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MICB 402 > Paper 1: Commensal Microflora, TLRs, Intestinal Homeostasis > Flashcards

Paper 1: Commensal Microflora, TLRs, Intestinal Homeostasis Flashcards

1
Q

what do TLR (toll-like receptors) bind to/recognize (2)

A
  • microbial ligands present on pathogens and commensal microorganisms (LPS, LTA, etc)
  • bacterial ligands recognized by TLRs are not unique to only pathogens, but rather shared by entire classes of bacteria (which include commensal bacteria)
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2
Q

before this paper, what was previously though about the inflammation and commensal bacteria

A
  • inflammatory response to commensal bacteria is avoided due to sequestration of microflora by surface epithelia
  • inflammatory response to pathogenic bacteria is initiated because they contain virulence factors that allow them to pass epithelial barriers where they can be detected by TLRs on macrophages/DCs
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3
Q

what were the main findings of this paper (4)

A
  • TLR signalling protects against DSS colitis
  • commensal bacteria are recognized by TLRs under normal steady-state conditions and interaction is critical for intestinal homeostasis
  • activation of TLRs by commensal microflora is critical for protection against gut injury and associated mortality/induce tissue protective factors
  • loss of commensals by antibiotic treatment results in impaired gut integrity
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4
Q

what TLR recognizes LPS

A
  • TLR4
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5
Q

what TLR recognizes LTA

A
  • TLR2
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6
Q

TLR function (2)

A
  • sensors of microbial infection
  • critical in initiation of inflammatory and immune defence responses
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7
Q

what was the suggested cause of inflammation after intestinal epithelial injury before this study was conducted (2)

A
  • disruption of mucosal barrier upon injury led to exposure of TLR ligands from commensals to TLR-expressing cells (macrophages, DCs, etc)
  • result in potent inflammatory response, intestinal inflammation, and corresponding injury
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8
Q

what is the purpose of dextran sulfate sodium (DSS) (2)

A
  • DSS is known to be directly toxic to colonic epithelium
  • oral administration of DSS models intestinal injury and inflammation and allows commensals to access lamina propia
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9
Q

myD88

A
  • adaptor molecule (in signal transduction pathway) essential for TLR-mediated induction of inflammatory cytokines
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10
Q

commensalism

A
  • a relationship between two organisms where one benefits and the other is not significantly harmed or helped
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11
Q

how are our gut microflora commensal to us (5)

A
  • required for gut-associated lymphoid tissue
  • primes the systemic immune system
  • regulates intestinal epithelial homeostasis
  • aids extraction of energy from food
  • promotes colonization resistance to pathogens
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12
Q

what relevant TLRs expressed on epithelial cells (2)

A
  • low levels of TLR2 and TLR4 expressed on apical side (side facing lumen) of intestinal epithelial cells
  • gut lamina propria contains large number of leukocytes which have TLRs on them
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13
Q

the balance of inflammation: too little inflammation (2)

A
  • recurring, uncontrolled infections
  • autoinflammatory disorders
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14
Q

the balance of inflammation: balanced inflammation (3)

A
  • effective tissue repair
  • clearance of infections
  • reversal of inflammation
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15
Q

the balance if inflammation: too much inflammation (2)

A
  • sepsis/organ failure
  • chronic inflammatory diseases
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16
Q

inflammatory bowel disease (IBS)

A
  • consequence of breakdown of immune homeostasis in the gut
  • increased frequency of colorectal cancer in patients with IBC
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17
Q

DSS colitis - experimental method (2)

A
  • acute colitis induced by administering low % DSS in water over 7 days
  • monitor weight loss and disease activity index (DAI) daily
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18
Q

DSS colitis - markers of inflammation

A
  • colon length
  • histology scoring/IF
  • cytokine production
  • cytokine analysis by qPCR
  • protein analysis/signalling
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19
Q

AOM/DSS model of colon cancer (2)

A
  • carcinogen and chronic colitis induce development of colon tumours
  • achieved after 3 cycles (14 days/cycle; 7 days of DSS and 7 days of recovery) of DSS following carcinogen (AOM) injection at day 1
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20
Q

gene targeting in embryonic stem (ES) cells to create knockout mice (6)

A
  1. ES cells cultivated from mouse embryos
  2. targeting vectors are constructed which contain new DNA that can be selected for in vitro
  3. ES cell transfection occurs target vector finds and recombines with target gene
  4. selection for targeted ES cells and proliferation of those cells
  5. targeted ES cells injected into embryo to form a mosaic with cells, then injected into surrogate where mosaic mice are born
  6. mosaic mouse mates with WT mouse to create fully KO mice and fully WT mice
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21
Q

what was the purpose of figure 1

A
  • to study the effects of intestinal epithelial injury on survival and weight change of different mice strains through induced DSS colitis
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22
Q

what did the paper observe in figure 1 (3)

A
  • MyD88 -/- mice showed severe mortality and morbidity compared to wt mice which had 100% survival and minimal morbidity
  • MyD88 -/- mice and TLR2 and TLR 4 showed severe weight loss compared to wt mice
  • MyD88-/- showed more severe mortality and morbidity compared to TLR2 and TLR4-deficient mice
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23
Q

what are the findings from figure 1 (2)

A
  • defective signalling of TLRs induced by commensal-derived products increase susceptibility to DSS-induced disease
  • signalling of multiple TLRs induced by commensal-derived products are involved in protection against DSS-induced disease; TLR2 and TLR-deficient mice were not as affected as MyD88-deficient mice which have defect in many TLR signalling pathway
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24
Q

what was the purpose of figure 2

A
  • to analyze the cause of death and morbidity seen in MyD88-/- mice using several parameters
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25
Q

what were the parameter used to analyze results in figure 2 (3)

A
  • colonic blood scoring
  • histological scoring
  • analysis of RBC
26
Q

what did the paper observe in figure 2 (2)

A
  • bleeding was occurring in the colons of MyD88-/- mice, which occurred with more rapid onset and with greater severity compared to wt mice
  • MyD88-/- mice became severely anemic with time (using histological scoring and RBC analysis) compared to wt mice
27
Q

what are the findings from figure 2

A
  • MyD88-/- mice were dying of severe colonic bleeding and anemia upon administration of DSS, although the cause of the bleeding is not yet clear
28
Q

what is the purpose of figure 3

A
  • to investigate possible mechanisms of colonic bleeding in MyD88-/- mice
29
Q

what did the paper observe in figure 3

A
  • microscopic evaluation of colons showed severe and extensive erosion of surface epithelium and mucodepletion of glands compared to wt control mice
  • histopathological scoring revealed severe ulceration and epithelial injury in MyD88-/- compared to wt control
30
Q

what are the findings from figure 3

A
  • the cause of colonic bleeding in MyD88-/- mice results from increased ulceration and epithelial injury
31
Q

what were the possible theories suggested for the increased epithelial injury in MyD88-/- colons (4)

A
  • loss of MyD88 may impair the ability to control commensal bacteria populations
  • loss of MyD88 might results in much greater inflammation and systemic bacterial spread
  • MyD88 may have altered the gut microflora
  • MyD88 ad TLRs may have a role in resistance to intestinal injury/be required for repair
32
Q

how did the paper determine if commensal overgrowth was the reason for mortality in MyD88-/- mice (3)

A
  • pretreatment of mice with antibiotics did not prevent DSS-induced damage in MyD88-/- mice and mice died in similar time-course
  • loss of MyD88 doesn’t result in systemic spread of bacteria as there was no increase in bacteria in spleen
  • titers of anaerobes and aerobes in fecal material of wt and MyD88-/- mice were similar
33
Q

how did the paper determine if increased inflammation from hyper-infiltrating leukocytes was the reason for mortality in MyD88-/- mice (2)

A
  • studied leukocyte infiltration at different times post DSS-treatment and found no differences in overall infiltrating leukocytes between MyD88-/- and wt mice
  • leukocyte infiltration not evident until day 5, but colonic blood was already detected at day 3
34
Q

what factors determine protection from intestinal injury (3)

A
  • balance of proliferation and differentiation along crypt axis
  • production of mediators involved in protecting epithelial cells from initial injury
  • things involved in orchestrating repair response mechanisms after injury
35
Q

how did the paper determine if TLR signalling controlling homeostasis of intestinal epithelium was the reason for mortality in MyD88-/- mice (2)

A
  • examined baseline proliferative state of colonic crypts of wt and MyD88-/- mice
36
Q

what was the purpose of figure 4

A
  • to determine if TLR signalling was controlling homeostasis of the intestinal epithelium
37
Q

what did the paper observe in figure 4A (3)

A
  • proliferating cells were labelled and it was found that there was an increased number of proliferating cells in MyD88-/- mice compared to wt controls
  • proliferating cells were present in the middle and upper regions of the the crypt in MyD88-/- mice, areas of crypt remote from stem cell area and normally fully differentiated and non-proliferating
  • 2 hour graph was was used to show that cells did not migrate up from the stem cell area
38
Q

what were the findings from figure 4A

A
  • expanded proliferative zone and increase in proliferating cells in MyD88-/- mice suggest dysregulated proliferation and differentiation of intestinal epithelium in absence of TLR signals
39
Q

what method was used for figure 4A(2)

A
  • immunohistochemical staining with BrDU
  • BrDU positive cells are cells that are actively proliferating and will appear brown on photomicrographs
40
Q

what did the paper observe in figure 4B and 4C

A
  • the average total number of cells per crypt were higher in MyD88-/- mice compared to wt
  • markers for cycling cells, such as cyclin D1, were up-regulated in these cells
41
Q

what method was used for figure 4C

A
  • western blot
42
Q

what did the paper observe in figure 4D and 4E (2)

A
  • MyD88-/- mice showed severe mortality upon radiation compared to wt controls (proliferating cells more susceptible to radiation)
  • MyD88-/- mice sustained more severe radiation-induced epithelial damage and showed pronounced defects in crypt repopulation and compensatory proliferation compared to wt mice (from decreased proliferating cells after radiation and shortened villus length)
43
Q

what were the papers findings from figure 4D and 4E

A
  • although proliferative status is a major determinant of radiation sensitivity, compromised expression of cytoprotective and repair factors by MyD88-/- epithelium likely contributes to increased susceptibility to radiation-induced injury
44
Q

what was the purpose of figure 5
- general
- A, B, C (3)

A
  • to investigate whether commensals and TLR are responsible for induction of cytokines known for cytoprotective and reparative factors under normal conditions or during intestinal epithelial injury
  • IL-6, KC-1, and TNF at steady state
  • IL-6 and KC-1 after DSS administration
  • hsp25 and hsp72 at steady state
45
Q

what was observed in figure 5A

A
  • colons of wt mice produced TNF, IL-6, and KC-1 prior to DSS administration, while MyD88-/- mice produced low levels of these factors in uninjured state
  • removal of microflora with antibiotics eliminated MyD88-dependent production of cytokines by the colon
46
Q

what was observed in figure 5B

A
  • production of IL-6 ad KC-1 were significantly up-regulated after DSS administration in wt mice, while MyD88-/- mice were unable to induce these factors after DSS administration despite severe intestinal injury
47
Q

what method was used for figure 5B to quantify the cytokine amounts

A
  • ELISA
48
Q

what were the papers findings from figure 5A and 5B

A
  • commensal bacterial products stimulate TLRs under normal conditions with intact epithelium to secrete cytokines with protective responses
49
Q

which cytokines play a role in inflammation, host defence, and protecting various cell types (3)

A
  • IL-6
  • TNF
  • KC-1
50
Q

hsp25 and hsp72

A
  • heat-shock proteins that play cytoprotective role in intestinal epithelial cells
51
Q

what was observed in figure 5C

A
  • expression of hsp25 and hsp72 were severely diminished in colonic epithelium of MyD88-/- mice at steady state compared to wt mice
52
Q

what are the findings from figure 5C

A
  • hsp25 and hsp72, cytoprotective proteins, are constitutively induced by commensal products through TLRs at steady state in wt mice
53
Q

what method was used for figure 5C

A
  • western blot
54
Q

what is the purpose of figure 6

A
  • to investigate if commensal bacterial is responsible for triggering TLRs and conferring protection from direct epithelial damage
55
Q

what was observed from figure 6 (and antibiotic treatment in figure 5A) (3)

A
  • mice that received full antibiotic combo showed severe mortality and morbidity after DSS treatment compared to wt mice
  • commensal-depleted mice showed no detectable levels of IL-6, TNF, or KC-1 at steady state
  • mice with incomplete depletion of commensals showed 100% survival with minimal bleeding/colonic bleeding, similar to wt animals
56
Q

what were the findings from figure 6 (2)

A
  • commensals are required for protection of intestinal epithelial injury
  • it is not any particular group of commensal bacteria that provides protection; commensals not depleted by antibiotic can still activate TLRs and induce protective and repair responses
57
Q

what is the purpose of figure 7

A
  • to investigate whether metabolic activity from commensals themselves was required for intestinal homeostasis or is it was just the microbial products from commensals (LPS, LTA, etc) that were inducing TLR signalling that is required
58
Q

what procedure was used to investigate figure 7 (3)

A
  • mice had their microflora depleted using antibodies
  • microflora-depleted animals were given purified LPS or LTA in drinking water before and during DSS treatment to mimic microbial gram + and gram - commensal products
  • this would trigger TLRs, but not metabolic or bioactivity from microflora themselves
59
Q

what were the observations of figure 7 (3)

A
  • microflora-depleted mice with LPS or LTA treatment were completely protected from DSS-induced mortality, morbidity and severe colonic bleeding seen microflora-depleted mice without the treatment
  • expression of hsp25 and hsp72 were up-regulated after LPS/LTA treatment in microflora-depleted mice
  • TLR4-/- mice were not rescued by LPS (detected by TLR2), but TLR2-/- was
60
Q

what were the findings from figure 7

A
  • the recognition of commensal bacterial products by TLRs is responsible for protection from mortality caused by intestinal epithelial bleeding

MICB 402 (11 decks)

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