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Mechanisms of Mucosal Defense. Soma Jyonouchi, M.D. January 24, 2008. Mucosal Surfaces. The GI mucosal surfaces cover 400 m ² Thin – facilitate nutrient absorption. The Gut Associated Lymphoid Tissue (GALT) - Organized T and B cell areas

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mechanisms of mucosal defense

Mechanisms of Mucosal Defense

Soma Jyonouchi, M.D.

January 24, 2008

mucosal surfaces
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.

galt architecture
GALT Architecture

Lamina Propria

** Dome structures extend into the lumen of the intestine.

lamina propria effector site
Lamina Propria – effector site

Inductive Site

Effector Site

enormous antigen load
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
innate defense i barrier fxn
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.

i barrier fxn
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

ii proteolytic enzymes
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.
iii antimicrobial molecules
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.

iv commensal organisms
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
iv commensal organisms1
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.

slide12
Mucosal Immune

System:

Adaptive Response

common mucosal immune system
“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

common mucosal system
Common Mucosal System?

IgA response for different routes of vaccination

Holmgren et al. Nature Medicine. 2005

galt vs peripheral lymphoid tissue
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.
galt unique epithelium
GALT – Unique Epithelium
  • The epithelium overlying the peyer’s patches is composed of cells that differ from the surrounding enterocytes.
m cells microfold cells
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)

m cells microfold cells1
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

galt vs peripheral lymphoid tissue1
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.

intraepithelial lymphocytes
Intraepithelial Lymphocytes
  • Strategically located to respond to antigenic stimulation
  • Most T-cells are CD8+
  • Mainly αβ TCR (In mice, γδ TCR predominates).
iel cd8 t cells
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.

van kerckhove et al 1992
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
slide23

Vβ1, Vβ2, Vβ3, and Vβ6 families

frequently shared among IEL from

different individuals

lamina propria lymphocytes
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
malt vs peripheral lymphoid tissue
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
b cell response s iga
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

secretory iga function
Secretory IgA Function
  • Inhibits microbial adherence
  • Neutralizes viruses and toxins
  • Neutralizes catalytic activity of microbial enzymes.
secretory iga transport
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

secretory iga transport1
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.
iga subtypes
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)

b cell isotype switching cytokine stimulation
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
van ginkel et al 1999
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)

elson et al 1979 t cell regulation of iga
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.
elson et al
Elson et al:

IgM IgG IgA

IgA

Baseline

Addition of Con A

elson et al unique environment vs unique t cell subset
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

galt vs peripheral lymphoid tissue2
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.
gut anti inflammatory mechanisms secretory iga
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

wolf et al iga induces il 1 receptor antagonist
Wolf et al: IgA induces IL-1 Receptor antagonist
  • IgA induces IL-1 R antagonist from monocytes.

IL-1 IL-1 Ra

t regulatory cells
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.

regulatory cytokines
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.
antigen response
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

shigella infection
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

tien et al lactobacillus
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

kelly et al bacteriodes
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.

summary
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.
references
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.
references1
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.
antigen load
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.