Lecture 18 - Mucosal Immunity II Flashcards
Outline the gross anatomy and function of the GIT
Stomach:
• Digestion of food
• Neutralisation
Small intestine:
• Duodenum, jejunum, ileum
• Digestion
• Absorption of nutrients
Large intestine:
• Absorption of water and vitamins, storage
Describe the tissue structure of the intestine
1. Mucosa: • Epithelium Form villi, have microvilli on apical surface • Lamina propria • Muscularis mucosae
- Submucosa
- Adventitia
• Most external
• Adjacent to perineal cavity
How does the small intestine have such a great surface area?
Valves of Kerking (folds)
Villi
Microvilli
→ 200 square metres
What sort of antigens is the gut mucosa exposed to?
Gut mucosa is exposed to innocuous antigens, as well as pathogenic ones
The gut immune system has to differentiate between these things
Describe the presence of immune cells in the gut
‘Physiological inflammation’
There are numerous immune cells present in the steady state:
Innate immunity: • Epithelial cells • Mast cells • Eosinophils • Macrophages • DCs • Innate lymphoid cells • Unconventional T cells
Adaptive immunity:
• Plasma cells
• T cells
Describe the role of epithelial cells in mucosal immunity
Epithelial cells in the GIT mucosa play an important role:
- Physical barrier
• Tight junctions
2. Mucous: • From goblet cells Functions: • Physical barrier against bacteria • Matrix for IgA • Contains decoy molecules that further interfere with bacterial invasion
- Antimicrobial peptides
• Paneth cells - Defensins
• Cathelicidins, lectins
4. Microbial sensing and inflammation • Epithelial have PRRs that detect bacteria and trigger inflammation through NFKB signalling • TLRs (luminal, basolateral) • NODs • Ligation of PRRs results in the production of pro-inflammatory cytokines, chemokines and defensins: - IL-1 - IL-6 - CXCL8 - CCL1 etc. • Also important for gut homeostasis
What mediates the immune function of epithelial cells in the gastric mucosa?
Immune mediators
• IL-22
• Bacterial recognition (PRR-PAMPs)
What effect do bacteria have on the immune function of the epithelial cells?
Microbial products (from commensal microbiota) stimulate TLRs and NOD2
This results in a ‘tolerising’ response in the cells
What observation has been made in Crohn’s disease?
NOD2 receptor mutations have been associated with Crohn’s disease
What effect can antibiotics have on the gut?
Antibiotics can wipe out commensal bacteria, allowing for the overgrowth of pathogenic bacteria (e.g. C. difficile)
Can lead to colitis (pseudomembranous colitis)
Describe the compartmentalisation of the adaptive immune system in the gut
- Inductive sites
- where Ags are sampled and responses are induced
GALT: gastric associated lymphoid tissue: • Organised tissues • Peyer's patches • Isolated lymphoid follicles (ILFs) • Mesenteric lymph nodes
- Effector sites
- where effect cells perform their action after activation and differentiation
• Lamina propria
• Surface epithelium
Describe the regional differences in lymphoid tissue along the GIT
Isolated lymphoid follicles:
• Found along in both small and large intestines
PPs:
• Only found in small intestine (usually distally)
Duodenum:
• Intra-epithelial lymphocytes
• LP lymphocytes
Large intestine:
• ILFs
Compare the numbers of PP and ILFs in the average human
PPs: 100-200
(Each contains 5-200 follicles)
ILFs: Thousands
Compare the development of PPs and ILFs
PP Anlagen develop before birth
ILFs: develop after birth
Shows that the development of PP is not dependent on exposure to commensal bacteria, whereas the development of ILFs is dependent on exposure to commensal bacteria after birth
Describe the structure of PPs
Resemble LNs, but there are more B cells
- FAE: follicle associated epithelium
• M cell at apex
• Enterocytes - Sub-epithelial dome (SED)
• DCs
• T cells
• B cells - Follicles
• Germinal centres
• B cells, plasma cells - T cell area
• Under the follicles
What are M cells?
Describe their function and structural features
Microfold cells
Cells that are specialised in Ag uptake and handover:
• Do not process and present Ag
• Continually transcytose Ag from the lumen of the gut (take up by endocytosis, then exocytose from basal membrane)
• Major pathway of Ag sampling in GALT
• Intimate contact with DCs, T and B cells on basal side
Structure:
• Folded luminal surface
• No microvilli
• No glycocalyx underlying the cells → greater access for lumen Ags
What are LP-DCs?
What is their function?
How do they access antigen?
Lamina propria DCs
• Take up Ag (through a number of routes), process, drain to mesenteric LNs for activation of naïve T cells
• ? Like M cells outside of the PPs
Antigen capture:
• ? Transepithelial processes for capturing luminal Ag
• Endocytosis of apoptotic epithelial cells
• Antibody mediated (FcRn-dep.) transepithelial transport: Ab bound to FcR on luminal side that have bound antigen are transcytosed
Describe how DCs ‘condition’ for gut-specific responses
CD103+ DCs (of gastric mucosa) produce certain molecules: • TGF-β • IL-10 • Retinoic acid (RA) • TSLP (thymic stromal lymphopoietin)
This results in the DCs having a largely tolerising effect on lymphocytes:
• FoxP3 up-regulation in T cells → Tregs
Where does retinoic acid come from?
What does it bring about in the gut?
DCs produce RA from dietary vit A:
Dietary vitamin A + Retinol → RA
Functions: 1. IgA class switching
- i/Treg induction
- Gut homing of lymphocytes:
• Imprinting of gut-specific homing receptors:
- α4β7
- CCR9 - Inhibition of Th1 and Th17 phenotypes
What is oral tolerance?
How is it induced?
Non-responsiveness to the antigens present in food
Food Ags are foreign, yet the immune system does not respond to them
Originates from mesenteric LNs
Induction of oral tolerance:
- Food Ag in gut
- Tregs are induced
- Tregs secrete TGF-β and IL-10
- Suppression of effector T cells and IgE production
Compare the immune response in the gut to commensal and pathogenic bacteria
Commensals:
• Generally do not invade past the mucosal epithelium
• No detection of foreign Ags by DCs (no PRR ligation)
• No inflammation
• DCs have tolerising effect on naïve T cells in mesenteric LNs (TGF-beta, RA, TSLP)
→ Tregs
Pathogenic bacteria:
• Invade past mucosa to LP, where they are sensed by DCs
• DCs express co-stimulatory molecules and cytokines
• DCs activate naïve T cells in mesenteric LNs
→
Th1 (viruses)
Th2 (parasites)
Th17 (bacteria)
Describe how activated lymphocytes ‘home’ to effector sites in the gut
Lymphocytes are stimulated to express certain surface molecules:
• CCR9
• α4β7
CCR9:
• Chemokine receptor for chemokine expressed in the gut
• Receptor for CCL25
• CCL25 expression by small intestine epithelial cells
α4β7:
• Binds MAdCAM-1 on mucosal vascular endothelium
Describe the compartmentalisation of CD4 and CD8 T cells in the gut mucosa
CD4 T cells: LP
CD8 T cells: epithelial layer, (intraepithelial)
Compare the immune cells in the LP and the mucosal epithelium
LP and mucosal epithelium have distinct populations of immune cells (despite being so close)
LP: • Plasma cells • CD4 T cells • Innate cells • (much more heterogeneous than epithelium)
Epithelial layer:
• CD8 T cells
• γδ T cells
What are intra-epithelial cells?
Describe which cells are IELs
Which molecules do they express?
Cell types:
• >90% T cells, 80% of those are CD8+:
- type a: α:β TCR, CD8α:β T cells
- type b: γ:δ TCR, CD8α:α T cells
• T(RM)
These cells are lodged in between the epithelial cells of the intestinal mucosa
Express:
• αEβ7 (ligand for E-cadherin, expressed on epithelium)
• CCR9
Describe Ab protection in the gut mucosa
How is the isotype selected?
How does this Ab help against infection?
IgA is the predominant isotype
Activated B cells home to gut and differentiate into plasma cells
IgA class switching:
• The microenvironment in the GALT stimulates class switching to the IgA isotype:
- TGF-β
- RA
- NO
( CD40-CD40L interactions with ‘helper’ T cells)
sIgA protection:
• Inhibition of microbial adherence
• Neutralisation of toxins / enzymes
Describe the structure of sIgA, and the process of secretion
Structure:
• Dimer, linked by a J chain
• Secretory component
Secretion:
- IgA synthesised by plasma cells in the LP
- IgA dimers bind poly-IgR (polymeric Ig receptor) through the J chain on basolateral side of enterocytes
- IgA transcytosed in vesicles to the luminal side of the enterocyte
- IgA dimer + secretory component released into the gut lumen
What is the secretory component?
Where does it come from?
What role does it serve?
Part of the pIgR that remains bound to IgA dimers when delivered into the gut lumen
Function: prevents degradation of IgA in the gut lumen
Describe the pathogenesis of coeliac disease
- Gluten protein in food is proline rich and avoids proteolysis in the intestines
- Gliadin peptides absorbed by enterocytes
- Deamidation: tTG converts glutamine residues in gliadin to glutamic acid (-ve charge)
- Individuals with HLA-DQ2 and DQ8, these altered peptides bind strongly to HLA (due to the negative charge)
- Deamidated gliadin presented by APCs in LP
- CD4 T cells are activated by APCs
- CD4 T cells release cytokines that damage the enterocytes
- Villous atrophy; crypt hyperplasia; intraepithelial lymphocytosis
NB Auto-Abs against tTG are present
They are not pathogenic, but they are a diagnostic marker
What are the features of coeliac disease?
- Villous atrophy
- Crypt hyperplasia
- Intraepithelial lymphocytosis
Describe the role of NK cells in coeliac disease
- Gliadin peptides in epithelial cells induces stress proteins (MIC-A,B)
- NK cells recognised stressed epithelial cells through NKG2D and become activated
- NK cells kill epithelial cells (perforin dep.)
What is the role of IL-15 in coeliac disease?
Intraepithelial cells over express IL-15 in the coeliac intestine
This results in accumulation and activation of lymphocytes in the gut mucosa
Describe the importance of IL-10 in the gastric mucosa
IL-10 is a suppressive cytokine that prevents exuberant inflammation in the GIT in the steady state
- IL-10 deficient mouse:
• Develop chronic enterocolitis - Inflammatory bowel disease
• Mutations in IL-10 receptor gene
List some forms of chronic bowel disease
Crohn’s disease
Ulcerative colitis
What is Crohn’s disease?
What is the role of genetics?
Describe the pathological features of CD
A form of inflammatory bowel disease
Overactive inflammatory response to commensal bacteria in genetically susceptible individuals
Genetic susceptibility:
- LOF mutation in NOD2 → decreased defensin production
- Autophagy genes → ?
- IL-23R gene polymorphisms → over-active Th17 responses
Pathological features: • Chronic transmural inflammation anywhere along GIT, but predominantly distal ileum • Fistulae • Stenoses • Granulomas • Strictures
What are some novel therapeutic strategies targeting chronic inflammation in the gut?
- Anti-TNFα
- Anti-p40
- Enhancing tolerance
- Costimulation blockade
- Anti-α4 or α4β7
Compare the tissue structure in the small intestine and the colon
Small intestine: villi + crypts
Colon: crypts
When do gastric epithelial cells produce antimicrobial products?
Constitutively
However, production can be altered / upregulated when TLRs / NODs are stimulated by microbial products
Describe how basolateral recognition of bacteria can occur
TLR5 is present on the basolateral side of enterocytes
Bacteria that penetrate the epithelial layer, and gain access to the LP can be recognised by this TLR
Where are Peyer’s patches found?
Mainly in the small intestine
Describe the lymphoid tissue in the large intestine
Do not have PPs
Do have IELs and ILFs
Which chemokines do M cells secrete?
Why?
CCL20
CCL9
These chemokines bind receptors (CCR6, CCR1) on DCs, recruiting them for uptake of transcytosed Ag
Compare DCs in various locations in the gastric mucosa and skin
There are different subpopulations of DCs in different locations; they express different cell surface markers
- Skin DCs
- PP DCs
- ILF DCs
- LP DCs
- Intraepithelial LP DCs
Are PPs organised lymphoid tissue?
Yes
This means that there are distinct B and T cell areas within the tissue
PPs have distinct B cell follicles which overly the T cell areas
Describe how Ag is presented to lymphocytes in PPs
- M cells continuously transcytose Ag
- DCs recruited to basal side of M cells by chemokines
- DCs take up Ag and process for presentation
- DCs migrate to T cell area and present Ag to naïve T cells
- Activated DCs and T cells help B cells to become activated in follicles
- B cell IgA class switching
What is unusual about the presence of leukocytes in mucosal tissue?
There are great numbers of these cells, even in the absence of disease
Which leukocyte is not observed in the gastric mucosa in the steady state?
Neutrophils
Compare chemokines and their receptors that cause lymphocytes to home to the skin and the gastric mucosa
Skin:
• CCR4 - CCL17
• CCR10 - CCL27
Gastric mucosa:
• CCR9 - CCL25 (PP)
• CD103+ (LP)
What is MAdCAM1?
Adressin
Expressed on mucosal vascular endothelium
Binds α4β7 on gut-homing lymphocytes
Where is CCL25 expressed?
Expressed by epithelium of gastric mucosa
Binds CCR9 on gut homing lymphocytes, recruiting them to the gastric mucosa
What are CX3CR1+ DCs?
CX3CR1+ DCs:
• Probably not classical DCs
• Do not migrate to mesenteric LNs in steady state
• Apart from in dysbiosis
• Role in inflammation ? (through the production of cytokines)
Compare surface marker expression of DCs in the PPs and the LP
SED of PP:
• CD11b+
• CD8α-
• CCR6 (CCL20 from M cells)
LP:
• CD103+
Briefly characterise the effect of gut DCs on T cells
Anti-inflammatory:
Due to production of:
• TGF-beta
• RA
• TSLP
Why don’t effector T cells cause disease in the gut mucosa?
The presence of Th1, Th2 and Th17 is balanced by Tregs which produced IL-10
When an infection occurs, the balance shifts towards the effector T cells, and the function / differentiation of Tregs is diminished
Describe homing of B cells to the colon
B cells are imprinted to express CCR10, which binds CCL28 released from colonic epithelial cells
CCR10 - CCL28
What is CD103 also known as?
αEβ7
Compare the location of TLRs and NODs in gastric epithelial cells
TLRs:
• Associated with membrane:
• Apical, basolateral, endosomal
NODs:
• Cytosolic
How do activated T cells get back into the LP?
Activated T cells are in the blood
They move through HEV into the LP
This recruitment is mediated by α4β7 binding to MAdCAM-1 on HEV endothelium
What is pseudomembranous colitis?
Infection of the large intestine with an overgrowth of C. difficile.
Which enzyme makes retinoic acid?
RALD: Retinal dehydrogenase
What function does luminal mucus serve?
Where does it comes from?
- Physical barrier against bacteria
- Matrix for IgA
- Contains decoy molecules that further interfere with bacterial invasion
Secreted by goblet cells
List factors that limit exposure of bacteria to the gastric mucosal epithelium
- IgA
- Mucus
- Anti-microbial peptides (defensins, cathelicidins)
Describe ILFs
Where are they found?
Isolated lymphoid follicles
• Organised lymphoid structure
• Contain predominantly B cells and CD4 T cells
• Contain M cells and follicle associated epithelium
• Role in production of antigen specific IgA
Distribution:
• Small and large intestine
Describe the relationship between M cells and lymphocytes in PPs
Intimate contact between M cells and B, T and DCs at basolateral surface
Compare antigen sampling across various sites in the gastric mucosa
- PPs
• Sampling performed by M cells - Villi
• Sampling performed by LP-DCs
Which antibody is secreted into the intestinal lumen?
- IgA (dimer)
* IgM (pentamer)
What are the functions of sIgA?
- Neutralisation of toxins / enzymes
* Inhibition of microbial adherence
