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

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.

The association of immune-related genes and the potential role of IL10 with biliary atresia.

BACKGROUND: Biliary atresia (BA) is a severe immune-related disease that is characterized by biliary obstruction and cholestasis. The etiology of BA is unclear, our aim was to explore the relationship between biliary tract inflammation and immune-related genes. METHODS: We selected 14 SNPs in 13 immune-related genes and investigated their associations with BA by using a large case‒control cohort with a total of 503 cases and 1473 controls from southern China. RESULTS: SNP rs1518111 in interleukin10 (IL10) was identified as associated with BA (P = 5.79E-03; OR: 0.80; 95% CI: 0.68-0.94). The epistatic effects of the following pairwise interactions among these SNPs were associated with BA: signal transducer and activator of transcription 4 (STAT4) and chemokine (C-X-C motif) ligand 3 (CXCL3); STAT4 and damage-regulated autophagy modulator1 (DRAM1); CXCL3 and RAD51 paralog B (RAD51B); and interferon gamma (IFNG) and interleukin26 (IL26). Furthermore, we explored the potential role of IL-10 in the pathogenesis of the neonatal mouse model of BA. IL-10 effectively prevented biliary epithelial cell injury and biliary obstruction in murine BA as well as inhibit the activation of BA-related immune cells. CONCLUSIONS: In conclusion, this study provided strong evidence implicating IL10 as a susceptibility gene for BA in the southern Chinese population. IMPACT: This study provided strong evidence implicating IL10 as a susceptibility gene for BA in the southern Chinese population. This study could infer that IL-10 may play a protective role in BA mouse model. We found that four SNPs (rs7574865, rs352038, rs4622329, and rs4902562) have genetic interactions.

CD177+ cells produce neutrophil extracellular traps that promote 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.

Down-regulation of STAT3 enhanced chemokine expression and neutrophil recruitment in biliary atresia.

Biliary atresia (BA) is an immune-related disorder and signal transducer and activator of transcription 3 (STAT3) is a key signalling molecule in inflammation. The present study was designed to clarify the function of STAT3 in BA. STAT3 expression was examined in patients and a mouse BA model in which STAT3 levels were further altered with a specific inhibitor or activator. Neutrophil accumulation and the levels of the neutrophil chemoattractants (C-X-C motif) ligand 1 (CXCL1) and IL-8 were determined. The effects of STAT3 inhibition on IL-8 expression were examined in human biliary epithelial cell (BEC) cultures. Functional changes in liver STAT3+ neutrophils in the mouse model were analysed with 10× single cell RNA-seq methods. Results showed STAT3 and p-STAT3 expression was reduced in BA liver tissue compared with control samples. Administration of a STAT3 inhibitor increased jaundice and mortality and reduced body weight in BA mice. In contrast, the STAT3 activator ameliorated BA symptoms. Extensive neutrophil accumulation together with CXCL1 up-regulation, both of which were suppressed by an anti-CXCL1 antibody, were observed in the STAT3 inhibitor-treated group. Recombinant IL-8 administration increased disease severity in BA mice, and the STAT3 activator had the reverse effect. Inhibiting STAT3 increased apoptosis of human BECs together with up-regulated IL-8 expression. RNA-seq analysis revealed reduced the numbers of STAT3 expressing neutrophil in BA which was accompanied by marked enhanced interferon-related antiviral activities. In conclusion, STAT3 reduction, enhanced IL-8 and CXCL1 expression and promoted the accumulation of interferon-responsive neutrophils resulting in BEC damage in BA.

Irrelevance of USF2 rs916145 polymorphism with the risk of biliary atresia susceptibility in Southern Chinese children.

BACKGROUNDS: Biliary atresia (BA) is a very rare neonatal disease, however, it has been the most common cause of obstructive jaundice in infancy. The complex pathogenesis of BA is not entirely clear and a lot of possible pathogenic mechanisms have been proposed to explain the etiology of BA, including genetic, inflammatory, environmental and developmental abnormalities. As a transcription factor, USF2 gene rs916145 polymorphism has been shown to be related to the risk of BA. METHODS: We examined the USF2 rs916145 genotype in a large case-control study consisting of 506 BA patients and 1473 healthy controls, using the MassARRAY iPLEX Gold system (Sequenom). Odds ratios (ORs) and 95% confidence intervals (CIs) were used to evaluate the association between the USF2 gene rs916145 polymorphism and BA susceptibility. RESULTS: The frequency of different genotypes showed no statistical significance (GG/GC, OR: 1.09, P=0.470, 95% CI: 0.87-1.35; GG/CC, OR: 0.86, P=0.378, 95% CI: 0.62-1.20). No obvious association was revealed between the USF2 gene rs916145 polymorphism and BA susceptibility. CONCLUSION: USF2 rs916145 polymorphism may not be the best predictor of BA.