fetal interface and pregnancy

outcome]. Zhongguo Zhong Yao Za Zhi 35:3065-3068.

Lamarca B, Brewer J, Wallace K (2011) IL-6-induced pathophysiology during pre-

eclampsia: potential therapeutic role for magnesium sulfate? International journal

of interferon, cytokine and mediator research : IJIM 2011:59-64.

Laresgoiti-Servitje E, Gomez-Lopez N, Olson DM (2010) An immunological insight into

the origins of pre-eclampsia. Hum Reprod Update 16:510-524.

93

Laurence JA, Fatemi SH (2005) Glial fibrillary acidic protein is elevated in superior

frontal, parietal and cerebellar cortices of autistic subjects. Cerebellum 4:206-

210.

Leigh R. Guerin LMM, Jelmer R. Prins, John J. Bromfield, John D. Hayball, Sarah A.

Robertson (2011) Seminal fluid regulates accumulation of FOXP3+ regulatory T

cells in the pre-implantation mouse uterus through expanding the FOXP3+ cell

pool and CCL19-mediated recruitment. Biology of reproduction.

Li H, Li Y, Shao J, Li R, Qin Y, Xie C, Zhao Z (2008) The association analysis of RELN

and GRM8 genes with autistic spectrum disorder in Chinese Han population. Am

J Med Genet B Neuropsychiatr Genet 147B:194-200.

Li Q, Cheung C, Wei R, Hui ES, Feldon J, Meyer U, Chung S, Chua SE, Sham PC, Wu

EX, McAlonan GM (2009) Prenatal immune challenge is an environmental risk

factor for brain and behavior change relevant to schizophrenia: evidence from

MRI in a mouse model. PLoS One 4:e6354.

Liu X, Liu Y, Ding M, Wang X (2011a) Reduced expression of indoleamine 2,3-

dioxygenase participates in pathogenesis of preeclampsia via regulatory T cells.

Molecular medicine reports 4:53-58.

Liu Y, Chen H, Sun Y, Chen F (2012) Antiviral role of toll-like receptors and cytokines

against the new 2009 H1N1 virus infection. Molecular biology reports 39:1163-

1172.

94

Liu YS, Wu L, Tong XH, Wu LM, He GP, Zhou GX, Luo LH, Luan HB (2011b) Study on

the relationship between Th17 cells and unexplained recurrent spontaneous

abortion. Am J Reprod Immunol 65:503-511.

Lockwood CJ, Yen CF, Basar M, Kayisli UA, Martel M, Buhimschi I, Buhimschi C, Huang

SJ, Krikun G, Schatz F (2008) Preeclampsia-related inflammatory cytokines

regulate interleukin-6 expression in human decidual cells. Am J Pathol 172:1571-

1579.

Makhseed M, Raghupathy R, Azizieh F, Farhat R, Hassan N, Bandar A (2000)

Circulating cytokines and CD30 in normal human pregnancy and recurrent

spontaneous abortions. Hum Reprod 15:2011-2017.

Marti J, Wills KV, Ghetti B, Bayer SA (2002) A combined immunohistochemical and

autoradiographic method to detect midbrain dopaminergic neurons and

determine their time of origin. Brain research Brain research protocols 9:197-205.

Matousek SB, Ghosh S, Shaftel SS, Kyrkanides S, Olschowka JA, O'Banion MK (2011)

Chronic IL-1beta-Mediated Neuroinflammation Mitigates Amyloid Pathology in a

Mouse Model of Alzheimer's Disease without Inducing Overt Neurodegeneration.

Journal of neuroimmune pharmacology : the official journal of the Society on

NeuroImmune Pharmacology.

McCracken SA, Gallery E, Morris JM (2004) Pregnancy-specific down-regulation of NF-

kappa B expression in T cells in humans is essential for the maintenance of the

cytokine profile required for pregnancy success. J Immunol 172:4583-4591.

95

Meyer U, Engler A, Weber L, Schedlowski M, Feldon J (2008) Preliminary evidence for a

modulation of fetal dopaminergic development by maternal immune activation

during pregnancy. Neuroscience 154:701-709.

Meyer U, Yee BK, Feldon J (2007) The neurodevelopmental impact of prenatal

infections at different times of pregnancy: the earlier the worse? The

Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry

13:241-256.

Mo Z, Zecevic N (2008) Is Pax6 critical for neurogenesis in the human fetal brain? Cereb

Cortex 18:1455-1465.

Monji A, Kato TA, Mizoguchi Y, Horikawa H, Seki Y, Kasai M, Yamauchi Y, Yamada S,

Kanba S (2011) Neuroinflammation in schizophrenia especially focused on the

role of microglia. Progress in neuro-psychopharmacology & biological psychiatry.

Mori T, Miyamoto T, Yoshida H, Asakawa M, Kawasumi M, Kobayashi T, Morioka H,

Chiba K, Toyama Y, Yoshimura A (2011) IL-1beta and TNFalpha-initiated IL-6-

STAT3 pathway is critical in mediating inflammatory cytokines and RANKL

expression in inflammatory arthritis. Int Immunol 23:701-712.

Morrison PJ, Ballantyne SJ, Kullberg MC (2011) Interleukin-23 and T helper 17-type

responses in intestinal inflammation: from cytokines to T-cell plasticity.

Immunology 133:397-408.

Norton MT, Fortner KA, Oppenheimer KH, Bonney EA (2010) Evidence that CD8 T-cell

96

homeostasis and function remain intact during murine pregnancy. Immunology

131:426-437.

Pekkanen-Mattila M, Pelto-Huikko M, Kujala V, Suuronen R, Skottman H, Aalto-Setala

K, Kerkela E (2010) Spatial and temporal expression pattern of germ layer

markers during human embryonic stem cell differentiation in embryoid bodies.

Histochem Cell Biol 133:595-606.

Petroff MG (2005) Immune interactions at the maternal-fetal interface. J Reprod

Immunol 68:1-13.

Piccinni MP (2002) T-cell cytokines in pregnancy. Am J Reprod Immunol 47:289-294.

Piccinni MP (2005) T cells in pregnancy. Chemical immunology and allergy 89:3-9.

Piccinni MP (2007) T cells in normal pregnancy and recurrent pregnancy loss. Reprod

Biomed Online 14 Spec No 1:95-99.

Piccinni MP (2010) T cell tolerance towards the fetal allograft. J Reprod Immunol 85:71-

75.

Piccinni MP, Maggi E, Romagnani S (2000) Role of hormone-controlled T-cell cytokines

in the maintenance of pregnancy. Biochem Soc Trans 28:212-215.

Piccinni MP, Romagnani S (1996) Regulation of fetal allograft survival by a hormone-

controlled Th1- and Th2-type cytokines. Immunol Res 15:141-150.

97

Poole JA, Claman HN (2004) Immunology of pregnancy. Implications for the mother.

Clin Rev Allergy Immunol 26:161-170.

Pousset F (1994a) Cytokines as mediators in the central nervous system. Biomed

Pharmacother 48:425-431.

Pousset F (1994b) Developmental expression of cytokine genes in the cortex and

hippocampus of the rat central nervous system. Brain research 81:143-146.

Radyushkin K, Hammerschmidt K, Boretius S, Varoqueaux F, El-Kordi A, Ronnenberg

A, Winter D, Frahm J, Fischer J, Brose N, Ehrenreich H (2009) Neuroligin-3

deficient mice: Model of a monogenic heritable form of autism with an olfactory

deficit. Genes, brain, and behavior.

Raghupathy R, Al-Azemi M, Azizieh F (2012) Intrauterine growth restriction: cytokine

profiles of trophoblast antigen-stimulated maternal lymphocytes. Clinical &

developmental immunology 2012:734865.

Rezai-Zadeh K, Ehrhart J, Bai Y, Sanberg PR, Bickford P, Tan J, Shytle RD (2008)

Apigenin and luteolin modulate microglial activation via inhibition of STAT1-

induced CD40 expression. J Neuroinflammation 5:41.

Robertson SA, Christiaens I, Dorian CL, Zaragoza DB, Care AS, Banks AM, Olson DM

(2010) Interleukin-6 is an essential determinant of on-time parturition in the

mouse. Endocrinology 151:3996-4006.

98

Romero R, Chaiworapongsa T, Espinoza J (2003) Micronutrients and intrauterine

infection, preterm birth and the fetal inflammatory response syndrome. The

Journal of nutrition 133:1668S-1673S.

Romero R, Maymon E, Pacora P, Gomez R, Mazor M, Yoon BH, Berry SM (2000)

Further observations on the fetal inflammatory response syndrome: a potential

homeostatic role for the soluble receptors of tumor necrosis factor alpha. Am J

Obstet Gynecol 183:1070-1077.

Rothwell NJ, Luheshi G, Toulmond S (1996) Cytokines and their receptors in the central

nervous system: physiology, pharmacology, and pathology. Pharmacol Ther

69:85-95.

Saini V, Arora S, Yadav A, Bhattacharjee J (2011) Cytokines in recurrent pregnancy

loss. Clin Chim Acta 412:702-708.

Saito S, Nakashima A, Shima T, Ito M (2010) Th1/Th2/Th17 and regulatory T-cell

paradigm in pregnancy. Am J Reprod Immunol 63:601-610.

Sansom SN, Griffiths DS, Faedo A, Kleinjan DJ, Ruan Y, Smith J, van Heyningen V,

Rubenstein JL, Livesey FJ (2009) The level of the transcription factor Pax6 is

essential for controlling the balance between neural stem cell self-renewal and

neurogenesis. PLoS Genet 5:e1000511.

Sauvageot CM, Stiles CD (2002) Molecular mechanisms controlling cortical gliogenesis.

Current opinion in neurobiology 12:244-249.

99

Schluns KS, Lefrancois L (2003) Cytokine control of memory T-cell development and

survival. Nature reviews Immunology 3:269-279.

Schoeters GE, Den Hond E, Koppen G, Smolders R, Bloemen K, De Boever P, Govarts

E (2011) Biomonitoring and biomarkers to unravel the risks from prenatal

environmental exposures for later health outcomes. The American journal of

clinical nutrition 94:1964S-1969S.

Seelinger G, Merfort I, Wolfle U, Schempp CM (2008) Anti-carcinogenic effects of the

flavonoid luteolin. Molecules 13:2628-2651.

Shi L, Fatemi SH, Sidwell RW, Patterson PH (2003) Maternal influenza infection causes

marked behavioral and pharmacological changes in the offspring. J Neurosci

23:297-302.

Shima T, Sasaki Y, Itoh M, Nakashima A, Ishii N, Sugamura K, Saito S (2010)

Regulatory T cells are necessary for implantation and maintenance of early

pregnancy but not late pregnancy in allogeneic mice. J Reprod Immunol 85:121-

129.

Sicca F, Imbrici P, D'Adamo MC, Moro F, Bonatti F, Brovedani P, Grottesi A, Guerrini R,

Masi G, Santorelli FM, Pessia M (2011) Autism with seizures and intellectual

disability: possible causative role of gain-of-function of the inwardly-rectifying K+

channel Kir4.1. Neurobiol Dis 43:239-247.

Siddiquee K, Zhang S, Guida WC, Blaskovich MA, Greedy B, Lawrence HR, Yip ML,

100

Jove R, McLaughlin MM, Lawrence NJ, Sebti SM, Turkson J (2007) Selective

chemical probe inhibitor of Stat3, identified through structure-based virtual

screening, induces antitumor activity. Proceedings of the National Academy of

Sciences of the United States of America 104:7391-7396.

Silbergeld EK, Patrick TE (2005) Environmental exposures, toxicologic mechanisms,

and adverse pregnancy outcomes. Am J Obstet Gynecol 192:S11-21.

Smith SE, Li J, Garbett K, Mirnics K, Patterson PH (2007) Maternal immune activation

alters fetal brain development through interleukin-6. J Neurosci 27:10695-10702.

Somerset DA, Zheng Y, Kilby MD, Sansom DM, Drayson MT (2004) Normal human

pregnancy is associated with an elevation in the immune suppressive CD25+

CD4+ regulatory T-cell subset. Immunology 112:38-43.

Suter DM, Tirefort D, Julien S, Krause KH (2009) A Sox1 to Pax6 switch drives

neuroectoderm to radial glia progression during differentiation of mouse

embryonic stem cells. Stem Cells 27:49-58.

Sutton P, Giudice LC, Woodruff TJ (2010) Reproductive environmental health. Current

opinion in obstetrics & gynecology 22:517-524.

Sweeney SE (2011) Hematopoietic stem cell transplant for systemic lupus

erythematosus: Interferon regulatory factor 7 activation correlates with the IFN

signature and recurrent disease. Lupus 20:975-980.

101

Tetreault NA, Williams BA, Hasenstaub A, Hakeem AY, Liu M, Abelin ACT, Wold BJ,

Allman JM (2009) RNA-Seq studies of gene expression in fronto-insular (FI)

cortex in autistic and control subjects reveal gene networks related to

inflammation and synaptic function. In: Society for Neuroscience Chicago, Illinois.

Tosun M, Celik H, Avci B, Yavuz E, Alper T, Malatyalioglu E (2010) Maternal and

umbilical serum levels of interleukin-6, interleukin-8, and tumor necrosis factor-

alpha in normal pregnancies and in pregnancies complicated by preeclampsia. J

Matern Fetal Neonatal Med 23:880-886.

Tower C, Crocker I, Chirico D, Baker P, Bruce I (2011) SLE and pregnancy: the potential

role for regulatory T cells. Nature reviews Rheumatology 7:124-128.

van Zoelen EJ, Twardzik DR, van Oostwaard TM, van der Saag PT, de Laat SW, Todaro

GJ (1984) Neuroblastoma cells produce transforming growth factors during

exponential growth in a defined hormone-free medium. Proceedings of the

National Academy of Sciences of the United States of America 81:4085-4089.

Vargas DL, Nascimbene C, Krishnan C, Zimmerman AW, Pardo CA (2005) Neuroglial

activation and neuroinflammation in the brain of patients with autism. Ann Neurol

57:67-81.

Virkud YV, Todd RD, Abbacchi AM, Zhang Y, Constantino JN (2009) Familial

aggregation of quantitative autistic traits in multiplex versus simplex autism. Am J

Med Genet B Neuropsychiatr Genet 150B:328-334.

102

Vitoratos N, Economou E, Iavazzo C, Panoulis K, Creatsas G (2010) Maternal serum

levels of TNF-alpha and IL-6 long after delivery in preeclamptic and

normotensive pregnant women. Mediators Inflamm 2010:908649.

Wang Y, Lewis DF, Gu Y, Zhao S, Groome LJ (2011) Elevated maternal soluble Gp130

and IL-6 levels and reduced Gp130 and SOCS-3 expressions in women

complicated with preeclampsia. Hypertension 57:336-342.

Warren N, Caric D, Pratt T, Clausen JA, Asavaritikrai P, Mason JO, Hill RE, Price DJ

(1999) The transcription factor, Pax6, is required for cell proliferation and

differentiation in the developing cerebral cortex. Cereb Cortex 9:627-635.

Wen Z, Zhong Z, Darnell JE, Jr. (1995) Maximal activation of transcription by Stat1 and

Stat3 requires both tyrosine and serine phosphorylation. Cell 82:241-250.

Wigle DT, Arbuckle TE, Walker M, Wade MG, Liu S, Krewski D (2007) Environmental

hazards: evidence for effects on child health. Journal of toxicology and

environmental health Part B, Critical reviews 10:3-39.

Winter C, Reutiman TJ, Folsom TD, Sohr R, Wolf RJ, Juckel G, Fatemi SH (2008)

Dopamine and serotonin levels following prenatal viral infection in mouse--

implications for psychiatric disorders such as schizophrenia and autism. Eur

Neuropsychopharmacol 18:712-716.

Zenclussen AC (2006a) A novel mouse model for preeclampsia by transferring activated

th1 cells into normal pregnant mice. Methods Mol Med 122:401-412.

103

Zenclussen AC (2006b) Regulatory T cells in pregnancy. Springer Semin Immunopathol

28:31-39.

Zenclussen AC, Gerlof K, Zenclussen ML, Ritschel S, Zambon Bertoja A, Fest S, Hontsu

S, Ueha S, Matsushima K, Leber J, Volk HD (2006) Regulatory T cells induce a

privileged tolerant microenvironment at the fetal-maternal interface. Eur J

Immunol 36:82-94.

Zhang X, Blenis J, Li HC, Schindler C, Chen-Kiang S (1995) Requirement of serine

phosphorylation for formation of STAT-promoter complexes. Science (New York,

NY 267:1990-1994.

Zhao B, Schwartz JP (1998) Involvement of cytokines in normal CNS development and

neurological diseases: recent progress and perspectives. Journal of

neuroscience research 52:7-16.

Zhou CC, Irani RA, Dai Y, Blackwell SC, Hicks MJ, Ramin SM, Kellems RE, Xia Y

(2011) Autoantibody-mediated IL-6-dependent endothelin-1 elevation underlies

pathogenesis in a mouse model of preeclampsia. J Immunol 186:6024-6034.

Zhu J, Paul WE (2010) Peripheral CD4+ T-cell differentiation regulated by networks of

cytokines and transcription factors. Immunological reviews 238:247-262.

Zhu LH, Bi W, Qi RB, Wang HD, Wang ZG, Zeng Q, Zhao YR, Lu DX (2011) Luteolin

reduces primary hippocampal neurons death induced by neuroinflammation.

Neurol Res 33:927-934.

104

Zuckerman L, Rehavi M, Nachman R, Weiner I (2003) Immune activation during

pregnancy in rats leads to a postpubertal emergence of disrupted latent

inhibition, dopaminergic hyperfunction, and altered limbic morphology in the

offspring: a novel neurodevelopmental model of schizophrenia.

Neuropsychopharmacology 28:1778-1789.

Zwaigenbaum L, Bryson S, Rogers T, Roberts W, Brian J, Szatmari P (2005) Behavioral

manifestations of autism in the first year of life. Int J Dev Neurosci 23:143-152.

Zwirner NW, Domaica CI (2010) Cytokine regulation of natural killer cell effector

functions. Biofactors 36:274-288.

105

APPENDIX 1

PUBLICATION CONTRIBUTING TO DISSERTATION

Parker-Athill, E., D. Luo, et al. (2009). "Flavonoids, a prenatal prophylaxis via targeting

JAK2/STAT3 signaling to oppose IL-6/MIA associated autism." J Neuroimmunol

217(1-2): 20-27. [Figures 3.1-3.2, 4.5-4.9]

106

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