Metabolic dysfunction–associated steatohepatitis exacerbated by Clostridium perfringens–derived ammonia is attenuated by tripeptide DT-109
Pengxiang Qu, Shusi Ding, Yanru Zhang, Yang Zhao, Erfei Song, Liangshuo Hu, Ruike Ding, Wenbin Cao, Yiting Hou, Jia Qi, Juan Zhao, Chenjing Duan, Shuangqing Liu, Chong Shen, Ying Zhao, Yanhong Guo, Zuowen Zheng, Shiwei Luo, Huizhong Hu, Liang Bai, Sihai Zhao, Bo Wang, Shuixiang He, Yi Wu, Xuelian Xiong, Qiutong Wu, Weiwang Gu, Oren Rom, Aimin Xu, Lemin Zheng, Jifeng Zhang, Enqi Liu, Y. Eugene Chen
Pengxiang Qu, Shusi Ding, Yanru Zhang, Yang Zhao, Erfei Song, Liangshuo Hu, Ruike Ding, Wenbin Cao, Yiting Hou, Jia Qi, Juan Zhao, Chenjing Duan, Shuangqing Liu, Chong Shen, Ying Zhao, Yanhong Guo, Zuowen Zheng, Shiwei Luo, Huizhong Hu, Liang Bai, Sihai Zhao, Bo Wang, Shuixiang He, Yi Wu, Xuelian Xiong, Qiutong Wu, Weiwang Gu, Oren Rom, Aimin Xu, Lemin Zheng, Jifeng Zhang, Enqi Liu, Y. Eugene Chen
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Research Article Gastroenterology Hepatology

Metabolic dysfunction–associated steatohepatitis exacerbated by Clostridium perfringens–derived ammonia is attenuated by tripeptide DT-109

Abstract

The global prevalence of metabolic dysfunction–associated steatohepatitis (MASH) is rising, driven by a complex interplay of metabolic disturbances, inflammation, and fibrosis, yet effective treatment options remain limited. This study examined the relationships among intestinal microbial dysbiosis, ammonia production, and hepatic CD8+ T cell activity in MASH, then assessed the therapeutic potential of DT-109, a glycine-based tripeptide. We investigated the gut/liver axis across human cohorts and both nonhuman primate and mouse MASH models. Multiomics approaches were used to characterize ileal microbiota, ammonia levels, and hepatic immune and metabolic pathways. Causality was verified through microbiota transplantation, C. perfringens NirA-knockout mutants, and functional validation in vitro and in vivo. The efficacy of DT-109 was evaluated in nonhuman primates and mice. Our results revealed a significant increase in the ammonia-producing gut bacterium C. perfringens, which led to elevated intestinal ammonia and disruption of the intestinal barrier in MASH. Elevated ammonia levels triggered FosB-mediated upregulation of CCL5 in CD8+ T cells, which in turn drove T cell cytotoxicity in the liver. Notably, DT-109 effectively lowered C. perfringens abundance, reduced intestinal ammonia, restored intestinal barrier integrity, and alleviated CD8+ T cell dysregulation in MASH. These results identify a distinct mechanism in which gut-derived ammonia drives CD8+ T cell–mediated MASH and demonstrate that DT-109 effectively targets this axis by inhibiting C. perfringens and reducing ammonia, ultimately ameliorating MASH.

Authors

Pengxiang Qu, Shusi Ding, Yanru Zhang, Yang Zhao, Erfei Song, Liangshuo Hu, Ruike Ding, Wenbin Cao, Yiting Hou, Jia Qi, Juan Zhao, Chenjing Duan, Shuangqing Liu, Chong Shen, Ying Zhao, Yanhong Guo, Zuowen Zheng, Shiwei Luo, Huizhong Hu, Liang Bai, Sihai Zhao, Bo Wang, Shuixiang He, Yi Wu, Xuelian Xiong, Qiutong Wu, Weiwang Gu, Oren Rom, Aimin Xu, Lemin Zheng, Jifeng Zhang, Enqi Liu, Y. Eugene Chen

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Figure 7

Elevated ammonia triggers FosB-mediated CCL5 expression in CD8+ T cells, driving MASH progression.

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Elevated ammonia triggers FosB-mediated CCL5 expression in CD8+ T cells,...
Relative mRNA expression levels of FosB (A) and Ccl5 (B) in mouse CD8+ T cells in the control and ammonia groups. (C) ChIP-seq results of active histone modification markers H3 trimethylated at lysine 4 (H3K4me3) and H3 acetylated at lysine 27 (H3K27ac) and RNA-seq results for CCL5 and its adjacent DNA sequences in monkey livers from the MASH group (marked in red) and normal group (marked in blue) (n = 2/group). (D and E) FosB ChIP-qPCR analyses of CD8+ T cells of mice in the control and ammonia groups (n = 6/group). FosB binding signals were detected at the promoter region (D) and the enhancer region (E) of Ccl5. IgG was used as a negative control. mRNA expression of FosB (F) and Ccl5 (G) in mouse CD8+ T cells transfected with control siRNA (siCtrl) or FosB-specific siRNA (siFosB) under ammonia exposure (n = 6). (H) Schematic of adoptive transfer experiment: CD8+ T cells from 3 groups were adoptively transferred into nude mice: untreated controls (UT, without ammonia exposure or siRNA transfection), scramble siRNA–transfected cells, and siFosB-transfected cells. Cells in the latter 2 groups were exposed to ammonia for 24 hours in vitro prior to transfer. (I) Plasma ALT levels in recipient nude mice 48 hours after transfer of CD8+ T cells. (J and K) Hepatic Ccl5 and Prf1 in recipient nude mice 48 hours after transfer. Data (A, B, D–G, and I–K) are presented as means ± SEM. Statistical differences were analyzed using Kruskal-Wallis test followed by Dunn’s post hoc test (I), 1-way ANOVA followed by Dunnett’s post hoc test (J and K), Mann-Whitney U test (A, B, D, and E), or unpaired t test (F and G). Values above brackets in figures represent P values unless indicated as FDR.