Intestinal Ecosystem & Immunology Flashcards
What are the different parts of the intestine?
- Duodenum (nutrient digestion)
- Jejunum (nutrient digestion)
- Ileum (more complex microbiome)
- Colon w/ caecum and appendix (more complex microbiome)
What are the 3 main function of the intestine?
- Absorption
- Excretion
- Providing a residence for the commensal microbiome → has an immune impact in priming and educating the immune system
What is the importance of the microbiome in the intestine?
Colonization resistance: all microbes living in the intestine take up space and nutrients, preventing accessibility for incoming pathogens
Why is the microbial load so much higher in the bottom of the intestine than the top?
What are the metabolites produced in the top vs bottom?
Top (and stomach) → highly acidic for nutrient digestion, too acidic for microbial growth
- Vitamin A and AHR ligands
Bottom → high microbial load, complex microbiome
- Short-chain fatty acids (SCFAs) → produced by gut bacteria through the fermentation of dietary fiber, are key immunomodulatory factors, impacting the host’s immune response and overall health
How the the intestinal topography change from the duodenum all the way to the colon?
Duodenum → nutrient absorption
- More rigid and diversed topography
- Increased surface area for absorption
- Thin mucus layer
Ileum/Colon
- Smoother
- Water absorption & movement of the bolus
- Thin inner mucus layer + Thick outer mucus layer
*All of 1 layer of epithelial cells, which if dammaged exposes the inside of the body to many many pathogens and microbes
- Protection by mucosal layer + microbial peptides & antibodies sitting in it
What is the epithelial escalator? How does it relate to colon cancer?
The epithelial escalator is the constant movement of epithelial cells from the crypts to the villi, to then be turned over
- Part of the weep and sweep response
- Stem cells at the bottom of the crypts give rise to diversed lineages of epithelial cells → absorptive, secretory (ex: goblet cells), etc.
Mutations don’t really accumulate as cells are turned over so quickly. Cells causing colon cancer must be retained in the crypts → block the turnover
What is the immune cell presence like across the intestine?
- Antigenic content goes down across the intestine (almost 0 at the Ileum)
- macrophage content is pretty high throughout
- Eosinophils & Mast cells are highest in the duodenum and go down (increased inflammatory and tissue repair functions)
- Tregs are higher in the large intestine
- Th1 cells are medium throughout
- Th17 cells are highest in the duodenum and jejunum and medium after
*More immune cells in the intestine than all other organs combined
What are the 3 types of immune responses? (And their respective pathogenic defense functions)
Type 1:
- IFNy
- protective against viruses and intracellular pathogens
- Th1, NK, gd T cells
Type 2:
- protective against extracellular pathogens, can cause allergies (Ex: helminths)
- IL-13, Th2 cells
Type 3:
- IL-17
- against extracellular pathogens (ex: bacteria, fungi)
- Th17, gd T cells
What are the organ function specific roles of the 3 types of immune responses?
Type 1 → Vascular integrity & tissue remodelling
Type 2 → Wound healing / tissue repair
Type 3 → Epithelial barrier integrity
- Antimicrobial peptides
- Stem cell differentiation
*These may have evolved even before the host defense functions
What study was done on early human life immune-microbiome?
They wanted to characterize immune system development from birth to death and relate it to changes in microbiome throughout life
Took stool samples (microbiome) and blood drops (blood) every single day from day 1 of life
What are gd T cells?
They are T cells expressing a TCR, but they also have an innate like function and can be activtaed by inflammatory cytokines without engagement of the TCR
What was observed in the human immune-microbiome study at the time of switch between breast feeding and real food? (~8 weeks?)
In the blood:
Increase in total gd T cells, specifically CD161+ gd T cells (associated with a population living in the lower intestine)
Increase in IL-17 (Type 3 response)
- Associated with the exposure to new pathogens of a real diet? disruption of the homeostasis?
In the stool:
Shift in microbiome at the same time period
Proposition from this data: IL-17 response was pathogenic and if we can prevent this response, if may protect us from the development of diseases later in life
How did the mouse colon change between week 2, week 3 and week 10?
How did the intestinal bacterium change?
*Week 3 = weaning (breast fed → normal diet)
Weight to length ratio increased significantly (more density) between week 2 and week 3
- Increase in epithelial, mucus, presence of immune cells
They than did RNAseq to characterize these changes → gut transcriptome changes drastically (PCA analysis)
Exponential increase in bacterial abundance between weeks 2 → 3 + big change in bacterial transccriptoe (rDNA sequencing)
What cytokine increases in the colon during early life in mice?
How does it correlate to colon size?
IL-17 (same as human)
- No change seen in type 1 and 2 responses
- IL-17 is much higher at weaning than as adult baseline → type 3 immune response at the time of weaning
- Produced by gd17 T cells
IL-17 KO mice have smaller weight to length ratio
Conclusion: IL-17 is important for rapid development of the gut in early life
How did they atest that early life gd17 T cell activation is microbiome-dependent?
Made germ-free mice (treated with antibiotics) → measured cytokine production at weaning → absence of IL-17 production
They than colonized microbiomes with different microbial sources from different facilities → only microbiome from this facility induced IL-17 response
Suggests some specific microbes are responsible for inducing this IL-17 response
How can the early life IL-17 microbiome-dependent immune response have implication in adulthood? What experiment tested this?
Induced colotis in mice → gave them Dextran sodium sulfate (DSS) which degraded the colon barrier
Colitis was more pathogenic for mice which had early life IL-17 responses (lost more weight)
What is an example of the gut-brain axis?
The gut produces up to 90% of serotonin in the body which is a potent regulator of brain activity/state
What different organs are comprised/refered to in mucosal immunity?
What are the Anatomical/Physical barriers related to them?
Comprises the «surface exposed» parts of the body (gut, mouth, genital tract, lung, eye, etc.)
Skin → acidic,anti-microbialpeptides
Mouth → enzymes,anti-microbialpeptides, directional flow of fluid to stomach
Stomach → lowpH,digestiveenzymes,fluidflowto intestine
Largeintestine → normalfloracompeteforniches
Airways & lungs → cilia,mucus,coughing/sneezing, macrophages in lung alveoli
Why is mucosal immunity important to study appart from systemic immunity?
Systemic immune system = spleen, LN, blood, lymph
Mucosal immunity = surface exposed parts of the body
- Most infections start at mucosal surfaces
- Most evolutionarily ancient
- Enormous antigen load of the mucosa
What are the main induction sites of the «common» mucosal immune system?
BALT = bronchial associaed lymphoid tissues
GALT = gastrointestinal associated lymphoid tissues
What are unique features of the mucosa immune system?
- Close association of mucosal epithelial layer and lymphoid structures (less organized than 2ndary lymphoid organs and rest of immune system, more diffuse)
- Specialized Ag-uptake mechanisms (ex: Microfold cells in the gut)
- Effectors
- IgA
- Specialized T cells, activated even without infection - Strong immuno-regulatory envrionment → tightly regulated responses to commensals
What are the different Mucosal-Associated Lymphoid Tissue (MALT)?
- GALT (gut) : Peyer’s Patches (PP), Cryptopatches (CP), Isolated Lymphoid Follicles (ILF), Lamina Propria (LP)
- NALT (nasopharynx) : Adenoids, Tonsils
- BALT (bronchial) : iBALT
- Genital tract → not as well kown, no lymphoid follicles
What are features of Peyer’s Patch?
- Secondary lymphoid organs within wall of intestine
- Rich in B cell follicles, smaller T cell areas
- Surrounded by epithelium interspersed with «microfold» M cells
What are some innate defense mechanisms of the mucosal immune system?
- Barrier function
- Proteolytic enzymes
- Antimicrobial molecules
- Commensal organisms
What are features of the barrier function of innate defense of the mucosal immune system?
- 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. - Epithelial Cell Tight Junctions → prevent the passages of macromolecules through the single epithelial layer
What is Zonulin?
Protein that regulates intestinal permeability by modulating tight junctions/inducing tight junction disassembly
- Impacted in various diseases such as celiac disease
- Increased zonulin levels → loosening of tight junctions (more permeable tight junctions)
- Structural and functional homology with toxin secreted by Vibrio cholerae → loosens tight junctions
How do proteolytic enzymes act in innate defense of the mucosal immune system?
- 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.
**T cell epitopes need specific lengths of peptides to be presented on MHC. If the nutrients are broken down fast enough and short enough, they are not antigenic/immunogenic anymore (too short)
What are 3 major antimicrobial molecules of the innate defense of mucosal immune system?
- Lactoferrin → binds iron and inhibits bacterial growth
- Lysozyme → cleaves cell wall of gram positive bacteria
- Defensins → 30-40 aa peptides that disrupts the cell membranes of bacteria and fungi causing lysis (punch wholes into bacteria → causes them to burst/cytosolic release
How are commensal organisms an important part of the innate defense component of the mucosal immune system?
- > 400 species of commensal bacteria
- Provide enzymatic break down of food
- Competes with pathogenic bacteria for space and nutrients
- Prevents colonization of the gut
- Antibiotics disrupt homeostasis
**When taking antibiotics, helps to clear the pathogen, but also clears commensal bacteria which are good for gut health (not great)
What are 4 important differences between GALT and petipheral Lymphoid tissue?
- Unique epithelium for antigen uptake
- Unique lymphocyte repertoire
- IgA dominated humoral response
- A need to minimize injury to the mucosal tissue while providing protection
What are functions of M (microfold) cells?
- Transport antigens across the mucosal surface → via vesicles «Trancytosis»
- M cells take up antigen by endocytosis and phagocytosis
- Extensively folded cell membrane - large surface area
- Can be exploited by pathogens to gain entry into
Creates a «pocket» for DCs, lymphocytes Ag uptake on basolateral side → DC’s migrate to T cell areas in PP or mesenteric LNs
How are luminal antigens taken up for T cell presentation?
Luminal antigens are taken up by M cells and presented to T cells by macrophages
- DCs can reach (extend processes across epithelaial layer) through and direclty sample Ag from gut lumen
**Unique mechanism
What is the importance of IgA in the mucosal immune system? What form are they found in?
The dominant class of Ab in the mucosal immune system
- Can prevent pathogen adherence to epithelium → block the adhesion sites before they bind to the receptors on epithelial cells to get internalized
Can exist in multiple forms:
- Serum-IgA is mostly monomer
- Mucosa → IgA is dimer linked by J chain (joining peptide chain)
- After transcytosis, associated with secretory component
B cells (in ILF and PP) have to undergo class switching to be able to produce IgA, B cells in different regions release dimers/monomers → mediated by TGF-b !!!
Certain pathogens can cleave IgA as a defense mechanism → cleave in the hinge region (ex: influenza, pneumonia, meningitidis, gonorrhoeae, etc.)
What type of Antibody is majoritarily produced in mucosal tissue vs systemic tissue? What is the daily total production rate of IgA vs IgG?
Mucosal tissue → 80% IgA, 13% IgM, 6% IgG
Systemic tissues (BM, LN, spleen) → 52% IgG, 30% IgA, 17% IgM
IgG daily total production rate → 3000 mg (up to 5 grams)
IgA daily total production rate → 4800 - 9000 mg
How does IgA get across the epithelial layer to get to the lumen?
- IgA released as dimer in Lamina Propria binds to the Poly-Ig receptor on the basolateral surface of epithelial cells
- Internalized → vesicular transport (trancytosis)
- Release of IgA dimer at the apical face of epithelial cell (IgA dimer bound to secretory component)
- When tethered to «secretory component» = sIgA
What is an important marker of IgA-secreting B cells?
IgA-secreting B cells express a4b7, which binds to MAdCAM-1 and directs them to mucosal tissues
What are 3 examples of cytokines expressed by epithelial cells to attract lymphocytes?
Chemokine - Receptor (on lymphocytes)
CCL25 - CCR9 → small intestinal epithelium and endothelium
CCL28 - CCR10 → Salivary glands, trachea, bronchi, mammary glands, stomach, large intestine, SI, BM
CCL20 - CCR6 → Peyer’s patch subepithelial some and inflamed epithelium
What process/migration cycle do T cells undergo in the mucosal immune system?
- T cells enter Peyer’s patches from blood vessels, directed by homing receptors CCR7 and L-selectin
- T cells in the Peyer’s patch encounter antigen tranposrted acress m cells and become activated by DCs
- Activated T cells drain via mesenteric lymph nodes to the thoracic duct and return to the gut via the bloodstream
- Activated T cells expressing a4b7 integrin and CCR9 home to the lamina propria and intestinal epithelium of small intestine
What is the general pathway of the Common Mucosal Immune System?
Antigen presentation in Peyer’s patches → Mesenteric LN → (Thoracic Duct) → Blood Stream → Other mucosal organs (Resp. tract, Breast, Intestinal mucosa, Urinary tract, Salivary/Lacrimal gland)
Inductive Site = Afferent Limb, Peyer’s Patch (Antigen entry)
Effector Site = Efferent Limb, Lamina Propria Lymphocytes and Plasma Cells (Other immunized areas)
What are the different populations of Innate vs Adaptative T lymphocytes in the GALT?
Innate T lymphocytes:
- Intraepithelial → limited TCR repertoire, Ag-specific
- Innate lymphoid cells → no TCR, lineage negative, no Ag-specificity
Adaptative T lymphocytes: (MHC-restricted)
- Mostly CD4+ T helper cells; few CD8+ T cells
- Th1, Th2, Th17/22, Th9, Treg (LOTS), Tfh
- Ag-specific
What are features of intraepithelial lymphocytes?
- Strategically located to respond to antigenic stimulation
- Mostly CD8+ T cells
- Mainly ab TCR in humans // yg TCR in mice
- Intercalated in between epithelial cells above peyer’s patches
Features of 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
*Hard to study because it’s a rare population
How are intraepithelial CD8+ T lymphocytes involved in recognition of self-stress antigens?
Cell next to intraepithelial lymphocytes is not functional anymore → induces expression of MIC-A/MIC-B → recognized by IEL CD8+ T cells which induce apoptosis of the non-functional neighbouring cell (via perforin/granzyme pathway)
NKG2D on IELs bind to MIC-A/-B and activate IEL
what is CD45RO a marker of?
Marker of memory phenotype of mucosal T cells/IEL
Gut homing markers → CCR6, CCR9, a4:b7 integrins
What type of T cells are mainly found as Intraepithelial lymphocytes vs Adaptative T lymphocytes?
IEL → mostly CD8+ T cells
Adaptative T lymphocyte → CD4+ T cells
What are general features of GALT Adaptative T 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
What Adaptative T lymphocytes (CD4 T helper cells) are associated with the 3 different types of immune response?
And what are the 2 other phenotypes of adaptative T lymphocytes?
Type 1 - Intracellular pathogens (bacteria, parasites, viruses)
Th1 → secrete IFNy, TNFa
Type 2 - Worms, allergens
Th2 → secrete IL-5, IL-9, IL-13, IL-4 (humans)
Type 3 - Extracellular pathogens (bacteria, parasites, fungi)
Th17/22 → IL-17, IL-22
Follicular helper T cells (Tfh)
Tregs
What transcription factors are associated with differentiation of Th1 vs Th2 vs Th17/22?
IL-12, Type1 IFN, TNFa → T-bet → Th1
TSLP, IL-25, IL33 → GATA3 → Th2
IL-1, IL-6, IL-23, TGFb → RORyt → Th17/22
The cytokines that induce TF are produced DCs
What is IPEX?
Severe enteropathy caused by mutations in the Foxp3 gene that result in the defective development of CD4+CD25+ regulatory T cells
*CD25 = alpah chain of IL-2 receptor
What cytokine is required for Treg differentiation?
What is the importance of Tregs in the gut?
TGF-b
*Tregs respond to TGF-b and can produce it
Transfer of Tregs into mice with IBD can lead to resolution of colitis (lack of Tregs → colitis/inflammation in the gut)
What are the most important regulatory cytokines?
What is their effect?
IL-10
- Increased production of IgA
- Decreased cytokine production by DC, T-cells, macrophages
- Promotes TH2 response
- IL-10 knockout mice: severe enterocolitis
TGF-beta
- Increased IgA by promoting class switching
- Maintain functional CD4+CD25+cells in the periphery.
IL-10 and TGF-beta (TGFb) are involved in regulating B cell class switching and IgA production, with IL-10 inducing TGFb secretion and TGFb receptor upregulation, ultimately leading to increased IgA secretion
Priming T cells in the draining Lymph Nodes for adaptative immune response takes 3-4 days. Signature cytokines appear much before that. What cells are responsible for their production if they are not adaptative T lymphocytes?
How where these discovered?
Innate Lymphoid cells (ILC)
Visualized with reporter mice (fluorescent protein fused to specific signature cytokine promotors to visualize the cells that produce them)
What are general features and functions of Innate Lymphoid cells? (ILC)
How are they regulated?
General Features:
- Population induced at steady state & inflammation/infection states
- Lymphocyte morphology
- Common developmental origin - common lymphoid progenitor (CLP) ?
- Lack of rearranged antigen receptors
- Important sources of Th cell signature cytokines (IFNg, IL-5/IL-9/IL-13, IL-17A/IL22) Specific to type of immune response
Function:
- Innate immunity against microbes, immune surveillance (tumors)
- Lymphoid tissue formation, remodeling and repair
- Epithelia/Stroma function, homeostasis, remodeling and repair
- Strong immunopathology
- Many aspects unclear!!
- Often in close contact with epithelial layer
- Dysregulation leads to chronic inflammatory diseases
Regulation:
- Induced by the same/overlapping cytokines as T helper cells
- No MHC restriction (?) Co-stimulation (?)
What are the 3 main subclasses of ILCs? (Innate Lymphoid Cells?)
Type 1 → ILC1 (includes NK)
Type 2 → ILC2
Type 3 → ILC3 (also includes LTi cells)
*Other subsets as well inside these 3 groups
What are effects of the activation of ILC2 innate lymphoid cells?
Activated ILC2s, a type of innate lymphoid cell, produce cytokines like IL-4, IL-5 and IL-13, which promote type 2 inflammation, eosinophilia, mucus hypersecretion, and tissue remodeling, contributing to conditions like asthma and allergic diseases.
- Muscle contraction (IL-13)
- IL-13 → Goblet cell activation → mucus hypersecretion
- IL-4 → Th2 cell differentiation
- IL-5 → eosinophil chemoattractant
What are effects of the activation of ILC3 innate lymphoid cells?
Response to extracellular pathogens (bacterial infection) → ex: Helicobacter, Citrobacter, E. coli → trapped by DCs → secrete IL-23 to activate ILC3s
NCR- ILC3s → secrete IL-17
NCR+ iLC3s → secrete IL-22
Associated with neutrophil chemoattraction → professional phagocytes
IL-22 → production of antimicrobial peptides and proteins by epithelial cells
What are LTi cells?
A subtype of Innate Lymphoid cells
LTi (lymphoid tissue inducer) cells → essential for the development of lymph nodes and Peyer’s patches during embryonic development, and they are also involved in maintaining lymphoid tissue architecture and promoting innate immunity.
LTi cells constitutively produce lymphotoxin (LTα1β1) and tumor necrosis factor (TNF) → binds to LTbR on stromal cells which → stimulate mesenchymal cells to produce chemokines and adhesion molecules essential for lymphoid organogenesis
Due to chemokine production, they also attract naive B and T cells
What are Type I vs Type II mucosal surfaces?
Type I → covered by simple epithelium - expresses a simple polymeric Ig receptor (pIgR) that allows dimeric IgA to access the lumen
- Intestine, lungs, uterus
Type II → covered by stratified squamous epithelium which provides physical protective barriers for activities that are important for the host species
- Oral cavity, Vaginal cavity
What are the main important take away features of GALTs?
- Mechanism = Antigen Sampling
- PP locations (ILF, cryptopatches)
- Gut is the organ with the most lymphocytes in the whole body
- IgA is the dominant Ab (requires class switching of B cells by TGF-b + transport through epithelial layer by PIgR)
- Signature cytokines (Th, ILC)
What are the main features/functions of the commensal bacteria?
*More bacteria/microbes than cells in our body
Commensal = microbes in our body, but do not treaten us
- Prevent colonization by more pathogenic species → compete for space and nutrients
- Produce metabolites (ex: vitamins) that are used by the host
- Colonization of the gut begins immediately
after birth
– 10^13-10^14 microorganisms
– 400 to 500 different species
– Majority are obligate anaerobes → work in anaerobic conditions - Negative effects on normal bacterial flora may explain the rise of immune disorders (allergies and IBD) → antibiotics may help these patients
*We still need protection/barrier because if we put them in the blood, we would dye from sepsis due to huge inflammatory cascade
What are some beneficial effects of indigenous GI microflora? (How has the body evolve to adapt to them in a good way?)
*The gut has adapted to the presence of these commensal microbes → evolved to utilize them
- Formation of anatomical structures (Peyer’s patches)
- Expansion of germinla center reactions involving B and T cells
- Increased IgA production by intestinal B cells
- Expansion of IEL populations
- Bacterial antagonism
- Maintain GI tract peristalsis and intestinal mucosal
integrity - Convert dietary precarcinogens and carcinogens to
noncarcinogens - Synthesis of vitamin K and vitamin B complexes
**However, translocating bacteria can cause infections in debilitated patients
Some diseases are linked to imbalance in microbiota. What have these actually been found to relate to?
They often relate to the metabolites produced by the microbiota more than the actual composition of the microbiota → which comes down to what we eat
Ex: Cancer, obesity and metabolic syndrom, IBD, cardiovascular disease
What is the difference in the gut histology of germ-free vs conventionally raised mice?
Germ-free:
- Tinner/longer finger-like villi
- Lower number of cells
- Less IgA
- Decreased mucus thickness / altered mucus properties
- Less vascularized
