Lecture 17 - Immunity at Mucosal Surfaces

the mucosal immune system

protected by an epithelial barrier

enormous antigen load

key mucosal sites

• GI tract (largest immune organ)

• Respiratory tract

• urogenital tract

• lacrimal, salivary, mammary glands

the mucosal immune system

• covers ~400m² of surface area (vs 2m² of skin)

• constantly exposed to food ags, commensals, pathogens, and environmental particles

• requires tight epithelial barrier function

• must balance tolerance (food/microbiota) and immunity (pathogens)

core challenges of the mucosal immune system

• enormous ag load

• risk of inflammation → tissue damage → loss of barrier

different types of epithelium line barrier tissue

mucosae - simple columnar epithelium, pseudostratified columnar epithelium, nonkeratinized stratified squamous epithelium

skin - keratinized stratified squamous epithlium

what does the type of epithelium a tissue uses tell us

the type of epithelium a tissue uses is the first clue to the kind of immune defenses it needs - different types depending on the organ

all epithelia face what

enormous ag load - pathogens, food ags, commensal bacteria (friendly ags)

what type of epithelium does the intestine have and what are some features

intestine has simple columnar epithelium

• microvilli increase absorption

• goblet cells secrete mucus

what type of epithelium does the respiratory tract have and what are some features

respiratory tract has pseudostratified columnar epithelium

• ciliated epithelium + mucus clearance

what type of epithelium does the oral cavity and esophagus have and what are some features

nonkeratinized stratified squamous epithelium

• protects against friction, non-drying surfaces

what type of epithelium does skin have and some features

keratinized stratified squamous epithelium

• tough, water-resistant outer layer

distinctive features of the mucosal immune system

anatomic features

effector mechanisms

immunoregulatory environment

examples of anatomic features of the mucosal immune system

• intimate interactions between mucosal polarized epithelia and lymphoid tissues

• discrete compartments of diffuse lymphoid tissues and more organized structures such as peyer’s patches, isolated lymphoid follicles, and tonsils

• specialized antigen-uptake mechanisms, e.g. M cells in Peyers patches, adenoids, and tonsils

• broad surface area in contact with environmental agents/microbes

examples of effector mechanisms of the mucosal immune system

• activated/memory T cells predominate even in the absence of infection

• multiple activated ‘natural’ effector/regulatory T cells present

• production of mucins and mucus

• secretory IgA antibodies

• production of antimicrobial peptides (AMPs)

• presence of distinctive microbiota

examples of immunoregulatory environment in mucosal immune system

• active down-regulation of immune responses (e.g. to food and other innocuous antigens) predominates at homeostasis

• inhibitory macrophages and tolerance-inducing dendritic cells

• high number of FoxP3+Treg cells and FoxP3-Tr1 cells

mucosal infections are one of the biggest global health problems true or false

true

why are mucosal infections one of he biggest global health problems

many major pathogens enter through mucosal surfaces:

• respiratory tract (influenza, SARS-CoV-2, RSV, TB)

• gastrointestinal tract (cholera, rotavirus, norovirus)

• urogenital tract (HIV, HPV, gonorrhea, chlamydia)

most infectious diseases that kill humans do so how

because they breach a mucosal barrier first

Organization of the Mucosal Immune System

MALT (Mucosa-associated lymphoid tissues)

NALT

• tonsils, adenoids

• strong Th1/Th17 responses to respiratory viruses

BALT

• inducible in adults

• site of TB priming in lung

GALT

• peyers patches, isolated lymphoid follicles, and appendix

• oral tolerance, IgA, defense against enteropathogens

where else is MALT present

also present in urogenital tract (HIV entry & STI immunity), mammary gland, conjunctiva

induction sites of the mucosal immune system

where the mucosal immune response is initiated

• GALT

• BALT

• NALT

• urogenital tracy

• lacrimal glands

• salivary glands

• mammary glands

Effector sites of the mucosal immune system

where the mucosal immune response is carried out

• lamina propria (plasma cells, T cells, ILCs)

• epithelium (intraepithelial lymphocytes, goblet cells, Paneth cells)

• IgA-producing plasma cells

• mucus secretion, AMP production, barrier fortification

peyers patch → induction →

villus/crypt regions → effector response

specialized epithelium overlying intestinal lymphoid tissues takes up particulate antigens - examples

Peyer’s Patch

• M cell: microfold cell - transport ag from lumen

• FAE (follicle associated epithelium) lack thick mucus → easier to access for ag sampling

• subepithelial dome rich in DCs and lymphocytes

• leads to activation of B and T cells in germinal centers

Isolated Lymphoid follicle (ILF)

• smaller inducible lymphoid structures distributed along intestine

• also contain M cells overlying lymphoid follicles

• sample ag and deliver it to underlying DCs

• connect to mesenteric lymph nodes via lymphatics

isolated lymphoid follicles mech

• smaller inducible lymphoid structures distributed along intestine

• also contain M cells overlying lymphoid follicles

• sample ag and deliver it to underlying DCs

• connect to mesenteric lymph nodes via lymphatics

Peyer’s patch mech

• M cell: microfold cell - transport ag from lumen

• FAE (follicle associated epithelium) lack thick mucus → easier to access for ag sampling

• subepithelial dome rich in DCs and lymphocytes

• leads to activation of B and T cells in germinal centers

what do M cells do

take up commensal bacteria, pathogens, and particles from the gut lumen

transport (transcytose) them across the epithelium

deliver ags directly to B cells, T cells, and DCs in the subepithelial dome

enable rapid initiation of mucosal immune response

inductive sites of GALT

cryptopatches

ILFs

Peyers patches

cryptopatches of GALT

(present at birth)

• small clusters of DCs and lymphoid tissue-inducer (LTi) cells

ILFs of GALT

develop after birth

expand in response to microbial colonization

Peyers patches in GALT

present at birth but grow and mature postnatally

require microbial signals for full development

Germ-free mice have what

poorly developed Peyer’s patches

no ILFs

decreased IELs

decreased IgA-secreting plasma cells

decreased AMPs

decreased immune mediators (cytokines)

maturation of GALT is driven by what

acquisition of commensal microbiota

what may impair GALT impairment

early-life antibiotics may impair GALT development

cesarean delivery alters initial microbial colonization

dysbiosis can drive IBD, food allergies, asthma (systemic effects)

effector sites of GALT

intestinal epithelium

lamina propria

intestinal epithelium

(single cell layer)

IELs (or IETs) - mostly CD8+, no B cells, ILCs

produce AMP, cytokines

interacts directly with commensals, sIgA, nutrients

lamina propria

Rich in innate cells (macrophages, DCs, ILCs), CD4+ and CD8+ T cells

B cells and IgA-secreting plasma cells

Tregs

site of most IgA production and immune effector activity

which of the following is a characteristic feature of mucosal surfaces

a) low antigen exposure

b) highly keratinized epithelium

c) constant exposure to food and microbial antigens

d) absence of immune cells

c) constant exposure to food and microbial antigens

what is the major function of M cells

a) produce antimicrobial peptides

b) facilitate antigen transport to immune cells

c) maintain tight junctions

d) secrete mucus

b) facilitate antigen transport to immune cells

goblet cell-associated antigen passages primarily promote:

a) Th1 immunity

b) IgG class switching

c) Oral tolerance

d) viral cytotoxicity

c) oral tolerance

innate immune defenses of intestinal immune system

1. absorptive subsets

2. secretory subsets

absorptive subsets

a. enterocytes (nutrient absorption, sense microbes via TLRs, secrete cytokines)

b. M cells

Secretory subsets

a. goblet cells - mucus

b. paneth cells - AMPs

c. enteroendocrine cells - hormones and neuropeptides

d. tuft cells - cytokines and lipid mediators - imp. in parasite response and type 2 immunity

mucus in the gut is

• viscous, slippery and sticky

• negatively charged → retains sIgA and AMPs

• substrate for commensals

• major component - mucins (MUC2)

mucus in small int. compared to mucus in large int.

in small: thin

in colon: much thicker and organized into two layers:

• inner layer: highly cross-linked, dense, and impenetrable to bacters

• outer layer: looser, less cross-linked, and supports commensal (good) bacteria

glycocalyx

carbohydrate rich layer directly coating epithelial cells

intestinal epithelium is not just a barrier, but

an active immune organ composed of specialzed innate cell types

IELs provide what

rapid, front-line defense at the mucosal barrier

include conventional CD8+ T cells and unconventional (innate-like) T cells

Type A IEL

function like CTL (TCR activation → perforin, granzyme, FasL

Type B IEL

function like NK cells (act independently of TCR activation through NKG2D)

loss of dysfunction of IELs contributes to

Celiac disease

chronic infections

barrier breakdown in IBD

lymphocytes called intraepithelial lymphocytes (IELs) are positioned where

within the intestinal epithelium

ILCs in GALT respond to what

microbes that breach epithelium

ILCs

abundant in mucosal tissues (especially intestine)

mirror helper T cell subsets in cytokine profiles

activate without antigen-specific TCRs (respond to cytokines + epithelial signals)

excess ILC1 →

chronic inflammation, Crohn’s disease associatin

Excess ILC2 →

allergies, asthma, eosinophilic GI disease

excess ILC3 →

IL-17 driven inflammation, IBD

reduced ILC3 →

loss of IL-22 → weak barrier → dysbiosis

mucosal immune system establishes & maintains what

tolerance to harmless Ags

what do intestinal macrophages do

robust phagocytosis

• they’re excellent at engulfing bacteria and debris

But, they don’t trigger inflammation

• they don’t activate naïve T cells (red inhibitory lines)

• they produce very little proinflammatory cytokines

instead, they promote tolerance

how do intestinal macrophages promote tolerance

they produce IL-10, which:

• encourages Treg (regulatory T cell) development

• helps maintain balance between Th17 and Treg cells

they produce PGE2, which

• helps with epithelial repair (healing the gut lining)

overall, intestinal macrophages do what

overall, intestinal macrophages clean up microbes while preventing unnecessary inflammation

gut dendritic cells what do they do?

capture food antigens and commensal bacteria

present these antigens to CD4 T cells in a non-inflammatory way (teaches immune system these are safe)

promote

• Treg differentiation → more regulatory T cells = more tolerance

• IgA class switching in B cells → B cells make IgA, the antibody specialized for the gut → IgA coats microbes and food antigens without causing inflammation

tolerogenic phenotype

both macrophages and DCs in the gut are designed to prevent overreaction.

• promote IL-10, Tregs, and IgA

• avoid inflammation

• maintain gut homeostasis

• tolerate harmless substances (food + commensals)

this protects you from chronic inflammation and allows peaceful coexistence with the microbiome

immune priming and tolerance are what

different outcomes of intestinal exposure to Ag

the same ag delivered orally can generate immunity or tolerance depending on what

danger signals and context

Pathogens → PAMPs → danger → immunity

Food + commensals → no PAMPs → steady-state DC signals → tolerance

why commensals don’t trigger inflammation even though they have PAMPs

1. spatial segregation: mucus, sIgA, and AMPs keep commensals away

2. epithelial TLRs are compartmentalized

3. specialized tolerogenic phagocytes

4. microbial metabolites instruct tolerance

5. absence of contextual danger - danger is not just pathogen or PAMP, true danger: PAMP/DAMP + barrier breach + inflammatory cytokines

intestinal epithelial cells express TLRs in strategic, non-reactive ways:

• TLR5 on basolateral surface only

• TLR9 can produce tolerogenic signals (IL-10) when signaling from apical side

• TLR4 is often low or absent on apical membrane

Thus, commensal PAMPs hitting apical TLRs → tolerance, not inflammation

routes of antigen uptake in the intestine

*the gut does NOT rely on just one mechanism to sample antigens; it uses multiple, highly specialized routes depending on ag type

• M cells

• Paracellular transport

• FcRn: neonatal Fc receptor

• Goblet cells

• Apoptosis-Dependent Transfer

• TEDs

M cells

bulk and receptor-mediated transcytosis of microbes and particles.

deliver ags to CD103+ DCs beneath

FAE

paracellular transport

• limited ag diffusion between epithelial cells

• occurs when tight junction sare physiologically ‘open’ or mildly loosened

• controlled, not pathological

FcRn: neonatal Fc receptor

transports IgG-ag complexes across enterocytes

allows sampling of luminal antigens bound to maternal IgG (neonates) or endogenous IgG

goblet cells

can deliver soluble proteins to underlying tolerogenic DCs

important for oral tolerance to food ags

apoptosis dependent transfer

IECs undergoing apoptosis release antigenic material

phagocytosed by local DCs & macrophages

TEDs

CX3CR1+ macrophages extend dendrites between epithelial cells into the lumen

capture bacteria or particles directly

important for commensal sampling and early pathogen sensing

Tregs in mucosal Tissue balance what

active immunity vs. immunological tolerance

during pathogenic exposure

• pathjogens trigger innate and adaptive immune activation

• effector T cells, ILCs, macrophages, and antibodies clear infection

• Tregs prevent excessive tissue damage during the response

during exposure to innocuous Ags

• Tregs enforce tolerance and suppress unnecessary inflammation

• prevent local and systemic immune reactivity

• protect epithelial barrier integrity

Tregs suppress effector responses through:

• IL-10 (suppresses DCs, Th1, Th17, macrophages)

• TGF-B (promotes IgA class switching + epithelial repair)

• CTLA-4 (removes co-stimulatory molecules from DCs)

• IL-2 consumption (starves effector T cells)

• Amphiregulin (creates tissue repair microenvironment)

loss of mucosal Treg function contributes to

• IBD

• celiac disease

• food allergy

• autoimmune disorders

• chronic infections

• HIV mucosal dysfunction

which cytokines are characteristic of ILC3 responses?

a) IFN-gamma and TFN

b) IL-5 and IL-13

c) IL-22 and IL-17

d) IL-10 and TGF-B

c) IL-22 and IL-17

oral tolerance is primarily mediated by which cell type

a) Th1 cells

b) FoxP3+ Tregs

c) NK cells

d) ILC1

b) FoxP3+ Tregs

Transcytosis of IgA across epithelia is mediated by what

polymeric Ig receptor (pIgR)

steps of transcytosis of IgA across epithelia

1. binding (basolateral surface) - Dimeric IgA (with J chain) binds to plgR on the basolateral side of epithelial cells

2. Endocytosis - The plgR-IgA complex is internalized into the epithelial cell

3. Transcytosis - vesicles move the complex across the cell toward the apical surface

4. release (apical surface) - plgR is cleaved, IgA is released into the lumen as secretory IgA (sIgA). sIgA = IgA dimer + secretory component (cleaved part of the pIgR), which protects IgA from proteolysis

secretory IgA (sIgA):

IgA dimer + pIgR secretory component (soluble in lumen)

mucosal IgA has many functions

• prevents microbial attachment to epithelial cells

• blocks intracellular spread of viruses, toxins, and bacteria taken up by IECs

• Helps remove toxins/pathogens already present in tissues

• enhances ag sampling and promotes IgA class switching + immune surveillance

secretory IgA (sIgA) consists of:

a) monomeric IgA only

b) Dimeric IgA plus the secretory component

c) IgA bound to IgG

d) IgA attached to FcRn

b) dimeric IgA plus the secretory component

IgA provides protection at mucosal surfaces primarily by:

a) activating complement

b) neutralizing pathogens without inflammation

c) triggering mast cell degranulation

d) killing bacteria directly

b) neutralizing pathogens without inflammation