Background: Chronic otitis media (COM) is characterized by middle ear fluid predominantly containing cytokines, Nontypeable haemophilus influenzae (NTHi), the mucin MUC5B, and neutrophil extracellular traps (NETs). NETs consist of extracellular DNA coated with antibacterial proteins such as myeloperoxidase (MPO) and citrullinated histone 3 (CitH3). NETs can damage tissues and sustain inflammation. Our study aimed to develop an in vitro model of NETosis, testing COM inductors.
Methods: NETosis was evaluated in fresh blood human neutrophils attached to collagen-coated plates and in suspension exposed to phorbol myristate acetate (PMA) as a control, and COM relevant mediators. Confocal microscopy, DNA fluorescence assay and flow cytometry were used to quantify NETosis.
Results: PMA exposure induced DNA, MPO, and CitH3 by immunofluorescence (IF) most significantly at 3 hours (3.8-fold for DAPI, 7.6-fold for MPO, and 6.9-fold for CitH3, all P < .05). IL-8 and TNF-α cytokines showed milder increases of DAPI, MPO, and CitH3 positive cells. NTHi had no effect on these NETs markers. Purified salivary MUC5B (10 to 40 μg/mL) produced potent increases, comparable to PMA. A composite NET score summing the fold-increases for DAPI, MPO, and CitH3 demonstrated PMA at 13.6 to 19 relative to control set at 1; and MUC5B at 8.6 to 16.3 (all P < .05). IL-8 and TNF-α showed scores of 5.4 and 3, respectively, but these were not statistically significant.
Conclusion: We developed a reliable in vitro assay for NETosis which demonstrated that salivary MUC5B is a potent inductor of NETs whereas IL-8, TNF-α, live and lyzed NTHi demonstrated minimal to no NETosis.
Level of evidence: NA.
Keywords: citrullinated histone; confocal microscopy; extracellular DNA; inflammation; innate immunity; middle ear infection; myeloperoxidase.
© 2020 The Authors. Laryngoscope Investigative Otolaryngology published by Wiley Periodicals, Inc. on behalf of The Triological Society.