Mucosal Immunity Part 2

Sarah L. GaffenSpring, 2009

sig65@pitt.edu

Mucosal Immunity & Infections

• Balancing act– Tolerate commensals, food antigens– Protect against infectious agents

• Gut is most frequent site of infection against pathogenic bugs

• Emerging themes: Th17 cells in the mucosa

Mucosal pathogens exploit mucosal surfaces

• Enteric pathogens often enter via M cells– Salmonella – typhoid, food poisoning– Shigellae – dysentery– Yersiniae – plague

Oral Tolerance

• Oral tolerance is – A general immunosuppressive state in the oral

mucosa to prevent reaction to harmless Ags such as commensals or foods

– the generation of systemic immune unresponsiveness by feeding of antigen

• Necessary to prevent excessive response to normal flora and food antigens

A modern version of the same experiment:

o Mice are given naïve CD4+ T cells that transgenically express TCR specific for OVA peptide (OT-II mice)

o Mice are fed ovalbumin

o After tolerance induction, OVA-T cells are anergized - they do not proliferateto OVA/adjuvant in vitro.

o Smaller numbers of OVA-specific T cells suggest clonal deletion

Experiment to demonstrate Oral Tolerance

Limitations to Oral Tolerance

• Can be overcome with mucosal adjuvant (e.g. Cholera toxin)

• Alter physical characteristics of antigen: antigen in micro-spheres that target PP

• Feeding of attenuated enteric pathogen expressing the antigen (Salmonella)

Commensals• They outnumber us at least 10-100:1 (“you are only

1-10% human”)• Weight ~1 kg• Normal commensal flora maintain health

– Help metabolize cellulose, etc.– Fill niches to prevent pathogenic bacteria from colonizing– Required for immune development: germ-free rodents

have reduced lymphoid organs, low Ig, reduced immune responses of all types

– Help maintain tolerance

Commensals are immunosuppressive• Different microbiota in disease vs health• Intestinal epithelial cells play role in regulating response to

commensals– IECs deficient in SIGGIR (negative regulator of TLRs) show

increased cytokines, increased susceptibility to intestinal inflammation

• Immune status of host also influences makeup of commensal flora, thereby affecting immune system function– IgA-deficient mice have different microbial spp– Tbet KO/scid mice have increased “colitogenic” bacteria and UC

symptoms

Specific commensals determine inflammation vs. suppression

• Differentiation of lamina propria T cells into Treg vs Th17 cells is determined by flora– Bacterial DNA signals through TLR9 to suppress

Treg conversion, acts as a “natural adjuvant” (Hall, Immunity 2008)

– Specific types of flora promote Th17 vs Treg development in the intestine (Ivanov, Cell Host & Microbe, 2008)

Commensals & Pathogens influence NF-kB

Artis, Nat Rev Immunol 2008

Examples of mucosal diseases- role for Th17 cells

• Gut: IBD – autoimmune diseases of the gut• Vaginal mucosa:Th17 cells are protective against

Neisseria gonnorrheae• Lung: Th17 cells are protective against various

pneumoniaes, TB, etc., promote airway hyperreactivity/allergy

• Mouth: Th17 cells promote Sjogren’s (autoimmune disease of salivary gland, tear ducts), but protective in oropharyngeal candidiasis (“thrush”)

The Spectrum of Inflammatory Bowel Disease

Ulcerative colitis Crohn’s disease

IBD: Evidence of Genetic Influence

• Racial differences in incidence:White > Black > Asian

• Ethnic influences:Jewish > non-Jewish; Ashkanazi > Sephardic

• Twin studies: Monozygotic > dizygotic

• Association with specific HLA antigens

• Genes

– NOD2, IL-23RSartor RB. Inflammatory Bowel Dis. 1995;24:475.

NOD2• First identified Crohn’s disease

susceptibility gene.• Chromosome 16q12.• One frameshift mutation (Europe and USA)

and 2 point mutations (Europe) identified.• Allele frequency 8.2% in CD, 4.0% in

controls, 3.2% in UC.

Ougura et al; Nature 2001; 411: 603Hugot et al; Nature 2001; 411: 599

IL23R

ATG16L1

3p21

5p13

IBD5

IRGM 10q21 NKX2-3

NOD2

PTPN2MHC

SBNO2

5q33

Innate Mucosal defenses- Pattern Recognition Receptors

• TLRs – Toll-like receptors– Bind LPS, flagellin, bacterial (CpG) DNA, dsRNA, etc.– “extracellular” pattern recognition

• NLRs – NOD-like receptors– Bind muramyl di- and tri-peptides (from LPS)– “intracellular” pattern recognition

• C-type lectin receptors– Dectins– Clec’s

Kawai and Akira (2006) Cell Death and Differentiation

NODs complement function of TLRs

• NODs (nucleotide-binding oligomerization domain) recognize microbial components found in cytosol

• NODS are intracellular pattern recognition receptors• NODs bind peptidoglycan in bacterial cell walls• NODs lead to activation of Caspase-1, cleavage of inactive

forms of IL-1 (and other related cytokines)• NODs can also downregulate immune responses• Mutations in NOD genes associated with Crohn’s disease,

inflammatory bowel disease, asthma

NLRs

“Loss of Function” Mutations in NOD2

• How could this lead to IBD?– Bacterial products from commensal flora deliver

anti-inflammatory signals through NOD2– Intracellular defect in LPS signaling (NOD2) may

lead to increased extracellular signaling (TLRs)– Decreased apoptosis of bacterially activated cells– Defective killing of bacteria leading to persistent

immune response– Maeda et al, Science 2005

Local Environmental Factors in IBD

• Germ-free-derived colitis-prone animals do not develop IBD unless commensal enteric bacteria are re-introduced

• Restriction of bacterial colonization reduces the incidence of IBD

• Antibiotic administration reduces the incidence of IBD

• Enteric bacterial antigens activate T cells and induce colitis

• A selected subset of bacterial proteins triggers immune reactions

• No specific bacterial or viral infection has been identified

Cytokines in IBD

• Spontaneous IBD models– IL-10 -/-– IL-10R -/-– TGF -/-– IL-2 -/-– Transgenic TNF– IL-12p40-KO mice are resistant

• Long interpreted to mean that Th1 cells mediate pathology in IBD, CD

- anti-IL-12p40 used in humans to treat CD!

Chronic Inflammation: Imbalance Between Mediators

Pro-inflammatoryPro-inflammatory

Anti-inflammatory

Anti-inflammatory

TNF-TNF-IL-1IL-1

IL-17IL-17IL-12/23IL-12/23

IFN-IFN-TGF-TGF-

IL-10IL-10

IL-1raIL-1raIL-4/IL-13IL-4/IL-13

IL-10• Immunosuppressive cytokine

– Inhibits DC maturation– Inhibits TNF production by macrophages– Inhibits IL-12 production– Limits “collateral damage” in infection

• IL-10-KO mice have severe, spontaneous IBD• Produced by all Th subsets (not just Th2)• IL-23 inhibits IL-10 production in Th17 cells• O’Garra and Vieira, Nat Rev Immunol, June 2007

Cytokines in IBD

• Spontaneous IBD models– IL-10 -/-– IL-10R -/-– TGF -/-– IL-2 -/-– Transgenic TNF– IL-12p40-KO mice are resistant

• Long interpreted to mean that Th1 cells mediate pathology in IBD, CD

- anti-IL-12p40 used in humans to treat CD!

IL-17-producing T cellsTh1- IFN (Cell-mediated immunity)

IL-12p40/p35

Th2- IL-4, IL-5, IL-13 (Humoral immunity, allergy)

IL-4Thp

IL-2,TGF

Treg- IL-10, TGF(Immune Suppression)

Th17- IL-17, IL-17F, IL-21, IL-22, IL-26 (Inflammation, autoimmunity)

TGF+ IL-6, IL-1, IL-23 (p40/p19)

IL-23R

IL-12 family of cytokines

IL-23 rather than IL-12 per se in IBD

J. Clin. Invest. David Yen, et al. 116:1310 doi:10.1172/JCI21404

• IL-10KO– Strong disease

• IL-10KO x IL-12p35KO– Still get disease

• IL-10KO x IL-23p19KO– Protected from disease

IL23R

ATG16L1

3p21

5p13

IBD5

IRGM 10q21 NKX2-3

NOD2

PTPN2MHC

SBNO2

5q33

• Lactobacillus prevents colitis in mouse models of IBD; can inhibit NF-kB and hence inflammation

• E. coli as effective as mesalazine in remission maintenance in UC.

• Enteric helminths are anti-inflammatory; generate strong Th2 response (inhibits Th17?) (“eat worms!”)

• Epidemiologic data incidence of IBD increases with improved sanitation = “hygiene hypothesis” (more Th1 responses, inhibits Th17?)

Probiotics and IBD

IL-17-producing T cellsTh1- IFN (Cell-mediated immunity)

IL-12p40/p35

Th2- IL-4, IL-5, IL-13 (Humoral immunity, allergy)

IL-4Thp

IL-2,TGF

Treg- IL-10, TGF(Immune Suppression)

Th17- IL-17, IL-17F, IL-21, IL-22, IL-26 (Inflammation, autoimmunity)

TGF+ IL-6, IL-1, IL-23 (p40/p19)

IL-23R

Examples of mucosal diseases- role for Th17 cells

• Gut: IBD – autoimmune diseases of the gut• Vaginal mucosa:Th17 cells are protective against

Neisseria gonnorrheae• Lung: Th17 cells are protective against various

pneumoniaes, TB, etc., promote airway hyperreactivity/allergy

• Mouth: Th17 cells promote Sjogren’s (autoimmune disease of salivary gland, tear ducts), but protective in oropharyngeal candidiasis (“thrush”)

Immunity in the oral mucosa:Lick your wounds

Candida albicans

(Berman, 2002)

•Common fungal commensal of the human oral cavity

•Pathogenic when host immune response compromised

•HIV+•Cancer-radiation and chemotherapy•HIES (hyper-IgE syndrome)•Infants and elderly

•Progression from commensal state to pathogen is poorly understood

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CD4+ T cells

Immune Components:CD4+ T cells, neutrophils,

monocytes, macrophages, dendritic cells, T cells, mucosal epithelial cells, antimicrobial peptides,

cytokines

Systemic

Oropharyngeal candidiasis“OPC”

Vaginal

Mucocutaneous

CD4+ T cell are vital for protection against OPC

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IL-12

Th1

Thp

IL-23

IFN-

IL-17IL-17FIL-21IL-22

Cell-mediated Immunity

Intracellular bacteria,

protozoa, viruses

Acute InflammationAutoimmunityExtracellular

microbes

CD4+

CD4+

CD4+

Th2

CD4+IL-4

IL-4,5,13Humoral Immunity

Helminthes

Which CD4+ Th cell subset is involved in immunity to OPC?

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p40

p35

Th1 vs. Th17

p40

p19

IL-12Th1

IL-23Th17

vs.

IL-12p35KO mice IL-23p19KO mice

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Immunity to OPC• Long considered to be Th1-dependent

– IL-12p40KO (Th1-deficient) mice are susceptible– IL-4KO (Th2-deficient) mice are resistant

• BUT…– IFNKO mice (Th1-deficient) are resistant– IL-12p40 is part of IL-23 (therefore, Th1 and Th17-deficient)

• AND …– IL-17RAKO mice are susceptible to disseminated candidiasis– IL-17 and IL-23 drive pathogenesis in gastric candidiasis

• SO… What is the role of Th17 cells in OPC?

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Experimental Mouse Model of OPC

Day: -1 0 1 2 3 4 5 6 7

Cortisone Cortisone Cortisone

Infection

Monitor weight, appearance, activity level, etc.

Sac micecollect blood,LN, spleen,

tongue

Tongue-YPDAgar plates

Colony Count

Mean: 3 6.4X105 47

Th17-deficient mice are more susceptible to OPC than Th1-deficient mice

Conclusion:Th17 cells, not Th1 cells, are essential for mucosal host

defense against oral candidiasis

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Th17-deficient mice are more susceptible to OPC than Th1-deficient mice

IL-23KO + CandidaIL-12KO + Candida

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WT-No Cort, Sham

Th17 cells control PMNs, anti-microbial defenses

(Kolls et al, Nature Reviews Immunology Nov 2008) 50

Host defense against oral C. albicans is mediated mainly by -T cells, rather than -T cells

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Affymetrix GeneChip Mouse Genome 430 2.0 Array analysis of tongue tissue

Comparisons:WT Day 0 vs. WT Day 1IL-17RAKO Day 0 vs. IL-17RAKO Day 1WT Day 0 vs. IL-17RAKO Day 0WT Day 1 vs. IL-17RAKO Day 1

Day: -1 0 1 2 3 4 5 6 7

Cortisone Cortisone Cortisone

Infection

Monitor weight, appearance, activity level

Sac micecollect blood,LN, spleen, tongue, etc.

Tongue-YPDAgar plates

Colony Count

Microarray Microarray

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IL-17 induces expression of antimicrobial proteins

-defensin 3 (BD3, DEFB3)• small inducible cationic protein expressed by salivary gland and

epithelial cells• Interacts with CCR6• Exerts potent candidacidal activity in vitro• Induced by IL-17 and IL-22 in the gut and lungs

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Anti-microbial peptides

• Small, cationic peptides (6-60 a.a.) typically found in mucosal surfaces and skin, can directly kill microbes

• Found in frogs, flies, humans, plants, etc.• Regulated by TLRs, NODs, inflammation• Often work by disrupting target membranes,

mechanisms not full known

IL-17RAKO mice show decreased expression of mBD3

(Experiment performed in collaboration with Jianing Sun)56

IL-23 and IL-17, not IL-12, regulate the antimicrobial activity of saliva

(Experiment performed in collaboration with Namrata Nayyar)

+ carbachol

+

Add Candida (104 cells)

Incubate 1 hour, 37°C

Plate 500 cells on YPD agar

Count colonies 48 hours later 57

Hyper-IgE syndromeHIES or ‘Job’s syndrome’

•Rare multi-system disorder (incidence <1/106) with immunologic and non-immunologic features:

•Extremely elevated serum IgE

•Staphylococcal abscesses

•Mucocutaneous and oral candidiasis

•Bone and connective tissue abnormalities

•Characteristic facial features (prominent forehead, fleshy nasal tip)

•Pathological fractures

•Retention of primary teeth

•Lesions of hard palate and dorsal tongue 58

Defective response to IL-6, IL-10, IL-2159

Th1

ThpTh2

STAT1,4T-bet

STAT6GATA3

RORtSTAT3

Th cell subset defining transcription factors

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Th17 cells/IL-17 receptor signaling and not Th1 cells are essential for host defense against OPC

Oral CavityAdapted from O’quinn et al. Advances in Immunonolgy Vol 99, 2008

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