ERG lacking endothelium identifies IL8-CXCR2 axis as a therapeutic target for resolving neutrophilic lung vascular injury

Neutrophilic inflammation and hyperpermeable lung microvasculature are consequential to lethal acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). The vascular endothelium is the first point of entry for neutrophils to breach and cause tissue injury. In this study we demonstrate that conditional deletion of ERG (ETS related gene) in adult endothelium of iEC-ERG-/- mice, spontaneously induces accumulation of neutrophils in the lungs. The infiltrated neutrophils showed altered transcriptome, enriched with genes that induce neutrophil recruitment and activation. These activated neutrophils induced lung vascular inflammatory injury. This study shows that endothelial ERG, instructs EC anti-inflammatory niche and thereby controls neutrophil accumulation and activation in the lungs. As the loss of ERG is observed in chronic lung injury, including ARDS, pulmonary hypertension (PAH) and fibrosis, our findings will have implications for understanding and therapeutically mitigating neutrophil function in inflammatory diseases. Overall design: RNA sequencing (RNA-seq) was performed to investigate the endothelial ERG deletion effect of pulmonary neutrophils lungs in ERGfl/fl & iEC-ERG-/- mice. Lung neutrophils were flow sorted using anti-CD45 and anti-Ly6G antibodies after tamoxifen injection and drug washout period from both ERGfl/fl & iEC-ERG-/- mice (six mice per group pooled together). Total RNA was extracted using the RNeasy mini kit (Qiagen USA), and ribosomal RNA was depleted using the RiboMinus™ Eukaryote kit v2 (Thermo Fisher Scientific). RNA integrity was assessed using an Agilent 2100 Bioanalyzer; only samples with RNA integrity number (RIN) > 8 were used. RNA libraries were then constructed with the TruSeq RNA library prep kit v2 (Illumina) and sequenced using the NovaSeq 6000 platform with paired-end 150 bp reads.