Table 1.
The disease activity index score (DAI).
Table 2.
Sequence of primer pairs used in real-time quantitative PCR.
Fig 1.
AZD8055 attenuates the development of intestinal inflammation.
When the mice developed clinical signs of disease, they were sacrificed. (A) During the course of colitis, body weights were recorded and calculated as percentage of the initial weight at day 1 (n = 8 for AZD, n = 8 for control). (B) Disease activity index (DAI) of each group. (C) Gross morphology of each group. (D) The length of colon of each group. (E) Histological observations of colon sections with H&E staining (Original magnifications, 200×magnification). (F) Histological score of each group. All data represented the mean±SEM. Statistical significance was assessed by t-test. *P<0.05, **P<0.01.
Fig 2.
AZD8055 treatment suppresses the expression of pro-inflammatory mediators.
(A-F) The expression profiles of IFN-γ, TNF-α, IL-1β, IL-6, IL-17A and IL-10 were determined in the colon of emulsifier or AZD8055-treated mice by real-time PCR. Data was normalized to the expression of HPRT mRNA (n = 8/group). All data represented the mean±SEM. Statistical significance was assessed by t-test. *P<0.05, **P<0.01.
Fig 3.
AZD8055 treatment leads to a decrease in the percentage of CD4+ T cells and CD8+ T cells in vivo.
(A) The percentage of CD4+ T cells in the spleen, lymph nodes and peripheral blood of mice treated with AZD8055 or emulsifier. (B) The percentage of CD8+ T cells in the spleen, lymph nodes and peripheral blood of mice treated with AZD8055 or emulsifier. (C) The cell number of CD4+ T cells and CD8+ T cells in the spleen, lymph nodes and peripheral blood of mice treated with AZD8055 or emulsifier. All data represented the mean±SEM. Statistical significance was assessed by t-test. *P<0.05, **P<0.01.
Fig 4.
AZD8055 inhibits the proliferation of CD4+ T cells and CD8+ T cells in vitro.
CD4+ T cells and CD8+ T cells isolated from spleen and mesenteric lymph node of mice were stimulated with anti-CD3/CD28 and labeled with CFSE in the presence of AZD8055 (10–50nM) for two or three days. (A) The proliferation of CD4+ T cells in the presence of AZD8055 were examined by flow cytometry. (B) The proliferation of CD8+ T cells in the presence of AZD8055 were examined by flow cytometry. Results were from three independent experiments.
Fig 5.
AZD8055 inhibits TH1 and TH17 cell polarization and expands Treg cell polarization in vivo.
Lymphocytes were isolated from the spleen, mesenteric lymph nodes and lamina propria and analyzed by flow cytometry on day7. (A) The percentage of TH1 cells (CD4+IFN-γ+), TH17 cells (CD4+IL-17+) and Treg cells (CD4+FOXP3+) in spleen treated with AZD8055 or emulsifier. (B) The percentage of TH1 cells, TH17 cells and Treg cells in mesenteric lymph nodes treated with AZD8055 or emulsifier. (C) The percentage of TH1 cells, TH17 cells and Treg cells in lamina propria treated with AZD8055 or emulsifier. (D) The cell number of CD4+ T cells and CD8+ T cells in the mesenteric lymph nodes and lamina propria treated with AZD8055 or emulsifier. The data were expressed as mean±SEM. Statistical significance was assessed by t-test. *P<0.05, **P<0.01.
Fig 6.
AZD8055 inhibits the proliferation and differentiation of naive CD4+ T cells in vitro.
Naive CD4+ T cells were isolated from spleen and lymph nodes of mice, CFSE-labeled, and activated in the presence of AZD8055 (0nM, 20nM, 50nM) under TH0, TH1 and TH17 conditions for 4 days. (A) TH1 cells were activated with PMA + Ionomycin and stained for CD4 and intracellular expression of IFN-γ in the presence of AZD8055. Dot-plots showed the expression of CD4+ IFN-γ+ TH1 cells under TH0 condition (Upper panels). Dot-plots showed the differentiation of TH1 cells under TH1 condition (Middle panels). Scatterplot displayed the proliferation of TH1 cells under TH1 condition (Lower panels). (B) TH17 cells were stimulated with PMA + Ionomycin and stained for CD4 and intracellular expression of IL-17 in the presence of AZD8055. Dot-plots showed the expression of CD4+ IL-17 + TH17 cells under TH0 condition (Upper panels). Scatterplot displayed the differentiation of TH17 cells under TH17 condition (Lower panels). (C) Results were from three independent experiments. All results showed the mean±SEM. Statistical significance was determined by student’s t-test. *P<0.05, **P<0.01, ***P<0.005.
Fig 7.
AZD8055 expands the proliferation and differentiation of Treg cells in vitro.
Naive CD4+ T cells were isolated from spleen and lymph nodes of mice, CFSE-labeled, and activated in the presence of AZD8055 (0nM, 20nM) under TH0 and Treg conditions for 4 days. (A) Treg cells were permeabilized and stained for CD4 and intracellular expression of Foxp3 in the presence of AZD8055 (0nM, 20nM). Dot plots showed the differentiation of CD4+ Foxp3+ Treg cells under TH0 and Treg conditions (Upper panels). Scatterplot displayed the proliferation of Treg cells under TH0 and Treg conditions treated with AZD8055 (Lower panels). (B) Results were from three independent experiments. All results showed the mean±SEM. Statistical significance was evaluated by student’s t-test. *P<0.05, **P<0.01, ***P<0.005.