IL-2–mTORC1 signaling coordinates the STAT1/T-bet axis to ensure Th1 cell differentiation and anti-bacterial immune response in fish
Fig 5
STAT1/T-bet axis regulates the Th1 differentiation in tilapia.
(A) Spleen leukocytes were stimulated with PHA for 12 h. Relative mRNA levels of T-bet, STAT1 and STAT4 were examined by qPCR, n = 4. (B) Flow cytometry showed the phosphorylation level of STAT1 in lymphocytes that stimulated with PHA. (C) Transcriptional levels of IFN-γ in HEK 293T cells that transfected with tilapia T-bet, STAT1 and STAT4, n = 6. (D) HEK 293T cells were co-transfected with tilapia T-bet, STAT1 or STAT4, and pGL3-IFN-γ promoter. The LUC activities were assessed at 48 h post-transfection, n = 4. (E-G) Spleen leukocytes from tilapia that i.p. injected with T-bet-specific, STAT1-specific or control siRNA for 2 days were harvested and stimulated with CD3ε mAb for 12 h. Relative mRNA levels of STAT1, T-bet and IFN-γ (E, F, n = 4), and the percentage of CD3+CD4-1+IFN-γ+ T cells (G) were examined. (H-L) Tilapia i.p. injected with T-bet-specific, STAT1-specific or control siRNA were infected with E. piscicida. Tilapia was i.p. injected with BFA 6 h before sacrifice, and spleen leukocytes were harvest for assay. Western blot assay showed the expression of IFN-γ at 48 h post infection (H). Flow cytometry and scatter plot figures showed the percentage and absolute numbers of CD3+CD4-1+ T cells (I, K) and CD3+CD4-1+IFN-γ+ T cells (J, L) on 5 DPI, n = 4. (M-O) Tilapia individuals that infected with E. piscicida were injected with STAT1 inhibitor Fludarabine or PBS, and animals were i.p. injected with BFA 6 hours before sacrifice. (M) Western blot assay showed the protein levels of IFN-γ in spleen leukocytes on indicated days. (N) Flow cytometry showed the percentage of IFN-γ in spleen CD4-1+ T cells on day 7 post-infection. (O) Kaplan-Meyer survival plot showed the survival percentage of tilapia, n = 25. These experiments were repeated for at least two independent times. *: p<0.05, **: p<0.01, ***: p<0.001, determined by a two-tailed Student’s t-test. The accession numbers of selected sequences were listed in S1 Table.