RNA-sequencing analysis of lung primary fibroblast response to eosinophil-degranulation products predicts downstream effects on inflammation, tissue remodeling and lipid metabolism.

ABSTRACT

BACKGROUND: The association of eosinophils with inflammation and tissue remodeling is at least partially due to their release of toxic granule proteins and other mediators, including cytokines. Tissue remodeling and consequent functional defects are affected by activity of connective tissue fibroblasts. Exaggerated fibroblast activation, accumulation and change of phenotype may lead to fibrosis and loss of tissue function. So far, little information has been reported on how eosinophils affect inflammation and tissue remodeling via the activation of fibroblasts. We have recently shown that eosinophil activation with IL-3 led to a robust eosinophil degranulation on immunoglobin-G (IgG) coated plates. Thus, in the present study, we analyze the effects of IL-3-activated eosinophil degranulation products on primary human lung fibroblasts (HLF) using whole transcriptome sequencing. METHODS: Conditioned media was obtained from eosinophils that were pre-activated with IL-3 or IL-5 and subsequently cultured for 6 h on IgG to induce degranulation. This conditioned media was added on human lung fibroblasts (HLF) for 24 h and the cell lysates were then subjected to whole transcriptome sequencing to identify global changes in gene expression. Differentially expressed genes were analyzed using the Ingenuity Pathway Analysis (IPA), and validated by qPCR. RESULTS: In HLF, the expression level of 300 genes was changed by conditioned media from IL-3-activated eosinophils compared to control fibroblast cultures. Among these 300 genes, the expression level of 35 genes coding for known proteins was upregulated by IL-3- versus IL-5-pre-activated eosinophils. Of the 35 upregulated genes, IPA identified C3, CH25H, CXCL1, CXCL8, CYP1A1, ICAM1, IL6 and UCN2 as having downstream functions on inflammation, tissue remodeling and lipid synthesis. This analysis combined with previous RNA sequencing analyses of eosinophils suggest IL-1ß, OSM and TNFSF12 as potential upstream regulators of fibroblasts. CONCLUSIONS: This study has identified several novel pro-inflammatory and pro-remodeling mediators produced by fibroblasts in response to activated eosinophils. These findings may have significant implications on the role of eosinophil/fibroblast interactions in eosinophilic disorders.