Mechanisms of Mucosal Defense

1. Mechanisms of Mucosal Defense Soma Jyonouchi, M.D. January 24, 2008

2. Mucosal Surfaces • The GI mucosal surfaces cover 400 m² • Thin – facilitate nutrient absorption. • The Gut Associated Lymphoid Tissue (GALT) - Organized T and B cell areas - where antigen is collected and adaptive immune response is generated. - Tonsils, Peyer’s patches, appendix, solitary lymphoid follicles in the large intestine and rectum.

3. GALT Architecture Lamina Propria ** Dome structures extend into the lumen of the intestine.

4. Lamina Propria – effector site Inductive Site Effector Site

5. Enormous Antigen Load • Systemic Immune System – largely sterile environment. Vigorous response to microbial invasion. • Mucosal Immune System – Constant exposure to foreign matter • Human gut is exposed to an enormous amount commensal microorganisms (1 x 10 14) • Constant exposure to food matter

6. Innate Defense – I. Barrier Fxn 1. Glycocalyx – Goblet cells produce mucous to create a thick barrier that covers the GI epithelium and prevents easy access. - Pathogens become trapped in the mucous and are expelled via peristalsis. - Mucous also acts as a reservoir for secretory IgA.

7. I. Barrier Fxn Epithelial Cell Tight Junctions - prevent the passages of macromolecules. ** Zonulin – Homology to Vibrio cholera toxin. upregulated during the acute phase of celiac disease. - Induces tight junction disassembly and increased intestinal permeability. Drago et al. Scand J Gastroenterol. 2006 Fasano et al. Lancet 2000

8. II. Proteolytic Enzymes • Enzymes in the stomach (pepsin) and small bowel (trypsin, chymotrypsin, pancreatic proteases). • Break down large polypeptides into di-peptides and tri-peptides. • Peptides < 8-10 aa are poor immunogens. • Enzymes cytotoxic to pathogens.

9. III. Antimicrobial Molecules • 1. Lactoferrin – binds iron and inhibits bacterial growth. 2. Lysozyme – cleaves cell wall of gram positive bacteria. 3. Defensins – 30-40 aa peptides that disrupts the cell memebranes of bacteria and fungi causing lysis.

10. IV. Commensal Organisms • >400 species of commensal bacteria • Provide enzymatic breakdown of food • Competes with pathogenic bacteria for space and nutrients • Prevents colonization of the gut • Antibiotics disrupt homeostasis

11. IV. Commensal Organisms • Germ free mice – no commensal microflora. - Pups delivered by C-section and raised in sterile conditions. • Hypoplastic peyer’s patches with scant germinal centers. - decreased IgA plasma cells - decreased lamina propria CD4+ cells - Abnormalities reversed by placing non-germ free mice in same cage.

12. Mucosal Immune System: Adaptive Response

13. “Common Mucosal Immune System” *Antigen Presentation* Peyer’s Patch Mesenteric Lymph Node Thoracic Duct Blood Stream Resp Tract Intestinal Mucosa Breast GU Tract Salivary/Lacrimal Gland

14. Common Mucosal System? IgA response for different routes of vaccination Holmgren et al. Nature Medicine. 2005

15. GALT vs peripheral Lymphoid tissue • 1)Unique epithelium for antigen uptake • 2) Unique lymphocyte repertoire • 3) IgA dominated humoral response • 4) A need to minimize injury to the mucosal tissue while providing protection.

16. GALT – Unique Epithelium • The epithelium overlying the peyer’s patches is composed of cells that differ from the surrounding enterocytes.

17. M-Cells (microfold cells) • M-cells lack microvilli • No glycocalyx coating • Designed to to interact directly with antigens in the gut – portal of entry into GALT. – some pathogens gain entry via M-cells (salmonella, shigella)

18. M-Cells (microfold cells) • Basolateral aspects are invaginated. • They contain T-cells, B-cells, Dendritic cells, and Macrophages. • Antigens from the lumen are taken up by endocytosis and presented directly to APCs • APCs migrate to germinal center Germinal Center

19. GALT vs peripheral Lymphoid tissue • 1) Unique epithelium for antigen uptake • 2) Unique Lymphocyte Repertoire • 3) IgA dominated humoral response • 4) A need to minimize injury to the mucosal tissue as well as development of tolerance.

20. Intraepithelial Lymphocytes • Strategically located to respond to antigenic stimulation • Most T-cells are CD8+ • Mainly αβ TCR (In mice, γδ TCR predominates).

21. IEL: CD8 + T-Cells • Limited Repertoire of TCR - marked difference compared to peripheral T-cells. • Recognize a limited # of antigens • Prevents indiscriminate inflammation • Recognition of self-stress antigens (MIC-A, MIC-B) - T-cells induce apoptosis of injured epithelial cells.

22. Van Kerckhove et al: 1992 • Analysis of T-cell receptor Vβ gene usage in IEL vs Peripheral lymphocytes • Quantitative PCR • Results: • PBL - fairly even distribution of Vβ gene usage • IEL - 1-3 Vβ families made up more than 43% of total Vβ transcripts detected in each individual

23. Vβ1, Vβ2, Vβ3, and Vβ6 families frequently shared among IEL from different individuals

24. Lamina Propria Lymphocytes • T-cells are predominantely CD4 + (95% CD45RO+) • Limited capacity to proliferate • Weak proliferative responses to mitogens or specific antigens. • Still act as helpers for B-cells

25. MALT vs peripheral Lymphoid tissue • 1) Unique epithelium for antigen uptake • 2) Unique Lymphocyte Repertoire • 3) IgA dominated humoral response • 4) A need to minimize injury to the mucosal tissue

26. B-Cell Response: S-IgA • Secretory IgA is the predominant Ig isotype in the gut. • Blood IgA exists mainly as a monomer • In the mucosa, IgA is exclusively dimeric J-Chain

27. Secretory IgA Function • Inhibits microbial adherence • Neutralizes viruses and toxins • Neutralizes catalytic activity of microbial enzymes.

28. Secretory IgA Transport • S-IgA is produced by plasma cells in the lamina propria. • S-IgA binds to polymeric Ig receptoron the basolateral surface of intestinal epithelial cells • It is transported to the intestinal lumen by transcytosis. Lamina Propria Lumen

29. Secretory IgA transport **Secretory Component (SC) of the receptor remains associated with IgA • SC protects IgA from proteolytic cleavage. • SC also acts as a “glue” to bind IgA to the glycocalyx.

30. IgA Subtypes • IgA 1 and IgA 2 mainly differ in their hinge regions • IgA 1 ab contain 13 additional aa in the hinge region. - More flexible - More susceptible to IgA1 specific proteases made by bacteria. • IgA 2 is resistant to proteases - Serum ratio 4:1 - Mucosal ratio 3:2 (even higher in colon)

31. B-Cell Isotype Switching: Cytokine Stimulation • IgA response is likely the result of the unique micorenvironment in the gut. • TGF-β + IL-10 induces sIGM+ B-cells to switch to sIgA+ B-cells • Addition of TGF-β to LPS triggered mouse B-cell cultures leads to increased IgA synthesis. • Mucosal epithelial cells are a major source of TGF-β and IL-10

32. Van Ginkel et al: 1999 • TGF-β knockout mice (-/-) • Significantly decreased IgA-committed B-cells in the gut and secretory IgA WT TGF-β -/- Blue stain - IgA Green stain - IgM Red stain - IgG Enhanced IgG and IgM response in the gut (fixes complement)

33. Elson et al. 1979T-cell regulation ofIgA • Antigen activated T-cells from peyer’s patches drive IgA synthesis but suppress IgM and IgG Synthesis. • Ig synthesis first from lymphoid cells stimulated by LPS • Con A was added to culture and the % change in IgG, IgM, IgA measured.

34. Elson et al: IgM IgG IgA IgA Baseline Addition of Con A

35. Elson et al: Unique environment vs. Unique T-cell Subset • T-cells from spleen or PP stimulated with con A then added back into tissue. IgA IgG IgM IgA PP T-cells added to spleen cell cx Spleen T-cells added to PP cell cx

36. GALT vs peripheral Lymphoid tissue • 1) Unique epithelium for antigen uptake • 2) Unique Lymphocyte Repertoire • 3) IgA dominated humoral response • 4) A need to minimize injury to the mucosal tissue.

37. Gut Anti-Inflammatory Mechanisms: Secretory IgA • IgA is unable to activate complement by classical or alternative pathways. • S-IgA can inhibit phagocytosis and chemotaxis of neutrophils, macrophages • Can down regulate synthesis of TNF-α and IL-6

38. Wolf et al: IgA induces IL-1 Receptor antagonist • IgA induces IL-1 R antagonist from monocytes. IL-1 IL-1 Ra

39. T-Regulatory Cells • IPEX – severe enteropathy results from lack of CD4+CD25+ Foxp3+ T Regs. • Naïve T-cells can differentiate into T regs in the presence of TGF-β¹ • Transfer of Tregs into mice with IBD can lead to resolution of colitis² 1. Chen et al. Journal of Experimental Medicine. 2003. 2. Mottet et al. Journal of Immunology. 2003.

40. Regulatory Cytokines • IL-10 – Increased IgA • Decreased cytokine production by DC, T-cells, macrophages • Promotes TH2 response • IL-10 knockout mice: severe enterocolitis • TGF-beta – Increased IgA • Maintain functional CD4+CD25+ cells in the periphery.

41. Antigen Response • Pathogen vs. Commensal response • Both pathogens and commensals often share similar PAMPs • Commensals may be contained by IgA and innate barriers. - Pathogens have additional virulence factors (adhesion molecules, toxins) - commensals also endocytoced by M-cells and engage TLRs

42. Shigella Infection • Nod 1 (aka CARD 4) – Binds shigella endotoxin • Nod 1 dimerization allows binding to RICK protein kinase • Activation of NF-κB Pathway Release of IL-8 attracts Neutrophils

43. Tien et al: Lactobacillus • Mucosal Epithelial cells challenged with shigella then infected with lactobacillus • Macroarray DNA chips used to compare gene expression vs. control • Proteins involved in degradation of I-κBα down-regulated - Result: Inhibition of the NF-κB pathway

44. Kelly et al: Bacteriodes • Rel A: member of NF-κB complex • Intestinal cells cultured with Salmonella • Bacteriodes induced nuclear clearance of Rel A limiting the duration of NF-κB action Immunoflourescence at 2 hrs Medium Salm Salm + Bact Bact Kelly, D. Nature Immunology. 2004.

45. Summary • Mucosal immune system needs to selectively respond to pathogens • Humoral immune response is IgA dominated. • Unique lymphocyte repertoire and cytokine environment limit inflammation • Commensal organisms act to maintain the mucosal immune system and have mechanisms to limit inflammation.

46. The End!

47. References • Mayer, L. Mucosal Immunity. Pediatrics. 111, 1595-1600. 2003. • Janeway. Immunobiology. 2005 • Macpherson, A. Interactions between commensal intestinal bacteria and the immune system. Nature Reviews Immunology. 4; 478-485. 2004. • Fasano, A. Zonulin, a newly discovered modulator of intestinal permeability, and its expression in coeliac disease. Lancet. 355; 1518 – 1519. 2000. • Drago, S. Gliadin, zonulin and gut permeability: Effects on celiac and non-celiac intestinal mucosa and intestinal cell lines. Scandinavian Journal of Gastroenterology. 41; 408 – 419. 2006. • Van Ginkel, F. Partial IgA deficiency with increased Th-2 Type Cytokines in TGF-β1 knockout mice. Journal of Immunology. 163; 4. 1999. • Wolf, H.M. Anti-inflammatory proterties of human IgA. Clinical Experimental Immunology. 105; 537-543. 1996.

48. References • Macpherson, A. Interactions between commensal intestinal bacteria and the immune system. Nature Reviews Immunology. Vol 4. June 2004. • Tien, MT. Anti-Inflammatory Effect of Lactobacillus casei on Shigella-Infected Human Intestinal Epithelial Cells. The Journal of Immunology. 176; 1228. 2006. • Coombes, Janine. Control of Intestinal Homeostasis by regulatory T-cells and dendritic cells. Seminars in Immunology. 19; 116-126. 2007. • Van Kerckhove, Catherine. Oligclonality of Human Intestinal Intraepithelial T-cells. Journal of Experimental Medicine. 175; 57-63. 1992.

49. Antigen Load • GALT must selectively respond to certain pathogens while ignoring other antigens. • Food Proteins – DCs produce IL-10 to produce a TH2 response and suppression of inflammatory response. • Pathogens – TLR ligands sensed by APCs favor pro-inflammatory response. - Humoral and cellular immune response.