C107-09 Bronchial and Alveolar Epithelial Cell-specific Deficiency of Zfhx3 Protects Against Hypercapnia-induced Increases in Viral Replication and Lung Injury in Mice Infected With Influenza A

Abstract Rationale Hypercapnia is associated with poor outcomes of pulmonary infection and advanced lung disease. We have shown that hypercapnia increases viral replication and mortality in mice infected with influenza A virus (IAV). We recently identified the zinc finger homeodomain transcription factor, Zfhx3, as a mediator of CO2-induced suppression of macrophage immune and antiviral gene expression, and showed that myeloid-specific deletion of Zfhx3 partially protects mice against CO2-induced suppression of antiviral host defense. Further, we showed that hypercapnia decreased expression of antiviral effectors and increased expression of viral proteins in bronchial and alveolar epithelium of IAV-infected mice. In the current study, we explored whether bronchial epithelial cell (BEC)- and alveolar type 2 cell (AT2)-specific deficiency of Zfhx3 protects against effects of hypercapnia in mice infected with IAV. Methods Wild-type mice and Zfhx3fl/flScgb1a1CreER or Zfhx3fl/flSftpcCreER mice with tamoxifen-induced BEC- or AT2-specific Zfhx3 deficiency, respectively, were pre-exposed to normoxic hypercapnia (10% CO2/21% O2) for 3 days, or air as control, then infected intratracheally with 3 pfu IAV and maintained in 10% CO2 or air, until sacrifice 4 days post infection. Gene expression in lungs from wild-type mice was analyzed using the Xenium spatial transcriptomics platform. Lung tissue from mice with tamoxifen-induced BEC- or AT2-specific Zfhx3 deficiency, and non-tamoxifen-treated controls, was stained with H&E to determine histologic lung injury, or immunostained to assess viral NS1 and M2 protein expression by immunofluorescence microscopy. Results Spatial transcriptomic analysis of lungs from IAV-infected wild-type mice showed that hypercapnia decreased expression of antiviral genes and increased expression of IAV genes in BEC and AT2. Tamoxifen-induced BEC- and AT2-specific Zfhx3 deficiency in Zfhx3fl/flScgb1a1CreER and Zfhx3fl/flSftpcCreER mice, respectively, each attenuated the increase in histologic lung injury caused by hypercapnia following IAV infection. BEC- and AT2-specific Zfhx3 deficiency also protected against hypercapnia-induced increases in viral NS1 and M2 expression in the lungs of IAV-infected mice, and in cultured BEC isolated from tamoxifen-treated Zfhx3fl/flScgb1a1CreER infected with IAV in vitro. Conclusion Hypercapnia decreases expression of antiviral genes and increases expression of viral genes in BEC and AT2 in mice infected with IAV. BEC- and AT2-specific Zfhx3 deficiency protects against hypercapnia-induced increases in viral replication and histologic lung injury following IAV infection. Thus, Zfhx3 is a mediator of hypercapnic suppression of antiviral host defense not only in macrophages, but also in BEC and AT2. This abstract is funded by: Department of Veterans Affairs and NIH