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, Peyers 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 lt 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

• gt400 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 peyers 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
Peyers Patch
Mesenteric Lymph Node
Thoracic Duct
Blood Stream
Resp Tract
Breast
Intestinal Mucosa
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 peyers 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 aß TCR (In mice, ?d 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 Vß gene usage in IEL
  vs Peripheral lymphocytes
• Quantitative PCR
• Results
• PBL - fairly even distribution of Vß gene usage
• IEL - 1-3 Vß families made up more than 43 of
  total Vß transcripts detected in each individual

23
Vß1, Vß2, Vß3, and Vß6 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
• 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 41
• - Mucosal ratio 32 (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-ß IL-10 induces sIGM B-cells to switch to
  sIgA B-cells
• Addition of TGF-ß to LPS triggered mouse B-cell
  cultures leads to increased IgA synthesis.
• Mucosal epithelial cells are a major source of
  TGF-ß and IL-10

32
Van Ginkel et al 1999

• TGF-ß knockout mice (-/-)
• Significantly decreased IgA-committed B-cells in
  the gut and secretory IgA

WT
TGF-ß -/-
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 of IgA

• Antigen activated T-cells from peyers 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-a 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
  CD4CD25 Foxp3 T Regs.
• Naïve T-cells can differentiate into T regs in
  the presence of TGF-ß¹
• 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 CD4CD25 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-?B 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-?Ba down-regulated
• - Result Inhibition of the
• NF-?B pathway

44
Kelly et al Bacteriodes

• Rel A member of NF-?B complex
• Intestinal cells cultured with Salmonella
• Bacteriodes induced nuclear clearance of Rel A
  limiting the duration of NF-?B action

Immunoflourescence at 2 hrs
Salm
Salm Bact
Medium
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-ß1 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.

50
(No Transcript)