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CD177+ cells produce neutrophil extracellular traps that promote biliary atresia

  • Author Footnotes
    # Co-first authors.
    Ruizhong Zhang
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    # Co-first authors.
    Affiliations
    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, Guangzhou, 510623, Guangdong, China
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    # Co-first authors.
    Liang Su
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    # Co-first authors.
    Affiliations
    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, Guangzhou, 510623, Guangdong, China
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    # Co-first authors.
    Ming Fu
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    # Co-first authors.
    Affiliations
    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, Guangzhou, 510623, Guangdong, China
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  • Author Footnotes
    # Co-first authors.
    Zhe Wang
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    Affiliations
    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, Guangzhou, 510623, Guangdong, China
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  • Ledong Tan
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    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, Guangzhou, 510623, Guangdong, China
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  • Hongjiao Chen
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    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, Guangzhou, 510623, Guangdong, China
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  • Zefeng Lin
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    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, Guangzhou, 510623, Guangdong, China
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  • Yanlu Tong
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    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, Guangzhou, 510623, Guangdong, China
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  • Sige Ma
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    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, Guangzhou, 510623, Guangdong, China
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  • Rongchen Ye
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    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, Guangzhou, 510623, Guangdong, China
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  • Ziyang Zhao
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    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, Guangzhou, 510623, Guangdong, China
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  • Ziqing Wang
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    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, Guangzhou, 510623, Guangdong, China
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  • Weiyi Chen
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    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, Guangzhou, 510623, Guangdong, China
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  • Jiakang Yu
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    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, Guangzhou, 510623, Guangdong, China
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  • Wei Zhong
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    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, Guangzhou, 510623, Guangdong, China
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  • Jixiao Zeng
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    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, Guangzhou, 510623, Guangdong, China
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  • Fei Liu
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    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, Guangzhou, 510623, Guangdong, China
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  • Chenwei Chai
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    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, Guangzhou, 510623, Guangdong, China
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  • Xisi Guan
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    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, Guangzhou, 510623, Guangdong, China
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  • Tao Liu
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    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, Guangzhou, 510623, Guangdong, China
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  • Jiankun Liang
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    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, Guangzhou, 510623, Guangdong, China
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  • Yun Zhu
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    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, Guangzhou, 510623, Guangdong, China
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  • Xiaoqiong Gu
    Affiliations
    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, Guangzhou, 510623, Guangdong, China
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  • Yan Zhang
    Affiliations
    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, Guangzhou, 510623, Guangdong, China
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  • Vincent C.H. Lui
    Affiliations
    Department of Surgery, The University of Hong Kong, Hong Kong SAR, China
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  • Paul K.H. Tam
    Affiliations
    Department of Surgery, The University of Hong Kong, Hong Kong SAR, China

    Faculty of Medicine, Macau University of Science and Technology, China
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  • Jonathan R. Lamb
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    Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, SW7 2AZ UK
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  • Zhe Wen
    Correspondence
    Corresponding authors. Address: Provincial Key Laboratory of Research in Structure Birth Defect Disease, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, No. 9, Jinshui Road, Guangzhou, 510623, Guangdong, China. Tel: +86 3807 6560. Fax. +86 3836 7693.
    Affiliations
    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, Guangzhou, 510623, Guangdong, China
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  • Yan Chen
    Correspondence
    Corresponding authors. Address: Provincial Key Laboratory of Research in Structure Birth Defect Disease, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, No. 9, Jinshui Road, Guangzhou, 510623, Guangdong, China. Tel: +86 3807 6560. Fax. +86 3836 7693.
    Affiliations
    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, Guangzhou, 510623, Guangdong, China

    Department of Surgery, The University of Hong Kong, Hong Kong SAR, China

    Faculty of Medicine, Macau University of Science and Technology, China
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  • Huimin Xia
    Correspondence
    Corresponding authors. Address: Provincial Key Laboratory of Research in Structure Birth Defect Disease, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, No. 9, Jinshui Road, Guangzhou, 510623, Guangdong, China. Tel: +86 3807 6560. Fax. +86 3836 7693.
    Affiliations
    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, Guangzhou, 510623, Guangdong, China
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  • Author Footnotes
    # Co-first authors.
Open AccessPublished:July 05, 2022DOI:https://doi.org/10.1016/j.jhep.2022.06.015

      Highlights

      • CD177+ cells express interferon-stimulated and neutrophil degranulation genes in biliary atresia.
      • CD177 knockout delays the onset of illness and reduces morbidity and mortality in mice.
      • High levels of mitochondria and ROS in CD177+ cells lead to NET production and cause cholangiocyte death.
      • N-acetylcysteine results in a decline in CD177+ cell count, ROS and NETs in patients with biliary atresia.

      Background & aims

      We have previously reported on the potential pathogenic role of neutrophils in biliary atresia (BA). Herein, we aimed to delineate the role of CD177+ neutrophils in the pathogenesis of BA.

      Methods

      Immune cells from the livers of mice with rhesus rotavirus-induced BA were analysed. Single-cell RNA-sequencing was performed to specifically analyse Gr-1+ (Ly6C/Ly6G+) cells in the liver. Gene expression profiles of CD177+ cells were analysed using the Smart-Seq RNA-sequencing method, and the pathogenesis of BA was examined in Cd177-/- mice. Neutrophil extracellular trap (NET) inhibitors were used to determine the role of CD177+ cell-derived NETs in BA-associated bile duct damage, and a pilot clinical study evaluated the potential effects of N-acetylcysteine on NET release in BA.

      Results

      Increased levels of Gr-1+ cells were observed in the livers of mice with rhesus rotavirus-induced BA. RNA-sequencing analysis revealed that CD177+ cells were the main population of Gr-1+ cells and expressed elevated levels of both interferon-stimulated and neutrophil degranulation genes. Cd177-/- BALB/c mice exhibited delayed disease onset and reduced morbidity and mortality. High numbers of mitochondria were detected in CD177+ cells derived from mice with BA; these cells were associated with increased levels of reactive oxygen species and increased NET formation, which induced the apoptosis of biliary epithelial cells in cocultures. In a pilot clinical study, the administration of N-acetylcysteine to patients with BA reduced CD177+ cell numbers and reactive oxygen species levels, indicating a potential beneficial effect.

      Conclusions

      Our data indicate that CD177+ cells play an important role in the initiation of BA pathogenesis via NET formation.

      Clinical trial registration

      The pilot study of N-acetylcysteine treatment in patients with BA was registered on the Chinese Clinical Trial Registry (ChiCTR2000040505).

      Lay summary

      Neutrophils (a type of innate immune cell, i.e. an immune cell that doesn’t target a specific antigen) are thought to play a role in the development of biliary atresia (a rare but potentially lethal condition of the bile ducts that occurs in infants). Herein, we found that neutrophils expressing a particular protein (CD177) played an important role in bile duct damage by releasing a special structure (NET) that can trap and kill pathogens but that can also cause severe tissue damage. A pilot study in patients with biliary atresia showed that inhibiting NETs could have a beneficial effect.

      Graphical abstract

      Keywords

      Introduction

      Biliary atresia (BA) is a neonatal disease that is associated with a poor prognosis without surgical intervention (Kasai portoenterostomy or liver transplantation as a last resort). The aetiology and pathogenesis of BA remain unclear, but dysfunctional immune cells, such as natural killer (NK) cells, B cells, CD4+ and CD8+ T cells, are associated with direct or indirect biliary epithelial cell (BEC) damage, which leads to bile duct injury.
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      Accumulation of neutrophils is observed around the bile ducts of patients with BA,
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      Macrophages are targeted by rotavirus in experimental biliary atresia and induce neutrophil chemotaxis by Mip2/Cxcl2.
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      Loss of interleukin-12 modifies the pro-inflammatory response but does not prevent duct obstruction in experimental biliary atresia.
      and we previously found that the depletion of neonatal Gr-1+ (a neutrophil marker comprising Ly6C/Ly6G) cells prevents the initiation of BA in a mouse disease model,
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      The role of neonatal Gr-1(+) myeloid cells in a murine model of rhesus-rotavirus-induced biliary atresia.
      which suggests a pathological role for these cells. Because neonatal Gr-1+ cells are a mixed population,
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      Understanding the multiple functions of Gr-1+ cell subpopulations during microbial infection.
      further analysis is needed to define the exact roles of the cellular component(s) of the Gr-1+ cell population.
      CD177 is specifically expressed on a neutrophil subset, and CD177+ neutrophil accumulation is reportedly associated with a variety of autoimmune diseases.
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      However, in the pathogenesis of inflammatory bowel disease, CD177+ neutrophils play a protective role by releasing IL-22 and neutrophil extracellular traps (NETs).
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      CD177+ neutrophils as functionally activated neutrophils negatively regulate IBD.
      NETs are 3D structures consisting of chromatin DNA filaments and multiple bactericidal effector proteins, such as histones, neutrophil elastase (NE), myeloperoxidase (MPO), cathepsin G and lactoferrin, that can capture and kill pathogens
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      Neutrophil extracellular traps kill bacteria.
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      NETs can act as autoantigens, inducing the production of autoantibodies and inflammatory products during the development of systemic lupus erythematosus and rheumatoid arthritis.
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      It has been suggested that NETs may be associated with BA, but the mechanism(s) underlying their involvement in the disease process has not been fully explored.
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      Correlation of immune markers with outcomes in biliary atresia following intravenous immunoglobulin therapy.
      In this study, we first established a model of BA in NOD/SCID neonatal mice, which lack an adaptive immune system, to determine the function of innate immune responses in BA. The composition of Gr-1+ cells was then analysed using the 10x Genomics single-cell RNA-sequencing (scRNA-seq) platform, and the identity and function of CD177+ cells were determined. Studies in CD177 gene-deficient mice revealed the importance of CD177+ cells in the development of BA. In vitro, CD177+ cells isolated from patients with BA released NETs, and their role in the apoptosis of BECs was examined. In addition, the effects of NET inhibitors were examined in the BA mouse model. RNA-sequencing was used to determine the gene expression profile in CD177+ cells from patients with BA. In a pilot clinical study, patients with BA were administered N-acetylcysteine (NAC). Collectively, this study evaluated the role of CD177+ cells and the NETs that they produced in the pathogenesis of BA.

      Materials and methods

      BA mouse model analysis

      The pregnant BALB/c mice and NOD/SCID mice were purchased from Guangdong Animal Experimental Center, China. Cd177 null mice (Cd177-/-) were generated by Cyagen (Cyagen US Inc., CA, USA) and backcrossed onto a BALB/c genetic background. Newborn mice were injected intraperitoneally with 20 μl of 1.5 × 106 plaque-forming units/ml of rhesus rotavirus (RRV) within 24 hours of birth, and the normal control group mice were injected with the same dose of saline. Mice had oily hair and yellow skin staining on days 5-6 after RRV injection. The experimental protocols were approved by the Animal Protection and Use Committee of Guangzhou Medical University Experimental Animal Center (IACUC-DB-16-0602). Details regarding animal treatments and the methods of analysis of mouse liver Gr-1+ cells, including liver Gr-1+ cell isolation, 10x Genomics scRNA-seq of Gr-1+ cells, histology and immunohistochemistry are described in the supplementary materials and methods.

      Patient samples and analysis

      Patients and controls were recruited from the Department of Surgery of Guangzhou Women and Children’s Medical Center (Guangzhou, China), including control group (infants with haemangioma without evidence of liver disease), intrahepatic cholestasis (IHC) group (patients with IHC, defined by serum bilirubin levels higher than 17 μM but without bile duct atresia) and BA group (defined by intraoperative cholangiography and postoperative liver histopathological examination). The human BA pathogenesis study procedures were approved by the institutional review boards of Guangzhou Women and Children’s Medical Center, China (ID: 34500). Detailed methods for sample collection, flow cytometric analysis, cell isolation, SMART-Seq2 analysis of BA CD177+ cells and coculture with cholangiocytes are provided in the supplementary materials and methods.

      NAC treatment assay in patients with BA

      To determine whether NAC treatment has any effects on CD177+ cell accumulation and the formation of NETs, a pilot study was performed in patients with BA. For the clinical treatment trial, the study procedures were approved (ID: 62001), and the trial was registered with the Chinese Clinical Trial Registry (ChiCTR2000040505). The legal guardians of all participants signed the consent forms. Before Kasai surgery, the patients were given an intravenous injection of NAC (Hangzhou Minsheng Pharmaceutical Co. Ltd, patch # 2006231. 150 mg/kg qd, n = 6) for 1 week. To explore the effects of NAC after the operation, 4 patients continued to receive the same dose of NAC for another week. Changes in routine blood measures and liver function indices were recorded and compared. More detailed information and analysis are provided in the supplementary materials and methods.

      Results

      Gr-1+ cells are sufficient to induce bile duct obstruction in RRV-inoculated neonatal NOD/SCID mice

      In a previous study, we demonstrated that the deletion of Gr-1+ cells prevented the initiation of BA in the BALB/c BA mouse model. To investigate whether the function of these cells is dependent on adaptive immunity, NOD/SCID mice were intraperitoneally injected with RRV. The results showed that 49.2% (30/61) of the NOD/SCID mice and 100% (50/50) of the BALB/c mice developed BA. The appearance of jaundice (Fig. 1A), weight loss and alcoholic white stools, were similar between the 2 groups. Cholangiography indicated extrahepatic bile duct blockage (Fig. 1B). The RRV-inoculated BALB/c mice exhibited increased numbers of B and T cells, but as expected, these cells were not detected in NOD/SCID mice (data not shown). No significant difference in Gr-1+ cells was found between untreated NOD/SCID mice and untreated BALB/c mice. However, RRV inoculation increased the numbers of Gr-1+ cells in both the NOD/SCID and BALB/c mice by approximately 3-fold compared with the control numbers (22.32 ± 4.48% and 19.03 ± 1.89% vs. 7.43 ± 0.98% and 6.27 ± 1.42%, respectively, p <0.001 in both groups) (Fig. 1C). Similar levels of CD11b+F4/80+ macrophages were found in the NOD/SCID and BALB/c mice, but a reduction in the percentage of cells was observed after RRV inoculation in these 2 types of mice (p <0.001) (Fig. S1A). An increase in NK cells was observed at day 6 in both NOD/SCID and BALB/c mice with BA. Taken together, the results suggest that Gr-1+ cells may be the main innate immune cell population that responds to virus infection and mediates bile duct damage. More details on CD11b+F4/80+ macrophages and NK cells in BA are reported in the supplementary results.
      Figure thumbnail gr1
      Fig. 1Establishment of a BA model in NOD/SCID and BALB/c mice through RRV inoculation.
      (A) Comparison of the RRV-inoculated BALB/c and NOD/SCID groups at day 12 after viral inoculation with the control group (saline). (B) Liver dissection showing the surface and bile ducts at day 12 after viral injection. The black triangle indicates the inflammatory spots in the liver surface, and the blue dashed line marks the gallbladder and the outline of the bile ducts. (C) Flow cytometric analysis and quantification of liver Gr-1+ cells in BALB/c and NOD/SCID mice at day 6 with and without RRV inoculation (n = 6 per group). (D) 10x Genomics scRNA-seq analysis of liver-isolated Gr-1+ cells (5 mice were mixed per group) from RRV-infected and control (saline) BALB/c mice. The data were analysed using the UMAP method with K-means = 2. The number indicates the percentage of each cluster in the Gr-1+ cell population. (E) The percentage of CD177+ cells in the Gr-1+ cell population is indicated. (F) CD177+ cells in the saline and RRV groups are shown with the same colour as in the UMAP method analysis. (G) The 8 genes showing the highest upregulation in CD177+ cells in the RRV group compared with the saline group are presented. (H) Metascape analysis showing the enrichment of gene expression in CD177+ cells in different signalling pathways and the interaction of molecules in terms of highly differentially expressed genes between the 2 groups. (I) Analysis of the most highly expressed genes in CD177+ cells and comparison of the 2 groups to determine the functional differences. ∗∗∗p <0.001. BA, biliary atresia; RRV, rhesus rotavirus.
      Gr-1+ cells were further isolated from the livers of BALB/c mice with BA at day 5 after RRV inoculation, analysed using the scRNA-seq system and compared with those of control mice administered a saline injection. The UMAP (uniform manifold approximation and projection) clustering method was used to analyse mixed populations of Gr-1+ cells. The results from analyses using the K-means algorithm with 2 clusters showed that when Gr-1+ cells were divided into 2 subclusters, the cell number in subcluster 1 (K2-C1, 76.3%) was 3.2-fold greater than that in subcluster 2 (K2-C2, 23.7%) (Fig. 1D). Gene expression profiles revealed that the cell surface molecule CD177 was highly expressed in the Gr-1+ cell population (70.9% in Gr-1+ cells); more specifically, 90.4% of K2-C1 cells in Gr-1+, which suggests that CD177+ cells could be considered a major functional subpopulation of Gr-1+ cells and that CD177 could be a cell marker for K2-C1. In contrast, only 8.1% of K2-C2 cells were CD177+ cells, indicating cluster-specific expression; hence, K2-C2 cells can be considered to consist of CD177- cells (Fig. 1E). Further CD177+ cluster analysis by separating the saline group from the RRV group revealed that the number of CD177+ cells in the Gr-1+ population decreased slightly (from 74.5% to 66.7%) after viral infection (Fig. 1F). A comparison of the variation in expression between the RRV-treated and control CD177+ cells showed that the cells expressed many molecules related to interferon-stimulated genes, such as Isg15, Ifit1, and Ifi27l2a, after RRV infection (Fig. 1G). Viral infection markedly changed the gene expression profile of CD177+ cells, as demonstrated by 1,911 upregulated and 2,086 downregulated genes (Fig. S5A). A functional analysis showed that the activated signalling pathways included those associated with antiviral and interferon responses as well as antigen processing and presentation (Fig. 1H). An analysis of the genes that were more highly expressed in CD177+ cells from RRV-infected than saline-treated mice showed that many genes were associated with antiviral functions. Notably, genes involved in neutrophil degranulation showed increased expression, which may be related to the tissue damage observed during the BA disease process (Fig. 1I). These data suggest that CD177+ cells are activated and exhibit intense antiviral responses after RRV infection in the neonatal period. Further subcluster analysis of cluster 1 is shown in the supplementary results.

      Accumulation of CD177+ cells in patients with BA and a BA mouse model

      CD177+ cells in the peripheral blood and livers of children with BA were analysed by flow cytometry and immunohistochemical staining. The results showed that the number of CD177+ cells in the peripheral blood of patients with BA (n = 41) was significantly higher than in the control group (deformity without liver disease, n = 40) and in patients with IHC (n = 41; p <0.001, Fig. 2A). Similarly, liver biopsies from patients with BA also revealed significantly higher numbers of CD177+ cells (p <0.001, Fig. 2B) compared to those from children with IHC and the disease control group, namely patients with portal hypertension (control n = 7). Most CD177+ cells were found in portal areas (Fig. 2C). In BA mice, CD177+ cells in the liver and spleen were also detected by flow cytometric analysis and immunohistochemical staining. The results showed that the percentage of CD177+ cells was significantly increased at all time points starting from day 3 (p <0.001, Fig. 2D,E) compared with that in the saline-treated mice. Immunohistochemical staining detected CD177+ cells mostly in the periductal area in the saline- and RRV-inoculated group, indicating a close relationship with bile duct pathogenesis (Fig. 2F). In NOD/SCID mice, a higher proportion of CD177 cells was only observed in mice with jaundice (Fig. 2G).
      Figure thumbnail gr2
      Fig. 2Detection of CD177+ cells in patients with BA.
      (A) The proportion of CD177+ cells in the peripheral blood of children was analysed by flow cytometry, control (n = 40): infants with haemangioma without evidence of liver disease; IHC (n = 41): patients with intrahepatic cholestasis (defined by serum bilirubin levels higher than 17 μM but without bile duct atresia); BA (n = 41): patients with biliary atresia. (B) The proportion of CD177+ cells in liver tissue of children was analysed by flow cytometry (control = 7, these individuals exhibit portal vein cavernous degeneration and liver and choledochal cysts without cholestasis and severe bile duct inflammation, as confirmed by pathological examination; IHC, n = 41; BA, n = 41). (C) Immunohistochemical staining and quantification of CD177 in the livers of children in the control, IHC and BA groups (n = 6 per group). Scale bar: 100 μm. (D–E) The proportion of CD177+ cells in the liver and spleens of the saline and RRV groups on days 3, 6, 9 and 12 (Saline n = 21, 20, 20 and 20; BA n = 22, 22, 22 and 20 respectively). (F) Immunohistochemical staining and quantification of CD177 in the BA mouse livers and the saline mouse livers starting from day 3 (n = 6 per group). Scale bar: 50 μm. (G) The proportion of CD177+ cells in the liver in NOD/SCID mice with or without jaundice (saline, n = 5; RRV without jaundice, n = 6; RRV with jaundice, n = 7) was detected by flow cytometric analysis. ∗∗p <0.01; ∗∗∗p <0.001; n.s., not statistically significant. BA, biliary atresia; Cont., control; IHC, intrahepatic cholestasis; RRV, rhesus rotavirus.

      Cd177 knockout alleviates the symptoms of RRV-induced BA in mice

      To further define the function of CD177+ cells in BA, Cd177–/– BALB/c mice were compared with wild-type (WT) mice (Fig. 3A). All 23 mice in the WT+RRV group had jaundice (100%), but in the Cd177–/– +RRV group, only 6 of the 24 mice developed jaundice (25%). The appearance of jaundice in the Cd177–/– +RRV group was delayed until day 9 after RRV inoculation compared with day 6 in the WT mice (Fig. 3B). The average weight loss in the BA mice was lower in the Cd177–/– group (day 14: 6.68 ± 1.26 g in the Cd177–/– +RRV group vs. 3.86 ± 0.18 g in the WT+RRV group: p <0.001, Fig. 3C). None of the 23 mice in the WT+RRV group survived more than 14 days, and in the Cd177–/– +RRV group, 18 of 24 mice survived for more than 20 days (Fig. 3D). No inflammation or spots of necrosis were found on the liver surface at the time of dissection in the Cd177–/–+RRV group at days 6 and 12 (Fig. 3E). Cholangiography showed no blockage in the extrahepatic bile ducts (Fig. 3G) in the Cd177–/– +RRV group. Tissue section staining revealed no obvious infiltration of inflammatory cells around the intrahepatic bile ducts (Fig. 3F), and the intrahepatic bile ducts in the Cd177–/– +RRV group exhibited a tube-like structure (Fig. 3H). Laboratory examination showed no obvious changes between the Cd177–/– +RRV group and the corresponding control group other than slight increases in direct bilirubin and alkaline phosphatase at day 12, but a significant difference was observed in the WT+RRV group (Fig. 3I). The results indicate that CD177+ cells play a major role in the pathogenesis of BA in mice.
      Figure thumbnail gr3
      Fig. 3Clinical symptoms, pathology, and survival of Cd177–/– and WT mice inoculated with RRV.
      (A–D) Representative images of mice at day 12, Jaundice rate, body weight recorded and survival curve of mice recorded from days 1 to 20 in each group (WT+saline, n = 21; WT+RRV, n = 23; Cd177–/– +saline, n = 22; Cd177–/– +RRV, n = 24). (E) Representative image of the liver surface of each group at days 6 and 12. The black triangle (π) indicates the inflammatory spots in the liver surface. (F) Representative images of liver H&E staining and quantification for each group at days 6 and 12 (n = 6 per group). (G) Representative images of extrahepatic bile duct cholangiography of the mice in each group at days 6 and 12. (H) Representative images of anti-mouse CK19 staining of the intrahepatic bile ducts of mice and quantification in each group at days 6 and 12 (n = 6 per group). (I) Clinical parameter measurements of the liver function of WT and Cd177–/– mice at days 6 and 12 (n = 6 per group). Scale bar: 100 μm. ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001; n.s., not statistically significant. ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BA, biliary atresia; BD, bile duct; DBIL, direct bilirubin; GGT, gamma glutamyltransferase; PV, portal vein; RRV, rhesus rotavirus; TBA, total bile acid; TBIL, total bilirubin; WT, wild type.

      Increased oxidative phosphorylation level in BA CD177+ cells isolated from the liver

      To explore the function of CD177+ cells in patients with BA and patients with IHC (n = 41 in each group), cells were isolated from the liver, and their gene expression profiles were analysed using the Smart-Seq method. Total RNA was extracted, reverse transcribed to create the cDNA library and sequenced. The data were aligned to the human genome using HISAT2. The expression levels of mRNAs were measured by calculating the fragments per kilobase of transcript per million mapped reads (FPKM). Differentially expressed genes with −1.5>log2FC>1.5, adjusted p <0.05 and an average expression value higher than 1 FPKM were selected for further analysis. The results showed increased and decreased expression of 4,654 and 2,590 genes in CD177+ cells, respectively (Fig. 4A). Notably, the expression level varied among individuals in the BA groups. Gene expression profile-related functions were evaluated through gene ontology and KEGG enrichment analyses (Fig. 4B). Mitochondria- and neutrophil-related signalling pathways were the 2 main upregulated pathways with higher p values for the combined score from the enrichment analyses. In mitochondrial signalling pathways, genes related to translational regulation and positive regulation of mitochondrial outer membrane permeabilization, which is involved in apoptotic signalling, exhibited the strongest functional changes. Functional genes such as DAP3, HSD17B1, MRPS34, and SFN and the mitochondria-specific genes MT-ND3, MT-ND4, MT-CO1 and MT-CYB showed increased expression (Fig. 4C,D). Of the neutrophil-related signalling pathways, neutrophil-mediated immunity, neutrophil degranulation, and neutrophil activation were the most important functions, while neutrophil-mediated cytotoxicity, killing of symbiont cells and killing of bacteria were also involved to a lesser extent. Genes such as ELANE, FCRR2A, RHOA and VAMP8 were upregulated in CD177+ BA cells (Fig. 4E). Genes involved in oxidative phosphorylation and oxidative stress, such as CST3, CYBB (NOX2), IDH1 and MPO (Fig. 4F), were increased. The data presented here suggest a close relationship between enhanced mitochondrial activation and strong CD177+ cell immune function.
      Figure thumbnail gr4
      Fig. 4Smart-Seq analysis of CD177+ cells from the livers of patients with BA.
      (A) Heatmap showing the highly differentially expressed genes in CD177+ cells from patients with BA (n = 41) and IHC (n = 41). Hierarchical clustering was performed for both genes and samples. (B) The differential gene expression results were subjected to GO term functional enrichment and KEGG pathway enrichment analyses. The genes involved in the indicated biological functions are shown. (C–F) Violin plot showing examples of the expression profiles of mitochondrial function- and mitochondria-related genes, neutrophil activation, and oxidative phosphorylation. BA, biliary atresia; IHC, intrahepatic cholestasis.

      CD177+ cells of patients with BA promote BEC apoptosis via NETs

      To explore whether CD177+ cells from patients with BA can induce BEC damage, CD177+ cells were isolated from the peripheral blood (Fig. 5A), and Giemsa staining revealed that CD177+ cells from the BA group lacked nuclear segmentation and exhibited increased extracellular cytoplasmic extensions (Fig. 5B). The coculture of BECs with CD177+ cells isolated from the BA or control groups showed that cell death occurred only in the cultures containing CD177+ cells from the patients with BA (p <0.001) (Fig. 5C,D). However, flow cytometric analysis indicated a slight increase in the early apoptosis of BECs cocultured with CD177+ cells from either the BA or control groups, but late apoptosis of BECs was greatly augmented only in the BA group (Fig. 5E). The presence of NETs in CD177+ cells was then investigated. The NET activator phorbol myristate acetate (PMA) was added to the culture medium. The results demonstrate that CD177+ cells from the BA group can form NET filaments with and without PMA stimulation, but no NET filaments were detected in the control group, even with PMA (Fig. 5F). NET components such as double-stranded DNA, NE and MPO were significantly increased in the cell culture supernatants (11.2-, 2.9- and 2.7-fold, respectively; in the presence of PMA, 4.9-, 3.0- and 2.8-fold, respectively, p <0.001) (Fig. 5G–I). Different NET inhibitors, including NE inhibitor (sivelestat), PAD4 inhibitor (GSK484), ROS scavenger (N-acetylcysteine, NAC) and DNase I, were added to cultures of CD177+ cells, and the supernatants were harvested and added as conditioned medium (CM) to BECs. The morphology and the proliferation of BECs from mice with BA was impaired and the number of BECs was decreased by more than 50% (p <0.001) when compared with that in the control group; similar results were observed in the PMA-stimulated group (Fig. 5J,K and Fig. S10A,B). Consistently, increased BEC apoptosis was observed (with or without PMA stimulation) among cells cultured in CM derived from CD177+ cells from the BA group. However, this CM mainly affected early apoptosis and had less of an effect on late apoptosis. In contrast, the addition of PMA reduced the degree of early apoptosis but increased the degree of late apoptosis (Fig. 5L and Fig. S10C). The decreases of NE, ROS, PAD4 and DNase I all had similar effects in reducing both early and late apoptosis among BECs cultured with CM in the absence or presence of PMA. The results indicated that, in the context of BA, CD177+ cells could induce BEC apoptosis by releasing NETs.
      Figure thumbnail gr5
      Fig. 5CD177+ cells of patients with BA promote the apoptosis of BECs via NETs.
      (A) Detection of the purity of CD177+ cells isolated from the peripheral blood through flow cytometry after immunomagnetic bead separation. (B) Representative images of Giemsa-stained CD177+ cells from the peripheral blood of the control individuals and patients with BA. Scale bar: 5 μm. (C) Representative images of cell morphological changes in cocultures of BECs with CD177+ cells isolated from the peripheral blood at day 3, Scale bar: 50 μm. (D) Proliferation of BECs measured by the MTT assay. (E) The apoptosis of BECs in each group at day 3 was determined by flow cytometry and quantified. (F) Representative images of NET staining and quantification of control and BA CD177+ cells with Hoechst 33342 after culture with and without PMA stimulation for 12 h (n = 4 in each group). Scale bar: 10 μm. (G-I) Levels of dsDNA, MPO, and NE in the culture supernatants of CD177+ cells in the BA and control groups detected by ELISA (n = 6 in each group). (J) Representative images of BECs cultured with control and BA CD177+ cell culture supernatants with or without the inhibitors. Scale bar: 50 μm. (K) Proliferation of BECs detected by the MTT assay. (L) The apoptosis of BECs in each group was detected by flow cytometry and quantified. The data in A–E and J-L are representative of 3 biologically independent experiments. ∗∗p <0.01; ∗∗∗p <0.001; n.s., not statistically significant. BA, biliary atresia; CM, conditioned medium; Cont., control; MPO, myeloperoxidase; NAC, N-acetylcysteine; NE, neutrophil elastase; NETs, neutrophil extracellular traps; PAD4, peptidylarginine deiminase 4; PMA, phorbol 12-myristate 13-acetate; Veh., vehicle.

      Safety and efficacy of NAC in the treatment of BA

      The inhibition of NETs in cell culture and in the BA mouse model (supplementary results) prompted us to test whether N-acetylcysteine (NAC) has any effects on the number of CD177+ cells and the formation of NETs in patients with BA. Intravenous NAC was administered for 1 week before the Kasai procedure, and no relevant changes in the general condition of patients were observed after treatment. CD177+ cells were isolated from their peripheral blood and analysed. The results indicated that the number of CD177+ cells (33.94 ± 6.55% vs. 16.05 ± 2.60% in BA without [n = 10] vs. with NAC [n = 6], respectively) and the levels of intracellular ROS (8,309 ± 1,136 vs. 4,725 ± 786.40 in BA without [n = 10] vs. with NAC [n = 6], respectively) were reduced significantly by NAC treatment (p <0.001; Fig. 6A,B). NAC treatment also decreased the activity and number of mitochondria (Fig. 6C,D) and the formation of NETs (Fig. 6E). Furthermore, 4 patients were administered NAC for an additional week after Kasai surgery. These patients presented significantly decreased total bile acid and direct bilirubin levels compared with the non-treatment group (control group, n = 56) (Fig. 6F). For a better understanding of the possible signalling pathways involved, RNA-seq was performed in CD177+ cells isolated from patients in the BA (control, n = 4) and BA+NAC (n = 6) groups. Four and 41 genes exhibited increased and reduced expression in CD177+ cells, respectively (Fig. 6G). The downregulated genes were further analysed with Metascape, and the results showed that the pathways were related to neutrophil degranulation, regulation of the MAPK cascade and leukocyte migration (Fig. 6H). The genes included CEACAM1 and S100A12, which are involved in multiple pathways, STOM and HK3, which are involved in neutrophil degranulation, and GADD45A, which is involved in the MAPK cascade. Interestingly, NAC treatment downregulated CD177 expression and increased RASA4 gene expression (Fig. 6I). These data suggest that NAC can reduce the number and expression of CD177+ cells, probably through inhibition of neutrophil degranulation, suppress the formation of NETs and ameliorate the pathological conditions of BA.
      Figure thumbnail gr6
      Fig. 6Effect of NAC in the treatment of patients with BA.
      (A-B) The proportion and ROS level of CD177+ cells in the peripheral blood of children with BA treated with NAC (BA+NAC) or treated without NAC (BA) for 7 days before Kasai surgery were detected by flow cytometric analysis and compared with those of the control group (control, haemangioma). (C-D) Representative image of CD177+ cell mitochondrial fluorescent staining with MitoTracker Red CMXRos (MT-Red) and MitoTracker Green FM (MT-Green) and quantification in the 3 groups. Scale bar: 10 μm. (E) Representative images of NET staining of the 3 groups of CD177+ cells with Hoechst 33342 and quantification. Scale bar: 10 μm. (F) Levels of total bile acid and direct bilirubin measured after 1 more week of NAC treatment after Kasai surgery. The significant results are indicated (n = 56 in the control group, n = 4 in the treatment group). (G) The volcano plot shows the highly differentially expressed genes in CD177+ cells isolated from the blood of children with BA treated with NAC (n = 6) and not treated with NAC (n = 4) detected by RNA-seq. (H) The downregulated genes in CD177+ cells after NAC treatment were subjected to GO term functional enrichment analysis performed with Metascape. (I) Violin plot showing examples of gene expression profiles. The data in A-E (Cont, n = 9; BA, n = 10; BA+NAC, n = 6), ∗∗∗p <0.001. BA, biliary atresia; Cont., control; MFI, mean fluorescence intensity; NAC, N-acetylcysteine; NETs, neutrophil extracellular traps; ROS, reactive oxygen species.

      Discussion

      In this study, NOD/SCID mice inoculated with RRV developed BA, which suggests that both innate and adaptive immunity are involved in BA pathogenesis. CD177+ cells responded strongly to RRV, as illustrated by their elevated expression of interferon-stimulated genes, and thus have the potential to cause bile duct damage. RNA-seq analyses revealed that antigen processing and presentation pathways are active in CD177+ cells, indicating that they might interact with the adaptive immune system. Collectively, these observations suggest that CD177+ cells are a key element in the initiation of BA pathology. However, the depletion of CD177+ cells does not fully prevent BA, indicating that Gr-1+CD177- cells or other cell types are involved in BA pathology.
      Interpretation of the function of CD177+ cells from patients with BA based on RNA-seq data using gene ontology enrichment and KEGG pathway analyses (Fig. S11A) showed that common genes related to multiple pathways were modulated. The analysis of the first 10 genes revealed that they were grouped into 4 functional pathways associated with oxidative phosphorylation, mitochondrial function, neutrophil function, and the cell cycle. The results showed that the common genes between oxidative phosphorylation and mitochondrial function, such as ATP5MC3, ATP5PD, BCL2, and CYC1, were the most abundant. Some of the genes, such as CST3, CYBB, and TRPM2, have functions in both oxidative phosphorylation and neutrophil activity. These genes have been shown to directly regulate NET formation.
      • Hook J.S.
      • Cao M.
      • Potera R.M.
      • Alsmadi N.Z.
      • Schmidtke D.W.
      • Moreland J.G.
      Nox2 regulates platelet activation and NET formation in the lung.
      • Tripathi J.K.
      • Sharma A.
      • Sukumaran P.
      • Sun Y.
      • Mishra B.B.
      • Singh B.B.
      • et al.
      Oxidant sensor cation channel TRPM2 regulates neutrophil extracellular trap formation and protects against pneumoseptic bacterial infection.
      • Xiao Y.
      • Cong M.
      • Li J.
      • He D.
      • Wu Q.
      • Tian P.
      • et al.
      Cathepsin C promotes breast cancer lung metastasis by modulating neutrophil infiltration and neutrophil extracellular trap formation.
      Genes such as CCNA2, CDK1 and PCNA play a role in both oxidative phosphorylation and the cell cycle. E2F1 is a common gene between mitochondrial function and the cell cycle. Interestingly, TP53 functions in 3 signalling pathways: oxidative phosphorylation, mitochondrial function, and the cell cycle (Fig. S11B–F). However, we did not detect changes in genes common to mitochondrial and neutrophil function, or to neutrophil function and the cell cycle. Whether the interaction of these pathways occurs through oxidative phosphorylation warrants further analysis.
      Mitochondria are an important source of ROS in most mammalian cells,
      • Murphy M.P.
      How mitochondria produce reactive oxygen species.
      and the presence of a highly activated interferon signalling pathways may be partially due to the release of interferogenic mitochondrial DNA.
      • Lood C.
      • Blanco L.P.
      • Purmalek M.M.
      • Carmona-Rivera C.
      • De Ravin S.S.
      • Smith C.K.
      • et al.
      Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease.
      Furthermore, a ROS scavenger (NAC) that diminishes mitochondrial ROS activity,
      • He Y.
      • Zhou L.
      • Fan Z.
      • Liu S.
      • Fang W.
      Palmitic acid, but not high-glucose, induced myocardial apoptosis is alleviated by N-acetylcysteine due to attenuated mitochondrial-derived ROS accumulation-induced endoplasmic reticulum stress.
      alleviates BA, suggesting that mitochondria are involved in NET formation and eventually mediate bile duct damage. A correlation analysis of mitochondrial gene upregulation in the CD177+ cells of patients with BA was performed using RNA-seq data. CD32A and CD64 are associated with inflammation-activated neutrophils and are related to TNF-α and IFN-γ/G-CSF, respectively,
      • Wang Y.
      • Jönsson F.
      Expression, role, and regulation of neutrophil Fcγ receptors.
      though only the increased expression level of CD32A was statistically significant (p <0.05) (Fig. S12A). Survivin and XIAP are expressed in neutrophils and are associated with the inhibition of neutrophil apoptosis.
      • Geering B.
      • Simon H.U.
      Peculiarities of cell death mechanisms in neutrophils.
      Delayed neutrophil apoptosis reportedly enhances NET formation in cystic fibrosis.
      • Gray R.D.
      • Hardisty G.
      • Regan K.H.
      • Smith M.
      • Robb C.T.
      • Duffin R.
      • et al.
      Delayed neutrophil apoptosis enhances NET formation in cystic fibrosis.
      Only the increased expression of Survivin was significant (p <0.05) (Fig. S12A). No significant correlation for mitochondrial DNA expression was found in the IHC group, but a strong correlation was found in the BA group, which suggested that these molecules may contribute to BA pathogenesis (Fig. S12B,C). However, mitochondrial DNA can be released not only by neutrophils but also by B cells, T cells and NK cells,
      • Ingelsson B.
      • Söderberg D.
      • Strid T.
      • Söderberg A.
      • Bergh A.C.
      • Loitto V.
      • et al.
      Lymphocytes eject interferogenic mitochondrial DNA webs in response to CpG and non-CpG oligodeoxynucleotides of class C.
      which highlights the complexity of the mechanisms that drive BA pathogenesis.
      In the mouse BA model, NAC treatment ameliorated BA in this and other studies.
      • Luo Z.
      • Shivakumar P.
      • Mourya R.
      • Gutta S.
      • Bezerra J.A.
      Gene expression signatures associated with survival times of pediatric patients with biliary atresia identify potential therapeutic agents.
      In human liver diseases, NAC has been used in the treatment of non-acetaminophen-induced acute liver failure in children;
      • Kortsalioudaki C.
      • Taylor R.M.
      • Cheeseman P.
      • Bansal S.
      • Mieli-Vergani G.
      • Dhawan A.
      Safety and efficacy of N-acetylcysteine in children with non-acetaminophen-induced acute liver failure.
      intravenous NAC administration is safe in the treatment of pregnancy and neonatal diseases.
      • Wiest D.B.
      • Chang E.
      • Fanning D.
      • Garner S.
      • Cox T.
      • Jenkins D.D.
      Antenatal pharmacokinetics and placental transfer of N-acetylcysteine in chorioamnionitis for fetal neuroprotection.
      Our clinical study, which included a limited number of patients with BA, indicated that NAC treatment has a potential beneficial clinical effect. However, it has been shown that NAC can enhance the cytotoxic activity of NK cells,
      • Tsuyuki S.
      • Yamauchi A.
      • Nakamura H.
      • Nakamura Y.
      • Kinoshita K.
      • Gomi T.
      • et al.
      N-acetylcysteine improves cytotoxic activity of cirrhotic rat liver-associated mononuclear cells.
      and NK cells reportedly promote BA pathogenesis. NAC has anticoagulant and platelet-inhibiting properties,
      • Niemi T.T.
      • Munsterhjelm E.
      • Pöyhiä R.
      • Hynninen M.S.
      • Salmenperä M.T.
      The effect of N-acetylcysteine on blood coagulation and platelet function in patients undergoing open repair of abdominal aortic aneurysm.
      so caution is needed regarding the use of NAC in BA, particularly late stage disease. Furthermore, the long-term effects of NAC on liver function, cholangitis ratios, survival times, and liver transplantation are unclear and need to be investigated. Large-scale, multicentre, prospective, double-blind controlled studies will be important to evaluate the potential clinical utility of NAC in the management of BA.
      In conclusion, our study provides further information on the contribution of innate immunity to the induction of BA. CD177+ cells play a pivotal role in BEC damage through NET formation and possibly the accumulation of mitochondria in these cells. Anti-ROS NET treatment might aid in the management of BA.

      Abbreviations

      BA, biliary atresia; BECs, biliary epithelial cells; CM, conditioned medium; FPKM, fragments per kilobase of transcript per million mapped reads; IHC, intrahepatic cholestasis; NAC, N-acetylcysteine; NE, neutrophil elastase; NET, neutrophil extracellular trap; MPO, myeloperoxidase; PMA, phorbol myristate acetate; RRV, rhesus rotavirus; scRNA-seq, single-cell RNA-sequencing.

      Financial support

      National Natural Science Foundation of China (81770510) to RZ, National Natural Science Foundation of China (81671498) to YC, National Natural Science Foundation of China (81974056 and 81771629) and Science and Technology Planning Project of Guangdong Province (No. 2019B020227001) to HX.

      Authors’ contributions

      R.Z., L.S., M.F., L.T., H.C., Z.L, Y.T., S.M., Z.Z. and Ziqing Wang performed the experiments. Zhe Wang, J.Y., W.Z., J.Z., F.L., C.C., Xisi Guan, T.L., and J.L recruited patient, provided clinical information and performed clinical care. Y.T. and Xiaoqiong Gu were responsible for samples collection. W.C., R.Y. Yun Zhu., and Yan Zhang performed bioinformatic and statistical analysis. R.Z., L.S., J.R.L. and Y.C. wrote the manuscript with significant input from V.L. and P.T. Zhe Wen, Y.C. and H.X. conceived and supervised the project.

      Data availability statement

      Lead Contact: Further information and request for resources and reagents should be directed to the Lead Contact Professor Huimin Xia ([email protected]).
      Materials Availability: This study did not generate unique reagents. Data and Code Availability: The raw sequencing data that support the findings of this study were deposited into the Genome Sequence Archive of Beijing Institute of Genomics, Chinese Academy of Sciences: GSA CODE (CRA007360), website (https://bigd.big.ac.cn/gsa/browse/CRA007360); GSA-Human CODE (HRA002607), website (https://bigd.big.ac.cn/gsa-human/browse/HRA002607). For data access, please follow the guidelines of the Genome Sequence Archive (http://bigd.big.ac.cn/gsa-human). Publicly available databases and software are specified in the supplementary materials and the key resources in supplementary CTAT table.

      Conflict of interest

      The authors declare no conflicts of interest that pertain to this work.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Acknowledgements

      The authors would like to thank the Clinical Biological Resource Bank of Guangzhou Women and Children’s Medical Center for providing the clinical samples.

      Supplementary data

      The following are the supplementary data to this article:

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