Hypercapnia Inhibits Host Defense Against Influenza A Virus by Potentiating Virus‐induced Activation of the PI3K/Akt Pathway

Rationale Hypercapnia, the elevation of CO 2 in blood and tissue, commonly develops in advanced COPD and is associated with frequent and potentially fatal pulmonary bacterial and viral infections, including influenza. We previously reported that hypercapnia downregulates several innate immune genes and increases the mortality of bacterial pneumonia and influenza A virus (IAV) infection in mice. High CO 2 also increases viral nucleoprotein (NP) expression in the mouse lung, primarily in alveolar macrophages (MØs). In the current study, we explored the effect of hypercapnia on the PI3K/Akt pathway during IAV infection in MØs. In addition, using pharmacologic PI3K and Akt inhibitors we studied the role of hypercapnia‐induced activation of Akt on the expression of antiviral proteins, including MX dynamin like GTPase 1 (MX1), 2′‐5′‐oligoadenylate synthetase 1 (OAS1) and viperin; viral nonstructural 1 protein 1 (NS1) and NP. Methods Human alveolar and PMA‐differentiated THP‐1 MØs as well as murine alveolar and bone marrow derived MØs were cultured in 5% CO 2 (PCO 2 36 mmHg; normocapnia) or 15% CO 2 (PCO 2 108 mmHg; hypercapnia) and infected with IAV. Akt activation was assessed by immunoblot and immunofluorescence microscopy (IF). MX1, OAS1 and viperin mRNA and protein levels were determined using qPCR and immunoblot or IF, respectively. IAV replication was assessed using MDCK plaque assay, and NS1 and NP expression were determined by immunoblot or IF. For in vivo studies, mice were pre‐exposed to normoxic hypercapnia (10% CO 2 /21% O 2 ) for 3 days, or air as control, then infected with IAV. Mice were sacrificed 4 days after infection for determination of antiviral and viral proteins expression in lung tissue by immunoblot and IF. Results Hypercapnia potentiated IAV‐induced Akt activation in MØs in vivo and in vitro , an effect blocked by the PI3K inhibitor LY294002 and the Akt inhibitor MK2206. In addition, elevated CO 2 decreased MX1, OAS1 and viperin mRNA and protein expression and increased viral replication and NS1 and NP proteins in IAV infected human and mouse MØs. Hypercapnia also increased viral protein expression in alveolar MØs, and decreased antiviral proteins and augmented viral titers in the lungs of IAV‐infected mice. We also found that the PI3K and Akt inhibitors blocked the hypercapnia‐induced suppression of antiviral proteins and the increase on viral proteins and IAV replication in vitro . Conclusion Hypercapnia decreases antiviral proteins and increases viral NS1 and NP proteins and virus replication in MØs following IAV infection in vivo and in vitro . The effect of elevated CO 2 on the macrophage antiviral response and virus production can be blocked by small molecule inhibitors of PI3K and Akt, suggesting a link between hypercapnia‐induced activation of the PI3K/Akt pathway and an impaired antiviral host response. Therefore, PI3K/Akt inhibitors may hold promise as pharmacotherapy to improve antiviral host defense in patients with advanced lung disease and hypercapnia. Support or Funding Information R01HL107629, R56HL131745, P01AG049665