Figure 3.
Th1 and Th17 differentiation was seen in TMAO-fed GVHD mice but not in the TMAO-supplemented in vitro culture system. (A) Experimental scheme of BMT mice or GVHD mice protocol. Recipients were fed with TMAO or vehicle from day −14 and thereafter. Data were collected on day 7 and day 14. (B) Representative in vivo fluorescence image of skull BM in recipient mice on day 7 and day 14. The BALB/c recipients were injected with B6-Tg(CAG-EGFP) TCD-BM cells to generate the BMT model or B6-Tg(CAG-EGFP) TCD-BM cells and B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato-EGFP)/Nju (mT/mG) splenic CD3+ T cells to generate GVHD mice. Grafted tdTomato+ T cells (red) and GFP+ TCD-BM cells (green) were detected. The intravital vessels were stained with dextran-Cy5 (blue) by tail vein injection. Scale bar, 200 μm. (C-E) TCD-BM cells from C57BL/6 mice with splenic T cells from B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato-EGFP)/Nju (mT/mG) mice were coinjected IV into lethally irradiated BALB/c mice. (C) Representative image of the allogeneic tdTomato+ T cells in murine femurs stained with tdTomato-recognizable anti–red fluorescent protein (red, Alexa Fluor 594) antibody and anti-CD3 (green, Alexa Fluor 488) antibody. The cellular nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 100 μm. (D) Representative in vivo intravital image of recipients’ spleen on day 7 and day 14. Grafted tdTomato+ T cells (red) and vasculature (blue) were imaged in the presence or absence of TMAO treatment. Scale bar, 200 μm. (E) The mRNA expression of IFN-γ (n = 6), IL-4 (n = 6), IL-17 (left to right column, n = 5, 4), T-bet (n = 6), RORγt (left to right column, n = 5, 6), and STAT3 (n = 6), STAT4 (n = 4), STAT6 (n = 7) in allogenic splenic tdTomato+ T cells were determined. The T cells were isolated from GVHD spleen on day 14 with or without TMAO treatment. Data were pooled from 2 independent experiments. *P < .05, **P < .01. (F) CD4+ T cells isolated from C57BL/6 mice were stimulated with anti-CD3 and anti-CD28 antibodies, in the presence or absence of TMAO treatment (300 μM). The mRNA expression of IL-4, IL-17, IFN-γ, Gata3, and STAT3, STAT4, STAT6 were determined 24 hours after TMAO treatment. N = 3 in each group.

Th1 and Th17 differentiation was seen in TMAO-fed GVHD mice but not in the TMAO-supplemented in vitro culture system. (A) Experimental scheme of BMT mice or GVHD mice protocol. Recipients were fed with TMAO or vehicle from day −14 and thereafter. Data were collected on day 7 and day 14. (B) Representative in vivo fluorescence image of skull BM in recipient mice on day 7 and day 14. The BALB/c recipients were injected with B6-Tg(CAG-EGFP) TCD-BM cells to generate the BMT model or B6-Tg(CAG-EGFP) TCD-BM cells and B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato-EGFP)/Nju (mT/mG) splenic CD3+ T cells to generate GVHD mice. Grafted tdTomato+ T cells (red) and GFP+ TCD-BM cells (green) were detected. The intravital vessels were stained with dextran-Cy5 (blue) by tail vein injection. Scale bar, 200 μm. (C-E) TCD-BM cells from C57BL/6 mice with splenic T cells from B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato-EGFP)/Nju (mT/mG) mice were coinjected IV into lethally irradiated BALB/c mice. (C) Representative image of the allogeneic tdTomato+ T cells in murine femurs stained with tdTomato-recognizable anti–red fluorescent protein (red, Alexa Fluor 594) antibody and anti-CD3 (green, Alexa Fluor 488) antibody. The cellular nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 100 μm. (D) Representative in vivo intravital image of recipients’ spleen on day 7 and day 14. Grafted tdTomato+ T cells (red) and vasculature (blue) were imaged in the presence or absence of TMAO treatment. Scale bar, 200 μm. (E) The mRNA expression of IFN-γ (n = 6), IL-4 (n = 6), IL-17 (left to right column, n = 5, 4), T-bet (n = 6), RORγt (left to right column, n = 5, 6), and STAT3 (n = 6), STAT4 (n = 4), STAT6 (n = 7) in allogenic splenic tdTomato+ T cells were determined. The T cells were isolated from GVHD spleen on day 14 with or without TMAO treatment. Data were pooled from 2 independent experiments. *P < .05, **P < .01. (F) CD4+ T cells isolated from C57BL/6 mice were stimulated with anti-CD3 and anti-CD28 antibodies, in the presence or absence of TMAO treatment (300 μM). The mRNA expression of IL-4, IL-17, IFN-γ, Gata3, and STAT3, STAT4, STAT6 were determined 24 hours after TMAO treatment. N = 3 in each group.

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