Defactinib inhibits PYK2 phosphorylation of IRF5 and reduces intestinal inflammation

Grigory Ryzhakov,Kristina Zec,Tariq Khoyratty,Dorothée L Berthold,Simon P L Travis,Irina A Udalova,Hannah Almuttaqi,Hayley L Eames,Zhichao Ai,Roman Fischer,Samuel Bullers,Benedikt M Kessler,Sarah Bonham,Claire Pearson,Luke Jostins-Dean,Alastair L Corbin

doi:10.1038/s41467-021-27038-5

Abstract

Interferon regulating factor 5 (IRF5) is a multifunctional regulator of immune responses, and has a key pathogenic function in gut inflammation, but how IRF5 is modulated is still unclear. Having performed a kinase inhibitor library screening in macrophages, here we identify protein-tyrosine kinase 2-beta (PTK2B/PYK2) as a putative IRF5 kinase. PYK2-deficient macrophages display impaired endogenous IRF5 activation, leading to reduction of inflammatory gene expression. Meanwhile, a PYK2 inhibitor, defactinib, has a similar effect on IRF5 activation in vitro, and induces a transcriptomic signature in macrophages similar to that caused by IRF5 deficiency. Finally, defactinib reduces pro-inflammatory cytokines in human colon biopsies from patients with ulcerative colitis, as well as in a mouse colitis model. Our results thus implicate a function of PYK2 in regulating the inflammatory response in the gut via the IRF5 innate sensing pathway, thereby opening opportunities for related therapeutic interventions for inflammatory bowel diseases and other inflammatory conditions.

Highlights

Interferon regulating factor 5 (IRF5) is a multifunctional regulator of immune responses, and has a key pathogenic function in gut inflammation, but how IRF5 is modulated is still unclear
We have previously established an in vitro reporter system for measuring IRF5-dependent transcription based on the TNF (IRF5-dependent gene)-promoter-driven luciferase construct, which contains a number of interferonstimulated response elements (ISREs)[24]
When we overexpressed the kinases with IRF5 and TNF-luciferase reporter in HEK-293 TLR4/CD14/MD-2 cells, we found that overexpression of PYK2, JNK2 or MARK3, boosted IRF5-dependent TNF-reporter activation (Fig. 1b)

Summary

Introduction

Interferon regulating factor 5 (IRF5) is a multifunctional regulator of immune responses, and has a key pathogenic function in gut inflammation, but how IRF5 is modulated is still unclear. A recent singlecell transcriptomic analysis of colon biopsies from patients with ulcerative colitis (UC) provided a framework for linking GWAS risk loci with specific cell types and functional pathways and helped to nominate causal genes across GWAS loci[3], amongst them Interferon regulatory factor 5 (IRF5). We used reporter-based screening of a kinase inhibitor library to identify PTK2B/PYK2 as an upstream regulator of IRF5. We identify PTK2B/PYK2 as an upstream regulator of IRF5 based on a systematic screen of kinase inhibitors in macrophages. CRISPR-Cas9-mediated knockout of PYK2 in murine RAW264.7 macrophages impairs LPS-induced IRF5 activation and IRF5-dependent expression of pro-inflammatory cytokines. This study demonstrates a major role for PYK2 as a key regulator of IRF5 activation, macrophage inflammatory response, and intestinal pathology and may facilitate the development of new therapeutics

Methods

Results

Conclusion