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Characterization and Function of Neutrophils in Alcoholic Hepatitis in Humans and Mice: Roles for Neutrophilic p47phox in Disease Progression

ABSTRACT: Intrahepatic neutrophil infiltration has been implicated in the pathogenesis of severe alcoholic hepatitis (SAH), a disease with high short-term morality; however, how neutrophils contribute to SAH progression remain obscure. This study aimed to characterize intrahepatic neutrophil infiltration and its involvement in AH pathogenesis. We found that hepatic expression of neutrophil cytosolic factor 1 (NCF1), a key factor in controlling neutrophilic ROS production, was upregulated and correlated with neutrophil number, inflammation and ROS-associated genes in SAH patients. Ncf1 floxed mice were generated using CRISPR/Cas9 technology by inserting two LoxP sequences into intron 1 and intron 8 of the mouse Ncf1 gene, then crossed with Lyz Cre mice to generated myeloid cell-specific Ncf1 knockout (Ncf1Lyz-/-) mice. The LyzCre negative Ncf1 floxed mice were used as corresponding WT littermate control. The chronic-plus-binge ethanol feeding model was used in this study. The total RNAs were extracted from ETOH-fed 5 WT and 4 Ncf1Lyz-/- mouse livers and submitted for a Poly(A) RNA sequencing. Genetic deletion of the Ncf1 gene in neutrophils abolished hepatic ROS, inflammation, and fibrosis induced by ethanol feeding. RNA-sequencing analysis and the data from experimental models revealed that neutrophilic NCF1-dependent ROS promoted alcoholic liver injury by inhibiting AMP-activated protein kinase (a key regulator of lipid metabolism) and microRNA-223 (a key anti-inflammatory and anti-fibrotic microRNA). Our data suggest that divergent pathogeneses exist in SAH and neutrophilic NCF1-dependent ROS promotes alcoholic liver injury by inhibiting AMPK and microRNA-223.

ORGANISM(S): Mus musculus

PROVIDER: GSE192720 | GEO | 2022/07/16

REPOSITORIES: GEO

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Project description:Background: MicroRNAs are potent regulators of biologic systems that are critical to tissue homeostasis. Individual microRNAs have been identified in airway samples. However, a systems analysis of the microRNA-mRNA networks present in the sputum that contribute to airway inflammation in asthma has not been published. Methods: We conducted a genome-wide analysis of microRNA and messenger RNA (mRNA) in the sputum from patients with asthma and correlated expression with clinical phenotypes. Weighted gene correlation network analysis (WGCNA) was implemented to identify microRNA networks (modules) that significantly correlate with clinical features of asthma and mRNA expression networks. MicroRNA expression in peripheral blood neutrophils and lymphocytes, and in situ hybridization of the sputum were used to identify the cellular sources of microRNAs. MicroRNA expression obtained before and after ozone exposure was also used to identify changes associated with neutrophil counts in the airway. Results: Six microRNA modules were associated with clinical features of asthma. A single module (nely) was associated with a history of hospitalizations, lung function impairment, and numbers of neutrophils and lymphocytes in the sputum. Of the 12 microRNAs in the nely module, hsa-miR-223-3p was the highest expressed microRNA in neutrophils and was associated with increased neutrophil counts in the sputum in response to ozone exposure. Multiple microRNAs in the nely module correlated with two mRNA modules enriched for toll-like receptor (TLR) and Th17 signaling, and endoplasmic reticulum stress. Hsa-miR-223-3p was a key regulator of the TLR and Th17 pathways in the sputum of subjects with asthma. Conclusions: This study of sputum microRNA and mRNA expression from patients with asthma demonstrates the existence of microRNA networks and genes that are associated with features of asthma severity. Among these, hsa-miR-223-3p, a neutrophil-derived microRNA, regulates TLR/Th17 signaling and endoplasmic reticulum stress.

Project description:Acute lung injury (ALI) is characterized by acute respiratory failure in the setting of non-cardiogenic pulmonary edema, causing acute respiratory distress syndrome (ARDS) in patients and contributes significantly to mortality of critically illness. The main goal of our study is to elucidate the role of miRNAs in neutrophil-epithelial communication during pulmonary inflammation and thereby identifying novel targets for therapy of acute lung injury (ALI). In our studies we identified a miR-223-dependent neutrophil-epithelial crosstalk during ALI. Activated neutrophils (PMN) and pulmonary epithelial cells come into a close spatial relationship during ALI. And, since previous studies had indicated the possibility that inflammatory cell-dependent release of miRNA-containing microvesicles could function as a means of exchanging genetic information from a donor to a target cell, we assessed PMN-elicited alterations of pulmonary epithelial miRNA expression in an experimental co-culture setup. This approach provided a selective and extremely robust readout: While other miRNAs were not or only moderately altered in their expression, pulmonary-epithelial-expressed miR-223 was significantly induced after 4 or 6h of co-incubation. Additional in vitro and in vivo studies clearly demonstrate that this increase of epithelial miR-223 is not due to miR-223 transcriptional induction, but instead PMN-dependent and caused by shuttling miR-223 from PMN into pulmonary epithelial cells. To address the functional role of miR-223-dependent neutrophil-epithelial crosstalk during ALI, we exposed mice to ventilator-induced ALI and observed robust induction of pulmonary miR-223 during ALI, while increases of miR-223 were completely abolished after antibody-depletion of PMN. Moreover, studies of alveolar epithelial cells isolated from mice with ALI showed robust increases of miR-223, indicating that miR-223 is shuttled from PMN towards alveolar epithelia during ALI in vivo. Functional studies revealed that gene-targeted mice for miR-223 experience a more severe phenotype during ALI as compared to controls, while their phenotype could be resuscitated by nanoparticle-mediated overexpression of miR-223 in the lungs. In summary, these studies reveal a novel role of miR-223-dependent neutrophil-epithelial crosstalk representing an anti-inflammatory pathway that can be targeted for ALI treatment.

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Characterization and Function of Neutrophils in Alcoholic Hepatitis in Humans and Mice: Roles for Neutrophilic p47phox in Disease Progression

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