16361 Uncovering the regulatory effects of exogenous thymic stromal lymphopoietin on the keratinocyte transcriptome

Abstract

Thymic stromal lymphopoietin (TSLP) is a cytokine produced by various cell lines; including epidermal keratinocytes, which induces TH2 mediated inflammation. TSLP has been shown to activate immune cells to produce high concentrations of IL-13, IL-5, and TNF-α, while inhibiting expression of the anti-inflammatory cytokine IL-10. While TSLP represents a key keratinocyte-derived cytokine, its direct effect on keratinocytes is unclear. To investigate the effect of TSLP on keratinocyte signaling, we treated primary human keratinocytes with active recombinant TSLP. Primary keratinocytes were grown in serum-free, low-calcium media to subconfluence. Subsequently, they were treated for 24 hours with recombinant TSLP or control media in triplicate. After treatment, RNA was extracted, following quality control, enrichment, and library preparation. Sequencing was performed on an Illumina HiSeq PE150. We used DESeq to analyze differentially expressed genes followed by Gene Ontology (GO) enrichment analysis. Keratinocyte stimulation with recombinant TSLP resulted in 167 differentially expressed genes. GO analysis demonstrated significant impact on gene clusters involving epidermal development and keratinocyte differentiation. Fos proto-oncogene was significantly up-regulated as expected. RT-PCR was used to confirm differentially expressed genes from RNA-seq experiments, with further investigation into specific genes differentially expressed in various keratinocyte processes. Of interest, we noted significant up-regulation of late cornified envelope protein complex (LCE1A, LCE1B, LCE1C, LCE1E, LCE2A, and LCE2D), consistent with epidermal cell differentiation. Our results demonstrate a significant role of TSLP binding in altering the keratinocyte transcriptome, namely related to epidermal cell differentiation. The role of this altered expression in atopic dermatitis pathogenesis remains unknown.