Lecture 11: Mucosal Immunity(based on lecture by Dr. Betsy Herold, Einstein)

Questions to Consider

How is the mucusal

immune system different from the

systemic immune system?

How does the immune system prevent overreaction to antigenic loads?

How does the mucosal immune system protect itself from infection?

How do pathogens bypass mucosal immunity?

What Th

subtypes are preferentially activated in the

mucosal immune system?

Overview of Mucosal Immune System

Major components•

GI tract•

Respiratory tract•

Genital tract

Unique attributes•

First line of defense•

Constantly exposed to Ag

Worldwide Mortality From Mucosal Infections

Components of the Mucosal Immune System

GALT: Gut Associated Lymphoid Tissue: Anatomic Sites

Peyer’s patches

Aggregates of lymphoid cells with B cell follicles & smaller T cell areas

Lymphoid follicles

Smaller; mainly B cells

Also found in respiratory tract (BALT) , lining of nose (NALT)-

together referred to as MALT-

mucosa associated

Appendix

(Tonsils, adenoids)

Mesenteric nodes

Peyer’s patches & lymphoid follicles connected by lymphatics

to draining nodes

Peyer’s patches, mesenteric nodes differentiate independently of systemic immune system during fetal development (control chemokines)

M cells: Microfold

Cells: Specialized Epithelial Cells

Epithelial cells covering Peyers

Patches

Differ from epithelial cells (enterocytes)•

No microvilli; broader microfolds•

Do not secrete enzymes, mucus, and no thick surface glycocalyx•

Transport organisms from gut lumen to immune cells across epithelial barrier•

Endocytose

or phagocytose

Ag at AP surface & deliver it to DCs

or T cells via transcytosis

at BL surface•

Note: some pathogens (Shigella, Salmonella, Yersinia) exploit M cells as a way to penetrate the intestinal epithelium

•

CXCR4 HIV strains bind to M cells and may get transported across

the epithelium to infect immune cells

Uptake and Transcytosis

of Antigen Across M Cells

Pocket in basal membrane of M cell encloses T cells and DCs

DCs

recruited to the mucosa in response to chemokines constitutively expressed by epithelial cells

Extend processes across epithelium to capture Ag in lumen

DCs

also prevalent within lamina propria•

CCL20 (MIP-3α) & CCL9 (MIP-1γ) bind to receptors on DCs

(CCR6 and CCR1, respectively)

•

Interference with CCL20 signaling blocks recruitment & may prevent HIV in macaques; Nature 2009 Apr 23;458(7241):1034-8.

•

Ag loaded DC migrate from dome region of Peyer’s patch to T cell area or to draining lymphatics

to mesenteric nodes

Dendritic Cells

Effector T Cells

Resident T cells found in epithelium and lamina propria

Epithelium contains mostly CD8 T cells, whereas lamina propria

is more heterogenous

(CD4, CD8, plasma cells,

macrophages, DCs, eosinophils

and mast cells)In intestine and respiratory tract, plasma cells predominately IgAIn genital tract: IgG> IgA

Neutrophils

are found usually only in response to inflammation/infection

T cells in lamina propria

of small intestine express integrinα4β7

and CCR9, which attracts them into the tissue from bloodstream;

Epithelial cell T cells express integrin

αE

β7, which binds to E-cahedrin

on epithelial cells.

Mucosal Lymphocyte Life Cycle and Gut-specific Homing Receptors

Naïve T & B cells emanate from thymus & bone marrow & circulate in bloodstream

Enter Peyer’s patches (or nodes) through endothelial venules

directed by homing receptors, CCR7 & L-selectin

If no Ag is encountered, exit via efferent lymphatics

& return to bloodstream

If Ag is encountered, cells become activated, exit via lymph nodes to thoracic duct & recruited back to gut

T cells that first encounter Ag in GALT express gut-specific homing receptors (α4β7 and CCR9)

Homing ReceptorsExpression of homing receptors triggered by GALT DCα4β7 binds to the mucosal vascular addressin

(MAdCAM-1) expressed on gut endothelial cells

CCR9 binds to CCL25(TECK) on gut epithelium (small intestine)Priming explains why vaccination by mucosal route against intestinal

infections (e.g. Rotavirus) ensures imprinting to the gut. MAdCAM-1 also expressed in other mucosal sites: T cells primed in GALT

can recirculate

as effector cells to respiratory, genital or lactating breast tissue: “common mucosal immune system”Vaccines: Mucosal route can be used to protect multiple mucosal

sites

Homing Receptors- Role of MAdCAM-1 and Chemokines

Secretory IgADominant class in gut & respiratory tract (not genital

tract)In blood, IgA

mostly monomer (IgA1:IgA2=10:1)

In mucosa, dimer

linked by J chain (IgA1: IgA2=3:2)Class switching from IgM

to IgA

producing cells

occurs in response to TGFβCommon intestinal pathogens can cleave IgA1IgA2 more resistant

IgA

in Gut

Activated B cells (like T cells) express homing integrin

(α4β7) & CCR9/10, which localizes them to gut

IgA

producing plasma cells secrete IgA

dimers, bind to poly-Ig

receptor expressed on BL surface of immature epithelial cells at base of intestinal crypts

Bound complex taken up by cells; traversed to AP surface by transcytosis

Poly-Ig

receptor cleaved releasing IgA

dimer

& secretory component at luminal surface; secretory IgA

IgA

binds to mucins

at epithelial surface via carbohydrates on secretory component;

•

Retention of IgA

at epithelial surface prevents adherence of microbes & neutralizes toxins, etc.

Intracellular IgA

Neutralizes Ags

(e.g. LPS)

IgA

does not activate complement pathway

Does not trigger inflammatory response

Restricts commensal

flora to the lumen

IgA

Deficiency

Common: 1:500-1:700 in Caucasian population

Most individuals have no clinical problems

Associated with IgG2 subclass deficiency→↑

risk

infections

IgM

may replace IgA

in secretions; IgM

also is J-chain

linked & binds poly-Ig

receptor• IgM-producing plasma cells are ↑

in IgA

deficiency

KO mouse model: KO poly-Ig

receptor susceptible to

mucosal infections; KO IgA, no ↑

susceptibility

Mucosal T cells

Most T cells in lamina propria

CD45RO+ (similar to effector/memory T cells)

Express gut homing markers

Express receptors for inflammatory chemokines e.g CCL5 (RANTES)

Proliferate poorly in response to Ag or mitogens

Secrete large amounts of cytokines (IL-10, IL-5 and IFN-γ

constitutively

Function in healthy gut uncertain•

? regulatory role

IEL: Intraepithelial Lymphocytes

10-15 lymphocytes/100 epithelial cells

90% are T cells; 80% CD8+

Express homing markers CCR9 &αE

β7; binds E- cadherein

on epithelial cells

Activated

+ perforin and granzyme

in intracellular granules

Relatively restricted use of V(D)J gene segments; responsive to limited Ag repertoire

Type A: conventional CD8 cytotoxic effectors

MHC-restrictedexpress CD8α:β

Functions of IEL

Type B; Express CD8α:αExpress NKG2D(activating C-type lectin NK receptor) which binds to 2 MHC-like-molecules; MIC-A, MIC-B that are expressed on epithelial cells in response to stress/damage & killed via perforin/granzyme

pathwayActivation of these IEL cells mediated byIL-15↑in celiac disease

IEL Kill Infected Epithelial Cells

IEL Kill Stressed Epithelial Cells

Mucosal Response to Infection

Mucosal surfaces are not sterile

Mucosal immune system must differentiate harmless (endogenous flora) from pathogenic microbes and respond differently

Gut is most frequent site of infection

Epithelial Cells are Immune Cells

Mucosal epithelial cells are polarized

Apical surface faces the intestinal lumen; BL surface faces the adjacent epithelial cells and underlying basement membrane

Polarized expression of different receptors/proteins/channels

EXPRESS Toll-like Receptors (TLRs) at both membranes, but responses differ

Activation by commensal

bacteria has an essential

role in maintaining colonic homeostasis

Pathogen-related Specificity of TLR Molecules

Nat Cell Biol. 2006 Dec;8(12):1327-36 .

Polarity of TLR Responses

Interaction of TLR with microbes activates signaling complex (NFkB, MAPK, IFNs)→ transcription of inflammatory and immunoregulatory

genes (chemokines, cytokines and costimulatory

molecules, defensins)

Human IECs

express a spectrum of TLRs, including TLR2, TLR4, TLR5, and TLR9

•

Genital tract epithelial cells express full array of TLRs

Polarized responses differ & may explain differential response to microbes:•

BL TLR9 signals IkB

degradation & activation of NF-

kB•

AP TLR9 stimulation invokes a unique response in which ubiquitinated

IkB

accumulates in the cytoplasm preventing NF-

kB

activation. •

AP TLR9 stimulation confers intracellular tolerance to subsequent TLR challenges

•

TLR9-deficient mice display a lower NF-

kB

activation threshold & are highly susceptible to experimental colitis.

Nat Cell Biol. 2006 Dec;8(12):1327-36 .

Epithelial Cells Also Have Intracellular Sensors for Infection

TLRs

within intracellular vesicles

NOD1/NOD2 (nucleotide-binding oligomerization

domain)

NOD1 recognizes muramyl

tripeptide

on GNR

NOD2 recognizes muramyl

dipeptide

in peptidoglycan

of most bacteria

Signaling activates NFkB

pathways

Chemokines, cytokines, defensins

Activation of signaling pathways: double-

edged sword

Facilitate further invasion (e.g. IL-1β

and TNFα

disrupt tight junctions

Inflammation causes symptoms, but also recruits immune cells & initiates adaptive immune response to eliminate microbe

Salmonella Invade Epithelium by Three Routes

Adhere to M cells, cause apoptosis of M cell, infect macrophages and epithelial cells; trigger TLR5(flagellin) at BL membrane and trigger NFkB

inflammatory pathways

Invade by direct adherence of fimbriae

to luminal epithelial surface

Enter DCs

that sample gut luminal contents

Balance: Tolerance vs. Immune Response

Mechanisms of oral toleranceDeletion of Ag-specific T cells?Regulatory T cells (TH

3)?Produce TGFβ; immunosuppressive

Consequences of Mucosal Tolerance Breakdown

Celiac disease

Genetically susceptible (HLADQ2)

Generate IFNγ-CD4 T cell response to protein gluten (gliaden) leading to inflammation

? Food allergies

Crohn’s

disease

Overresponsiveness

to commensal

gut flora

NOD2 mutations and uncommon polymorphic variants

Commensal

Bacteria Prevent Disease

Normal gut flora maintains health

Compete with pathogenic bacteria prevent them from colonizing & invading

Directly inhibit proinflammatory

signaling pathways

TLR response to commensals

controls inflammation

Loss of normal gut flora (i.e. in response to antibiotics) allows other bacteria to grow: C difficile

Integrity of intestinal epithelium disrupted (trauma, infection, vascular disease)

Nonpathogenic commensal

invade blood stream→disease

Immune Response to Endogenous Flora

Recognized by adaptive immune system

sIgA

and T cells recognize commensals

Effect responses not typically elicited

Do not typically invade: compartmentalized response

Animals raised in germfree (gnotobiotic) environment

Reduced size of lymphoid organs

Low Ig

levels

Reduced immune responses

DCs

loaded with commensal

Activate B cells into IgA

producers-

redistributed to lamina propria

Epith

cells produce TGFβ, TSLP, PGE2-

maintain DCs

in quiescent state

When present Ag to naive T cells, generate Treg

response (anti-

inflammatory)

Commensals

do not penetrate intact epithelium, do not activate NFkB, lack virulence factors

If regulatory mechanisms fail, systemic immune responses generated (TH1)→disease

DC Response to Pathogens and Commensals

Protective and Pathological Responses to Intestinal Helminths

TH

2 responses protective

TH

1 responses produce inflammatory reaction that damages mucosa

IL3 and IL9 recruit mucosal mast cells-produce PGS, leukotrienes

and proteases-

remodel intestinal mucosa, create hostile environment to parasite.

Host response to parasites involves turnover of epithelial cells which helps eliminate parasite: double edged sword as compromises intestinal function as newly produced epithelial cells defective in absorption

Parasites evolved mechanisms to modulate immune response

Response to Invasive Pathogens

The predominantly tolerant microenvironment “changed”

in

response to pathogens

DCs

now become fully activated and present Ag to T cells

to generate effector T cell response

Both DC populations –

inflammatory and regulatory may

exist simultaneously: state of physiological inflammation

Hygiene hypothesis: absence of exposure to helminths

and other Ags

results in hypersensitivity responses to harmless environmental Ags

and increased autoantigen

responses

Summary

Mucusal

immune system avoids making active responses to majority of Ags

encountered but recognizes both pathogenic and non-pathogenic Ags.

Disruption of this balance leads to disease

Local DCs

play key role•

DCs

in Peyer’s patches in almina

propria

produce IL-10, rather than pro-inflammatory IL-12

•

Response to Ag is local IgA

and induction of tolerance•

This tolerant response maintained by TSLP, TGFβ, PGE2 produced by local epithelial & stromal

cells•

Thus DCs

migrate to mesenteric node but lack co-stimulatory molecules to activate naïve T cells into effectors

•

Induce gut homing molecules on T cells, to restrict any response to mucosa

Questions to Consider

How is the mucusal

immune system different from the

systemic immune system?

How does the immune system prevent overreaction to antigenic loads?

How does the mucosal immune system protect itself from infection?

How do pathogens bypass mucosal immunity?

What Th

subtypes are preferentially activated in the

mucosal immune system?