Hypercapnia, elevated PCO2 in blood and tissue, commonly develops in patients with severe acute and chronic pulmonary disease. Hypercapnia is a risk factor for mortality in COPD, community-acquired pneumonia, cystic fibrosis and adenoviral lung infection. We have shown that hypercapnia inhibits transcription of NF-κB- regulated innate immune and host defense genes, independent of pH, in human, mouse and Drosophila cells, and that it increases the mortality of bacterial infections in mice and Drosophila. We have also reported that hypercapnia inhibits type I interferon (IFN) pathway antiviral gene expression in alveolar macrophages (AM) and increases viral replication, lung injury and mortality in mice infected with influenza A virus (IAV). Based on previous studies in which we identified the zinc finger homeobox transcription factor zfh2 as a mediator of CO2-induced immune suppression in Drosophila, we bred a mouse lacking Zfhx3, a mammalian ortholog of zfh2, in the myeloid lineage. Our recent studies show that myeloid Zfhx3 deficiency protects against hypercapnia-induced suppression of antiviral genes and increased IAV growth in macrophages, and that it reduces the hypercapnia-induced increase in mortality of IAV infection in mice. We also show that hypercapnia increases and Zfhx3 deficiency decreases methylation of multiple genes in AM, including host genes that are essential for IAV replication. Recent studies from our laboratories have established that monocyte-derived AM (MoAM) recruited to the alveolar space in response to diverse insults, including IAV infection, are pro-inflammatory and drive lung injury. This is in contrast to tissue resident AM (TRAM), which are protective against IAV-induced injury. Thus, we hypothesize that hypercapnia worsens outcomes of IAV infection by suppressing antiviral genes and increasing expression of inflammation/injury-associated genes in TRAM and MoAM, and that this results from hypercapnia-induced alterations in methylation of regulatory regions of these genes. We further hypothesize that genetic deficiency of Zfhx3 in macrophages mitigates hypercapnia-induced changes in DNA methylation, and that this preserves antiviral gene expression and reduces lung injury caused by IAV in the setting of hypercapnia. To test these hypotheses, we will determine whether hypercapnia worsens outcomes of IAV infection by decreasing expression of antiviral genes and increasing expression of inflammation/injury-associated genes in TRAM and MoAM in mice; whether hypercapnia-induced changes in DNA methylation cause these changes in antiviral and inflammation/injury-associated gene expression; and whether hypercapnia is associated with similar changes in DNA methylation and gene expression in AM from humans with severe viral pneumonia. This investigation will define novel mechanisms by which elevated levels of CO2 suppress antiviral host defense, a previously-unrecognized adverse consequence of hypercapnia, and will lay the...