Mechanisms of Mucosal Defense


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Mechanisms of Mucosal Defense

  1. 1. Mechanisms of Mucosal Defense Soma Jyonouchi, M.D. January 24, 2008
  2. 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. 3. GALT Architecture  Lamina Propria ** Dome structures extend into the lumen of the intestine.
  4. 4. Lamina Propria – effector site Inductive Site Effector Site
  5. 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. 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. 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. 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. 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. 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. 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. 12. Mucosal Immune System: Adaptive Response
  13. 13. “Common Mucosal Immune System” *Antigen Presentation* Peyer’s Patch Mesenteric Lymph Node Thoracic Duct Blood Stream Resp Breast Intestinal Mucosa GU Salivary/Lacrimal Tract Tract Gland
  14. 14. Common Mucosal System? IgA response for different routes of vaccination Holmgren et al. Nature Medicine. 2005
  15. 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. 16. GALT – Unique Epithelium  The epithelium overlying the peyer’s patches is composed of cells that differ from the surrounding enterocytes.
  17. 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. 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. 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. 20. Intraepithelial Lymphocytes  Strategically located to respond to antigenic stimulation  Most T-cells are CD8+  Mainly αβ TCR (In mice, γδ TCR predominates).
  21. 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. 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. 23. Vβ1, Vβ2, Vβ3, and Vβ6 families frequently shared among IEL from different individuals
  24. 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. 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. 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. 27. Secretory IgA Function  Inhibits microbial adherence  Neutralizes viruses and toxins  Neutralizes catalytic activity of microbial enzymes.
  28. 28. Secretory IgA Transport  S-IgA is produced by plasma cells in the lamina propria.  S-IgA binds to polymeric Ig receptor on the basolateral surface of intestinal epithelial cells Lamina Lumen Propria  It is transported to the intestinal lumen by transcytosis.
  29. 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. 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. 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. 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. 33. Elson et al. 1979 T-cell regulation of IgA  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. 34. Elson et al: IgM IgG IgA IgA Baseline Addition of Con A
  35. 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 Spleen T-cells added PP T-cells added to to PP cell cx spleen cell cx
  36. 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. 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. 38. Wolf et al: IgA induces IL-1 Receptor antagonist  IgA induces IL-1 R antagonist from monocytes. IL-1 IL-1 Ra
  39. 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. 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. 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. 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. 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. 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. 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. 46. The End! 
  47. 47. References 1. Mayer, L. Mucosal Immunity. Pediatrics. 111, 1595-1600. 2003. 2. Janeway. Immunobiology. 2005 3. Macpherson, A. Interactions between commensal intestinal bacteria and the immune system. Nature Reviews Immunology. 4; 478-485. 2004. 4. Fasano, A. Zonulin, a newly discovered modulator of intestinal permeability, and its expression in coeliac disease. Lancet. 355; 1518 – 1519. 2000. 5. 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. 6. Van Ginkel, F. Partial IgA deficiency with increased Th-2 Type Cytokines in TGF-β1 knockout mice. Journal of Immunology. 163; 4. 1999. 7. Wolf, H.M. Anti-inflammatory proterties of human IgA. Clinical Experimental Immunology. 105; 537-543. 1996.
  48. 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. 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.