Introduction: SARS-CoV-2 is the pathogen causing COVID-19. Young age and high vitamin D plasma levels have been associated with reduced infection risk and favourable disease outcomes.

Aim: Understand the molecular mechanisms associated with differential responses to SARS-CoV-2 across age groups and potential effects of vitamin D.

Methods: Airway epithelial cells (AECs) from children (7 months - 14 years, N=8) and adults (>50 years, N=8) were cultured for 4 weeks at the air-liquid interface, with or without vitamin D, and infected with SARS-CoV-2 for 48 hours. Differential gene expression signatures and DNA methylation were investigated using the NanoString and MethylationEPIC arrays respectively. Cell surface expression of CD13 was quantified via immunofluorescence, while vitamin D receptor (VDR) recruitment to the DNA and its interactions with DNMTs were interrogated using protein-IP and ChIP.

Results: When compared to children, AECs from adults showed higher SARS-CoV-2 recovery following infection. This associated with higher ANPEP/CD13 and reduced type I interferon (IFN) expression, and differential DNA methylation. Culture of AECs from adults with vitamin D increased DNA methylation and reduced the expression of TTLL12. This was mediated by VDR recruitment to its proximal promoter, where it instructs methylation through DNMT1. TTLL12 is a negative regulator of innate anti-viral responses, and its expression inversely correlated with IFN expression in AECs and HEp-2 cells.

Conclusion: This study links differential expression of CD13 and IFN with age differential virus recovery in AECs. It provides molecular evidence for vitamin D to reduce virus replication through inhibition of TTLL12 expression.