Human Microbiome

MALT - def

mucosal associated lymphoid tissue

GALT - def

gastrointestinal associated lymphoid tissue

structure of gastrointestinal system (4)

• 3-4m long

• organised into folds which form many projections → villi

• villi are lined by epithelial cells (enterocytes) with microvilli on apical surface

• lamina propria = area under epithelial cell lining where lots of immunec ells found

important cells of gastrointestinal system (3)

• Cell division occurs in crypts

• Goblet cells = found between epithelial cells and secrete mucous, lysoszyme and lactoferrin -> protects epithelial cells from bacterial adhesion

• Paneth cells = secrete small molecules and peptides (defensins) -> protect against microorganisms

innate defences - peristaltic action

continuous contraction of muscles to prevent microorganism to adhere to and penetrate epithelial cells

innate defences - list

1. peristaltic action

2. secretion of acid

3. mucous layer

4. enterocytes, goblet cells and paneth cells

5. macrophages and dendritic cells

6. innate lymphoid cell populations

innate defences - enterocytes, goblet cells and paneth cells (4)

• Tight junctions between enterocytes

• Regular replacement enterocytes

• Antimicrobial factors -> bile, defensins, lactoferrin, complement

• Cytokines and chemokines produced by enterocytes and other cells

how does adaptive immune system of mucosa differ from that of systemic - list (4)

• Lymphoid tissue

• Antigen access

• Lymphocyte activation and circulation

• Secretion of antibodies

location of lymphocyte activation (3)

• Peyer's Patches -> collections of B and T cells and APCs

• (Mesenteric) Lymph nodes (MLN)

• Antigen accessed directly from mucosal tissues

effector lymphocytes - location

found in lamina propria → lymphocytes scattered throughout tissue

migration of immune cells from Peyer’s patches

no direct traffic from Peyer’s patches to lamina propria → all cells leave via lymphatics to bloodstream

mechanisms for controlled antigen access - list (2)

1. peyer’s patches

2. M cells -. microfold cells

mechanisms for controlled antigen access - Peyer’s patches

epithelial layer that line svili continues over the top of Peyer’s patches → cells that cover dome of patch = follicle associated epithelial cells

mechanisms for controlled antigen access - M cells (4)

• Found in between epithelial cells and cover area of lymphoid follicles

• Derived from enterocytes but no microvilli on surface and no glycocalyx

• Do not express MHC-II -> cannot activate adaptive immune cells (CD4+ cells not activated)

• Sample antigen from lumen and transport antigens from lumen to subepithelial space (Peyer's Patch)

M cells - consequence of no microvilli and no glycocalyx

Some microorganism deliberately target M cells to gain access to basal lamina -> easier to get to cell surface and trigger uptake because no microvilli and no glycocalyx

Eg. Salmonella

innate defences - dendritic cells in mucosa

Can extend processes across epithelial layer to capture antigen from lumen of gut → present antigens in Peyer's patches

location of naive lymphocyte activation -list (2)

• Peyer's patches -> APCs take up antigens delivered by M cells

• Mesenteric lymph nodes (MLN) -> APCs migrate from Peyer's patches or from lamina propria

location of antigen uptake by APCS - list (2)

• Peyer's patches -> DC present to local T cells or in mesenteric lymph nodes

• Lamina propria -> DCs directly sample antigen and migrate to mesenteric lymph node where they activate local T cells

how are mucosal dendritic cells specialised to direct T cell and B cell activation - list (4)

1. Cytokine production skews towards T reg (TGFß) and Th2 pathway

   • Under inflammatory conditions, may induce Th1 and Th17

2. Bias B cell isotype switching to secretory IgA

3. Induce mucosal integrin on activated lymphocytes -> binds mucosal endothelial addressin MAdCAM1

4. Induce receptors for mucosal chemokines -> lymphocytes activated in mucosa can migrate back to mucosa

“homing” system for activated mucosal lymph nodes to return to their site of action

immune system uses molecule postcodes to direct cells to specific tissues → billions of lymphocytes in random circulation so would be inefficient and different tissues require different immune populations

Naïve lymphocytes migrate between lymph nodes and blood stream

Activated lymphocytes migrates from lymph node -> blood stream -> tissues via complimentary receptors

IgA production - steps (4)

1. B cells encounter antigens in Payer's patches -> gut lumen antigens enter via M cells

2. APCs and T cells provide appropriate signals for B cell -> proliferation and IgA production via TGFß secretion

3. Lymphocytes acquire homing receptors  integrins and chemokine receptors specific for lamina propria chemokines

4. Activated IgA producing B cells enter blood -> bind endothelium of lamina propria and secrete IgA into mucosal lumen

structure of IgA in lamina propria vs circulation

In lamina propria: IgA found as a dimer -> monomer in circulation

Monomers joined together by J chain

secretion of secretory IgA - steps (4)

1. Binding of IgA to polymeric Ig receptor (pIgR) on basolateral face of epithelial cell

2. Endocytosis

3. pIgR binds to J chain -> pIgR/ dimeric IgA molecule transcytoses to apical face of epithelial cell

4. Release of IgA dimer at apical face of epithelial cell -> part of pIgR remains associated with dimeric IgA (secretory component, SC)

SC helps protect secretory IgA by anchoring it in mucous and protecting from proteolysis

J chain in IgM

Pentameric IgM also held together by J chain and secreted via pIgR -> secretory component (SC) helps anchor secreted Abs to mucin and protects from degradation

functions of secretory IgA - list (6)

1. neutralisation on surface of epithlelium → block epithelial attachment and toxin attachment

2. Does not trigger complement cascade efficiently -> reduce inflammation and weak opsonin

3. neutralisation inside epithelial cells → eg. rotavirus, HIV

5. antigen export from lamina propria to lumen -> avoid some immune responses

6. Long half life because secretory component protects against enzymatic degradation

importance of secretory IgA not activating complement efficiently - summary (2)

• Inflammation impedes nutrient absorption and promotes leaky gut -> loss of fluids

• Inflammation can also damage the gut and offer opportunities for pathogens to enter tissues

homing molecules for recirculation of acivated lymphocytes to lamina propria via blood stream - list (3)

• lymphocytes → a4b7

• endothelial cells → MadCAM

• chemokine receptor → CCR9

• chemokine → CCL25

normal human mcirobiota - summary (3)

diverse group of microorganisms that live with us from birth until death

each site had high level of diversity -> unique site-specific fingerprint

numbers also vary by site

Eg. anaerobes in stomach are rare in stomach but present in intestines

factors affecting microbiome

• age → increase in diversity with age

• mode of delivery → babies delivered by C-sec have restricted diversity

• breast feeding

• diet → major determinant of diversity and relative numbers

• antibiotics → relatively short-term use can have long-term changes

stable in a healthy host except at extremes of life

benefits of gut microbiome - list (3)

1. Enhance host digestive efficiency by degrading polysaccharides

   • Synthesise essential metabolites

   • Break down plant fibres in food

   • Inactivate toxic substances in food or made by pathogens

2. Intestinal development -> epithelial cell maturation, angiogenesis, lymphocyte development

3. Protection against pathogenic microbes -> competition for nutrients and commensals stimulate the immune system

benefits of gut microbiome - example aids for metabolism (3)

• Synthesise essential metabolites

• Break down plant fibres in food

• Inactivate toxic substances in food or made by pathogens

impact of intestinal microflora on gut immune system - list (4)

• Mucous layer

• Intestinal epithelial cells

• Development of lymphoid structures

• Development of lymphocyte subsets

direct effects of microflora on innate immunity - list (3)

• Block binding/ interaction with epithelial cells

• Production of bacteriocins and other antimicrobial peptides

• Competition for nutrients

indirect effects of microflora on innate immunity - pathways list (2)

1. via interactions with PRR expressed on epithelial cells

2. via metabolites (short chain fatty acids) produced by microflora

indirect effects of microflora on innate immunity - via interaction with PRR expressed on epithelial cells (2)

• PAMPs/ butyrate signalling stimulates mucin production by Paneth cells

• PAMP signalling stimulates proliferation of crypt enterocytes and Paneth cells, release of antimicrobial peptides, induction of regulatory cytokines and IL-22 prodcution which supports epithelial barrier integrity

indirect effects of microflora on innate immunity - via metabolites produced by microflora (2)

short chain fatty acids inhibit NF-κB and production of inflammatory cytokines

Maintain epithelial integrity

evidence of microbiota’s effects on immune system - summary list (3)

1. germ free animals

2. antibiotic treated animals

3. humans

evidence of microbiota’s effects on immune system - germ free animals summary (5)

• Lower levels of IgA

• Poorly developed Peyer's patches

• Fewer intra-epithelial lymphocytes (IELs)

• More susceptible to infection

• Prone to developing allergic responses -> poor Treg, unwanted Th2 differentiation

evidence of microbiota’s effects on immune system - antibiotic treated animals summary

impaired T cell response against transplants

evidence of microbiota’s effects on immune system - humans summary (4)

• Peyer’s patches and MLN develop prenatally byt lymphoid follicles in colon develop postnatally → Peyer’s patches further develop after birth

• neonates do not produce IgA → commensal flora induce low levels of specific IgA which can cross react with pathogen antigens and provide protection

• develop tolerance to commensal flora through induction of T reg cells

• helminth infections protect against inflammatory conditions through induction of T reg cells

how are interactions between the PRR of epithelial cells and commensals prevented without impairing recognition of pathogens (3)

1. PRR are restricted on the apical membrane →

2. Commensals are found at luminal edge of mucous layer → dont interact with PRR to induce immune response

3. epithelial PRR will detect invasion via TLR5 at basolateral surface or via intracellular PRR if cell damaged

impact of immune response induced by normal microbiota via dendritic cells in the gut (2)

prevents strong immune response via promoting Treg

promotes immunity via promoting Th2 → supports isotype switching to IgA

impact of immune response induced by normal microbiota via dendritic cells in the gut - via epithelial cells (3)

1. Microbiota metabolites and retinoic acid act on epithelial cells

2. Epithelial cells produce TGFß and IL-10

3. TGFß and IL-10 create environment which influences T cell differentiation to promote Treg

impact of immune response induced by normal microbiota via dendritic cells in the gut - via dendritic cells (2)

1. Microbiota metabolites can directly act on DC

2. DC activates naïve T cells and can induce Treg and Th2 response via cytokines

maintenance of olerogenic DC

intact epithelial lining restricts exposure to antigenic pathogens

physiological vs pathological inflammation

• Physiological inflammation in response to commensals = inducing an immune response in a way that doesn't cause any damage

• Pathological inflammation = inducing immune response to pathogenic bacteria that can cause damage

important differences between systemic and mucosal immunity

systemic: surface wound introduces bacteria that activate macrophages to produce inflammatory cytokines

mucosal: bacteria in lamina propria via endocytosis activate macrophages but do not cause inflammation

dysbiosis - def

out of balance microbiota

directly and indirectly associated with disease

can becaused by usage of broad spectrum antibiotic use

dysbiosis - direct associations with disease (3)

• Infectious disease caused by pathogenic invaders → eg. Salmonella spp.

• Infectious disease caused by overgrowth of gut microbiota → eg. Candida or Clostridium difficile

• Nutritional -> malabsorption, steatorrhea, vitamin deficiencies

dysbiosis - indirect associations with disease (5)

• Obesity and metabolic syndromes

• Inflammatory bowel diseases

• Allergies

• Autoimmune diseases

• Undernutrition

• Others -> eg. colon cancer

clostridium difficile - summary (4)

• pseudomembranous colitis

• carried by approx 3% of healthy people → higher in neonates

• overgrows when gut microbiota is altered → eg by antibiotics or cytotoxic drugs

• may spread in hospitals → some patients develop recurrent C. diff infections

clostridium difficile - pathogenesis via antibiotic use (5)

1. Colon is colonised by large numbers of commensal bacteria

2. Antibiotics kill many of the commensal bacteria

3. C. Difficile gains foothold to adheres to epithelium and produces toxins that cause mucosal cell death, inflammation, and bowel necrosis

4. Neutrophils and RBCs leak into gut between injured epithelial cells

5. Connective tissue degradation leads to colitis and pseudo membrane formation

cause of recurrent C. diff infections

can be caused by reduced diversity and microbiota -> studied via analysis of faecal microbiota in patients with antibiotic associated diarrhoea due to C. difficile

Dysbiosis can make an individual more susceptible to C. difficile infections

treatment of persistent dysbiosis due to C. diff exposure (2)

bacteriotherapy → faecal microbiota transplant (FMT)

currently developing a licenced therapeutic to avoid transplant

treatment of persistent dysbiosis due to C. diff exposure - developement of licensed therapeutic steps (4)

1. Faecal extract

2. Rational selection of bacteria

3. Clinical trials

4. Licensed therapeutic

impact of diet on microbiota - case studies (2)

1. gnotobiotic mice with increasing protein diet → altered microbiota in faeces

2. twin studies on prolonged undernutrition → altered celular pathways and therapeutic diet insufficient for sustaining increased microbiota diversity

impact of diet on microbiota - gnotobiotic mice study summary

Gnotobiotic mice given diet with protein (casein), sucrose (simple sugar), corn oil (fat) and cornstarch (polysaccharide) and increasingly given more protein

Altered microbiota in faeces

germ free lab animals and energy intake requirements

require higher energy intake to maintain body weight in lab experiments

implications of germ free lab animals requiring higher energy intake on possible roles of microbiota (2)

1. Directly provide nutrients from dietary substances → eg. short-chain fatty acids from mucins, vitamin K, biotin, folate, CHO from plant sugars

2. Microbiota can alter the metabolic machinery of host cells by:

• Inducing changes in host genes involved in CHO and lipid metabolism

• Maintaining enterocyte differentiation and function -> short-chain fatty acids

metabolic activity of intestinal microbiota - examples list (4)

1. carbohydrates

2. vitamines

3. bile acids

4. amino acids

metabolic activity of intestinal microbiota - carbohydrates summary (4)

lactose especially in early life

cellulose

mucins broken down to short chain fatty acids

contributes 10% of calories

metabolic activity of intestinal microbiota - vitamin examples (4)

Vitamine B3

Vitamine K

biotin

folate

metabolic activity of intestinal microbiota - bile acids summary

dihydroxylation of cholic acid in gut where desoxycholic acid allows resorption back into liver → enterohepatic circulation

metabolic activity of intestinal microbiota - amino acids examples (3)

Break down of urea to produce ammonia → allows nitrogen recycling

produce lysine and threonine

metabolic pathways provided by microbiota - list (5)

• Bacterial degradation of host glycans and elicits synthesis of new glycans by host

• Produce short chain fatty acids from undigestible carbohydrates that maintain enterocyte differentiation

• Induces changes in host genes affecting angiogenesis

• Induces changes in host genes involved in CHO and lipid metabolism

• Induce changes in host genes that contribute to adiposity

impact of diet on microbiota - twin study aim

healthy twins and twin pairs discordant for Kwashiorkor to determine variability in microbiome genetic level over defined period and investigate effect of therapeutic food on microbiome

impact of diet on microbiota - twin study finding summary

Overall gene content of faecal microbiota in kwashiorkor-affected children fails to develop with increasing age

Therapeutic food did not sustain an initial change in gene content for kwashiorkor co-twin -> maintained for heathy co-twin

impact of diet on microbiota - twin study to mice transition summary

microbiota is associated with health status → transplantation of faecal microbiota into germ-free mice and feeding mice "Malawian diet" and therapeutic food

measure body weight measurements to indicate undernutrietion

impact of diet on microbiota - twin study to mice transition findings (2)

• Switching diet cases rapid change in microbiota

• Analysis of urinary and faecal metabolites shows inhibition of TCA cycle in mice with Kwashiorkor microbiota on Malawian diet -> indicates impaired cellular metabolism and energy production of host

impact of diet on microbiota - twin study conclusion

Kwashiorkor microbiota generated chemical products that result in selective inhibition of TCA cycle enzymes

effects on energy metabolism for children on Malawian diet