Single cell RNA-sequencing analysis reveals that N-acetylcysteine partially reverses hepatic immune dysfunction in biliary atresia

Rongchen Ye; Sige Ma; Yan Chen; Jiarou Shan; Ledong Tan; Liang Su; Yanlu Tong; Ziyang Zhao; Hongjiao Chen; Ming Fu; Zhipeng Guo; Xiaoyu Zuo; Jiakang Yu; Wei Zhong; Jixiao Zeng; Fei Liu; Chenwei Chai; Xisi Guan; Zhe Wang; Tao Liu; Jiankun Liang; Yan Zhang; Hongguang Shi; Zhe Wen; Huimin Xia; Ruizhong Zhang

doi:10.1016/j.jhepr.2023.100908

Single cell RNA-sequencing analysis reveals that N-acetylcysteine partially reverses hepatic immune dysfunction in biliary atresia

JHEP Rep. 2023 Sep 12;5(11):100908. doi: 10.1016/j.jhepr.2023.100908. eCollection 2023 Nov.

Authors

Rongchen Ye¹, Sige Ma¹, Yan Chen^{2

1}, Jiarou Shan¹, Ledong Tan¹, Liang Su¹, Yanlu Tong¹, Ziyang Zhao¹, Hongjiao Chen¹, Ming Fu¹, Zhipeng Guo¹, Xiaoyu Zuo¹, Jiakang Yu¹, Wei Zhong¹, Jixiao Zeng¹, Fei Liu¹, Chenwei Chai¹, Xisi Guan¹, Zhe Wang¹, Tao Liu¹, Jiankun Liang¹, Yan Zhang¹, Hongguang Shi³, Zhe Wen¹, Huimin Xia¹, Ruizhong Zhang^{1

3}

Affiliations

¹ Guangdong Provincial Key Laboratory of Research in Structure Birth Defect Disease and Department of Pediatric Surgery, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangdong Provincial Clinical Research Center for Child Health, Guangzhou, 510623, China.
² Faculty of Medicine, Macau University of Science and Technology, Macau, 999078, China.
³ Department of Pediatric Surgery, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, China.

Abstract

Background & aims: Our previous study indicated that CD177⁺ neutrophil activation has a vital role in the pathogenesis of biliary atresia (BA), which is partially ameliorated by N-acetylcysteine (NAC) treatment. Here, we evaluated the clinical efficacy of NAC treatment and profiled liver-resident immune cells via single cell RNA-sequencing (scRNA-seq) analysis to provide a comprehensive immune landscape of NAC-derived immune regulation.

Methods: A pilot clinical study was conducted to evaluate the potential effects of intravenous NAC treatment on infants with BA, and a 3-month follow-up was carried out to assess treatment efficacy. scRNA-seq analysis of liver CD45⁺ immune cells in the control (n = 4), BA (n = 6), and BA + NAC (n = 6) groups was performed and the effects on innate cells, including neutrophil and monocyte-macrophage subsets, and lymphoid cells were evaluated.

Results: Intravenous NAC treatment demonstrated beneficial efficacy for infants with BA by improving bilirubin metabolism and bile acid flow. Two hepatic neutrophil subsets of innate cells were identified by scRNA-seq analysis. NAC treatment suppressed oxidative phosphorylation and reactive oxygen species production in immature neutrophils, which were transcriptionally and functionally similar to CD177⁺ neutrophils. We also observed the suppression of hepatic monocyte-mediated inflammation, decreased levels of oxidative phosphorylation, and M1 polarisation in Kupffer-like macrophages by NAC. In lymphoid cells, enhancement of humoral immune responses and attenuation of cellular immune responses were observed after NAC treatment. Moreover, cell-cell interaction analysis showed that innate/adaptive proinflammatory responses were downregulated by NAC.

Conclusions: Our clinical and scRNA-seq data demonstrated that intravenous NAC treatment partially reversed liver immune dysfunction, alleviated the proinflammatory responses in BA by targeting innate cells, and exhibited beneficial clinical efficacy.

Impact and implications: BA is a serious liver disease that affects newborns and has no effective drug treatment. In this study, scRNA-seq showed that NAC treatment can partially reverse the immune dysfunction of neutrophil extracellular trap-releasing CD177+ neutrophils and Kupffer cells, and lower the inflammatory responses of other innate immune cells in BA. In consequence, intravenous NAC treatment improved the clinical outcomes of patients with BA in term of bilirubin metabolism.

Keywords: Biliary atresia; Innate immune response; N-acetylcysteine; Neutrophil; Oxidative phosphorylation; scRNA-seq.