Gut microbial metabolite trimethylamine N-oxide aggravates GVHD by inducing M1 macrophage polarization in mice.

The diversity of human microbiome heralds the difference of impact that gut microbial metabolites exert on allogenic graft-versus-host disease (GVHD), even though short-chain fatty acids and indole were demonstrated to reduce its severity. In this study, we dissected the role of choline-metabolized trimethylamine N-oxide (TMAO) in GVHD process. Either TMAO or high choline diet enhanced allogenic GVH reaction, while the analog of choline, 3,3-dimethyl-1-butanol reversed TMAO-induced GVHD severity. Interestingly, TMAO-induced alloreactive T cell proliferation and differentiation into T helper (Th) subtypes was seen in GVHD mice but not in in vitro cultures. We thus investigated the role of macrophage polarization which was absent from in vitro culture system. F4/80+CD11b+CD16/32+ M1 macrophage and signature genes, IL-1beta, IL-6, TNF-alpha, CXCL9 and CXCL10 were increased in TMAO-induced GVHD tissues and in TMAO-cultured bone marrow derived macrophages (BMDMs). Inhibition of NLRP3 inflammosome reversed TMAO-stimulated M1 features, indicating that NLRP3 is the key proteolytic activator involved in macrophage's response to TMAO stimulation. Consistently, mitochondrial reactive oxygen species and enhanced NF-kappaB nuclear re-localization were investigated in TMAO-stimulated BMDMs. In vivo depletion of NLRP3 in GVHD recipients not only blocked M1 polarization but also reversed GVHD severity in the presence of TMAO treatment. In conclusion, our data revealed that TMAO-induced GVHD progression is resulted from Th1 and Th17 differentiation, which is mediated by polarized M1 macrophage requiring NLRP3 inflammasome activation. It provides the link among the host choline diet, microbial metabolites and GVH reaction, shedding light on alleviating GVHD by controlling choline diet.