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Mucosal Immunity Part 2 Sarah L. Gaffen Spring, 2009 [email protected] 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 AExperiment modern version of the to demonstrate Oral Tolerance 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 proliferate to OVA/adjuvant in vitro. o Smaller numbers of OVA-specific T cells suggest clonal deletion 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-23R Sartor 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: 603 Hugot et al; Nature 2001; 411: 599 IL23R NOD2 ATG16L1 5p13 IRGM IBD5 NKX2-3 5q33 MHC 3p21 10q21 PTPN2 SBNO2 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 -/– 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! Chronic Inflammation: Imbalance Between Mediators IFN-g IL-12/23 IL-17 IL-1b TNF-a IL-10 TGF-b IL-1ra IL-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 -/– 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! IL-17-producing T cells Th1- IFNg (Cell-mediated immunity) IL-12 p40/p35 Thp IL-2, TGFb IL-4 Th2- IL-4, IL-5, IL-13 (Humoral immunity, allergy) TGFb + IL-6, IL-1, IL-23 (p40/p19) Th17- IL-17, IL-17F, IL-21, IL-22, IL26 (Inflammation, autoimmunity) IL-23R Treg- IL-10, TGFb (Immune Suppression) IL-12 family of cytokines 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 IL23R NOD2 ATG16L1 5p13 IRGM IBD5 NKX2-3 5q33 MHC 3p21 10q21 PTPN2 SBNO2 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?) IL-17-producing T cells Th1- IFNg (Cell-mediated immunity) IL-12 p40/p35 Thp IL-2, TGFb IL-4 Th2- IL-4, IL-5, IL-13 (Humoral immunity, allergy) TGFb + IL-6, IL-1, IL-23 (p40/p19) Th17- IL-17, IL-17F, IL-21, IL-22, IL26 (Inflammation, autoimmunity) IL-23R Treg- IL-10, TGFb (Immune Suppression) 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 •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) 42 CD4+ T cell are vital for protection against OPC Mucocutaneous Systemic Immune Components: CD4+ T cells, neutrophils, monocytes, macrophages, dendritic cells, gd T cells, mucosal epithelial cells, antimicrobial peptides, cytokines Vaginal CD4+ T cells Oropharyngeal candidiasis “OPC” 43 Which CD4+ Th cell subset is involved in immunity to OPC? Th1 CD4+ IFN-g Cell-mediated Immunity Intracellular bacteria, protozoa, viruses IL-12 Thp Th2 IL-4 CD4+ IL-23 CD4+ Th17 CD4+ IL-4,5,13 Humoral Immunity Helminthes IL-17 IL-17F IL-21 IL-22 Acute Inflammation Autoimmunity Extracellular microbes 44 Th1 vs. Th17 p40 p40 p35 IL-12 Th1 IL-12p35KO mice p19 vs. IL-23 Th17 IL-23p19KO mice 45 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 Th17deficient) • 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? 46 Experimental Mouse Model of OPC Monitor weight, appearance, activity level, etc. Sac mice collect blood, LN, spleen, tongue Infection Cortisone Day: -1 Cortisone 0 1 Tongue-YPD Agar plates Cortisone 2 3 4 Mean: 5 3 Colony Count 6 6.4X105 7 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 48 Th17-deficient mice are more susceptible to OPC than Th1-deficient mice IL-12KO + Candida IL-23KO + Candida WT-No Cort, Sham 49 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 ab-T cells, rather than gd-T cells 51 Affymetrix GeneChip Mouse Genome 430 2.0 Array analysis of tongue tissue Monitor weight, appearance, activity level Sac mice collect blood, LN, spleen, tongue, etc. Infection Cortisone Day: -1 Cortisone 0 1 Tongue-YPD Agar plates Cortisone 2 3 4 5 Colony Count 6 7 Microarray Microarray Comparisons: WT Day 0 vs. WT Day 1 IL-17RAKO Day 0 vs. IL-17RAKO Day 1 WT Day 0 vs. IL-17RAKO Day 0 WT Day 1 vs. IL-17RAKO Day 1 52 IL-17 induces expression of antimicrobial proteins b-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 53 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 KO IL-17RA mice show decreased expression of mBD3 56 (Experiment performed in collaboration with Jianing Sun) IL-23 and IL-17, not IL-12, regulate the antimicrobial activity of saliva + carbachol + Add Candida (104 cells) Incubate 1 hour, 37°C Plate 500 cells on YPD agar Count colonies 48 hours later 57 (Experiment performed in collaboration with Namrata Nayyar) Hyper-IgE syndrome HIES or ‘Job’s syndrome’ •Rare multi-system disorder (incidence <1/106) with immunologic and nonimmunologic 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-21 59 Th cell subset defining transcription factors Th1 STAT1,4 T-bet Th2 Thp STAT6 GATA3 Th17 RORgt STAT3 60 Th17 cells/IL-17 receptor signaling and not Th1 cells are essential for host defense against OPC Adapted from O’quinn et al. Advances in Immunonolgy Vol 99, 2008 Oral Cavity 62