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 1

Transcriptomic analysis reveals enhanced CD8+ T cell–mediated cytotoxicity in the monkey liver with MASH.

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Transcriptomic analysis reveals enhanced CD8+ T cell–mediated cytotoxici...
(A) Heatmap based on gene set enrichment analysis (GSEA) of monkey liver RNA-seq data comparing the DT-109, MASH, and normal groups at endpoint. Significantly upregulated pathways are shown in red and downregulated pathways in blue, and pathways with no significant changes are displayed without color. Scale bars, normalized enrichment score (NES) of the GSEA. (B) A bubble plot illustrates the proportion of immune cell abundance in monkey livers, comparing the DT-109, MASH, and normal groups using ImmuCellAI tool. Circle size represents the proportion of cells, and circle color indicates the P value. Red circles in the normal and DT-109 groups indicate cell types that differ significantly from the MASH group. Immunohistochemistry (C) for CD45 and CD3 in monkey livers from the normal, MASH, and DT-109 groups (scale bars, 100 μm). Ratio of CD45-positive (D) and CD3-positive (E) areas in monkey livers are compared among the normal (n = 6), MASH (n = 10), and DT-109 (n = 10) groups. Data (D and E) are presented as means ± SEM. Differences (D and E) were analyzed using 1-way ANOVA with Dunnett’s post hoc test. (F) UMAP visualization showing the clustering of CD8+ T cells from scRNA-seq analysis of monkey livers in the normal, MASH, and DT-109 groups (n = 3/group), with cells colored by their 6 subtypes. (G) Volcano plot depicting DEGs in CD8+ T cells for 2 comparisons (MASH vs. normal and DT-109 vs. MASH) based on scRNA-seq. Red dots represent upregulated genes, while blue dots represent downregulated genes in CD8+ T cells. Y axis denotes −log10 FDR; x axis shows log2fold-change values. (H) Bubble plot of enriched KEGG pathways for DEGs in CD8+ T cell subtypes (CD8+ Temra cells, CD8+ Tem cells, MAIT cells, CD8+ IEL cells, CD8+ T cycling cells, and CD8+ T naive cells) in 2 comparisons (MASH vs. normal, and DT-109 vs. MASH). KEGG pathways have an FDR of less than 0.05. DEGs, differentially expressed genes; scRNA-seq, single-cell RNA-seq; UMAP, uniform manifold approximation and projection. Values above brackets in figures represent P values unless indicated as FDR.