TMAO treatment enhanced macrophage infiltration and caused M1 polarization both in vitro and in vivo. (A-E) Lethally irradiated BALB/c recipients were infused with TCD-BM and splenic CD3⁺ T cells from C57BL/6 donors. (A) Splenic CD16/32⁺ M1 population was sorted from F4/80⁺CD11b⁺ macrophages in GVHD mice on day 14, in the presence or absence of TMAO treatment. (B) The percentages of splenic CD16/32⁺ M1 phenotype out of F4/80⁺CD11b⁺ macrophages were determined. Data were pooled from 2 independent experiments. N = 9 in each group. **P < .01. ●, TCD-BM+T; ▪, TCD-BM+T+TMAO. (C) Representative image of infiltrative F4/80⁺ (red, Alexa Fluor 594) iNOS⁺ (green, Alexa Fluor 488) M1 macrophages in ileum, colon, and liver tissues from GVHD recipients with or without TMAO treatment. The cellular nuclei were stained by DAPI. Scale bar, 10 μm. (D-E) Splenic F4/80⁺ cells were sorted from GVHD mice. (D) Expression of IL-6, CXCL9, and CXCL10 was determined by reverse transcription PCR (RT-PCR). Data were pooled from 3 independent experiments. N = 6 in each group. **P < .01. (E) Expression of NLRP3, IL-1β, and cleaved IL-1β was determined by western blotting analysis. (F) Expression of IL-1β (n = 6), IL-6 (n = 3), TNF-α (n = 6), CXCL9 (n = 5), and CXCL10 (n = 5) in BMDMs was analyzed by RT-PCR in the presence or absence of TMAO (300 μM) treatment. **P < .01. (G) Cytokines released into BMDM supernatants, including IL-1β (n = 4), IL-6 (left to right column, n = 5, 4) and TNF-α (left to right column, n = 3, 4), were detected by enzyme-linked immunosorbent assay (ELISA) after TMAO (300 μM) treatment. **P < .01.