ABSTRACT
Rationale: Individuals with COPD who develop COVID-19 are at increased risk of hospitalization, ICU admission and death. COPD is associated with increased airway epithelial expression of ACE2, the receptor mediating SARS-CoV-2 entry into cells. Hypercapnia commonly develops in advanced COPD and is associated with frequent and potentially fatal pulmonary infections. We previously reported that hypercapnia increases viral replication, lung injury and mortality in mice infected with influenza A virus. Also, global gene expression profiling of primary human bronchial epithelial (HBE) cells showed that elevated CO2 upregulates expression of cholesterol biosynthesis genes, including HMGCS1, and downregulates ATP-binding cassette (ABC) transporters that promote cholesterol efflux. Given that cellular cholesterol is important for entry of viruses into cells, in the current study we assessed the impact of hypercapnia on regulation of cellular cholesterol levels, and resultant effects on expression of ACE2 and entry of Pseudo-SARS-CoV-2 in cultured HBE, BEAS-2B and VERO cells, and airway epithelium of mice. Methods: Differentiated HBE, BEAS-2B or VERO cells were pre-incubated in normocapnia (5% CO2, PCO2 36 mmHg) or hypercapnia (15% CO2, PCO2 108 mmHg), both with normoxia, for 4 days. Expression of ACE2 and sterol regulatory element binding protein 2 (SREPB2), the master regulator of cholesterol synthesis, was assessed by immunoblot or immunofluorescence. Cholesterol was measured in cell lysates by Amplex red assay. Cells cultured in normocapnia or hypercapnia were also infected with Pseudo SARS-CoV-2, a Neon Green reporter baculovirus. For in vivo studies, C57BL/6 mice were exposed to normoxic hypercapnia (10% CO2/21% O2) for 7 days, or air as control, and airway epithelial expression of ACE2, SREBP2, ABCA1, ABCG1 and HMGCS1 was assessed by immunofluorescence. SREBP2 was blocked using the small molecules betulin or AM580, and cellular cholesterol was disrupted using MβCD. Results: Hypercapnia increased expression and activation of SREBP2 and decreased expression of ABC transporters, thereby augmenting epithelial cholesterol levels. Elevated CO2 also augmented ACE2 expression and Pseudo-SARSCoV- 2 entry into epithelial cells in vitro and in vivo. These effects were all reversed by blocking SREBP2 or disrupting cellular cholesterol. Conclusion: Hypercapnia augments cellular cholesterol levels by altering expression of cholesterol biosynthetic enzymes and efflux transporters, leading to increased epithelial expression of ACE2 and entry of Pseudo-SARS-CoV-2 into cells. These findings suggest that ventilatory support to limit hypercapnia or pharmacologic interventions to decrease cellular cholesterol might reduce viral burden and improve clinical outcomes of SARSCoV- 2 infection in advanced COPD and other severe lung diseases.
ABSTRACT
Rationale: Individuals with chronic obstructive pulmonary disease (COPD) who develop 2019 coronavirus disease (COVID-19) are at increased risk of hospitalization, intensive care unit admission and death. COPD is associated with increased airway epithelial expression of angiotensin converting enzyme 2 (ACE2), the cell surface receptor to which the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds and which mediates entry of the virus into cells. Hypercapnia, the elevation of CO2 in blood and tissue, commonly develops in advanced COPD and is associated with frequent and potentially fatal pulmonary infections. We previously showed that normoxic hypercapnia alters expression of innate immune genes, including multiple viral response genes, in primary human bronchial epithelial (HBE) cells (Sci Reports 8:13508, 2018). Thus, in the current study, we explored the effect of hypercapnia on expression of ACE2 and uptake of a Pseudo-SARS-CoV-2 baculovirus by airway epithelial cells. Methods: HBE cells (Lonza) differentiated at air-liquid interface or immortalized BEAS-2B cells were pre-incubated in normocapnia (NC, 5% CO2, PCO2 36 mmHg) or normoxic hypercapnia (HC, 15% CO2, PCO2 108 mmHg) for 4 days. ACE2 protein expression was assessed by immunoblot or immunofluorescence (IF). In addition, BEAS-2B cells pre-exposed to NC or HC for 2 days were infected with Pseudo SARS-CoV-2 for an additional 2 days. Pseudo SARS-CoV-2 (Montana Molecular) is a reporter baculovirus whose surface is decorated with SARS-CoV-2 spike protein, and which induces expression of Neon Green protein in the nucleus of host cells 24 h after viral entry. For in vivo studies, C57BL/6 mice were pre-exposed to normoxic hypercapnia (10% CO2/21% O2) for 7 days, or air as control, and ACE2 expression in lung tissue was assessed by IF. Results: Compared to culture in NC, HC increased ACE2 protein expression by ∼4-fold in HBE cells and ∼2.5-fold in BEAS-2B cells. Likewise exposure of mice for 7 days to 10% CO2, as compared to air, markedly increased airway epithelial cell expression of ACE2 (Figure 1). Additionally, culture in HC, as compared to NC, increased Pseudo SARS-CoV-2 entry to BEAS-2B cells. Conclusion: Elevated CO2 increases airway epithelial cell expression of the SARS-CoV-2 receptor, ACE2, in vitro and in vivo. This may lead to a greater burden of SARS-CoV-2 infection in patients with hypercapnia, and in part account for worse clinical outcomes of COVID-19 pneumonia in advanced COPD and other severe lung diseases.