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

Journal: bioRxiv

Article Title: DIA-based quantitative proteomics reveal the protective effects of quercetin against atopic dermatitis via attenuating inflammation and modulating immune response

doi: 10.64898/2026.01.08.698465

Figure Lengend Snippet: WB verified the key differential expression proteins. HaCaT cells were pretreated with quercetin (40 or 80 μM) for 30 min and then stimulated with TNF-α/IFN-γ for 24 h. Protein expression levels of IKBKE, IDO1, CXCL9, and RANTES were detected by western blotting and quantified relative to GAPDH.

Article Snippet: Antibodies against IL-1α, IL-1β, RANTES, IDO1, CXCL9, IKBKE, ICAM-1 and GAPDH were purchased from Proteintech (Wuhan, China).

Techniques: Quantitative Proteomics, Expressing, Western Blot

Journal: Cell & Bioscience

Article Title: A CCL5 + CD20 + CD8 + T cell subset drives pathogenic transformation of synoviocytes in rheumatoid arthritis via JAK-STAT signaling

doi: 10.1186/s13578-025-01516-5

Figure Lengend Snippet: Single-cell profiling of PBMCs from RA patients and HC revealed distinct clustering patterns of CD20 + T cell populations. a t-SNE plot of CD20 + T cells from PBMCs of RA patients ( n = 9) and HC ( n = 3), colored by group. b tSNE plot (left panel) and subgroup proportion pie chart (right panel) of CD20 + T cells from all donors. Subgroups were divided based on the expression levels of marker genes CD4 or CD8A . c tSNE plot of CD20 + T cells from PBMCs of all donors, showing 3 clusters based on the unsupervised graph-based clustering. d Dot plot for expression of marker genes for three CD20 + T cell subtypes. The color of the dots shows the relative expression levels of genes, and the size of the dots shows the percentage of expressed cells. e Heatmap showing expression levels of discriminative genes for each cluster. Cluster 0 prominently features high expression of CCL5 , NKG7 and GNLY ; Cluster 1 is defined by combined enrichment of CCR7 , IL6ST and TRABD2A ; Cluster 2 shows distinct expression of GATA3 , ITGB1 and S100A11 . f Feature plots of single-cell expression for identified genes across three CD20 + T cell clusters

Article Snippet: Briefly, RA-FLSs were serum-starved for 4–6 h and then pretreated for 30 min under the following conditions [ ]: (1) IL-1β (5 ng/mL); (2) CCL5 (2 ng/mL; MCE, Cat#HY-P70450) + IL-1β (5 ng/mL); (3) 25% (v/v) CD20 + CD8 + T-CM + IL-1β (5 ng/mL).

Techniques: Expressing, Marker

Journal: Cell & Bioscience

Article Title: A CCL5 + CD20 + CD8 + T cell subset drives pathogenic transformation of synoviocytes in rheumatoid arthritis via JAK-STAT signaling

doi: 10.1186/s13578-025-01516-5

Figure Lengend Snippet: The proportion of CCL5 + T cells in CD20 + CD8 + T cells was significantly elevated in naïve RA patients. a Schematic representation of gating strategy for flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) derived from HC and RA patients. b The frequencies of CD20 + CD8 + T cells were analyzed in PBMCs samples collected from HC ( n = 63) and naïve RA patients ( n = 69). Data shown as median with interquartile range (IQR), Mann-Whitney U test, **** P < 0.0001. c The frequencies of CCL5 + cells within CD20 + CD8 + T cells were shown for HC ( n = 32) and naïve RA patients ( n = 35). Data shown as median with interquartile range (IQR), Mann-Whitney U test, **** P < 0.0001. d The proportion of CCL5 + cells was significantly higher in CD20 + CD8 + T cells than in CD20 – CD8 + T cells in both HC and RA patients. Data shown as median with interquartile range (IQR), Wilcoxon matched-pairs signed-rank test, **** P < 0.0001. e The proportion of CCL5 + cells within CD20 + CD8 + T cells positively correlated with inflammation and disease activity of RA. Data shown as individual scatter points, Spearman’s rank correlation, P < 0.05. f KEGG pathway analysis revealed that the top five most significantly enriched pathways for DEGs between CD20 + CD8 + T and CD20 – CD8 + T cells included the chemokine signaling pathway, and CXCR3 and CCR5 were identified as genes significantly enriched in this pathway. g Violin plots (upper panel) demonstrated that the expression levels of CXCR3 and CCR5 genes were significantly upregulated in CD20 + CD8 + T cells compared to CD20 – CD8 + T cells. **** P < 0.0001; * P < 0.05. UMAP plot (lower panel) visualized the distribution of CXCR3 + or CCR5 + cells among CD8 + T cells. ( h Flow cytometric analysis demonstrated significantly higher proportions of CXCR3 + or CCR5 + cells among CD20 + CD8 + T cells compared to CD20 – CD8 + T cells in PBMCs from RA patients ( n = 27 in each group). Wilcoxon matched-pairs signed-rank test, **** P < 0.0001

Article Snippet: Briefly, RA-FLSs were serum-starved for 4–6 h and then pretreated for 30 min under the following conditions [ ]: (1) IL-1β (5 ng/mL); (2) CCL5 (2 ng/mL; MCE, Cat#HY-P70450) + IL-1β (5 ng/mL); (3) 25% (v/v) CD20 + CD8 + T-CM + IL-1β (5 ng/mL).

Techniques: Derivative Assay, MANN-WHITNEY, Activity Assay, Expressing

Journal: Cell & Bioscience

Article Title: A CCL5 + CD20 + CD8 + T cell subset drives pathogenic transformation of synoviocytes in rheumatoid arthritis via JAK-STAT signaling

doi: 10.1186/s13578-025-01516-5

Figure Lengend Snippet: CD20 + CD8 + T cells may promote the expansion of invasive FAPα + FLS through CCL5-mediated activation of the JAK-STAT signaling pathway. a Gating strategy for identifying FLS involved sequential selection of CD45-negative (CD45 – ) cells followed by positive selection for PDPN (podoplanin) expression. The frequencies of FAPα + cells in the PDPN + CD45 – CD31 – population were quantified and compared between normal human controls (NH, n = 9) and RA patients ( n = 9). (mean ± SEM, unpaired two-tailed t -test, *** P < 0.001). b Representative flow cytometry plots depict FAPα + cells (gated on PDPN + CD45 – CD31 – populations) with corresponding quantitative analysis following 48-hour treatment under various experimental conditions. (mean ± SEM, n = 7 in each group, one-way ANOVA with Tukey’s post hoc test, * P < 0.05, **** P < 0.0001). c Representative images from the Transwell assay demonstrate the migratory capacity of FLS under the experimental conditions corresponding to panel (b). Scale bar: 50 μm. (mean ± SEM, n = 3 in each group, one-way ANOVA with Tukey’s post hoc test, * P < 0.05; *** P < 0.001). d The frequency of FAPα-expressing FLS was assessed by flow cytometry following 48-hour incubation with CCR1 antagonist BX471 (5 µM) or CCR5 antagonist maraviroc (5 µM) in IL-1β (5 ng/mL) + CD20 + CD8 + T-CM cultures. (mean ± SEM, n = 6 in each group, one-way ANOVA with Tukey’s post hoc test, * P < 0.05; ** P < 0.01). e Representative histogram plots demonstrate FAPα expression in FLS following stimulation with IL-1β (5 ng/mL) in combination with either CD20 + CD8 + T-CM alone, or T-CM + tofacitinib (JAK inhibitor). (mean ± SEM, n = 6 in each group, paired t- test, *** P < 0.001). ( f ) ELISA was performed to quantify CCL5 concentrations in culture supernatants under experimental conditions corresponding to panels (B) and (C). (mean ± SEM, n = 6 in each group, one-way ANOVA with Welch’s correction, followed by Dunnett’s T3 post hoc test, ** P < 0.01). g Heatmap depicting transcriptomic profiling results, showing expression levels of differentially expressed genes across experimental groups. h GO (upper panel) and KEGG pathway (lower panel) analyses of upregulated genes in FLS treated with CD20 – CD8 + T-CM + IL-1β (left), and FLS treated with CD20 + CD8 + T-CM + IL-1β (right), both compared to IL-1β alone control (CTL). q -value < 0.05

Article Snippet: Briefly, RA-FLSs were serum-starved for 4–6 h and then pretreated for 30 min under the following conditions [ ]: (1) IL-1β (5 ng/mL); (2) CCL5 (2 ng/mL; MCE, Cat#HY-P70450) + IL-1β (5 ng/mL); (3) 25% (v/v) CD20 + CD8 + T-CM + IL-1β (5 ng/mL).

Techniques: Activation Assay, Selection, Expressing, Two Tailed Test, Flow Cytometry, Transwell Assay, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: Cell & Bioscience

Article Title: A CCL5 + CD20 + CD8 + T cell subset drives pathogenic transformation of synoviocytes in rheumatoid arthritis via JAK-STAT signaling

doi: 10.1186/s13578-025-01516-5

Figure Lengend Snippet: Schematic of CCL5 + CD20 + CD8 + T cells driving synoviocyte transformation. In RA, circulating CD20 + CD8 + T cells with high co-expression of chemokine receptors CXCR3 and CCR5 are chemoattracted from peripheral blood into synovial tissues by cognate chemokines (such as CXCL9 and CCL5). CD20 + CD8 + T cells further engage in crosstalk with fibroblast-like synoviocytes (FLS) through CCL5 secretion. This chemokine-dependent signaling engages cognate receptors (i.e., CCR1, CCR5) on FLS, subsequently activating the JAK/STAT signaling cascade, inducing phenotypic transformation of FLS (PDPN + CD45 – CD31 – ) into pathogenic FAPα + subpopulation. The FAPα + subpopulation demonstrates enhanced proliferative capacity, increased invasive migration, and robust secretion of synovial inflammatory cytokines, chemokines, and pro-angiogenic factors, collectively driving pannus formation and progressive joint destruction. ECM: extracellular matrix; MMPs: matrix metalloproteinases; TNFα: tumor necrosis factor α.

Article Snippet: Briefly, RA-FLSs were serum-starved for 4–6 h and then pretreated for 30 min under the following conditions [ ]: (1) IL-1β (5 ng/mL); (2) CCL5 (2 ng/mL; MCE, Cat#HY-P70450) + IL-1β (5 ng/mL); (3) 25% (v/v) CD20 + CD8 + T-CM + IL-1β (5 ng/mL).

Techniques: Transformation Assay, Expressing, Migration