Abstract
Protein kinase RNA activated (PKR) is a crucial mediator of anti-viral responses but is reported to be activated by multiple non-viral stimuli. However, mechanisms underlying PKR activation, particularly in response to bacterial infection, remain poorly understood. We have investigated mechanisms of PKR activation in human primary monocyte-derived dendritic cells in response to infection by Chlamydia trachomatis. Infection resulted in potent activation of PKR that was dependent on TLR4 and MyD88 signalling. NADPH oxidase was dispensable for activation of PKR as cells from chronic granulomatous disease (CGD) patients, or mice that lack NADPH oxidase activity, had equivalent or elevated PKR activation. Significantly, stimulation of cells with endoplasmic reticulum (ER) stress-inducing agents resulted in potent activation of PKR that was blocked by an inhibitor of IRE1α RNAse activity. Crucially, infection resulted in robust IRE1α RNAse activity that was dependent on TLR4 signalling and inhibition of IRE1α RNAse activity prevented PKR activation. Finally, we demonstrate that TLR4/IRE1α mediated PKR activation is required for the enhancement of interferon-β production following C. trachomatis infection. Thus, we provide evidence of a novel mechanism of PKR activation requiring ER stress signalling that occurs as a consequence of TLR4 stimulation during bacterial infection and contributes to inflammatory responses.
Highlights
Protein kinase RNA activated (PKR) was originally identified as a cytosolic kinase that was activated by double stranded RNA that could terminate protein translation by acting as an eIF2a kinase [1e3]
We found that higher multiplicity of infection (MOI) of 10e20 infectious units (IFU) per cell induced the greatest increase in PKR phosphorylation compared to the noninfected control and that this was reduced at lower MOIs
In this study we have demonstrated that infection of monocyte-derived DC with C. trachomatis or stimulation with LPS results in TLR4-dependent activation of the IRE1a branch of the unfolded protein response (UPR), and that an inhibitor of IRE1a RNAse activity blocks PKR phosphorylation
Summary
Protein kinase RNA activated (PKR) was originally identified as a cytosolic kinase that was activated by double stranded RNA (dsRNA) that could terminate protein translation by acting as an eIF2a kinase [1e3]. PKR activation occurs as a consequence of auto -phosphorylation at several serine and threonine residues following the binding of dsRNA within the N0 terminus and phosphorylation of Thr451 within. S.J. Webster et al / Microbes and Infection 18 (2016) 472e483 evidence that PKR is activated by a wide range of stimuli, the mechanism(s) of how this occurs, as a result of TLR stimulation and bacterial infection, is poorly understood. We have investigated mechanisms of PKR activation in response to a common intracellular bacterial infection; Chlamydia trachomatis, and the role that TLR4, ER stress and the NADPH oxidase system play in the process
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