Antimicrobial Peptide and Lipid Response of Alveolar Type II Model Cells to Cytokines of TH17 Signature

Abstract

Abstract Mycobacterium tuberculosis (Mtb) causes ~ 10 million deaths each year worldwide. The current BCG vaccine that targets TH1 cells and anti-tuberculosis drugs have limited efficacy. Novel preventative measures are in dire need. Epithelial cells in the lungs are among the first host cells to encounter inhaled Mtb. Epithelial cells produce antimicrobial peptides (AMPs) like human beta-defensins (HBDs) and antimicrobial lipids (AMLs). Microbial products such as lipopolysaccharide (LPS), IL-1β made by macrophages, and IL-17 and IL-22 secreted by TH17 cells are all known to increase production of AMPs in epithelial cells. However, their effect on the production of AMLs is unknown. We hypothesized that TH17 cytokines increase the production of AMPs and AMLs in lung alveolar type II cells, leading to increased antimycobacterial activity. As determined by immunofluorescence, IL-17 combined with IL-22 (IL-17/22) significantly increased intracellular HBD2 in A549 cells after 48 h. Using the lipid sensitive fluoroprobes Bodipy and Nile Red, we found that IL-1β combined with LPS significantly increased intracellular neutral lipids, while IL-17/22, IL-1β/LPS, and LPS alone significantly increased neutral and polar lipid secretion after 24h stimulation. To assess the antimycobacterial effect of A549 secretions, we developed a qPCR-based assay employing Mycolicibacterium smegmatis (Ms), an avirulent model organism for Mtb, bacterial cell lysis with TRI reagent and bead beating, gDNA extraction with Direct-zol™ DNA Miniprep, SYBR Green chemistry, and 16S rRNA gene targeting primers, and testing is ongoing. This research may lead to novel vaccine approaches for Mtb based on TH17-induced antimycobacterial activity of lung epithelial cells. This research was supported by NIH R25GM061331, NIH T34GM08228, and CalStateLA Provost’s Research Restart Grant.