Identification and validation of candidate genes dysregulated in alveolar macrophages of acute respiratory distress syndrome.

Yong Mao,Wei Xu,Zonghe Qin,Li Chen,Xin Lv,Handi Liao,Youguo Ying,Ya Liu

doi:10.7717/peerj.12312

Abstract

BackgroundAcute respiratory distress syndrome (ARDS) is a common cause of death in ICU patients and its underlying mechanism remains unclear, which leads to its high mortality rate. This study aimed to identify candidate genes potentially implicating in the pathogenesis of ARDS and provide novel therapeutic targets.MethodsUsing bioinformatics tools, we searched for differentially expressed genes (DEGs) in an ARDS microarray dataset downloaded from the Gene Expression Omnibus (GEO) database. Afterwards, functional enrichment analysis of GO, KEGG, GSEA and WGCNA were carried out to investigate the potential involvement of these DEGs. Moreover, the Protein–protein interaction (PPI) network was constructed and molecular complexes and hub genes were identified, followed by prognosis analysis of the hub genes. Further, we performed qRT-PCR, Western Blot and flow cytometry analysis to detect candidate genes of CCR2 and FPR3 in macrophage model of LPS-induced ARDS and primary alveolar macrophages(AMs). Macrophage chemotaxis was evaluated using Transwell assay.ResultsDEGs mainly involved in myeloid leukocyte activation, cell chemotaxis, adenylate cyclase-modulating G protein-coupled receptor signaling pathway and cytokine-cytokine receptor interaction. Basing on the constructed PPI network, we identified five molecular complexes and 10 hub genes potentially participating in the pathogenesis of ARDS. It was observed that candidate genes of CCR2 and FPR3 were significantly over-expressed in primary alveolar macrophages from ARDS patients and macrophgae model of LPS-induced ARDS. Moreover, in vitro transwell assay demonstrated that CCR2 and FPR3 down-regulation, respectively, inhibited LPS-triggered macrophage chemotaxis toward CCL2. Finally, a positive correlation between FPR3 and CCR2 expression was confirmed using pearson correlation analysis and Western Blot assay.ConclusionsOur study identified CCR2 and FPR3 as the candidate genes which can promote macrophage chemotaxis through a possible interaction between FPR3 and CCL2/CCR2 axis and provided novel insights into ARDS pathogenesis.

Highlights

Acute respiratory distress syndrome (ARDS) is a prevailing death cause of ICU patients
Using an in vitro migration assay, we demonstrated that LPS stimulation significantly enhanced chemotaxis of phorbol 12-myristate 13-acetate (PMA)-differentiated macrophages toward chemokine CCL2 and this effect can be abrogated by CCR2 silence, which suggested that CCL2/CCR2 axis may function in regulating macrophage chemotaxis in ARDS
Variance stabilization and quantile normalization was performed for the raw microarray data and screening and identification of the differentially expressed genes (DEGs) in ARDS alveolar macrophages compared to peripheral blood monocytes was accomplished using the limma package of R language

Summary

Introduction

As a form of non-cardiogenic pulmonary oedema, ARDS is characterized by diffusive alveolar damage, followed hyaline-membrane formation, epithelial-cell hyperplasia and interstitial edema. These pathological changes are secondary to inflammation process mainly caused by severe infection, trauma, burning and massive transfusion. This study aimed to identify candidate genes potentially implicating in the pathogenesis of ARDS and provide novel therapeutic targets. It was observed that candidate genes of CCR2 and FPR3 were significantly over-expressed in primary alveolar macrophages from ARDS patients and macrophgae model of LPS-induced ARDS. Our study identified CCR2 and FPR3 as the candidate genes which can promote macrophage chemotaxis through a possible interaction between FPR3 and CCL2/CCR2 axis and provided novel insights into ARDS pathogenesis

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