NLRP3 was indispensable for TMAO-enhanced GVHD. (A) Experimental scheme of GVHD mice protocol. B6 or B6 Nlrp3^−/− recipients were fed with TMAO or vehicle from day −14 and thereafter, lethally irradiated on day −1, and transfused with TCD-BM and splenic CD3⁺ T cells from BALB/c donors on day 0. Data were collected on day 14 except for survival curve analysis on day 60. (B) Kaplan-Meier survival curve was determined in GVHD B6 or B6 Nlrp3^−/− recipients (N = 8 in each group), in the presence or absence of TMAO treatment. Data were pooled from 2 independent experiments. *P < .05, **P < .01. (C) The percentages of splenic F4/80⁺CD11b⁺ macrophages (left panel) and splenic CD16/32⁺ M1 phenotype out of F4/80⁺CD11b⁺ macrophages (right panel) in B6 Nlrp3^−/− GVHD mice were determined. Data were pooled from 2 independent experiments. N = 6 in each group. ●, BALB/c→ B6 Nlrp3^−/−; ▪, BALB/c→ B6 Nlrp3^−/− +TMAO. (D) Representative H&E staining images of ileum, colon, skin, and liver tissues from B6 Nlrp3^−/− GVHD recipients with or without TMAO treatment. Scale bar, 200 μm. (E) Paraffin sections of ileum, colon, skin, and liver tissues from B6 Nlrp3^−/− GVHD recipients with or without TMAO treatment were analyzed for GVHD histological score. Data were pooled from 2 independent experiments. N = 6 in each group.