Abstract Rationale Influenza-induced cytokine storm causes lung injury and death. During influenza infection, the endoplasmic reticulum (ER) stress pathways in airway epithelial cells promote viral protein folding and cytokine production. We discovered that inhibiting PERK arm of ER stress pathway distinctly attenuates cytokine production without affecting the influenza protein production. PERK exerts transcriptional and translational control during ER stress, but it is not clear which process contributes to the decrease in cytokine production. Our objective is to determine how PERK inhibition reduces cytokines during influenza infection and propose that it limits cytokine storm driven lung injury and fibrosis in mice. Methods Human bronchial epithelial cells (HBECs) or mouse tracheal epithelial cells (MTECS) were infected with the H1N1 PR8. PERK phosphorylation was assessed by western blot analysis. PERK was inhibited by treating cells with GSK2606414, a selective PERK inhibitor (PERKi), and PERK siRNA. To delineate the specific role of PERK within the integrated stress response, an ISR inhibitor (ISRi) was employed. Cytokine levels were quantified by Luminex assay. Transcriptional and translational regulation under PERK modulation was evaluated using RT-qPCR and poly-(ribo)-some profiling together with RNA-seq respectively. Altered eukaryotic initiation factors (eIFs) associated with polysomes were identified using phospho-proteomics. Viral burden was determined via TCID50 assay. C57BL6/NJ mice were infected with PR8 and treated with GSK2606414 at various time points to profile cytokines, immune cells, %SPO2, and lung tissue damage. Results Cytokines such as IL-6, CCL20, and IL-8 were markedly elevated following PR8 infection in HBECs or MTECs but were significantly reduced upon PERK inhibition. RT-qPCR analysis showed no corresponding reduction in respective cytokine mRNAs by PERK inhibition. Since PERK exerts its effects on protein translation, we conducted poly ribosome (polysome) profiling, which revealed a significant decrease in the translational efficiency of these transcripts during PERK inhibition. Initially, we hypothesized that eIF2α mediated the selective translation of cytokines; however, it was challenged by the observation that additional eIFs displayed altered polysome association in response to PERK inhibition. Inhibition of PERK had no detectable effect on viral burden. Furthermore, inhibition of PERK in mice also reduced cytokine production, while its effects on lung damage and fibrosis are currently being evaluated. Conclusion Our work demonstrates that PERK activation selectively enhances cytokine translation during PR8 infection and represents a potential target for preventing cytokine-storm-driven lung injury. This abstract is funded by: NIH R21AI183025-01A1; ALA
Ruban et al. (Fri,) studied this question.