Differential gene regulation in miR-223 overexpressing pulmonary epithelial cell line compared to control cell line

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. Due to our observation that pulmonary epithelial cells at baseline express essentially no miR-223, and miR-223 expression is dramatically increased following co-culture with PMN, we decided to emulate the transcriptional changes of miR-223. To define a potential miR-223 target gene in pulmonary epithelial cells that could have functional relevance during ALI we pursued studies on differential gene expression in miR-223 by generating pulmonary epithelial cells with lentiviral-mediated overexpression of miR-223 and to subsequently expose those cells to a microarray analysis. We performed an expression profiling of 4 samples (n=2 for each cell line) using an industry-standard whole human gene array (Affymetrix Human Gene 1.0 ST Array). Quality analysis using Partek Genomics Suite 6.6 revealed high confidence in the quality of the microarray data and all samples met 'Quality Assurance/Quality Control' (QA/QC) criteria.