Small Molecule and Pooled CRISPR Screens Investigating IL17 Signaling Identify BRD2 as a Novel Contributor to Keratinocyte Inflammatory Responses
| Autor: | Min Hu, Ryan Duggan, Eric R. Goedken, Heath A. McDonald, Peter F. Slivka, J. Wetter, Marc H Domanus, Anthony Slavin, Sujatha M. Gopalakrishnan, Victoria E. Scott, Marian Namovic, Namjin Chung, Erin Murphy, John Locklear, Chen-Lin Hsieh, Charles Lu, Steven D. Pratt, Alex Lipovsky, Rebecca M. Edelmayer, Jacob King, Diana Donnelly-Roberts |
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| Rok vydání: | 2019 |
| Předmět: |
Keratinocytes
0301 basic medicine Chemokine Stromal cell Cellular differentiation Phenotypic screening Biology 01 natural sciences Biochemistry Small Molecule Libraries 03 medical and health sciences Psoriasis medicine Homeostasis Humans Clustered Regularly Interspaced Short Palindromic Repeats RNA Small Interfering Cells Cultured Inflammation Tumor Necrosis Factor-alpha 010405 organic chemistry Interleukin-17 Cell Differentiation General Medicine medicine.disease 0104 chemical sciences 030104 developmental biology Gene Knockdown Techniques biology.protein Cancer research Molecular Medicine Tumor necrosis factor alpha IL17A Signal transduction Signal Transduction Transcription Factors |
| Zdroj: | ACS Chemical Biology. 14:857-872 |
| ISSN: | 1554-8937 1554-8929 |
| Popis: | Interleukin-17A (IL17A) plays a critical role in the development of numerous autoimmune diseases, including psoriasis. The clinical success of IL17A neutralizing biologics in psoriasis has underlined its importance as a drug discovery target. While many studies have focused on the differentiation and trafficking of IL17A producing T-helper 17 cells, less is known about IL17A-initiated signaling events in stromal and parenchymal cells leading to psoriatic phenotypes. We sought to discover signaling nodes downstream of IL17A contributing to disease pathogenesis. Using IL17A and tumor necrosis factor α (TNF) to stimulate primary human epidermal keratinocytes, we employed two different phenotypic screening approaches. First, a library of ∼22000 annotated compounds was screened for reduced secretion of the pro-inflammatory chemokine IL8. Second, a library of 729 kinases was screened in a pooled format by utilizing CRISPR-Cas9 and monitoring IL8 intracellular staining. The highest-ranking novel hits identified in both screens were the bromodomain and extra-terminal domain (BET) family proteins and bromodomain-containing protein 2 (BRD2), respectively. Comparison of BRD2, BRD3, and BRD4 silencing with siRNA and CRISPR confirmed that BRD2 was responsible for mediating IL8 production. Pan-BRD inhibitors and BRD2 knockout also reduced IL17A/TNF-mediated CXC motif chemokines 1/2/6 (CXCL1/2/6) and granulocyte colony stimulating factor (G-CSF) production. In RNA-Seq analysis, 438 IL17A/TNF dependent genes were reduced in BRD2-deficient primary keratinocytes. KEGG pathway analysis of these genes showed enrichment in TNF signaling and rheumatoid arthritis relevant genes. Moreover, a number of genes important for keratinocyte homeostasis and cornification were dysregulated in BRD2-deficient keratinocytes. In IL17A/TNF/IL22 stimulated three-dimensional organotypic raft cultures, pan-BRD inhibition reduced inflammatory factor production but elicited aberrant cornification, consistent with RNA-Seq analysis. These studies highlight a novel role for BRDs and BRD2 in particular in IL17A-mediated inflammatory signaling. |
| Databáze: | OpenAIRE |
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