Abstract Rationale People of color experience more frequent and severe respiratory infections, likely due in part to increased prevalence of vitamin D deficiency (VDD). While vitamin D has been proposed as a prophylactic treatment for respiratory infections, studies of oral vitamin D supplementation have produced mixed results in preventing influenza infection. Inhalation offers the advantage of delivering vitamin D directly to the respiratory epithelium, the primary site of influenza infection, potentially providing superior protection compared to oral supplementation. We therefore investigated the role of vitamin D inhalation as a prophylactic strategy against H1N1 infection in human airway cells and evaluated potential antiviral and immunomodulatory mechanisms. Methods We exposed well-differentiated, primary human bronchial epithelial cells (HBECs) at air-liquid interface to H1N1 influenza virus (A/California/07/2009) at a multiplicity of infection of 0.01, followed by administration of 10 µL of 10 µM vitamin D to the apical compartment. Apical treatment was delivered via aerosol in a novel multi-well in vitro exposure system, On-Plate Aerosol Delivery Array (OPADA). Prophylactic effects of vitamin D were evaluated by measuring human and viral gene expression through bulk RNA-sequencing and gene set enrichment analysis (GSEA). Results We found that pre-treatment with aerosolized vitamin D significantly reduced H1N1 viral load in HBECs, as measured by decreased expression of influenza genes. Vitamin D pretreatment also reduced virus-induced expression of antiviral interferon-stimulated genes (ISGs) while modulating cytokine responses indicative of reduced inflammation. Similarly, GSEA revealed that vitamin D treatment attenuated viral inflammatory responses, immune dysfunction, and cellular damage pathways, while boosting pathways related to ciliary function. Vitamin D pretreatment alone increased expression of the antimicrobial peptide, cathelicidin (LL-37), with direct antiviral properties against influenza. Correlation analysis of human and viral gene expression showed that influenza and cathelicidin exhibited a significant inverse relationship. Conclusions Here, we provide studies demonstrating that inhaled vitamin D: 1) reduces H1N1 viral load, 2) decreases aberrant inflammatory responses in HBECs, and 3) likely acts through multiple mechanisms, including enhancement of ciliary function and induction of antimicrobial peptides. Our data suggest inhaled vitamin D may be a feasible prophylactic strategy for preventing influenza infection and may have utility in reducing health disparities associated with respiratory infections, such as the increased severity of influenza in people of color. Ongoing studies aim to further elucidate mechanisms by which inhaled vitamin D provides protection against influenza, and explore the utility of vitamin D inhalation in preventing influenza infections in humans in vivo. This abstract is funded by: 1F31HL179957-01
Schichlein et al. (Fri,) studied this question.