Journal: Clinical Science (London, England : 1979)

Article Title: CCL5 deficiency aggravates acute DSS-induced colitis by restricting IL-33-induced formation of Tregs in intestinal tract

doi: 10.1042/CS20256734

Figure Lengend Snippet: Wildtype (WT) and CCL5 knockout ( Ccl5 -KO) mice of both C57BL/6 and BalB/C backgrounds were subjected to 7-day 2.5% DSS administration in the drinking water. ( A ) Schematic of construction of DSS-induced colitis model. ( B ) Disease activity index (DAI), n = 25 per group. ( C ) Survival rate of WT ( n = 25) and Ccl5 -KO ( n = 25) mice was observed for 2 weeks, and the corresponding survival curves were plotted. ( D and E ) Gross anatomy of colons ( D ) and colon length ( E ) were monitored. Colon length was measured in both the C57BL/6 and BalB/C mouse strains after 7 days of 2.5% DSS treatment. The results are shown as the mean ± SEM, with statistical significance assessed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. ( F and G ) Representative H&E staining ( F ) of colon sections from WT and Ccl5 -KO mice 4 days after 7-day 2.5% DSS administration (scale bars: 50 μm, 100 μm). Histological score ( G ) was quantified. Results shown are the mean ± SEM (ns, nonsignificant; ** P <0.01, *** P <0.001, **** P <0.0001) of triplicate determination from three independent experiments.

Article Snippet: To rescue the phenotypes of enteritis, exogenous recombinant murine CCL5 protein (HY- P71890 , MCE) [ ] was intraperitoneally injected at a concentration of 350 ng/kg from the onset of DSS treatment.

Techniques: Knock-Out, Activity Assay, Staining

Journal: Clinical Science (London, England : 1979)

Article Title: CCL5 deficiency aggravates acute DSS-induced colitis by restricting IL-33-induced formation of Tregs in intestinal tract

doi: 10.1042/CS20256734

Figure Lengend Snippet: ( A and B ) Flow cytometric analysis of number of CD4 + and CD8 + T cells in intestinal lymphoid tissues from mice with indicated genotypes 4 days after 7-day 2.5% dextran sulfate sodium salt (DSS) administration. Graphs ( B ) show the relative percentage of, respectively, CD4 + and CD8 + T cells ( n = 6 per group). ( C and D ) Representative CD4 staining of distal colon sections from wildtype (WT) and CCL5 knockout ( Ccl5 -KO) mice 4 days after 7-day 2.5% DSS administration ( C ; scale bars, 20 μm), with quantitative analysis ( D , n = 50); black arrows indicate CD4-positive cells. ( E and F ) Flow cytometric plots ( E ) of FOXP3 + CD4 + T cell population in intestines from control or DSS-treated mice with indicated genotypes ( n = 6 per group). Percentage of FOXP3 + population among CD4 + T cells is shown ( F , n = 6). ( G and H ) Representative FOXP3 staining of distal colon sections from WT and Ccl5 -KO mice 4 days after 7-day 2.5% DSS administration ( G ; scale bars: 10 μm, 50 μm), with quantitative analysis ( H , n = 50); red arrows indicate FOXP3-positive cells. ( I ) Immunoblotting of FOXP3 expression in colonic tissues from WT and Ccl5 -KO mice 4 days after 7-day 2.5% DSS administration. Results shown are the mean ± SEM (ns, nonsignificant; * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001) of triplicate determination from three independent experiments.

Article Snippet: To rescue the phenotypes of enteritis, exogenous recombinant murine CCL5 protein (HY- P71890 , MCE) [ ] was intraperitoneally injected at a concentration of 350 ng/kg from the onset of DSS treatment.

Techniques: Staining, Knock-Out, Control, Western Blot, Expressing

Journal: Clinical Science (London, England : 1979)

Article Title: CCL5 deficiency aggravates acute DSS-induced colitis by restricting IL-33-induced formation of Tregs in intestinal tract

doi: 10.1042/CS20256734

Figure Lengend Snippet: ( A ) Enzyme-linked immunosorbent assay (ELISA) analysis of the level of IL-33 in colon explant cultures at the indicated time points (days 0, 2, 4, 6, 8) during the 7-day dextran sulfate sodium salt (DSS) treatment in wildtype (WT) and CCL5 knockout ( Ccl5 -KO) mice ( n = 6 per group). ( B ) Immunoblotting of IL-33 and ST2 in intestinal epithelial cells of WT and Ccl5 -KO mice 4 days after 7-day 2.5% DSS administration. ( C ) Immunoblotting of NF-κB ( P65 ) related pathway in intestinal epithelium of WT and Ccl5 -KO mice 4 days after 7-day 2.5% DSS administration. ( D ) Immunofluorescence staining for P65 (red) in colonic sections of WT and Ccl5 -KO mice 4 days after 7-day 2.5% DSS administration; DNA (DAPI, blue); scale bars, 50 μm. White arrows indicate P65-positive cells with nuclear translocation. ( E ) Immunoblotting of PI3K/Akt pathway in intestinal epithelium of WT and Ccl5 -KO mice 4 days after 7-day 2.5% DSS administration. ( F ) ELISA analysis of IL-33 levels in colon explant cultures of WT and Ccl5 -KO mice at the indicated time points (days 0, 2, 4, 6, 8) during 7-day DSS treatment with CCL5 small protein interventions ( n = 6 per group). ( G ) Colon length measurements on day 12 in Ccl5 -KO mice treated with different drug groups (control, CCL5 small protein, CCL5 small protein + BAY 11-7082, CCL5 small protein + Capivasertib); n = 6 per group. ( H ) Recording of DAI different time points ( D0, D3, D6, D9, D12 ) in different drug treatment groups during the treatment period. ( I ) Immunoblot analysis of corresponding protein levels in the intestinal epithelial tissues of Ccl5 -KO mice after treatment with different drug groups. ( J ) Immunofluorescence staining analysis of P65-positive (red) cells in the intestines of Ccl5 -KO mice treated with different drug groups (control, CCL5 small protein, CCL5 small protein + Capivasertib); DNA (DAPI, blue), scale bars, 50 μm. White arrows indicate P65-positive cells with nuclear translocation. Results shown are the mean ± SEM (ns, nonsignificant; * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001) of triplicate determination from three independent experiments.

Article Snippet: To rescue the phenotypes of enteritis, exogenous recombinant murine CCL5 protein (HY- P71890 , MCE) [ ] was intraperitoneally injected at a concentration of 350 ng/kg from the onset of DSS treatment.

Techniques: Enzyme-linked Immunosorbent Assay, Knock-Out, Western Blot, Immunofluorescence, Staining, Translocation Assay, Control

Journal: Clinical Science (London, England : 1979)

Article Title: CCL5 deficiency aggravates acute DSS-induced colitis by restricting IL-33-induced formation of Tregs in intestinal tract

doi: 10.1042/CS20256734

Figure Lengend Snippet: ( A ) Immunoblotting of IL-33, ST2, forkhead box protein 3 (FOXP3), and JAK1/STAT5 signaling in intestinal CD4 + T cells of wildtype (WT) and CCL5 knockout ( Ccl5 -KO) mice 4 days after 7-day 2.5% dextran sulfate sodium salt (DSS) administration. ( B and C ) Mice received daily intraperitoneal injections of rIL-33 protein (10 ng/µL) at the onset of the DSS regimen. The intraperitoneal administration of different therapeutic agents continued for an additional 4 days after 7-day DSS treatment. Subsequently, the mice were killed, and the affected intestinal tissues were analyzed. Disease activity index (DAI) ( B ) and colon length ( C ) were monitored, n = 6 per group. ( D and E ) Representative H&E staining ( D ) of colon sections in mice from different treated groups (scale bars, 50 μm). Histological score ( E ) was quantified. ( F and G ) Representative FOXP3 staining of distal colon sections from rescued DSS-treated mice with indicated genotypes after 8-day rIL-33/PBS treatment ( F ; scale bars, 20 μm) and quantitative analysis ( G , n = 50). ( H and I ) Flow cytometric plots ( H ) of FOXP3 + CD4 + T cell population in intestines from rescued DSS-treated mice with indicated genotypes after 8-day rIL-33/PBS treatment and quantitative analysis ( I , n = 6). ( J ) Immunoblotting of FOXP3 and JAK1/STAT5 signaling in intestinal CD4 + T cells, respectively, from rescued DSS-treated mice with indicated genotypes after 8-day rIL-33/PBS treatment. The results are shown as the mean ± SEM (ns, nonsignificant; * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001) of triplicate determination from three independent experiments, with statistical significance assessed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test.

Article Snippet: To rescue the phenotypes of enteritis, exogenous recombinant murine CCL5 protein (HY- P71890 , MCE) [ ] was intraperitoneally injected at a concentration of 350 ng/kg from the onset of DSS treatment.

Techniques: Western Blot, Knock-Out, Activity Assay, Staining

Journal: Clinical Science (London, England : 1979)

Article Title: CCL5 deficiency aggravates acute DSS-induced colitis by restricting IL-33-induced formation of Tregs in intestinal tract

doi: 10.1042/CS20256734

Figure Lengend Snippet: ( A ) Immunoblotting ( n = 12) of CCL5 and FOXP3 expression in inflamed tissue in ulcerative colitis (UC) patients. ( B ) Quantitative polymerase chain reaction (qPCR) analysis ( n = 32) of CCL5 expression, respectively, in intestinal epithelial (IECs) and stromal cells (SCs) from inflamed and adjacent normal tissue in UC patients. ( C and D ) Representative H&E staining (right), CCL5 (middle), and FOXP3 (left) staining of inflamed intestinal tissue from UC patients with different levels of CCL5 expression (CCL5 [high] vs. CCL5 [low]), ( C ; scale bars, 20 μm, 100 μm) and quantitative analysis of CCL5 and FOXP3 expression ( D , n = 32 per group) in inflamed (UC) and adjacent normal tissue (NC). ( E ) Correlation analysis between the proportion of CCL5-positive cells and FOXP3-positive cells in inflamed (UC) tissue from UC patients ( n = 32). ( F ) Quantitative real-time polymerase chain reaction (RT-qPCR) analysis of the correlation between CCL5 and FOXP3 expression levels in inflamed intestinal tissue ( n = 32) from UC patients with different levels of CCL5 expression. ( G ) Flow cytometric plots (right) of FOXP3 + CD4 + T cell population in intestines from CCL5-low and CCL5-high UC patient tissues with quantitative analysis (left, n = 6). Results shown are the mean ± SEM (ns, nonsignificant; *** P <0.001, **** P <0.0001) of triplicate determination from three independent experiments.

Article Snippet: To rescue the phenotypes of enteritis, exogenous recombinant murine CCL5 protein (HY- P71890 , MCE) [ ] was intraperitoneally injected at a concentration of 350 ng/kg from the onset of DSS treatment.

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Staining, Quantitative RT-PCR