Journal: Frontiers in Immunology

Article Title: Supersulfide controls intestinal inflammation by suppressing CD4 + T cell proliferation

doi: 10.3389/fimmu.2025.1506580

Figure Lengend Snippet: Cars2 +/- CD4 + T cells trigger exacerbated colitis in Rag2 -/- mice. (A) Cars2 +/- versus WT naïve CD4 + T lymphocytes induce exaggerated body weight loss in Rag2 -/- mice. Naïve CD4 + T cells derived from WT or Cars2 +/- mice were transferred into Rag2 -/- animals, and the hosts analyzed at different time points. The graph shows relative body weight of the recipient mice (n = 7 to 8). (B, C) Cars2 +/- CD4 + T cells trigger severe histological colitis. Donor cells were transferred into Rag2 -/- mice as described above, and the hosts analyzed 4 weeks later. The representative microscopic images of colonic sections display (B) H&E and (C) CD4-directed immunohistochemical staining while the bar graphs indicate (B) the histological scores as well as (C) quantification of CD4-positive cells in each group (n = 4 to 5). (D) Cars2 +/- CD4 + T lymphocytes accumulate in the intestine to a greater degree than do WT controls. The bar graph shows the absolute number of intestinal donor cells (CD3 + CD4 + Foxp3 - CD44 hi CD62L lo ) at the indicated time points (n = 3 to 5). (E) Th1 as well as Th17 differentiation rate is essentially the same between WT and Cars2 +/- donor cells. Several weeks after transfer into Rag2 -/- mice, donor cells were measured for IFN-γ as well as IL-17A expression. Bar graphs indicating (top) the frequency and (bottom) the absolute number of cytokine-producing cells among the total donor population accumulating in the colon are depicted (n = 3 to 5). (F) The rate of cell death is comparable between WT and Cars2 +/- donor cells. In the above experiments donor cells were stained with amine-reactive dye. The bar graph indicates the frequency of amine-reactive dye + (dead) cells among total donor population in the colon (n = 3 to 5). Data are (A, D-F) pooled from and (B, C) representative of two independent experiments performed. The data are shown as the mean ± standard deviation. Scale bars, 50 μm. * p < 0.05, ** p < 0.01.

Article Snippet: For Th17 condition, recombinant human (rh) IL-6 (30 ng/ml; ThermoFisher), rhIL-6R (66 ng/ml; ThermoFisher), anti-IFN-γ antibody (10 μg/ml; Biolegend) and anti-IL-4 antibody (5 μg/ml; Biolegend) were added.

Techniques: Derivative Assay, Immunohistochemical staining, Staining, Expressing, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Supersulfide controls intestinal inflammation by suppressing CD4 + T cell proliferation

doi: 10.3389/fimmu.2025.1506580

Figure Lengend Snippet: Treatment with GSSSG ameliorates colitis in Rag2 -/- mice that received Cars2 +/- CD4 + T cells. (A) GSSSG treatment inhibits body weight reduction of Rag2 -/- mice that have received Cars2 +/- naïve CD4 + T lymphocytes. Cars2 +/- naïve CD4 + T cells were transferred into Rag2 -/- hosts that were then daily treated with phosphate-buffered saline (PBS) or GSSSG. The graph shows relative body weight of the recipient mice at the indicated time points (n = 6 to 8). (B, C) Histological colitis triggered by Cars2 +/- naïve CD4 + T cells is ameliorated by supplementation with GSSSG. Representative microscopic images of colonic sections showing (B) H&E and (C) CD4-directed immunohistochemical staining together with bar graphs indicating (B) histological scores as well as (C) quantification of CD4-positive cells are displayed (n = 6 to 8). (D) GSSSG treatment inhibits accumulation of Cars2 +/- CD4 + T lymphocytes in the colon. The bar graph shows the absolute number of intestinal donor cells at 1 month post transfer (n = 6). (E) GSSSG inoculation does not affect differentiation of Th1 or Th17 cells. Four weeks after transfer into Rag2 -/- mice supplemented with PBS or GSSSG, donor cells were measured for IFN-γ as well as IL-17A expression. Bar graphs indicating the frequency and the absolute number of cytokine-producing cells among the total donor population accumulating in the colon are depicted (n = 6). (F) The rate of cell death in Cars2 +/- donor cells are comparable between PBS and GSSSG treated groups. In the above experiments donor cells were stained with amine-reactive dye. The bar graph indicates the frequency of amine-reactive dye + (dead) cells among total donor population in the colon (n = 6). (G) GSSSG administration inhibits cell cycle entry of Cars2 +/- CD4 + T cells at an early phase of colitis. The representative dot plots show expression levels of Ki67 and total amounts of DNA in donor cells while the bar graphs show the frequency of cells within the indicated cell cycle phases among total donor populations at 1 week and 4 weeks post transfer (n = 3 to 6). (H) GSSSG administration upregulates Trp53 expression in colonic Cars2 +/- T cells. A bar graph showing relative expression of Trp53 in donor cells at 1 week after transfer is displayed (n = 5 to 7). (I) GSSSG suppresses cell cycle entry of Cars2 +/- CD4 + T lymphocytes in vitro . Cars2 +/- naïve CD4 + T cells were stimulated with CD3 and CD28 in the presence or absence of GSSSG, and the cell cycle phases analyzed 3 days later. The representative dot plots show expression levels of Ki67 and total amounts of DNA in CD4 + T cells while the bar graphs show the frequency of cells within the indicated cell cycle phases (n = 4). Data are pooled from two or three independent experiments. The data are shown as the mean ± standard deviation. Scale bars, 50 μm. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For Th17 condition, recombinant human (rh) IL-6 (30 ng/ml; ThermoFisher), rhIL-6R (66 ng/ml; ThermoFisher), anti-IFN-γ antibody (10 μg/ml; Biolegend) and anti-IL-4 antibody (5 μg/ml; Biolegend) were added.

Techniques: Saline, Immunohistochemical staining, Staining, Expressing, In Vitro, Standard Deviation