Microbiota

Question 1

Architecture of the intestinal mucosal surface

Answer 1

Question 2

What are the extrisnic barrier defenses?

Answer 2

1. Mechanical/Involuntary Reflexes
   
   • Cough
   • Gag
   • Peristalsis
2. Structural
   
   • Mucus
3. Chemical
   
   • Acid
   • Enzymes
   • Antimicrobial peptides and polypeptides
4. Microbiological
   
   • Commensal microbiota

Question 3

1. What area do mucus/mucins define?
2. What secretes mucus/mucins?
3. What do mucus/mucins form?

Answer 3

1. Viscoelastic gel that defines structure of extrinsic barrier
2. Secreted by specialized Goblet cells
   
   • Secretion can be constitutive or regulated
   • Humans secrete 10 liters of mucus/day
3. Forms selectively permeable mucus blanket
   
   • Variable thickness depending on anatomic site and pathophysiology
   • Comprised of a variety of mucins, water, ions, proteins, and lipids
   • Contains antibodies, antimicrobial peptides, and bacteria
4. –Continuous turnover and changes in content contribute to dynamic system

Question 4

What is mucins role in host defense?

Answer 4

• Mucus-commensal interactions
  
  • Specific binding of some commensals via adhesins
  • “grazing” on mucus-cleaving specific sugars from tips of oligosaccharides
  • Small subset of commensals digest mucins
• Bacterial exclusion
  
  • Thickness and viscosity contribute to exclude bacteria
  • Bacteria and LPS have been shown to induce MUC gene expression
  • Pathogens have developed specific mechanisms to evade barrier (flagella, interference with exocytosis)
• Containment of secreted antibodies and antimicrobials
  
  • IgA and other secreted antibodies bind mucus through low affinity bonds, and interact with commensals and pathogens
  • Cationic AMP may be contained via electrostatic interactions with mucins

Question 5

How do microbes evade mucus?

Answer 5

1. degradation of mucin
2. avoidance of mucus
3. alterations in host cells

Question 6

What are the classes of mucosal defense mechanisms?

Answer 6

1. Acdification (pH ~ 3.5-4.0)
2. Toxic oxygen-derived products (O₂^-; H₂O₂; etc.)
3. Toxic nitrogen products (NO)
4. AMPs (defensins and cationinc proteins)
5. Enzymes (lysozyme; acid hydrolase)
6. Competitors (microbiota)

Question 7

• Where are AMPs found?
• What are the different AMPs?
  
  • What is the function of each?

long card, sorry : /

Answer 7

• AMPs are found in paneth cells
• Paneth cell AMPs:
  
  1. α-Defensins (cryptdins in mouse)
     
     • constitutively expressed cationic AMPs
     • anti-microbial and chemo-attractant properties
     • kill target microbes by forming pores in their cell membrane
  2. Lysozyme C
     
     • glycosidase
     • hydrolyzes peptidoglycan (bacterial cell wall)
  3. Phospholipase A₂
     
     • catalyzes hydrolysis of fatty acids (bacterial cell membrane)
     • bactericidal against G+ bacteria
  4. RegIIIγ
     
     • C-type lectin
     • binds to peptidoglycan
     • bactericidal against Gram-positive bacteria
     • inducibly expressed upon Toll-like receptor (TLR) activation
  5. Cryptdin related sequences (CRS)
     
     • antibacterial activity comparable to cryptdins

Question 8

How do pathogens evade AMPs?

Answer 8

1. Protease secretion
2. Surface charge modification
3. Capsule formation
4. Modulate AMP expression
5. Efflux pumps

Question 9

What are the secreted immunological defenses?

Answer 9

• sIgA
  
  • Predominant immunoglobulin in mucosal secretions, monomeric & polymeric
• IgM
  
  • Also associated with SC in mucosal secretions
  • May not be transported as well due to MW restrictions in SC dependent transport
  • Compensatory increase with IgA deficiency
• IgG
  
  • Found at same levels as IgM
  • Proportion of IgA to IgG varies by site and time of collection (ie: proportion varies through menstrual cycle)
  • Not selectively transported in humans
• **IgE **- Found in low concentration, associated with mucosal allergic responses
• IgD - Found in low concentration in milk and saliva

Question 10

Describe the IgA structure and biosynthesis:

Answer 10

• Serum IgA
  
  • Predominantly monomeric
  • Polymeric IgA-j chain containing polymers and variable but low amount of SIgA.
• Mucosal IgA
  
  • Predominantly polymeric
• Structure consists of alpha chain, J chain (only associated with pIgA), and secretory component
• Synthesized as monomer and forms pIgA prior to secretion
• Approximately 4 grams of IgA secreted daily
• Metabolized and cleared by liver

Question 11

How is mucosal IgA induced?

Answer 11

• Migrating DCs can induce B and T cell activation in the mesenteric lymph node
• DCs do not recirculate through lymph and blood
• Recirculation of B cells through lymph and blood

Question 12

What are the biological activities of IgA? (5)

Answer 12

• Inhibition of adherence
  
  • Surrounds microbe and repels attachment to mucosal surface
  • Agglutination of microbes by Fc-Fc interactions
• Mucus trapping
  
  • ​May associate with mucins, and trap microbes in mucus blanket
• Virus neutralization
  
  • Mechanism may depend on antibody specificity, isotype, and concentration
  • Inhibition of cellular attachment
  • Neutralization within epithelial cells
• Enzyme and toxin neutralization
  
  • In saliva, inhibition of enzymes from oral bacteria
  • In gut, neutralization of bacterial toxins
• Inhibition of antigen penetration
  
  • IgA deficient subjects show increased absorption of food antigens

Question 13

How do microbes evade IgA?

Answer 13

• Specific IgA proteases
  
  • Cleave one of several prolyl-seryl or prolyl-threonyl peptide bonds in hinge region. Cleave off intact Fab fragments that retain binding activity
  • Exquisitely substrate specific, not inhibited by protease inhibitors
  • Cause local IgA deficiency in vivo
  • Meningitis association (H. influenzae, N. meningitidis, S. pneumoniae)
• Other proteases
  
  • Wide spectrum protease activity can cleave IgA, noted in peridontal pathogen Porphyromonas gingivalis, and some intestinal Enterobacteriaceae
• Glycosidases
  
  • IgA is heavily glycosylated
  • subject to damage by bacterial glycosidases,
  • disrupting conformation, net charge, and resistance to proteolysis
• IgA binding proteins
  
  • Cell surface proteins that bind IgA non-specifically (ie: Fc)
  • Lectin binding of O-linked carbohydrate in IgA hinge region

Question 14

Healthy microbiota contains a balanced composition of three major classes of bacteria:

Answer 14

1. Symbionts
   
   • Share mutual relationship with the host, have known health promoting functions
2. Commensals
   
   • Permanent residents of this ecosystem and provide no benefit or detriment to the host
3. Pathobionts
   
   • Live as commensals but have the potential to induce pathology

• Note: Altered microbiota is associated with diseases (IBD, autoimmunity, obesity diabetes, etc.)

Question 15

Describe the duality of the commensal microbiome:

Answer 15

• Mucosal surfaces are in
  
  • constant contact with microbes
• Primary role of mucosal surfaces:
  
  • allow normal physiological function while protecting the host from infection
• Mucosal immune system:
  
  • protects the host from the microbiota
• Microbiota has a ….
  
  • symbiotic role in host protection and host physiology

Question 16

Microbial composition of the lower GI tract:

Answer 16

Question 17

______ ______ regulates microbiota composition

• What is the evidence for this?

Answer 17

Oxygen tension regulates microbiota composition

• Hyperbaric oxygen treatment raised tissue oxygen tension
• Changes in oxygen tension were associated with increased abundance of facultative anaerobic bacteria
• Changes in composition were limited to mucosal associated bacteria

Question 18

______ immune factors modulate intestinal colonization

Answer 18

Innate immune factors modulate intestinal colonization

Question 19

____ _______ selects for microbial composition

Answer 19

Host nutrition selects for microbial composition

Question 20

What is associated with distinct enterotypes?

Answer 20

Long-term dietary habits

Question 21

What are the protective functions of the intestinal microbiota?

Answer 21

• Pathogen displacement
• Nutrient composition
• Receptor competition
• Production of anti-microbial factors
  
  • bactieriocins & lactic acids

Question 22

What is the result of an absence of bacterial colonization (in mice)?

Answer 22

• Mice lack a mature mucosal immune system
  
  • Underdevelopment of lymphatic tissues
  • Delayed B cell migration in response to bacterial antigen
  • Reduced antibody diversity
  • Reduced lymphocyte responsiveness

Question 23

What was the result of** reconstitution of a normal microbiota** (in mice)?

Answer 23

• Mice develop normal mucosal immune function
  
  • Increased lymphocyte infiltration of gut mucosa
  • Germinal center formation in Peyer’s Patches
  • Induction of innate antimicrobial effector molecules
  • Treatment with bacterial polysaccharide from bacterial symbiont (B. fragilis) restores many immune functions

Question 24

Describe microbiota induction of epithelial antimicrobials:

Answer 24

• Luminal:
  
  1. TLR-dependent expression
  2. Activation of MYD88
  3. Upregulation of REG3γ
  4. Killing of bacteria
• Basolateral
  
  1. AMPs block bacteria
  2. Epithelium secretes IL6, IL23 and TGFβ
  3. Dendritic cells phagocytose microbe ⇒ secrete IL23
  4. DC IL23 secretion ⇒ induces IL22 secretion
  5. IL22 ⇒ causes epithelium to secrete chemokines
  6. Chemokines ⇒ activate endothelium

Question 25

Which T cell subsets does the microbiota specifically affect?

Answer 25

* iNKT cells * Innate lymphoid cells * Th17 cells * Tregs

Question 26

Describe the production and function of short chain fatty acids (SCFAs):

Answer 26

* Commensal bacteria _ferment nondigestible dietary polysaccharides to produce short chain fatty acids_ (SCFAs) * SCFAs regulate: * _PMNs, dendritic cells, macrophages/monocytes, and intestinal epithelial cells_ * SCFAs induce: * _regulatory T cell differentiation_ * SCFAs _regulate expression of virulence factors_ on bacterial pathogens

Question 27

What human diseases are caused by a **disruption in the microbiome**?

Answer 27

* Antibiotic associated diarrhea: * ***C. difficile* colitis** * Systemic infection: * **VRE**

Question 28

**Commensal microbiota prevent pathogen colonization via **(6)**: **

Answer 28

1. Bacteriocin production 2. SCFA production 3. Consumption of oxygen 4. Competition for nutrients and attachment sites 5. Induction of epithelial antimicrobials 6. Induction of mucus production and secretion

Question 29

Describe the effect of antibiotics on the gut microbiota:

Answer 29

Question 30

1. Commensal bacteria regulate digestion by: 2. What is the role of **commensal fermentation**?

Answer 30

1. **Commensal bacteria regulate digestion by:** * mediation bile acid synthesis * lipid absorption * amino acid metabolism * vitamin synthesis * SCFA production 2. Byproducts of commensal fermentation (metabolites) **regulate the immune system**

Question 31

(digestible/nondigestible) carbohydrates provide the most energy to the host

Answer 31

**digestible** carbohydrates provide the most energy to the host

Question 32

Be generally familiar with how the microbiota influences overall health. (it's a very dense chart)

Answer 32

Question 33

What are the disease associations with the microbiome?

Answer 33

1. Inflammatory Bowel Disease (Crohn’s Disease) 2. Obesity and obesity related diseases, including diabetes and non-alcoholic fatty liver disease 3. Cancer 4. Allergy/Asthma

Question 34

IBD patients show both abnormal ....

Answer 34

**bacterial colonization and immune function** * characteristic shift in microbial colonization moves from obligate anaerobic bacteria to facultative anaerobic species * predominantly proteobacteria * most likely associated with increased oxygen tension caused by inflammation

Question 35

A probiotic is ....

Answer 35

**“a viable microbial food supplement which beneficially influences the health of the host”**

Question 36

Why are probiotics used in treatment of immune disorders?

Answer 36

* Restoration of a “healthy” biota * Restoration of barrier function (prevent excess antigen transfer across skin and gut barriers) * Skew T cell immune responses to Th1 type