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

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 MALTmucosa 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

Dendritic Cells 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

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 IgA In 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 α E 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 Receptors Expression of homing receptors triggered by GALT DC α4β7 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 IgA Dominant 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 TGFβ Common intestinal pathogens can cleave IgA1 IgA2 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 &α E β 7; 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

Functions of IEL Type A: conventional CD8 cytotoxic effectors MHC-restricted express CD8α:β Type B; Express CD8α:α Express 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 pathway Activation of these IEL cells mediated by IL-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 NFkB 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: doubleedged 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 tolerance Deletion of Ag-specific T cells? Regulatory T cells (T H 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

DC Response to Pathogens and Commensals DCs loaded with commensal Activate B cells into IgA producersredistributed to lamina propria Epith cells produce TGFβ, TSLP, PGE2- maintain DCs in quiescent state When present Ag to naive T cells, generate Treg response (antiinflammatory) Commensals do not penetrate intact epithelium, do not activate NFkB, lack virulence factors If regulatory mechanisms fail, systemic immune responses generated (TH1) disease

Protective and Pathological Responses to Intestinal Helminths T H 2 responses protective T H 1 responses produce inflammatory reaction that damages mucosa IL3 and IL9 recruit mucosal mast cells-produce PGS, leukotrienes and proteasesremodel 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?