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A3852 - Hypercapnia Increases Apoptosis and Viral Replication in Influenza A-Infected Macrophages by a Caspase-Dependent Mechanism
Author Block: K. T. Ferguson1, F. Chen2, M. Casalino-Matsuda2, P. H. Sporn2; 1Pulmonary and Critical Care Medicine, Northwestern McGaw Medical Center, Chicago, IL, United States, 2Pulmonary and Critical Care Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, United States.
Rationale:
Hypercapnia, the elevation of CO2 in blood and tissue, commonly develops in advanced COPD and is associated with frequent and potentially fatal pulmonary respiratory infections, including influenza. We previously reported that hypercapnia inhibits antiviral gene expression and increases influenza A virus (IAV) replication in macrophages, which play a critical role anti-influenza host defense (AJRCCM 2017;195:A5264). Further, we showed that hypercapnia increases the mortality of IAV infection in mice. Following infection, IAV triggers apoptosis in target cells, but it is not clear whether the apoptotic response in macrophages promotes or limits viral replication. In the present study, we assessed the effect of hypercapnia on apoptosis in IAV-infected macrophages, and sought to determine whether changes in apoptosis induced by elevated CO2 contribute to enhanced viral pathogenicity.
Methods:
PMA-differentiated THP-1 macrophages were cultured in 5% CO2/95% air (PCO2 36 mmHg; normocapnia) or 15% CO2/21% O2/69% N2 (PCO2 108 mmHg; hypercapnia) and infected with IAV (A/WSN/33; MOI=1). Cells were harvested after incubation for 18 h. Apoptosis was assessed by immunoblot for cleaved caspase-3 and cleaved poly-ADP ribose polymerase (PARP), and by immunofluorescence microscopy to detect cleaved caspase-3-positive cells. Macrophage expression of the antiviral effector, viperin, and viral NS-1 was analyzed by immunoblot and immunofluorescence microscopy.
Results:
In the absence of infection, hypercapnia did not induce apoptosis in THP-1 macrophages. On the other hand, in cells cultured in normocapnia, infection with IAV triggered apoptosis, indicated by cleavage of both caspase-3 and PARP. IAV-induced apoptosis was augmented by hypercapnia, which increased cleavage of caspase-3 and PARP. As expected, the pan-caspase inhibitor Z-VAD-FMK blocked IAV-induced apoptosis. Interestingly, Z-VAD-FMK also blocked the hypercapnia-induced increase in NS-1 expression, while it did not reverse hypercapnic suppression of viperin.
Conclusion:
By itself, hypercapnia does not trigger apoptosis, whereas it augments cell death following IAV infection in the macrophage. The ability of Z-VAD-FMK to block the hypercapnia-induced increase in viral NS-1 expression suggests that apoptosis promotes IAV replication in macrophages, at least in the context of elevated CO2. Increased caspase-3 cleavage may also promote IAV replication independent of apoptosis, since activated caspase-3 is important for export of newly synthesized viral proteins from the host cell nucleus, a prerequisite for assembly of new virions. Our results suggest that caspase inhibition may be a useful strategy to ameliorate the deleterious effects of hypercapnia on host defense against influenza in patients with advanced lung disease.