RNA Seq profiling reveals a novel expression pattern of TGF-β target genes in human blood eosinophils

Abstract

Despite major advances in our understanding of TGF-β signaling in multiple cell types, little is known about the direct target genes of this pathway in human eosinophils. These cells constitute the major inflammatory component present in the sputum and lung of active asthmatics and their numbers correlate well with disease severity. During the transition from acute to chronic asthma, TGF-β levels rise several fold in the lung which drives fibroblasts to produce extracellular matrix (ECM) and participate in airway and parenchymal remodeling. In this report, we use purified blood eosinophils from healthy donors and analyze baseline and TGF-β responsive genes by RNA Seq, and demonstrate that eosinophils (PBE) express 7981 protein-coding genes of which 178 genes are up-regulated and 199 genes are down-regulated by TGF-β. While 18 target genes have been previously associated with asthma and eosinophilic disorders, the vast majority have been implicated in cell death and survival, differentiation, and cellular function. Ingenuity pathway analysis revealed that 126 canonical pathways are activated by TGF-β including iNOS, TREM1, p53, IL-8 and IL-10 signaling. As TGF-β is an important cytokine for eosinophil function and survival, and pulmonary inflammation and fibrosis, our results represent a significant step toward the identification of novel TGF-β responsive genes and provide a potential therapeutic opportunity by selectively targeting relevant genes and pathways.

Keywords: Asthma; Blood; Eosinophils; Gene expression; Human; RNA-Seq; TGF-β.

Figure 1.. Expression of TGF-β target genes by human eosinophils.

(A) Cells were left untreated or treated with TGF-β (1ng/ml) for 5 h before harvest. RNA quality was determined as described in Methods before RNA-Seq anaysis. M: size marker. (B) Genes altered (cut off: 1.5 fold) by TGF-β are shown in the circles. The expression level of 7981 genes are greater than 2.00 (PKAM). (C) Heat map of the TGF-β responsive genes (377 genes total) is shown. (D) Validation of RNA-Seq by qPCR with the 10 select transcripts. qPCR data were obtained from 13 eosinophil donors and normalized to GAPDH expression. (E) Validation of RNA-Seq by qPCR with the 6 transcripts associated with canonical TGF-β signaling. Smad6 was undetectable (not shown). The fold change for each gene was calculated and compared between the two detection methods.

Figure 2.. Analysis of pathways responsible for the expression of TGF-β target genes.

The annotated interactions and regulatory relationships were generated using IPA’s (Ingenuity Pathway Analysis) connectivity analysis. Hub genes (in white circles) were defined as those regulating or interacting with ≥5 TGF-β targeted transcripts. Representative genes immediately downstream of hub genes are shown (orange, upregulated; green, downregulated).