The effect of vitamin D supplementation on human airway epithelial antimicrobial activity, ion transport, and liquid absorption

Abstract

Vitamin D deficiency is implicated in chronic lung disease. Given its popularity as a respiratory season supplement, we sought to characterize the effect of active vitamin D (calcitriol) on human airway epithelial cell innate immune properties, including ion transport in vitro. We hypothesized calcitriol supplementation would enhance epithelial antimicrobial properties and alter airway surface liquid (ASL) composition and volume.We assayed primary human airway epithelial cells (hAECs) pretreated with calcitriol (10 -7 M) for 22h vs. paired controls. ASL bactericidal activity was tested by quantifying bioluminescent Staphylococcus aureus incubated with ASL from hAECs ±calcitriol. Epithelial transcriptional changes were measured by RNA sequencing and RT-qPCR. We measured the final volume of an apical 60 μL bicarbonate solution after 4h to assess the rate of epithelial fluid absorption ( J v ). To evaluate ion transport properties, epithelia were mounted in Ussing chambers and short-circuit current ( I sc ) and transepithelial conductance ( G t ) recorded. We quantified the half-life of ENaC by blocking protein translation with cycloheximide, then measured amiloride-sensitive I sc or G t in the presence of anion channels inhibitors.ASL from epithelia supplemented with calcitriol exhibited enhanced bactericidal activity against S. aureus, due to enhanced cathelicidin expression. RNA sequencing analysis revealed calcitriol decreased mRNA transcripts of proteins involved in airway epithelial sodium and chloride transport ( SCNN1G and ATP1B1). Consistent with RNA sequencing data, calcitriol-treated epithelia had decreased J v , amiloride-sensitive I sc , and amiloride-sensitive G t . Electrophysiological responses from anion channel blockers were unaffected. We obtained similar results with prolonged calcitriol incubation (7-day), while acute treatment (≤20 min.) did not affect I sc or G t . After apically treating epithelia with nystatin, no differences were observed in ouabain-sensitive I sc ±calcitriol, indicating calcitriol did not alter Na-K pump activity. Supplementation also did not alter the half-life of amiloride-sensitive I sc . RT-PCR experiments demonstrated both 3h and 22h calcitriol treatment increased CYP24A1 and CAMP expression (Vitamin D response element controls). However, ENaC genes only trended down for both timepoints, indicating the VDR likely indirectly decreased ENaC activity.In summation, calcitriol increased hAEC cathelicidin expression and ASL bacterial killing, while reducing sodium and liquid absorption across the epithelia via decreased ENaC activity. Calcitriol supplementation may be useful in airway diseases involving chronic bacterial infection and mucus dehydration. The Bowers Emphysema Fund and P01 HL091842/HL/NHLBI NIH HHS/United States This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.