Supplementation of αKG along with TGFβ/HIF1α antagonist rescues lung fibrosis in mice with severe SARS-CoV-2 infection

Abstract

Pulmonary fibrosis and acute respiratory distress syndrome (ARDS) are the clinical challenges among patients with post-acute sequelae of COVID-19 (PASC), affecting ~ 10% of the survivors worldwide. PASC manifestations include pulmonary fibrosis, impaired respiratory function, and long-term morbidity. Recent WHO reports from 83 countries also describe severe morbidity among COVID-19 survivors associated with PASC, indicating an unmet need to address the issue. We developed a mouse model showing the above disease pathophysiology, and investigated the related signalling molecules, with a focus on developing targeted treatment strategies against it. We developed the mild and severe SARS-CoV-2 infection mice models. We took the opportunity to transiently express high/low levels of hACE2 in the airways and lungs of the mice, to manipulate infection severity and longer survivability. The severe infection model developed ARDS pathogenesis and pulmonary fibrosis, limiting respiratory functions (assessed by dual-chamber plethysmography), and survived beyond 30 DPI. The signalling molecules of these pathogenic pathways were identified from related tissue/blood samples of the mice using mass spectrometry-based proteome analysis, histopathology, immunoblotting and flow cytometry. The proteomic analysis revealed key pathways driving epithelial-mesenchymal transition (EMT) and pulmonary fibrosis in mice lungs. Elevated expression of P-AKT, TGFβ and HIF1α supported the severity of ARDS and fibrosis in lungs at 15 as well as 30 DPI. We developed a treatment strategy by targeting the above signalling molecules. Treatment with dietary alpha-ketoglutarate (1% αKG daily), known suppressor of P-AKT, along with inhibitor to TGFβ (SB431542,10 mg/kg BW) or HIF1α (CAY10585, 10 mg/kg BW/daily, delivered intraperitoneally) till 7 DPI, significantly reduced EMT and fibrosis by suppressing the PAKT: TGFβ:HIF1α signalling, and finally improved mice survivability (50%) beyond 50 DPI with a restored pulmonary function. The study thus demonstrated the upregulation signalling molecules like P-AKT, TGFβ and HIF1α and their association with pulmonary fibrosis in mice after severe SARS-CoV-2 infection. Further, the inhibition of the above pathophysiology by the co-administration of αKG along with HIF1α/TGFβ antagonist highlighted a targeted therapeutic strategy against it. Importantly, for the first time, our study described the HIF1α axis as the therapeutic target in rescuing post-acute sequelae such as ARDS and fibrosis in mice with severe SARS-CoV-2 infection.