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 5

Ammonia produced by C. perfringens induces gut barrier disruption and hepatic inflammation.

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Ammonia produced by C. perfringens induces gut barrier disruption and he...
(A–H) Bacterial colonization experiment. (A) Schematic of experimental design: mice (C57BL/6) colonized with heat-inactivated bacteria (IC group), wild-type C. perfringens (C.p group), C. perfringens ΔNirA (C.p ΔNirA group), or C. ljungdahlii (C.lj group) for 8 weeks. (B) Ileal content ammonia concentrations. (C) Plasma endotoxin levels. (D) Histological and immunohistochemical sections in the ileum and liver (scale bars, 100 μm). Quantification of ileal mucosal damage by Chiu’s score (E) and MUC2 expression (F). Hepatic F4/80 macrophages (G) and CD8+ T cells (H). (I–S) Ammonium chloride treatment experiment. (I) Schematic of ammonium chloride treatment: mice (AM-T group) received oral gavage of ammonium chloride for 8 weeks vs. water (vehicle) control (WC group). (J) Histological and immunohistochemical sections in the ileum and liver (scale bars, 100 μm). Chiu’s score (K) and MUC2 expression (L) in the ileum. (M) Endotoxin levels in portal vein blood. Plasma ALT (N) and AST (O) levels. Hepatic F4/80 macrophages (P) and CD8+ T cells (Q). Hepatic Ccl5 (R) and Prf1 (S) concentrations. Data (B, C, E–H, K–S) are presented as means ± SEM. Statistical differences were analyzed using Kruskal-Wallis test followed by Dunn’s post hoc test (E, G, and H), 1-way ANOVA followed by Dunnett’s post hoc test (B, C, and F), unpaired t test (L, M, O, R, and S), or Mann-Whitney U test (K, N, P, and Q). Values above brackets in figures represent P values unless indicated as FDR.