Fig 1.
BJ-3105 decreases the percentage of Th1 and Th17 cells differentiation.
(A) Chemical Structure of BJ-3105 (2,4,5-trimethyl-6-(3-phenylpropoxy)pyridin-3-ol). (B) Purified naïve mouse CD4+ T cells from spleens and draining lymph nodes were stimulated in Th1 and Th17 polarizing conditions for 72 h and Treg for 96 h in the presence of BJ-3105 (1 μM) or tofacitinib (1 μM). CD4+ T cells were then restimulated with PMA, ionomycin and golgistop for 4 h and analyzed by intracellular cytokine staining by flow cytometer. The untreated controls were cultured in the presence of DMSO. Percentage of IFN-γ+ Th1 cells, IL-17A+ Th17 cells and Foxp3+ Treg cells were shown in bar diagram. (C) IFN-γ and IL-17A cytokine produced by CD4+ T cells were quantified by cytokine binding assay. (D) Th1, Th17 differentiation with different dose of BJ-3105. (E) Compilation of data from three individual experiments showing the inhibitory effect of different dose of BJ-3105 on Th1 (Upper) and Th17 (bottom) cytokine production were shown. For each cytokine, data were normalized to the percentage of cytokine producing cells in the absence of BJ-3105. Representative results of three experiments are shown. *p<0.05, **p<0.001 compared with drug untreated group. Data shown are mean±SEM.
Fig 2.
BJ-3105 decreases the percentage of antigen-specific Th1 and Th17 cell differentiation.
(A) Naïve CD4+ T cells and antigen presenting cells from spleens and lymph nodes were isolated by immunomagnetic positive selection from 6–10 weeks OT-II mice. CD4+ T cells and irradiated antigen presenting cells were cultured in Th1, Th17 and Treg cells differentiation conditions in the presence of OVA323–339 (0.1 μM) with BJ-3105 (1 μM) or tofacitinib (1 μM). CD4+ T cells were then restimulated with PMA, ionomycin and golgistop for 4 h and analyzed by intracellular cytokine staining by flow cytometer. The untreated controls were cultured in the presence of DMSO. (B) The cells were cultured in Th1 and Th17 cells differentiation conditions for 72 h with different concentration of BJ-3105 and analyzed by flow cytometer. (C) Compilation of data from three individual experiments showing the inhibitory effect of different dose of BJ-3105 on Th1 (Upper) and Th17 (bottom) cytokine production were shown. For each cytokine, data were normalized to the percentage of cytokine producing cells in the absence of BJ-3105. Representative results of three experiments are shown. *p<0.05, compared with drug untreated group. Data shown are mean±SEM.
Fig 3.
BJ-3105 slightly inhibits CD4+ T cell proliferation without inducing apoptosis.
CFSE-labeled naïve CD4+ T cells were activated with anti-CD3 anti-CD28 under the treatment of indicated dose of BJ-3105 for 3 days in (A) Th1 and (B) Th17 differentiation conditions. Cell proliferation was monitored by assessing CFSE dilution by flow cytometry. (C) Naïve CD4+ T cells and APCs from spleens and lymph nodes were isolated and activated under Th1 differentiation condition in the presence of BrdU (10 μM) with or without BJ-3105 (1 μM) and analyzed after 3 days by flow cytometer. Representative bar diagram for percentage of BrdU+CD4+ T cells were shown. (D) Representative Dot plots examining CD4+ T cell proliferation by detecting Ki-67 in Th1 differentiation condition with or without BJ-3105 by flow cytometer. Percentage of Ki-67 positive CD4+ T cells were shown. (E) Live cells were detected by Anexin-V and PI staining after cultured in Th1 differentiation conditions for 72 h and analyzed by flow cytometer. (F) Percentage of live cells on dose dependent treatment of BJ-3105. Representative results of three experiments are shown. *p < 0.05, compared with drug untreated group. Data shown are mean ± SEM.
Fig 4.
BJ-3105 treatment suppresses development of EAE by decreasing autoreactive Th1 and Th17 generation.
Active EAE was elicited in 8–12 weeks female C57BL/6 mice by immunization with MOG35-55. The mice were subcutaneously treated with vehicle or BJ-3105 (3 mg/kg/every other day) from day 1 post-immunization as detailed under materials and methods. Mice were monitored for signs and symptoms of EAE. (A) Clinical score were recorded every day. (B) Total cell count in spleen and CNS of drug treated and untreated EAE mice. (C) At 24 days after challenge, total mononuclear cell obtained from the brains and spinal cord of MOG35–55 immunized wild-type mice and BJ-3105-treated mice. Total percent of infiltrated CD4+ T cells and CD8+ T cells in CNS. (D) After 24 days of EAE, lymphocytes were isolated from the spleen, lymph nodes and spinal cord of mice. The fraction of IFN-γ and IL-17A producing CD4+ T cells from spleen, draining lymph node and CNS were determined by intracellular staining and analyzed by flow cytometry. Percentage of Th1 and Th17 cells were shown. (E) Flow cytometry analysis of CXCR3 expression on activated T cells under Th1 and CCR6 expression under Th17-polarizing conditions with or without BJ-3105 (1 μM). Bar diagram showing the mean fluorescence intensity (MFI) of CXCR3 and CCR6. Mean±SEM of multiple mice are shown. *p < 0.05, compared with drug untreated group.
Fig 5.
BJ-3105 controls T cell differentiation through inhibiting phosphorylation of JAK-STAT signaling pathway.
Naïve CD4+ T cells from spleens and draining lymph nodes were isolated and cells were cultured in Th1 and Th17 cells differentiation conditions with BJ-3105. Drug and cytokine untreated groups were used as control. (A) Naïve CD4+ T cells were polarized to Th1 cells with anti-CD3 (2 μg/mL), anti CD28 (1 μg/mL) and IL-12 (10 ng/mL) plus anti-IL4 Ab (5 μg/mL) for 72 h of culture. Th17 cells were differentiated with anti-CD3 (2 μg/mL), anti CD28 (1 μg/mL), TGF-β (1 ng/mL), IL-6 (10 ng/mL) plus anti-IL4 Ab (5 μg/mL) and anti-IFN-γ Ab (5 μg/mL) for 72 h of culture. Phosphorylated STAT1 and STAT4 from Th1 cells and STAT3 from Th17 cells were analyzed by flow cytometer. (B) Phosphorylated and total JAK1/2, STAT1 and STAT4 were detected by immuno-blotting under Th1 polarizing condition. (C) Phosphorylated and total JAK1/2 and STAT3 were detected by immuno-blotting under Th17 polarizing condition. (D) Western blot analysis of phosphorylated and total JAK1, JAK2, STAT1, STAT3 and STAT4 proteins in splenocytes and CNS infiltrated mononuclear cells from EAE induced mice. β-actin as loading control were detected by immune-blotting. Representative results of three experiments are shown.