Exosome swarms eliminate airway pathogens and provide passive epithelial immunoprotection through nitric oxide.

BACKGROUND: Nasal mucosa-derived exosomes (NMDEs) harbor immunodefensive proteins and are capable of rapid interepithelial protein transfer. OBJECTIVES: We sought to determine whether mucosal exposure to inhaled pathogens stimulates a defensive swarm of microbiocidal exosomes, which also donate their antimicrobial cargo to adjacent epithelial cells. METHODS: We performed an institutional review board-approved study of healthy NMDE secretion after Toll-like receptor (TLR) 4 stimulation by LPS (12.5 µg/mL) in the presence of TLR4 inhibitors. Interepithelial transfer of exosomal nitric oxide (NO) synthase and nitric oxide was measured by using ELISAs and NO activity assays. Exosomal antimicrobial assays were performed with Pseudomonas aeruginosa. Proteomic analyses were performed by using SOMAscan. RESULTS: In vivo and in vitro LPS exposure induced a 2-fold increase in NMDE secretion along with a 2-fold increase in exosomal inducible nitric oxide synthase expression and function through TLR4 and inhibitor of nuclear factor κB kinase activation. LPS stimulation increased exosomal microbiocidal activity against P aeruginosa by almost 2 orders of magnitude. LPS-stimulated exosomes induced a 4-fold increase in NO production within autologous epithelial cells with protein transfer within 5 minutes of contact. Pathway analysis of the NMDE proteome revealed 44 additional proteins associated with NO signaling and innate immune function. CONCLUSIONS: We provide direct in vivo evidence for a novel exosome-mediated innate immunosurveillance and defense mechanism of the human upper airway. These findings have implications for lower airway innate immunity, delivery of airway therapeutics, and host microbiome regulation.

Stereolithography-Based 3D Printed "Pillar Plates" that Minimizes Fluid Transfers During Enzyme Linked Immunosorbent Assays.

Enzyme linked immunosorbent assay (ELISA) is one of the most popular and indispensable tools in molecular biology. Despite numerous advances in ELISA methods that markedly improve the sensitivity and throughput of detection, a hallmark of all ELISA continues to be repeated pipetting of fluids that is not only cumbersome but can easily introduce errors or contaminations. Robotics, despite obvious advantages, remains expensive. Here, we designed and produced cheap "pillar plates" using stereolithography-based 3D printing that can be readily inserted into conventional 96- and 384- well plates and serve as the substrate for ELISA. We demonstrate that ELISA using these "pillar plates" affords comparable specificity and sensitivity of detection of serum antibodies to traditional sandwich ELISA, while markedly reducing the time and efforts associated with fluid transfer. These results underscore "pillar plates" as an attractive platform for rapid yet robotics-free ELISA.