Immunology of the Gut Flashcards
What is the surface area of the gut?
200 m^2
How long is the small and large bowel put together?
8 metres
What does the gut have to deal with?
Massive antigen load:
- Resident microbiota 10^14 bacteria
- Dietary antigens
- Exposure to pathogens
Has a responsibility to recognise, respond to, and adapt to countless foreign and self molecules
In what state is the gut in constantly to deal with the foreign and self molecules?
‘Restrained activation’
Has to balance tolerance (food antigens, commensal bacteria) VS active immune response (pathogens)
Dual immunological role
How are the gut microbiota and its role studied?
Gnotobiology
What is Gnotobiology?
Take germ free animals (e.g. mice)
Selectively colonise them with selective bacteria
Compare and observe differences in these mice to conventionally housed mice (with normal gut microbiota)
e.g. development of peyer’s patches in the small intestine of germ free mice are fewer and less cellular than those with conventional microbiota, paneth cells (important for defence against pathogens) are reduced in germ free mice compared to conventional
How many gut bacteria are there?
10^14 gut bacteria and 10^13 cells in body - most densely populated “ecosystem” on Earth
What are the 4 major phyla of bacteria?
Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria
Why is the gut microbiota important?
Provide traits we have not had to evolve on our own - genes in gut flora 100 times our own genome
Gut flora genes = 100x our own genome
What are their functions?
Provide essential nutrients we cannot manufacture
Metabolise what we find indigestible compounds
Act as a defence against colonisation of opportunistic pathogens
Contribute to intestinal structure
What is meant by the term microbiota?
What is meant by the term microbiome?
Mixture of microorganisms that make up a community within a particular anatomical niche
Collective genomes of all microbiota (of all anatomical niches)
What can increase numbers of microbiota?
Stimulatory factors:
Certain ingested nutrients
Secreted nutrients
What can decrease the numbers of microbiota?
Chemical digestive factors leading to bacterial lysis
Peristalsis and defecation
How does the bacterial content and chemical digestive factors produced by the host (humans) change along the digestive tract?
Stomach =
10^1
HCL, Pepsin and Gastic Lipase
Duodenum =
10^3
Bile acids from liver
Jejunum =
10^4
Trypsin, amylase, carboxypeptidase from pancreas
Ileum =
10^7
Brush border enzymes
Colon =
10^12
No host digestive factors
What is meant by the terms:
Symbiosis
Symbionts
Commensals
Pathobionts
Symbiosis = living together (does NOT imply either partner benefits)
Symbionts = organisms that live within the host without any benefits or harm to one and other
Commensals = micro-organism that benefits from association with the host, but has no effects on the host
Pathobionts = symbionts that do not normally elecit an inflammatory response, but under certain conditions (usually environmental) have the potential to cause dysregulated inflammation and disease
In a normal gut, how are symbionts, commensals and pathobionts balanced?
All equally balanced
________
^
What is dysbiosis?
Altered microbiota composition due to disturbance of the symbionts / commensals / pathobionts
—….__
^
e.g. something allowing pathobionts to start producing pathogens
What are the causes of dysbiosis?
Wide and varied:
Infection and inflammation Diet Xenobiotics - molecules that enter the gut unnaturally e.g. drugs and pollutants Hygiene Genetics
What is associated with dysbiosis?
Pathobionts produce bacterial metabolites and toxins which negatively affect us
What kind of metabolites and toxins can pathobionts produce?
- TMAO - trimethylamine-N-oxide = if increase leads to increased deposition of cholesterol in artery walls
- 4-EPS - 4-ethylphenylsulfate = associated with Autism
- SCFA - short chain fatty acids = if decreased can lead to IBD, if increased associated with neuropsychiatric disorders e.g. stress
- AHR - aryl hydrocarbon receptor ligands = associated with MS, RA and asthma
What is NAFLD?
Non-alcoholic fatty liver disease
What is NASH?
Non-alcoholic steatohepatitis
inflammation from fat
What factors help the gut defend itself?
Physical barriers
Commensal bacteria - occupy ecological niche
Immunological systems following invasion - MALT and GALT
What are physical barriers composed of?
Anatomical =
- Epithelial barrier
- Peristalsis
Chemical =
- Enzymes
- Acidic pH
What forms the epithelial barrier?
Mucus layer secreted by Goblet cells
Epithelial monolayer has tight junctions keeping pathogens out
Paneth cells in the small intestine are found in the bases of crypts of Ueberkuhn = secrete antimicrobial peptides )defensins) and lysozyme
What are the immunological defences of the gut if pathogens do invade into the gut/body?
MALT = mucosa associated lymphoid tissue
GALT = gut associated lymphoid tissue
Where is MALT found?
Found in the submucosa below the epithelium, seen as lymphoid mass containing lymphoid follicles
Follicles are surrounded by HEV (high endothelial venules) post-capillary venules, allowing easy passage of lymphocytes
Oral cavity rich in immunological tissue - esp. palatine and lingual tonsils, and at the back of the mouth pharyngeal tonsils (adenoids)
What is GALT comprised of?
Largest mass of lymphoid in the body
Consists of B & T lymphocytes, macrophages, APC (dendritic cells), and specific epithelial & intra-epithelial lymphocytes
What is GALT responsible for?
Both: adaptive & innate immune responses
In what two ways can the lymphoid tissue exist?
Non-organised
OR
Organised
What are the main features of non-organised GALT?
Intra-epithelial lymphocytes
Make up 1/5th of intestinal epithelium, consists of e.g. T-cells, NK cells
Also found within lamina propria lymphocytes
What are the main features of organised GALT?
Peyer’s patches (small intestine)
Caecal patches (large intestine)
Isolated lymphoid follicles
Mesenteric lymph nodes (encapsulated)
Describe the cells present in non-organised GALT e.g. in the ileum:
Stem cells produce enterocytes that rapidly migrate to apex (top of mirovilli)
Apoptosis of the intra-epithelial cells = apoptotic intraepithelial cells at the apex
Goblet cells formed at the base, they migrate north and produce mucus
Stem cells also produce paneth cells, which produce anti-microbial peptides (AMPs)
Within the epithelium itself, there are intra-epithelial lymphocytes - found lying between peithelial cells
Majority of immunological cells found in lamina propia (central part of villus) - including T cells, B cells, macrophages and DCs
How do the small and large bowel differ structurally?
Small bowel has villi, paneth cells
Large bowel has crypts, many goblet cells and intraepithelial lymphocytes
What is the main type of organised GALT?
Peyer’s patches - ‘immune sensors’
Where are peyer’s patches found?
Found in submucosa small intestine – mainly distal ileum
What are peyer’s patches comprised of?
Aggregated lymphoid follicles covered with follicle associated epithelium (FAE)
FAE contains no goblet cells, no secretory IgA, no microvilli
How are Peyer’s patches organised?
There is the FAE and M cell layer
Beneath the M cell is the peyer’s patch consisting of the:
Sub-epithelial zone - mainly dendritic cells (help transfer antigens from the gut lumen via M cells to the Peyer’s patch)
B-cell follicles
Interfollicular T-cells
All move towards mesenteric lymph nodes
What does peyer’s patches development require?
Development requires exposure to bacterial microbiota
e.g. 50 in last trimester foetus, 250 by teens
How do peyer’s patches work?
M (microfold) cells are found within the FAE
Antigen uptake via M cells within FAE
M cells express IgA receptors - facilitate the transfer of IgA bacteria complex into the Peyer’s patches
What is the route for preventing bacterial invasion separate to M cells?
Via dendritic cells
Open up tight junctions and send dendrites outside epithelium to retrieve / sample bacteria from lumen of gut
Bring back the bacteria and transport them to mesenteric lymph nodes
They are able to do this because they express tight junction proteins e.g. occludin, claudin-1 etc. so maintain the tight-junction barrier after taking sampled antigens from lumen of gut
Recap the M cell stuff:
M cells are found on the FAE layer
There are antigens present in the lumen of the gut
Antigens are taken up via the M cells
Dendritic cells, a type of antigen presenting cell (APC), engulf the antigen and present it on the MHC II molecules on the cell surface
The DCs then migrate to the Peyer’s patch, where there is further immunological response (as the Peyer’s patch contains T cells, B cells etc.)
What is the B-cell adaptive response in peyer’s patches?
Mature naïve B-cells express IgM in Peyer’s Patches
On antigen presentation class switches to IgA
T-cells & epithelial cells influence B cell maturation via cytokine production
Activated B cells further mature to become IgA secreting plasma cells
These populate lamina propria
Some enter lymphatic system
What happens to the immune cells that enter the lymphatic system?
Plasma cells migrate back to enterocytes
These are taken up into epithelial cells
There is enzymatic cleavage
And then they secrete IgA
What is the function of sIgA (secretory IgA)?
Up to 90% of gut B-cells secrete IgA
sIgA binds to luminal antigen preventing its adhesion and consequent invasion
Summarise lymphocyte honing and circulation:
Antigen presents in Peyer’s patch
Activation of T cells and B cells
These are transferred to mesenteric lymph node - lymphocyte proliferation
They then go into circulation via thoracic duct (main lymphatic duct for return to venous system)
Once it enters the venous system, it can enter the peripheral immune system
Or it can also exit back into the intestinal mucosa back to the lamina propria
What comprises peripheral immune system?
Skin
Tonsils
MALT and BALT (bronchus associated lymphoid tissue)
How do lymphocytes and B cells return to the Peyer’s patches?
HEV express MAdCAM1 which is a specialised adhesion molecule
Lymphocytes express alpha-4 beta-7 integrin
Lymphocytes roll along the HEVs until they get tethered by MAdCAM1
Leads to activation and rolling arrests of lymphocytes = migration back to lamina propria
Transports back to Peyer’s patches
Why do enterocytes and goblet cells have such a short lifespan?
Approx. 36 hrs (rapid turnover compared to other cells that last weeks / months)
Enterocytes are first line of defense against GI pathogens & are first to be directly affected by toxic substances in diet / pathoggens
Effects of toxic agents which interfere with cell function, metabolic rate etc. will be diminished
So any lesions that occur are short-lived
What is the mechanism of cholera infection?
Cholera - is an acute bacterial disease caused by Vibrio cholerae serogroups O1 & O139
Bacteria reaches small intestine and when it comes in contact with epithelium, it releases cholera enterotoxin
The cholera enterotoxin gets internalised via retrograde endocytosis into the enterocytes, activating adenylate cyclase
This increases cAMP
That causes active secretion of salt and fluid via the cystic fibrosis transmembrane conductance regulator (CFTR)
Leads to loss of salt, potassium, chloride, bicarbonate and water in the faeces
How is cholera transmitted?
Transmitted through faecal-oral route
Spreads via contaminated water & food
What are the symptoms of cholera?
Main symptoms =
Severe dehydration
Watery diarrhoea
Other symptoms =
Vomiting
Nausea
Abdominal pain
How is cholera diagnosed?
Bacterial culture from stool sample on selective agar is the gold standard, rapid dipstick tests also available
How is cholera treated?
Oral-rehydration is the main management - up to 80% of cases can be successfully treated
What are the main features of the cholera vaccine?
Dukoral, oral, inactivated
What are some other common causes of diarrhoea?
Viral =
Rotavirus (children)
Norovirus “winter vomiting bug”
Bacterial = Campylobacter jejuni Escherichia coli Salmonella Shigella Clostridium difficile
Protozoal parasitic =
Giardia lamblia
Entamoeba histolytica
What are rotaviruses?
How common are they?
RNA virus, replicates in enterocytes
5 types A – E, type A most common in human infections
Most common cause of diarrhoea worldwide in infants and young children
How are rotaviruses treated?
Oral rehydration therapy
Still causes up to 200,000 deaths/year.
Before vaccine, most individuals had recurrent infections by age 5, repeated infections develop immunity
What is the vaccine against rotaviruses?
Live attenuated oral vaccine (Rotarix) against type A introduced in UK July 2013
What are noroviruses? How common is it?
How is it transmitted?
RNA virus
Incubation period 24-48 hours
Estimated 685 million cases per year
Faecal-oral transmission.
Individuals may shed infectious virus for up to 2 weeks
Outbreaks often occur in closed communities
What are the symptoms of norovirus?
How is it treated?
Acute gastroenteritis, recovery 1 – 3 days
No specific treatment - just supportive
How is norovirus diagnosed?
Sample PCR
What are the 2 most common species of curved bacteria? How common is it?
How are they transmitted?
Campylobacter jejuni
Campylobacter coli
Estimated 280,000 cases per year in UK (food poisoning), 65,000 confirmed
Commonest cause of food poisoning in the UK
Undercooked meat (especially poultry), untreated water & unpasteurised milk Low infective dose, a few bacteria (<500) can cause illness
What is the treatment for campylobacter?
Not usually required - supportive e.g. rehydration
Azithromycin (macrolide) is standard antibiotic
Resistance to fluoroquinolones is problematic
What are the main features of E. coli?
Diverse group of Gram-negative intestinal bacteria
Most of the time it is harmless
6 ”pathotypes” associated with diarrhoea (diarrhoeagenic)
What are the 6 E.coli pathotypes associated with diarrhoea?
Enterotoxigenic E. coli (ETEC) - cholera like toxin, watery diarrhoea
Enteroinvasice E. coli (EIEC) - shigella like illness, bloody diarrhoea
Enterohaemorrhagic or Shiga toxin -producing E. coli (EHEC/STEC)
Enteropathogenic E. coli (EPEC)
Enteroaggregative E. coli (EAEC)
Diffusely adherent E. coli (DAEC)
Which of these is the most problematic?
Enterohaemorrhagic or Shiga toxin -producing E. coli (EHEC/STEC)
E. coli O157 serogroup, Shigatoxin/verotoxin
5-10% get haemolytic uraemic syndrome: loss of kidney function
Why is C diff. called its name (clostridium difficile)?
What often causes c. diff infections?
What is weird about C. diff?
It is difficult to grow in a lab
Long term antibiotics
Healthy microbiota can contain C. diff - so role in intestinal mucus is unclear, but can exist without causing harm
When does C. diff become a problem?
Intermediate dysbiotic state caused by a exogenous disturbance e.g. antibiotics
C. diff starts colonising enterocytes and you get an outgrowth in the distal gut, but still not producing toxins at this stage
Pathogen induced disturbance creates supportive environment for C. diff to continue dividing and surviving - C. diff then starts producing toxin
Causes inflammation of distal gut
At which stage of C. diff becoming an issue is there a chance to return to normal healthy gut microbiota?
Before C. diff begins producing toxins - so even when it has begun colonising enterocytes, if it is not producing toxins - there is a chance of returning back to normal state
What is the management in C. diff?
Isolate patient (very contagious) - tends to happen in hopsital
Stop current antibiotics
Start them on Metronidazole, Vancomycin
Recurrence rate 15-35% after initial infection, increasingly difficult to treat
Faecal Microbiota Transplantation (FMT) – 98% cure rate
What is weird about Metronidazole?
Can cause and treat C. diff
How can reccurent C. diff infections be treated?
Faecal microbiota transplantation - 90 to 98% cure rate
