Abstract Rationale Asthma is the most common pediatric lung disease in the U.S. and asthma endotypes exist on a spectrum of overlapping features from allergic driven type-2 (T2) to obesity-related non-T2 endotypes. The mechanisms of airway inflammation and signaling in non-T2 endotypes is less understood, particularly in children with obesity. The cytokine hormone, leptin, has gained attention as a potential mediator in the pathogenesis of complex obese-asthma endotypes. Leptin is primarily produced by adipocytes and is elevated in obesity, and higher leptin serum levels are associated with severe asthma and increased inflammatory markers including IL-6, G-CSF, and NF-κB. We hypothesized that leptin stimulation would alter epithelial structure and mucus secretion in pediatric bronchial epithelia. Methods Primary bronchial epithelial cells (BECs) from donors with pediatric asthma (n = 6) or healthy donors (n = 4) were differentiated at an air-liquid interface to an organotypic epithelium. Following differentiation, epithelia were stimulated with 1ng/ml leptin, 100ng/ml leptin, or vehicle control for 7 days and then infected with RV16 applied to the apical surface at a multiplicity of infection of 0.5. Epithelia were harvested for RNA isolation at 48 hours after infection for viral genome copy number assessment with qPCR and gene expression analysis with RNA-sequencing. Epithelia were also fixed in glutaraldehyde and processed for scanning electron microscopy and tunneling electron microscopy analysis of ciliary structure or fixed in formaldehyde for immunofluorescence staining for TUBA4A and MUC5AC. Results Leptin stimulation at both 1ng/ml and 100ng/ml for 7 days decreased surface ciliary coverage and increased surface secretory cell area. By immunofluorescence, no difference in number of MUC5AC secretory cells was found. Leptin stimulation did not alter viral genome copy number 48 hours after infection with RV16 and did not change interferon gene expression. Leptin stimulation decreased genes associated with protein translation, oxidative phosphorylation, and aerobic respiration. Leptin stimulation increased expression of differentiation markers and cell adhesion genes. Leptin stimulation led to increased metabolic and protein translation genes 48 hours after RV16 infection. Conclusion Leptin alters ciliary cell differentiation in pediatric bronchial epithelia leading to decreased surface ciliary cell coverage. Leptin stimulation does not change interferon gene expression after RV16 infection, but decreases protein translation and metabolic gene expression prior to infection and increases protein translation and metabolic gene expression after RV16 infection. These data suggest that leptin alters epithelial differentiation and ciliary structure which may contribute to pathophysiology in obese-asthma phenotypes. This abstract is funded by: SRS (WTP), Parker B Francis Fellowship (WTP), NIH K08HL179396 (WTP), NIH R01AI163160 (JSD), NIH K24AI150991 (JSD), NIH U19AI175089 (JSD).
Powell et al. (Fri,) studied this question.