IRAK4 kinase activity controls Toll-like receptor–induced inflammation through the transcription factor IRF5 in primary human monocytes

Leah Cushing,Aaron Winkler,Scott A Jelinsky,Katherine Lee,Wouter Korver,Rachael Hawtin,Vikram R Rao,Margaret Fleming,Lih-Ling Lin

doi:10.1074/jbc.m117.796912

Abstract

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in innate immune signaling by Toll-like receptors (TLRs), and loss of IRAK4 activity in mice and humans increases susceptibility to bacterial infections and causes defects in TLR and IL1 ligand sensing. However, the mechanism by which IRAK4 activity regulates the production of downstream inflammatory cytokines is unclear. Using transcriptomic and biochemical analyses of human monocytes treated with a highly potent and selective inhibitor of IRAK4, we show that IRAK4 kinase activity controls the activation of interferon regulatory factor 5 (IRF5), a transcription factor implicated in the pathogenesis of multiple autoimmune diseases. Following TLR7/8 stimulation by its agonist R848, chemical inhibition of IRAK4 abolished IRF5 translocation to the nucleus and thus prevented IRF5 binding to and activation of the promoters of inflammatory cytokines in human monocytes. We also found that IKKβ, an upstream IRF5 activator, is phosphorylated in response to the agonist-induced TLR signaling. Of note, IRAK4 inhibition blocked IKKβ phosphorylation but did not block the nuclear translocation of NFκB, which was surprising, given the canonical role of IKKβ in phosphorylating IκB to allow NFκB activation. Moreover, pharmacological inhibition of either IKKβ or the serine/threonine protein kinase TAK1 in monocytes blocked TLR-induced cytokine production and IRF5 translocation to the nucleus, but not nuclear translocation of NFκB. Taken together, our data suggest a mechanism by which IRAK4 activity regulates TAK1 and IKKβ activation, leading to the nuclear translocation of IRF5 and induction of inflammatory cytokines in human monocytes.

Highlights

Interleukin-1 receptor–associated kinase 4 (IRAK4)7 is a critical component of the Toll-like receptor/interleukin-1 receptor (TLR/IL1R) signaling pathway that plays a major role in innate immune responses and inflammation
To determine the mechanism by which IRAK4 inhibition is blocking interferon regulatory factor 5 (IRF5)-mediated transcription, we investigated IRF5 translocation to the nucleus of primary human monocytes stimulated with R848
Similar to IRAK4 kinase inhibition, we found that pharmacologically inhibiting IKK␤ [27] or TAK1 [28] in human monocytes blocks inflammatory cytokine production (Fig. 4B) and IRF5 translocation to the nucleus (Fig. 4C), but does not impact nuclear factor kappa B (NF␬B) p65 nuclear translocation (Fig. 4C)

Summary

Introduction

Work by multiple investigators has shown that inactivation of IRAK4 kinase activity has minimal effects on the activation of the canonical inflammatory pathways of NF␬B and the MAP kinases in mice and human cells [5,6,7]. These data lead to the question of how IRAK4 kinase activity controls pro-inflammatory cytokine production. IRAK4 activity controls TLR inflammation through IRF5 in monocytes has been implicated in TLR/IL1R signaling and is activated in a MyD88-dependent manner to induce cytokines and interferon [12]. Several groups have recently provided evidence in human myeloid cells that phosphorylation of IRF5 by IKK␤ results in IRF5 activation and nuclear translocation following TLR ligation [15, 16] and that this process involved TAK1 [17]

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