D18-07 IRE1 Signaling as a Regulator of Sialic Acid Production: Impacts on Influenza Infection in Asthma

Rationale Sialic acids (SAs) are sugar moieties found on the terminal ends of glycans and are crucial for immune cell recognition, inflammation regulation, and receptors for pathogens including influenza (IAV). However, the regulation of SA production in lung epithelial cells remains unclear. The unfolded protein response (UPR) transducer, IRE1, has been shown to modulate glycan composition in multiple cell types, but its effect in lung epithelial SA production has not been explored. Additionally, our retrospective RNA-seq analysis revealed IRE1 signaling is elevated in asthmatic lung epithelia, which correlates with sialic acid transferase upregulation, thus providing a potential link between SA production and UPR. Finally, literature has shown that IRE1 has been shown to be crucial for IAV replication, but its effects on receptor availability are unknown. We predict IRE1 signaling disruption in lung epithelia inhibits SA production and attenuates influenza burden in models of asthma and potentially is a therapeutic target. Methods RNA-seq from asthmatic patient bronchial brushings were analyzed for changes in IRE1 signaling and SA regulation. HBEC3KT cells were dosed with IRE1 inhibitors, 4u8c or MKC8866, in conjunction with HDM. LCMS analysis coupled with proteomics assessed changes in sialoglycans and glycoproteins. HBEC3KTs were then infected with H3N2 IAV to assess changes in viral titer via TCID50. In vivo, IRE1α and ATF6 were conditionally deleted in respective cohorts of mice. These mice were sensitized to HDM, then infected with H3N2 prior to HDM challenge. Percent SpO2 and weight were monitored throughout the experiment. Immune cell infiltration was assessed using flow cytometry. Influenza burden was assessed using RT-qPCR. SA changes were analyzed using immunofluorescence. Results Retrospective analysis revealed elevated markers of IRE1 signaling and SA regulation in asthmatic patient lung epithelial cells. IRE1 inhibition in vitro resulted in significant decreases in N-linked glycans, decreased SAs, and decreased influenza titers. In vivo, IREα knockout in lung epithelia resulted in decreased SAs, decreased recruitment of neutrophils and eosinophils, decreased viral RNA, and decreased injury markers. Additionally, IRE1α knockout prevented decreases in spO2 from influenza infection. Interestingly, ATF6 KO increased neutrophil recruitment, potentiated decreases in spO2, and resulted in increased viral RNA. Conclusions These findings suggest that IRE1 significantly impacts SA production in lung epithelial cells and inhibition or ablation of IRE1 α can reduce influenza burden and allergic inflammation in models of asthma. Conversely, ablation of ATF6 potentiates influenza burden, demonstrating differential impacts of the UPR pathways in influenza infection in asthma.