lecture 8: gut microbiota, health and disease

Question 1

What are diseases and disorders from 1950-2000?

Answer 1

• drop in many infectious diseases
• e.g. rates of tuberculosis have plummeted in developed countries
• common childhood diseases like mumps and measles have also decreased dramatically over the last couple of decades
• paralleled by a very marked increase in a number of autoimmune diseases such as MS, diabetes, and allergic diseases such as asthma and crohn’s disease
• not just due to improved reporting
• systemic studies have highlighted the strong correllation between the wealth of a society and the prevelance of these sorts of diseases

Question 2

What is the hygiene hypothesis?

Answer 2

• family size association with allergy - David Strachan, 1989
• extended to
  
  • cleanliness
  • vaccinations
  • antibiotic use
• reduced exposure to infectious agents
• dysregulated immune response
• while there are some pathogens that may enhance the allergic/autoimmune diseases, in fact both of these confitions involve completely different types of immune response
• autoimmune usually involves Th1, allergic Th2
• probably not an organism that is skewing the immune response in one particular direction
• but rather a much more complex process by which the entire immune system is somehow being modulated in a way that predisposes it towards both of these types of trends
• some bugs stimulate the autoimmune reaction through molecular mimicry → enhance autoimmunity rather than prevent it
• countries such as japan where the increase in sanition etc has not been associated with the increase in allergies and autoimmunity to the same extent as in many other western countries

Question 3

What is the old friends hypothesis?

Answer 3

• Graham Rook, 2003
• vital microbial exposure is NOT infectious
• dependent on our natural microbiota
• microbes co-evolved with the immune system
• humans are dependent on microbes for immune system
  
  • development
  • function

Question 4

What is the diet hypothesis?

Answer 4

• diet drives changes in the microbiota
• directs the development and function of the immune system

Question 5

What is nutritional transition?

Answer 5

• economic
  
  • increased wealth within developed societies
  • acquisition of various technological aids that make work easier
  • globalisation → makes us prone to influences that are developing around the planet
• demographic
  
  • changes in population dynamics
  • lower rates of population growth associated with decreased death rates
• epidemiological
  
  • lots of infectious disease, periodic famine vs good health care etc
• dietary activity
  
  • dramatic changes → western countries have transitioned to increase dependence on high fat/high sugar

Question 6

Is diet the basis of increased ‘western world’ disease?

Answer 6

• maybe
• high fat
• high sugar
• highly refined
• highly processed
• low in fibre
• idea is now that diet is a key factor that regulates the microbiota

Question 7

What influences microbial composition?

Answer 7

• diet
  
  • intake of fibre
  • obesity
• host genetics
• maternal transfer and early colonisation
• antibiotics and medications
• infection
• inflammation
• stress
• hygiene
• age
• symbiosis
  
  • SCFA
  • PSA
  • PTGN
  • (and so on)
  • → immune regulation → homestasis
• Dysbiosis
  
  • virulence factors
  • immune dysregulation
  • inflammation

Question 8

What is gut microbiota?

Answer 8

• human microbiome 1,000,000+ genes = 0.2-1.7kg
• human genome = 23,000 genes
• intestinal microflora
  
  • 10¹⁴ microorganisms, >500 different species
  • lactobacilli in stomach and duodenum
  • streptococci/lactobacilli in jejenum
  • enterobacteria, enterococcus, faecalis, bacteroides, bifidobacteria, peptococcus, peptosteptoccus, ruminococcus, clostridia, lactobacilli in ileum and colon with appendix
• 10² to 10³ in stomach
• duodenum/jejunum less than 10^4-5
• ileum - 10^{<span>3</span>} - 10⁷
• colon with appendix - 10⁹ to 10¹²
• also comprises fungi, protists and viruses
• 2 orders magnitude greater in terms of number of genes within our own genome
• 3 or 4 orders of enrichment in regards to genes that have to do with breakdown of fibre etc
• microbiota is massive in terms of biomass, genetic complement
• not uniform throughout the digestive tract
• species specific variation along the tract

Question 9

What are methodological advances in studying the gut microbiota?

Answer 9

• generation of germ free mice
  
  • different genetic backgrounds, mutants and in-bred congenic lines with different immune systems, and with selectively restored microbiotas
• high through-put DNA sequencing
  
  • taxonomic and metabolic classification of microbiota, generation of humanised microbiota
• transcriptional/metabolomic tools to measure impact of gut microbiota on host physiology, immunity and development
• development of other tractable experimental systems (invertebrates such as Caenorhabditis elegans and Drosophila melanogaster

Question 10

What is the human microbiome project?

Answer 10

• NIH
• first phase (2007-2012)
  
  • characterised composition and diversity (nose, mouth, skin, GIT, UGT)
  • evaluated genetic metabolic potential
• second phase (2013 - 2015)
  
  • “creation of the first integrated dataset of biological properties from both the microbiome and host from cohort studies of microbiome associated diseases”

Question 11

What is a healthy microbiome?

Answer 11

• each persons microbiome is unique
• two people may have different microbial communities but still be healthy
• certain communities can be used to predict characteristics e.g. breast feeding, level of education
• microbial communities on one site could predict another site e.g. gut predicted based on mouth communities (despite differences)

Question 12

Is gut microbiota variable or consistent?

Answer 12

• gut microbiota is highly variable
• but contains similar metabolic capabilites
• 16S RNA sequence analysis of microbiome from di/monozygotic twins in USA reveal high variability in bacterial species/Phylum composition
• no core microbiome (at least at species level)
• however, analysis by categories of genes (COG) indicated broadly similar metabolic capacity
• two major phylum of bacteria:
  
  • bacteroidetes (gram negative)
  • firmicutes (gram positive)

Question 13

What are diet-induced changes on gut bacteria?

Answer 13

• African children (Burkina Faso)
  
  • people eat as close as possible to pre-urban socities
  • largely bacteroidetes
  • low firmicutes
• European children (florence, Italy)
  
  • largely firmicutes
  • some bacteroidetes
• diet has dominant role in shaping gut microbiota over ethnicity, sanitation, hygiene, geography and climate

Question 14

What are functional classes of non-pathogenic members of microbiota?

Answer 14

• probiotics: transient, can confer health benefit and affect beneficial bacteria
  
  • e.g. Biffidobacterium spp, lactobacillus spp
• autobionts: permanent, symbiotic, immunomodulatory. part of normal microbiota; direct influence on host immune function
  
  • e.g. bacterioidies fragilis, Clostridium XIV
• pathobionts: permanent, parasitic/infectious; do not cause disease in presence of normal microbiota. cause disease when microbiota/immunity perturbed
  
  • e.g. clostridium difficle, Helicobacter hepaticus

Question 15

How are autobionts adapted to life in the gut?

Answer 15

• express polysaccharide utilisation loci (PUL)
• required for digestion of multiple plant polysaccharides that make up dietary fibre (humans can only digest starch, maltose, sucrose)
• selectively expressed when polysaccharide is in the diet
• Bacteroides ovatus xyloglucan PUL
  
  • 8 glycosyl hydrolases (HG)
  • 2 glycan transporters
  • 3 glycan chaperones
  • this a basic component of many vegetables that we are incapable of digesting
• plant polysaccharide
  
  • captured by chaperones on the surface of the bacteria
  • present it to various endoglycosidases that clip the polysaccharide along its chain length to generate small oligosaccharides
  • then bound by a second chaperone
  • deliver it into a transporter
  • glycosyl hydrolases selectively and sequentially remove these different sugars
• highly adaptive strategies

Question 16

What is SCFA production?

Answer 16

• digestion of dietary fibre
  
  • bacteroides → Acetate (2C), Propionate (3C)
  • Firmicutes → Butyrate (4C)
  • SCFA diffuse passively, are taken up by host transporters or bind to G-protein coupled receptors (Gpr-41, -43) on many cell types
• dietary fibre: fermentability
  
  • cellulose: 20 - 80
  • hemicellulose: 60 - 90
  • pectins: 100
  • guar gums: 100
  • ispaghula: 55
  • wheat bran: 50
  • resistant starch: 100
  • inulin, oligosaccharides: 100

Question 17

What is the role of SCFAs?

Answer 17

• fibre, indigestible starch → comensal bacteria → SCFAs:
  
  • stimulation of mucous production
  • IgA production
  • tissue repair
  • promotion of Treg cells, tolerance
  • inflammasome activation? IL-18 production, epithelial integrity
  • inhibition of inflammation/NFkB

Question 18

What is the importance of gut microbiota?

Answer 18

• digestion (10% of calorific intake from non-digestible fibre)
• production of vitamins (B and K)
• gut epithelial development (and nutrition)
• immune system development
• tolerance
• combat infections (barrier function, competitive exclusion)

Question 19

What is the role of gut bacteria in obesity and type-2 diabetes (T2D)?

Answer 19

• increased incidence of obesity underlies worldwide increase in T2D
• insulin resistance associated with T2D and metabolic inflammation occur in parallel
• several metagenomic-wide association studies (MGWAS) have identified significant changes in gut microbiota of obese and T2D patients, that might underlie increased chronic inflammation
• low gut diversity (gene count) associated with weight gain, obesity, insulin resistance, fatty liver and low-grade inflammation
• gut dybiosis in obese and T2D reflected in reduced butyrate-produces, increased opportunistic pathogens
• but beware of correlations
• gut microbiota from obese/lean human twins transferred to mice
• transfer of ‘obese’ microbiota resulted in greater body mass and adiposity
• cohousing experiments showed that obese phenotype in mice could be overcome by presence of mice with ‘lean microbiota’ or a ‘lean’ high-fibre diet
• diet if key determinant of microbiota effect

Question 20

What is the gut microbiota in pregnancy?

Answer 20

• gut microbiota changes during pregnancy
• linked to weight gain, insulin insensitivity and inflammation (cytokines)
• changes in immunity and dysbiosis reinforce each other
• similar changes occur in non-pregnant obesity
• transfer of microbiota from pregnant women to germ-free mice leads to weight gain/insulin insensitivity

Question 21

What is the modulation of host metabolism by SCFA?

Answer 21

• PYY = peptide YY
  
  • regulates gut motility, intestinal transit rate
• GLP-1 = hormone glucagon-like peptide
  
  • increases insulin sensitivity
• IGN = intestinal gluconeogenesis
  
  • uses SCFA as carbon source
• Fiaf = fasting induced adipose factor
  
  • inhibits lipoprotein lipase and fat accumulation in adipocytes
• SCFA are recognised by specific G protein coupled receptors
• the GPRC41 activates PYY
• GPRC43 activates signalling pathway that leads to secretion of GLP1 (increases insulin sensitivity)
• overall regulate intenstinal function and systemically lead to changes in liver function and the accumulation of fat deposits in adipocytes

Question 22

How are SCFA-GPRC receptors critical regulators of obesity?

Answer 22

• Gpr43-deficient mice are obese on a lean diet, and exhibit increased high-fat induced insulin resistance
• overexpression of Gpr43 in adipose tissue leads to leaness regardless of calorific intake
• these effects were dependent on prsence of gut microbiota (absent in GF-mice)

Question 23

What is diet induced gut dysbiosis?

Answer 23

• diet induced dysbiosis leads to local/systemic immunity and inflammation
• changes in balance of regulatory T-cells (Tregs) and CD4+ T-helper 17 cells
• changes in abundance of pathobionts and release of endotoxin (LPS)
• breakdown in gut barrier function and increase in gut bacteria and antigens (endotoxaemia) in blood

Question 24

What is the regulation of CD4+ T-cells by gut microbiota?

Answer 24

• immunosuppressive
  
  • Treg greater than Th17
  • butyrate promotes formation of regulatory T-cells (Treg)
  • IL-10 cytokine levels high
  • suppresses activation of Th17 cells
  • TLR5 signalling induced by capsular polysaccharide of autobionts (B. fragilis, Clostridia XIV and IV straings) contributes to intestinal homeostasis
• inflammatory
  
  • CD4+ Th17 cells activated by pathobionts
  • IL-17 cytokine levels high
  • required for mucosal immunity
  • associated with chronic autoimmunity (IBD, MS, arthritis, psoriasis, and some cancers)
  • Th17 absent in GF animals
  • induced by segmented filamentous bacteria (SFB, gram positive anaerobic bacteria)
  • SFB monocolonised mice do not induce inflammatory response or colitis

Question 25

What can contribute to the dysbiosis of the microbiota?

Answer 25

• induced by:
  
  • diet
  • antibiotics
  • inflammation
  • host genetics
• associated with
  
  • loss of beneficial bacteria
  • outgrowth of pathobionts
• once established
  
  • can become stable
  • transferable to healthy hosts
• can lead to:
  
  • immune response to influenza virus infection
  • behavioural problems
  • experimental immune encephalyomyelitis
  • development of the immune system
  • mucosal immunity
  • NAFLD → hepatobiliary-pancreatic autoimmune disease
  • autoimmune arthritis
  • obesity, metabolic syndrome, insulin resistance
  • diabetes
  • allergy
  • inflammatory bowel disease
  • asthma

Question 26

What is microbiota as immunotherapy?

Answer 26

• diet
  
  • less fat, more fibre (non-digestible carbohydrates)
• pro/prebiotics
  
  • pro: beneficial bacteria
  • pre: beneficial bacteria + non-digestible food
• foecal transplant
• targeted manipulation

Question 27

When do lifestyle factors exert their influence?

Answer 27

• at earliest stage of life
• risk of immunoallergic disorders is greater the earlier a migrant moves from region of low risk to region of high risk
• the two way interaction between microbiota and immune system is established early in life
• risk of misuse of antibiotics and earliest stages of life

Question 28

What are prebiotics/probiotics?

Answer 28

• alter growth and binding of pathogenic bacteria
• change in pH
• SCFAs
• do they work?
• require long term consumption

Question 29

What is foecal transplant in Clostridium difficile infections?

Answer 29

• foecal transplant from healthy donors effective at treating refractory C. difficile infections
• may be useful for other conditions (i.e. IBD)
• however, protocols are variable
• alternative approaches include:
  
  • use of cultured autobionts
  • inclusion of microbiota small molecules (i.e. acetate ?) in diet
  • identify antimicrobials from gut microbiota (i.e. Bacillus thuringiensis ‘thuricin’ kills C. difficile)