TMAO aggravated the severity and mortality of GVHD mice. (A) Experimental scheme of BMT or GVHD protocol. BALB/c recipients were fed with TMAO or vehicle from day −14 and, thereafter, lethally irradiated on day −1, and infused with C57BL/6 donor cells on day 0. The mice were injected with 5 × 10⁶ TCD-BM cells or 5 × 10⁶ TCD-BM cells and 2 × 10⁶ splenic CD3⁺ T cells, respectively. The data were collected on day 14 except for the survival curve analysis on day 50. (B) Serum TMAO concentration was determined on day 14 in BMT mice (TCD-BM) and GVHD mice (TCD-BM+T), with or without TMAO treatment. Data were pooled from 3 independent experiments. N = 8 in each group. **P < .01. (C) Kaplan-Meier survival curve was determined in BMT mice (n = 8) or in GVHD mice (n = 28). Graph represents pooled data from 2 (BMT) or 3 (GVHD) independent experiments, in the presence or absence of TMAO treatment. **P < .01. (D) GVHD scores were analyzed in BMT mice (n = 8) or GVHD mice (n = 28). Graph represents pooled data from 2 (BMT) or 3 (GVHD) independent experiments, in the presence or absence of TMAO treatment. **P < .01. (E) Representative hematoxylin-and-eosin (H&E) tissue staining of ileum, colon, skin, and liver from BMT mice or GVHD mice. Scale bar, 200 μm. (F) Histologic analyses of GVHD features in paraffin-sectioned ileum, colon, skin, and liver tissue were performed on BMT mice or GVHD mice, with or without TMAO treatment. N = 10 in each group. The pooled data were from 3 independent experiments. *P < .05; **P < .01.