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A3838 - Myeloid Zfhx3 Deficiency Prevents Hypercapnia-Induced Suppression of Antiviral Response and Increased Viral Replication Following Influenza A Virus-Infection in Mice
Author Block: M. Casalino-Matsuda1, F. Chen1, F. J. Gonzalez1, A. Nair1, G. J. Beitel2, P. H. S. Sporn1; 1Feinberg School of Med (Div of Pulm and Crit Care Med), Northwestern University, Chicago, IL, United States, 2Molecular Biosciences, Northwestern University, Evanston, IL, United States.
Rationale: Hypercapnia commonly occurs in advanced COPD and is associated with increased risk of pulmonary infections. We previously showed that hypercapnia impairs innate immunity and host defense in human, mouse and Drosophila systems, suggesting an evolutionarily conserved mechanism. Further, we conducted a genome-wide RNAi screen in Drosophila, which identified the transcription factor, zinc finger homeodomain 2 (zfh2), as a mediator of hypercapnic immunosuppression in cultured Drosophila cells; moreover, knockdown in adult flies confirmed that zfh2 is required for CO2-induced immunosuppression in vivo. Recently, we reported that hypercapnia suppresses the antiviral response and increases the mortality of influenza A virus (IAV) infection in mice. In the current study, we explored the role of Zfhx3, a mammalian ortholog of Zfh2, on the macrophage antiviral response and viral replication following IAV infection in mice. Methods: Global homozygous Zfhx3 deficiency is fetal lethal and heterozygosity results in pre-weaning mortality. We therefore crossed Zfhx3flox/flox (Zfhx3+/+) mice (J-T Dong, Emory University) with LysM-Cre mice to generate animals with myeloid-specific homozygous Zfhx3 deficiency (myeloid Zfhx3-/-). Macrophages from myeloid Zfhx3-/- and Zfhx3+/+ mice were exposed to hypercapnia and infected with IAV (A/WSN/33). For in vivo studies, mice were pre-exposed to normoxic hypercapnia (10% CO2/21% O2) for 3 days, or air as control, then infected with IAV. Expression of antiviral proteins OAS1 and viperin and of viral nucleoprotein (NP) and non-structural protein 1 (NS1) was analyzed in macrophages and lung tissue by immunoblot or immunofluorescence microscopy. Viral titers were determined by MDCK plaque assay. Results: We successfully bred the myeloid Zfhx3-/- mice, which in the unchallenged state appear phenotypically normal. While Zfhx3 is robustly expressed in Zfhx3+/+ macrophages, Zfhx3 mRNA and protein are absent in Zfhx3-/- macrophages. Remarkably, Zfhx3-/- macrophages were protected from hypercapnia-induced suppression of OAS1 and viperin, and increased viral NP and NS1 expression and IAV replication observed in Zfhx3+/+ control macrophages. Myeloid Zfhx3-/- mice exposed to 10% CO2 and infected with IAV in vivo were also protected from hypercapnia-induced suppression of antiviral proteins and enhanced viral growth observed in macrophages and lung tissue of Zfhx3+/+ mice. Conclusions: These data strongly support a specific role for Zfhx3 in mediating critical suppressive effects of hypercapnia on the macrophage antiviral response, leading to increased IAV replication in vitro and in vivo. These findings identify Zfhx3 as the first known component of a putative conserved pathway by CO2 suppresses innate immune function.