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, is postulated to promote middle-ear epithelial remodeling in the progression of OM from acute to chronic. The goal of this study was to examine early quantitative proteomic secretome effects of NTHi lysate exposure in a human middle-ear epithelial cell (HMEEC) line. NTHi lysates were used to stimulate HMEEC, and conditional quantitative stable isotope labeling with amino acids in cell culture of cell secretions was 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 in exosomes. After purification, the presence of exosomes in HMEEC secretions was 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 miRNA-laden exosomes from middle-ear epithelium in vitro. -Val, S., Krueger, A., Poley, M., Cohen, A., Brown, K., Panigrahi, A., Preciado, D. Nontypeable Haemophilus influenzae lysates increase heterogeneous nuclear ribonucleoprotein secretion and exosome release in human middle-ear epithelial cells.

CAF secreted miR-522 suppresses ferroptosis and promotes acquired chemo-resistance in gastric cancer

“…It has been reported that hnRNP family is required for the packaging into exosomes of a list of mRNAs and noncoding RNAs [17,37,38]. In the current study, hnRNPA1 has been screened out by MS for its up-regulation in gastric tumor tissues.…”

Role of Surfactant Protein D in Experimental Otitis Media

“…Cells were maintained in 1:1 of bronchial epithelial cell basal medium (Lonza, Walkersville, MD, USA) and DMEM (Thermo Fisher Scientific) and supplemented with the SingleQuots kit (Lonza) containing bovine pituitary extract, hydrocortisone, human epidermal growth factor, epinephrine, transferrin, insulin, tri-iodothyronine, retinoic acid, gentamycin, and amphotericin-B. We treated the cells with lipopolysaccharide (LPS) at a range of concentrations from 0 to 50 μg/mL and incubated for 24 h. Cells were deprived of SingleQuots 4 h before LPS challenge in bronchial epithelial basal medium/DMEM [11]. We used Western blot to measure the expression of SP-D and NLRP3 in response to different doses of LPS.…”

“…Both viral and bacterial agents can cause OM. Nontypeable Haemophilus influenzae (NTHi) is one of the most common infectious pathogens in OM [9-11]. The innate immune response plays a significant role in OM susceptibility in early life before the development of specific immunity [6].…”

The Emerging Role of Small Extracellular Vesicles in Inflammatory Airway Diseases

“…Not unexpectedly, the proteomic analysis showed an enrichment in neutrophil markers and molecules associated with innate immunity, such as immunoglobulins, MUC5B, and heat-shock proteins; also related to neutrophil stimulation were 29 enriched miRNA sequences unique to the middle ear samples, including the most abundant miR-223 [ 45 ]. On the cellular level, it was shown that human middle ear epithelial cells treated with Haemophilus influenzae lysate secrete sEVs containing heterogeneous nuclear ribonucleoproteins, such as hnRNP A2B1 and hnRNP Q, and also miRNAs that might be involved in immunity regulation [ 47 ].…”