Nontypeable Haemophilus influenzae lysates increase heterogeneous nuclear ribonucleoprotein secretion and exosome release in human middle-ear epithelial cells

Abstract

Nontypeable Haemophilus influenzae (NTHi), one of the most common acute otitis media (OM) pathogens, ispostulated topromote middle-ear epithelial remodeling inthe progression ofOMfrom acute to chronic. The goal of this studywas to examine earlyquantitativeproteomic secretome effects ofNTHi lysate exposure inahuman middle-ear epithelial cell (HMEEC) line.NTHi lysateswere used to stimulateHMEEC, and conditional quantitative stable isotope labelingwith amino acids in cell culture of cell secretionswas performed.Mass spectrometry analysis identified 766 proteins across samples. Of interest, several heterogeneous nuclear ribonucleoproteins (hnRNPs) were regulated by NTHi lysate treatment, especially hnRNP A2B1 and hnRNP Q, known to be implicated in microRNA(miRNA)packaging inexosomes.Afterpurification, thepresence of exosomes inHMEECsecretionswas characterized by dynamic light scattering (<100 nm), transmission electron microscopy, and CD63/heat shock protein 70 positivity. hnRNP A2B1 and hnRNP Q were confirmed to be found in exosomes by Western blot and proteomic analysis. Finally, exosomal miRNA content comprised 110 unique miRNAs, with 5 found to be statistically induced by NTHi lysate (miR-378a-3p + miR-378i, miR-200a-3p, miR-378g, miR30d-5p, and miR-222-3p), all known to target innate immunity genes. This study demonstrates that NTHi lysates promote release of miRNAladen exosomes from middle-ear epithelium in vitro.