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MICB 402 > Paper 3: Lyn activity, DSS colitis, IL-22 > Flashcards

Paper 3: Lyn activity, DSS colitis, IL-22 Flashcards

1
Q

IL-22 (2)

A
  • targets cells in barrier organs, such as intestinal epithelium, where it induces host defence, pro-survival, and proliferation factors
  • in colitis injury models, IL-22 antagonizes inflammation and promotes wound healing
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2
Q

which cells create IL-22 (3)

A
  • T cell subsets
  • multiple subsets of ILCs
  • production of IL-22 largely influenced by innate immune cells responses to TLR signals
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3
Q

Lyn (3)
- protein type
- expression in cells
- activation

A
  • Src-family tyrosine kinase (SFK)
  • expressed in all leukocytes except T cells
  • activated by ligand binding to adhesion molecules, cytokine receptors, immunoreceptors, and TLRs
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4
Q

does Lyn amplify or restrict signal transduction

A
  • depending on cell microenvironment, developmental stage, and type of stimulus, Lyn can either restrict or amplify signal transduction
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5
Q

Lyn^up mice (2)

A
  • contain mutation in endogenous Lyn gene at C-terminal negative-regulatory tyrosine phosphorylation site
  • leads to increased Lyn activity as inhibition mechanism is altered
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6
Q

what was the purpose of figure 1A - 1D

A
  • investigate how changes in Lyn activity and associated changes in responses to PAMPs might alter intestinal homeostasis and susceptibility to inflammation
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7
Q

what was observed in figure 1A, 1B, 1C

A
  • Lyn^up mice were protected from DSS compared with wt (less weight loss, longer colon length, less rectal bleeding)
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8
Q

what was observed in figure 1D

A
  • Lyn^up mice had fewer crypt loss and less epithelial sloughing and ulceration compared to WT mice
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9
Q

what were the findings of figure 1A - 1D

A
  • Lyn plays a protective role in DSS-induced colitis
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10
Q

what was the purpose of figure 1E - 1I

A
  • given Lyn’s protective role in acute colitis, to investigate outcome of chronic inflammation in Lyn^up mice
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11
Q

what was observed in figure 1E - 1I

A
  • Lyn^up could gain weight in recovery better than wt mice
  • Lyn^up mice had reduced morbidity than wt
  • Lyn^up mice had significantly longer colons compared to wt, indicated reduced chronic inflammation
  • Lyn^up mice had reduced tumour number and load compared to controls
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12
Q

what are the findings from figure 1E - 1I

A
  • Lyn activity protects mice from acute and chronic DSS-induced intestinal inflammation and reduced incidence of colitis-associated cancer
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13
Q

what is the purpose of figure 2

A
  • investigate contribution of cytokine responses in Lyn-mediated protection from DSS-colitis
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14
Q

how are IL-22 and IL-23 connected

A
  • IL-23 drives production of IL-22
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15
Q

what was observed in figure 2A, 2B, 2C (2)

A
  • Lyn^up colons had significantly more IL-22 and IL-23 protein and mRNA production than wt
  • increase seen as early as 2 days post-treatment, with significant increase in colon and detectable increase in serum
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16
Q

what was observed in figure 2D

A
  • colonic epithelial cells isolated from Lyn^up mice showed consistent increase in STAT3 activation (indicated by phosphorylation of tyrosines), which is done by IL-22
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17
Q

what was observed in figure 2E

A
  • elevated expression of antimicrobial lectins RegIIIg and RegIIIb and mucus protein Muc1 mRNAs, genes responses to STAT 3 which is induced by IL-22
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18
Q

what was observed in figure 2f

A
  • no differences in weight change during acute phase of DSS treatment
  • IL-22 neutralization resulted in significant delay in recovery of Lyn^up mice
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19
Q

what were the findings from figure 2

A
  • increased production of IL-22 in Lyn^up mice acts tp enhance intestinal repair and may contribute to protection from DSS colitis
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20
Q

what is the purpose of figure 3

A
  • to access the relative contributions of adaptive vs innate immune cells in limiting colitis in Lyn^up mice
21
Q

what was observed in figure 3 (3)

A
  • protection from DSS in Lyn^up mice was exaggerated in absence of adaptive immune system (Rag-/-)
  • Rag-/-Lyn^up mice showed less weight loss and rectal bleeding and had longer and less inflamed colons than Rag-/- mice
  • ceca of Rag-/- mice showed many more signs of inflammation and damage than Rag-/-Lyn^up mice
22
Q

what were the findings of figure 3

A
  • enhanced Lyn activity in innate immune cells is sufficient to protect mice from DSS-induced intestinal inflammation
23
Q

what was the purpose of figure 4

A
  • determine cell types responsible for increased IL-22 production of Lyn^up mice
24
Q

what was observed in figure 4A (3)

A
  • Lyn^up splenocytes produced more IL-22 than wt in response to IL-23 with or without LPS
  • adaptive immune cells were not required for enhanced IL-22 production
  • Lyn^up splenocytes IL-22 production was dependent on presence of CD90+ cells
25
Q

what was observed in figure 4B

A
  • increase in ILC number in Rag-/-Lyn^up compared to Rag-/- spleens
26
Q

what was observed in figure 4C

A
  • increased IL-22 production by Lyn^up splenocytes required presence of DCs as Lyn^up mice with depleted DCs failed to produce enhanced IL-22 compared with DC-depleted controls
27
Q

are DCs a source of IL-22 in splenic culture?

A
  • no, CD11c+ cells had no detectable IL-22, so DCs are likely an accessory cell that drives increase of IL-22 by Lyn^up ILCs
28
Q

what was observed in figure 4D

A
  • Lyn^up mice showed significant increased levels of IL-22 in blood and colon after LPS injection
29
Q

what was observed in figure 4E (2)

A
  • enhanced IL-22 production in blood and colons of Lyn^up mice was maintained in absence of adaptive immune system (Rag-/-) after LPS injection
  • enhanced IL-22 production by Rag-/-Lyn^up mice following LPS injection required presence of CD90+ cells as depletion of CD90+ cells results in reduction of IL-22 levels in blood and colon
30
Q

what was observed in figure 4F

A
  • unlike the spleen, increased colonic IL-22 production was not dependent on increased ILC numbers as ILC numbers had no difference between control and Lyn^up mice
31
Q

what was observed in figure 4G

A
  • IL-22 levels were reduced in the blood and the colon of LPS-injected DC-depleted Lyn^up and control mice
32
Q

what is brefeldin A used for

A
  • protein that stops Golgi maturation, causing detectable cytokine buildup
33
Q

what was observed in figure 4H (2)

A
  • Rag-/-Lyn^up mice had increased frequency of IL-22 producing cells and produced more IL-22 per cell
  • IL-22 was produced by a heterogenous population of colonic ILCs, including CD4+ and CD4- populations
34
Q

what were the findings from figure 4 (2)

A
  • Lyn activity enhances IL-22 production by ILCs in both gastrointestinal tract and systemically in response to TLR stimulation
  • this response is dependent on DCS
35
Q

what was the purpose of figure 5

A
  • to investigate the expression of Lyn in ILCs and colonic lamina propria (cLP) leukocytes
36
Q

what was observed in figure 5A

A
  • Lyn expression differed between ILC subsets; CD4-Sca-1+ ILCs expressed Lyn, whereas other ILCs did not
37
Q

what was observed in figure 5B

A
  • all DCs expressed Lyn, with expression varying between different subsets
  • CD11b+CD103- DCs exhibited the highest Lyn expression
38
Q

what was observed in figure 5C

A
  • in response to LPS, ILCs cultured with Lyn^up DCs produced more IL-22 than those cultured with wt DCs
39
Q

what was observed in figure 5D

A
  • Lyn^up DCs produced more IL-23 in response to LPS than wt DCs
40
Q

what was observed in figure 5E (2)

A
  • Lyn^up DCs increased expression of Il-12p40 mRNA compared to wt DC
  • there was no difference in Il-23p19 mRNA expression beween Lyn^up DCs and wt DCs
41
Q

what were the findings from figure 5 (2)

A
  • DC function is important in regulating ILC activity
  • increased Lyn activity in DCs regulates systemic responses to microbrial products like LPS, leading to enhanced IL-22 production by ILCs
42
Q

what was the purpose of figure 6

A
  • to investigate if internal hypersensitivity to LPS has a protective role following DSS treatment
43
Q

what was observed in figure 6B

A
  • antibiotic regime successfully reduced bacterial load in gut as assessed by fecal DNA and bacterial 16S rRNA
44
Q

what was observed in figure 6C

A
  • reduction in intestinal bacterial resulted in significant reduction in DSS-induced IL-22 production in wt and Lyn^up mice
45
Q

what was observed in figure 6D and 6E (4)

A
  • without LPS and with microbiota depleted, wt and Lyn^up mice showed similar weight loss
  • wt mice without LPS had high mortality, while microbiota reduced Lyn^up mice displayed more protection from DSS
  • presence of LPS in drinking water to mimic TLR interacting with microbiota did not rescue wt mice from DSS-induced weight loss or mortality
  • presence of LPS in drinking water rescued Lyn^up mice, rendering them with 100% survival and reduced weight loss
46
Q

what are the findings from figure 6

A
  • Lyn activity modulates intestinal responses to microbial products, which significantly impacts the outcome of disease following intestinal damage
47
Q

summary/conclusions from paper (4)

A
  • Lyn activity protects against acute and chronic DSS colitis
  • Lyn^up mice produce increased IL-22 in response to DSS
  • Lyn^up mice produce increased IL-22 in response to LPS
  • Lyn KO mice are susceptible to DSS colitis
48
Q

Lyn activity protects against acute and chronic DSS colitis details (2)

A
  • does not require adaptive immune system
  • hypersensitivity to LPS is sufficient to protect Lyn^up mice from DSS
49
Q

Lyn^up mice produce increased IL-22 in response to LPS details (4)

A
  • independent of the adaptive immune system
  • produced by lineage CD90+ innate lymphoid cells
  • requires DC presence
  • Lyn activity in DCs drives increased IL-22 production by ILCs

MICB 402 (11 decks)

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