C96-02 Proteasome Dysfunction Modulates Host Response to Influenza Infection

Rationale Influenza A virus (IAV) infection causes significant morbidity and mortality. Age is an independent risk factor correlating with poor outcomes from influenza infection, with elderly Americans disproportionately accounting for hospitalizations and deaths. Loss of proteostasis, including proteasome dysfunction, is a molecular hallmark of aging. However, how proteasome dysfunction affects host responses to infection is unknown. We propose that host responses to IAV infection are modified by proteasome dysfunction. Further, we test a novel small molecule D233-0877, a proteasome activator, during influenza infection in vitro and ex vivo in human precision-cut lung slices (PCLS). Methods Immortalized human AEC1 cell line (huARLO) cells were grown in submerged or air-liquid interface on transwell inserts. Cells were pretreated with carfilzomib (CFZ), a proteasome inhibitor, or vehicle for 72-hours before infection with 0.2 MOI IAV (H1N1). Transcription of proinflammatory genes (IL-6, IL-8, TNF-α), antiviral genes (IRF3, IRF7, IRF9), and influenza matrix protein (MP) was assessed by qRT-PCR 24-hours post infection (n = 3). Human PCLS (n = 12) were exposed to 105 PFU IAV (H3N2) or mock infection and treated with 2.5 μM D233-0877, a proteasome activator, or vehicle. PCLS lysates were assayed for IAV nucleoprotein (NP) by immunoblot and MP by qRT-PCR.Statistical testing was performed with GraphPad Prism. Results huARLO cells treated with 300 nM CFZ for 72-hours were viable and accumulated polyubiquitinated proteins, indicating proteotoxic stress (Figure A). CFZ-pretreated huARLO cells infected with IAV had increased expression of proinflammatory markers (IL6: 6.95-fold increased; IL8: 17.46-fold increased; TNF-α: 2.59-fold increased; p < 0.05) compared to IAV-exposed controls (Figure B). Antiviral pathway proteins (IRF3, IRF7, MX1) were unchanged or reduced. Human PCLS infected with IAV and co-treated with D233-0877 had unchanged NP transcript levels but reduced NP protein expression (2.53-fold reduction; p < 0.05) compared to IAV infection alone (Figure C). Conclusions Exposure of huARLO cells to CFZ induces proteotoxic stress, providing a model for age-related proteasome dysfunction. IAV infection increased proinflammatory programs, but abrogated antiviral gene expression. Thus, mitigating proteotoxic stress could be a novel host-directed therapeutic target in IAV infection. PCLS were successfully infected with IAV, providing an ex vivo model for IAV infection. PCLS infected with IAV and treated with D233-0877 had unchanged NP mRNA but showed reduced NP protein, suggesting pharmacologic proteasome activation partially attenuates IAV replication. As IAV NP is known to be degraded in the proteasome, further testing is warranted to assess if IAV infectivity, replication, and clearance is impacted by D233-0877. This abstract is funded by: Pitt KARAT, NIH 1R56HL174649 (JWE)