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
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
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
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
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
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
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)
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
9
Q
what were the findings of figure 1A - 1D
A
- Lyn plays a protective role in DSS-induced colitis
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
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
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
13
Q
what is the purpose of figure 2
A
- investigate contribution of cytokine responses in Lyn-mediated protection from DSS-colitis
14
Q
how are IL-22 and IL-23 connected
A
- IL-23 drives production of IL-22
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
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
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
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
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
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
