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Mucosal Immunity Part 2

Mucosal Immunity Part 2. Sarah L. Gaffen Spring, 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.

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Mucosal Immunity Part 2

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  1. Mucosal Immunity Part 2 Sarah L. Gaffen Spring, 2009 sig65@pitt.edu

  2. 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

  3. Mucosal pathogens exploit mucosal surfaces • Enteric pathogens often enter via M cells • Salmonella – typhoid, food poisoning • Shigellae – dysentery • Yersiniae – plague

  4. 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

  5. A modern version of the same experiment: Experiment to demonstrate Oral Tolerance • Mice are given naïve CD4+ T cells that transgenically express TCR specific for OVA peptide (OT-II mice) • Mice are fed ovalbumin • After tolerance induction, OVA-T cells are anergized - they do not proliferateto OVA/adjuvant in vitro. • Smaller numbers of OVA-specific T cells suggest clonal deletion

  6. 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)

  7. 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

  8. 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

  9. 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)

  10. Commensals & Pathogens influence NF-kB Artis, Nat Rev Immunol 2008

  11. 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”)

  12. The Spectrum of Inflammatory Bowel Disease Crohn’s disease Ulcerative colitis

  13. 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-23R Sartor RB. Inflammatory Bowel Dis. 1995;24:475.

  14. 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: 603 Hugot et al; Nature 2001; 411: 599

  15. IL23R NOD2 5p13 ATG16L1 IRGM 10q21 NKX2-3 IBD5 5q33 PTPN2 MHC SBNO2 3p21

  16. 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

  17. Kawai and Akira (2006) Cell Death and Differentiation

  18. 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

  19. NLRs

  20. “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

  21. 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

  22. Cytokines in IBD • Spontaneous IBD models • IL-10 -/- • IL-10R -/- • TGFb -/- • 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!

  23. Chronic Inflammation: Imbalance Between Mediators IFN-g IL-10 TGF-b IL-12/23 IL-1ra IL-17 IL-4/IL-13 IL-1b TNF-a Anti-inflammatory Pro-inflammatory

  24. 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

  25. Cytokines in IBD • Spontaneous IBD models • IL-10 -/- • IL-10R -/- • TGFb -/- • 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!

  26. TGFb + IL-6, IL-1, IL-23 (p40/p19) IL-2, TGFb Th17- IL-17, IL-17F, IL-21, IL-22, IL-26 (Inflammation, autoimmunity) Treg- IL-10, TGFb (Immune Suppression) IL-17-producing T cells Th1- IFNg(Cell-mediated immunity) IL-12 p40/p35 Thp IL-4 Th2- IL-4, IL-5, IL-13 (Humoral immunity, allergy) IL-23R

  27. IL-12 family of cytokines

  28. IL-23 rather than IL-12 per se in IBD • IL-10KO • Strong disease • IL-10KO x IL-12p35KO • Still get disease • IL-10KO x IL-23p19KO • Protected from disease J. Clin. Invest. David Yen, et al. 116:1310 doi:10.1172/JCI21404

  29. IL23R NOD2 5p13 ATG16L1 IRGM 10q21 NKX2-3 IBD5 5q33 PTPN2 MHC SBNO2 3p21

  30. Probiotics and IBD • 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?)

  31. TGFb + IL-6, IL-1, IL-23 (p40/p19) IL-2, TGFb Th17- IL-17, IL-17F, IL-21, IL-22, IL-26 (Inflammation, autoimmunity) Treg- IL-10, TGFb (Immune Suppression) IL-17-producing T cells Th1- IFNg(Cell-mediated immunity) IL-12 p40/p35 Thp IL-4 Th2- IL-4, IL-5, IL-13 (Humoral immunity, allergy) IL-23R

  32. 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”)

  33. Immunity in the oral mucosa:Lick your wounds

  34. Candida albicans • 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 (Berman, 2002)

  35. CD4+ T cells Immune Components: CD4+ T cells, neutrophils, monocytes, macrophages, dendritic cells, gd T cells, mucosal epithelial cells, antimicrobial peptides, cytokines Systemic Vaginal Oropharyngeal candidiasis “OPC” CD4+ T cell are vital for protection against OPC Mucocutaneous

  36. Th1 Th17 Th2 Thp Which CD4+ Th cell subset is involved in immunity to OPC? Cell-mediated Immunity Intracellular bacteria, protozoa, viruses IFN- CD4+ IL-12 Humoral Immunity Helminthes IL-4 CD4+ CD4+ IL-4,5,13 IL-23 IL-17 IL-17F IL-21 IL-22 Acute Inflammation Autoimmunity Extracellular microbes CD4+

  37. p40 p40 p19 p35 Th1 vs. Th17 vs. IL-23 Th17 IL-12 Th1 IL-12p35KO mice IL-23p19KO mice

  38. Immunity to OPC • Long considered to be Th1-dependent • IL-12p40KO (Th1-deficient) mice are susceptible • IL-4KO (Th2-deficient)mice are resistant • BUT… • IFNgKO 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?

  39. Monitor weight, appearance, activity level, etc. Sac mice collect blood, LN, spleen, tongue Infection Cortisone Cortisone Cortisone Tongue-YPD Agar plates ColonyCount Day: -1 0 1 2 3 4 5 6 7 Experimental Mouse Model of OPC Mean: 3 6.4X105

  40. 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

  41. Th17-deficient mice are more susceptible to OPC than Th1-deficient mice IL-12KO + Candida IL-23KO + Candida WT-No Cort, Sham

  42. Th17 cells control PMNs, anti-microbial defenses (Kolls et al, Nature Reviews Immunology Nov 2008)

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