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Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
Mechanisms of Mucosal Defense
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Mechanisms of Mucosal Defense

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  • 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 Breast Intestinal Mucosa GU Salivary/Lacrimal Tract Tract 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 receptor on the basolateral surface of intestinal epithelial cells Lamina Lumen Propria  It is transported to the intestinal lumen by transcytosis.
  • 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. 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. 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 Spleen T-cells added PP T-cells added to to PP cell cx spleen 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 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. 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.

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