Gastrointestinal Microbiota-host Interactions

Question 1

Describe the major metabolic functions of the colonic microbiota.

How does that function promote colonic health?

Answer 1

1. Fermentation of non-digestible dietary residues into short chain fatty acids (SCFA)
   
   • acetate, proprionate, butyrate
     
     • Butyrate is a major source of energy for colonocytes
2. Breakdown dietary carcinogens
3. Synthesis of
   
   • biotin, folate and vitamin K
4. Metabolise bile salts
5. Assist in the absorption of:
   
   • calcium, magnesium, iron
6. Regulation and efficiency of caloric intake and energy storage

Question 2

Identify and describe the trophic and protective effects conveyed to the intestinal epithelium by the microbiota

Answer 2

• Bacterial-induced expression of host genes has many influences on:
  
  • Nutrient uptake
  • Metabolism
  • Angiogenesis
  • Mucosal barrier integrity
  • Development of the enteric nervous system
• Ligands from the resident bacteria influence the development and function of mucosal immunity:
  
  • Modulate and regulate T cell and T-helper cell cytokine profiles
• Barrier to exogenous microbial colonisation:
  
  • Biofilm that provides a physical barrier - competing for binding sites
  • Competition for nutrients
  • Displacement
  • Porduction of anti-microbial substances

Question 3

Describe the concept of cross talk between the host and intestinal microbiota

Answer 3

• The innate mucosal immune system is in large part regulated by the microbiota.
• The microbiota interacts with mucosal associated lymphoid tissue and gut-associated lymphoid tissue early in life.
  
  • Microbiota is essential for appropriate development of both GALT and MALT
• Complex interactions between the GALT and MALT sample and sense changes within the microbiota / gut lumen
• Surface enterocytes, M cells and dendritic cells actively sample bacteria (pathogenic and microbiota related) and other antigens
  
  • M cells overlie lymphoid tissue
  • M cells deliver antigens to the DC’s which can store and transport bacteria to the local lymph nodes
  • Lymphoid tissue produces a local immune response
    
    • Helps to prevent access of the commensal and pathogenic bacteria to the host

Question 4

What are the two major Pattern Recognition Receptors in the gut?

What is their broadly defined role?

Answer 4

1. Nucleotide oligomerization domain molecules - NOD1 and NOD2 - intracellular PRR
2. Toll-Like Receptors (TLRs) - transmembrane PRR
3. Receptor of advanced glycation end products (RAGE)

• The pattern recognition receptors are mainly involed in the discrimination of host versus pathogenic microbes in the gut. They help to maintain hypo-responsiveness to the host microbiota while rapidly recognising pathogenic microbes

Question 5

Describe 3 main functions of the TLRs

Answer 5

1. Role in microbial recognition
2. Induction of anti-microbial genes
3. Control of the adaptive immune response

Question 6

Describe how the TLRs recognise and react to different microbes

Answer 6

• Each TLR binds to a specific PRR, thus making them specific for different classes of microbes
• The TLRs initiate signalling via conserved signal transduction pathways
  
  • mitogen activated protein kinases
  • NFkB - largely a pro-inflammatory pathway that leads to production of cytokines and chemokines

Question 7

Describe the major role of the NOD molecules

Answer 7

1. They exert an anti-microbial activity by induction of antimicrobial effector pathways
2. Help prevent host-cell invasion
3. Important role in autophagy to help degrade intracellular proteins - helps to form an autophagosome around the invading microbe
4. NOD2 can also inhibit, and thus regulate innate immune responses

Question 8

What role do receptors of advanced glycation end (RAGE) products perform in the gut?

Answer 8

1. RAGE bind several different ligands including advanced glycation end products such as:
   
   • AGE’s are a heterogeneous group of non-enzymatically altered proteins that accumulate at sites of inflammation
   • Bacterial LPS
   • Amyloid beta protein
2. Constituitively produced in the skin and lung. Induced by accumulation of its ligands +/or activation of transcription factors
   
   • RAGE expression in thus enhanced in a pro-inflammatory microenvironment
3. Upregulation of RAGE increases the recruitment of inflammatory cells via increased expression of endothelial adhesion molecules

Question 9

Note the major ways the NODs and TLRs participate in the host defense against GIT microbial pathogens

Answer 9

1. Recognition of molecular patterns on microbail pathogens
2. Expression at the interface of the GIT environment
   
   • On epithelial and non-epithelial cells
3. Induction of pro- and anti-inflammatory cytokines and chemokines that link to adaptive immunity
4. Create a chemotactic gradient for the entry of neutrophils, T-lymphocytes and monocytes into the mucosa
5. Induction of anti-microbial effector pathways
6. In health, NOD/TLR signalling promotes host defence and tissue repair responses.
7. Negative feedback regulatory pathways help blunt excessive immune responses and auto-inflammation

Question 10

What is Nuclear Factor - kappa B

Answer 10

• A protein complex found in almost all animal cell types that controls transcription of DNA, cytokine production and cell survival
• It is a rapid-acting transcription factor - present in cells in an inactive state
• Large variability in what can induce NF-kB
  
  • reactive oxygen species
  • TNF-alpha
  • IL-1 beta
  • bacterial LPS (via TLR 4)
  • radiation
  • cocaine
• NF-kB controls many genes involved in inflammation

Question 11

Describe the alterations in the various pattern recognition receptors in canine chronic enteropathy

Answer 11

1. Activation of NF-kB has been demonstrated in dogs with CE
   
   • No correlation betwen NF-kB and clinical signs or histology scores
   • NF-kB reduced with appropriate treatment and improvement in clinical signs
   • Dogs with FRD had higher NF-kB in the duodenum than those that required immunosuppressive therapy
2. TLRs (2, 4, 9) may show increased expression at the mucosa in dogs with CE
   
   • discordant results have been published
   • Only messenger RNA was assessed - may not directly correlate to number or type of inflammatory cells in the GI mucosa
3. Increased TLR 2 has been identified in colorectal polyps in miniature daschunds
4. Polymorphisms in TLR 4 and TLR 5 have been associated with increased or decreased risk of CE in German Shephed dogs
5. A mutation of TLR 5 was associated with IBD in Boxer dogs
6. Breed (GSD) and non-breeed associated NOD2 polymorphisms have been associated with CE
7. Both risk-associated and risk-protective polymorphisms of the TLRs and NODs have been demonstrated.

Question 12

Note the various perturbations of the microbial flora that have been identified in dogs with IBD

Answer 12

1. Flagellin derived from commensal bacteria is a dominant antigen in IBD
   
   • TLR-5 dependent recognition of flagellin plays a role in IBD
2. In numerous animal models, colitis and immune activation fail in the absence of commensal bacteria
3. Multiple animal models of IBD respond to antibiotic therapy
   
   • Primarily tylosin or metronidazole on dogs
4. Different duodenal microbial communities have been seen in helathy dogs and those with IBD
5. TLR 2, 4 and 9 upregulated in the inflamed duodenum and colonic mucosa of dogs with IBD
6. Granulomatous colitis associated with adherent/invasive E. coli in Boxer dogs

Question 13

Note the various perturbations of the microbial flora that have been identified in cats with IBD

Answer 13

1. Attenuation of microbial flora with metronidazole can improve clinical signs of IBD
2. Increased lamina propria myeloid/histiocyte antigen positive macrophages - similar to human IBD
3. Upregulated MHC class II - major role of presenting bacterial proteins/antigens to the CD4+ T cells
4. Increase antibody reaction to the normal flora has been associated with IBD
5. Increased mucosal Enteriobacteriacae correlated with:
   
   • Duodenal histology abnormalities
   • Upregulated mucosal cytokine RNA
   • Severity or number of clinical signs of IBD

Question 14

Define SIBO.

What are the major causes of small intestinal bacterial overgrowth?

Answer 14

1. Traditionally defined as an increase in the number of obligate anaerobic bacteria in the duodenal juice
   
   • Controvery exists over the normal numbers

Major causes:

• Exocrine pancreatic insufficiency
• Impaired clearance of bacteria:
  
  • Intestinal obstruction
  • Motility disorder
• Mucosal injury:
  
  • Ulcerative disease
  • Infiltrative mucosal disease

Question 15

List the mechanisms by which increased intestinal bacteria can contribute to diarrhoea

Answer 15

1. Malabsorption
   
   • Competition for nutrients such as cobalamin, the loss of which impairs intestinal absorption
2. Bacterial metabolism of nutrients into secretory products - increases colonic secretions
   
   • hydroxylated fatty acids
   • deconjugated bile salts
3. Biochemical injury to the intestinal brush border
   
   • Decreased enzyme activity
   • Decreased surface area for absorption

Question 16

What is the current diagnostic test of choice to “diagnose” antibiotic responsive diarrhoea?

Answer 16

• Antibiotic responsive diarrhoea is best presumptively diagnosed by the clinical response to an antibiotic trial.
• Specific tests of bacterial numbers and by-products are insensitive and non-specific and poorly reproducible
• Furthermore, Idiopathic IBD, infectious diarrhoea caused by bacteria and secondary SIBO due to another underlying disease can also improve or response positively to antibiotics.

Question 17

What laboratory tests may help to investigate antibiotic responsive diarrhoea? What is the usefulness of each?

Answer 17

1. Serum cobalamin
   
   • Can be reduced with ARD, IBD, FRD and EPI
   • Non-specific for ARD
2. Serum folate
   
   • Increased in 7/9 dogs with ARD in German 2003
     
     • Also increased in 6/11 dogs with either FRD or IBD
   • Not a specific test
3. Serum total unconjugated bile acids
   
   • No correlation between clinical and non-clinical dogs in a 2003 study by German et al.
   • Poor sensitivity for clinical bowel disease and poor specificity for antibiotic responsive diarrhoea

Question 18

What are thought to be the most common causes of bacterial enterocolitis

Answer 18

1. Clostridium perfringens
2. Clostridium difficile
3. Campylobacter spp
4. Pathogenic E. coli
5. Salmonella spp

Question 19

What are the major limitations to determining the prevalence of primary bacterial enteritis/enterocolitis?

Answer 19

• The majority of the bacterial organisms thought to cause diarrhoea are present in the normal flora
• The huge variation in the normal microbiota - from bacterial species to the quantities of each bacteria.
• There is vast inter-species differences so extrapolation from human data to dogs or cats is not viable

Question 20

What are the machanism for diarrhoea from primary bacterial enteritis/enterocolitis?

Answer 20

1. Proliferation and alteration of the host immune response
   
   • See previous section on microbial-host interactions
2. Enterotoxin production
   
   • Alters fluid homeostasis, primarily net reduced water absorption
   • CPE can cause significant histological change to the villus tips leading to cell death and flattening of the villi - reduced absorptive capacity.
3. Mucosal invasion
   
   • primary mucosal damange
   • Alteration to nutrient absorption
   • Increased secretion in response to the infection