IL6 Trans-signaling Increases Expression of Airways Disease Genes in Airway Smooth

23 Genetic data suggests that IL6 trans-signaling may have a pathogenic role in the lung; however, 24 the effects of IL6 trans-signaling on lung effector cells have not been investigated. In this study, 25 human airway smooth muscle (HASM) cells were treated with IL6 (classical) or IL6+sIL6R 26 (trans-signaling) for 24 h and gene expression measured by RNAseq. Intracellular signaling and 27 transcription factor activation were assessed by western blotting and luciferase assay respectively. 28 The functional effect of IL6 trans-signaling was determined by proliferation assay. IL6 trans29 signaling had no effect on PI3 kinase and Erk MAP kinase pathways in HASM cells. Both 30 classical and IL6 trans-signaling in HASM involves activation of Stat3. However, the kinetics of 31 Stat3 phosphorylation by IL6 trans-signaling was different than classical IL6 signaling. This was 32 further reflected in differential gene expression profile by IL6 trans-signaling in HASM cells. 33 Under IL6 trans-signaling conditions 36 genes were up-regulated, including PLA2G2A, IL13RA1, 34 MUC1, and SOD2. Four genes, including CCL11, were down-regulated ≥ 2-fold. The expression 35 of 112 genes was divergent between IL6 classical and trans-signaling, including the genes 36 HILPDA, NNMT, DAB2, MUC1, WWC1, and VEGFA. Pathway analysis revealed that IL6 trans37 signaling induced expression of genes involved in regulation of airway remodeling, immune 38 response, hypoxia, and glucose metabolism. Treatment of HASM cells with IL6+sIL6R induced 39 proliferation in a dose dependent fashion, suggesting a role for IL6 trans-signaling in asthma 40 pathogenesis. These novel findings demonstrate differential effect of IL6 trans-signaling on 41 airway cells and identify IL6 trans-signaling as a potential modifier of airway inflammation and 42 remodeling. 43