Analysis of Inflammatory Signaling in Human Middle Ear Cell Culture Models of Pediatric Otitis Media

Abstract

Objectives/Hypothesis Cell culture models are valuable tools for investigation of the molecular pathogenesis of diseases including otitis media (OM). Previous study indicates that age‐, sex‐, and race‐associated differences in molecular signaling may impact disease pathophysiology. Currently, a singular immortalized middle ear epithelial (MEE) cell line exists, HMEEC‐1, derived from an adult without known middle ear disease. In this study, HMEEC‐1 and primary MEE cultures from pediatric patients with and without OM were stimulated with inflammatory cytokines or OM‐pathogenic bacterial lysates to examine differences in the response of molecules associated with OM pathogenesis. Study Design Case–control series. Methods MEE cultures were established from patients aged <6 years: two with recurrent OM (ROM), two with OM with effusion (OME), and one patient without OM who was undergoing cochlear implant surgery control undergoing cochlear implantation (Peds CI). Primary MEE cultures and HMEEC‐1 cells were stimulated with tumor necrosis factor‐α, interleukin (IL)‐1β, or nontypeable Haemophilus influenzae lysate. TNFA, IL1B, IL6, IL8, IL10, and MUC5B were assayed via quantitative polymerase chain reaction. IL‐8 was assayed by enzyme‐linked immunosorbent assay. Results Gene/protein target expressions were frequently higher in pediatric OM lines than in HMEEC‐1 and Peds CI. HMEEC‐1 cells were frequently less responsive to stimuli than all pediatric lines. OME lines were often more responsive than ROM lines. Conclusions OM may be associated with specific molecular phenotypes that are retained in primary cell culture. Adult‐derived HMEEC‐1 cells differ significantly in baseline expression and response of OM‐associated molecules relative to pediatric MEE cells. Work is underway to immortalize pediatric OM MEE cultures as improved tools for the OM research community. Level of Evidence 4 Laryngoscope, 131:410–416, 2021

Association of Pepsin With Inflammatory Signaling and Effusion Viscosity in Pediatric Otitis Media

“…Following on work demonstrating that pediatric‐derived ME cell lines more accurately represent pediatric ME mucosa than the existing immortalized adult ME cell line, we are currently developing immortalized lines from pediatric ROM and OME patients. These prior experiments identified a hyperreactive cytokine response in ME epithelial cells from OME relative to ROM patients following treatment with OM‐relevant cytokines and pathogens 37 . We speculate that OME ME cells may exhibit heightened sensitivity to pepsin as well and work is underway in our laboratory to test this hypothesis.…”

“…These prior experiments identified a hyperreactive cytokine response in ME epithelial cells from OME relative to ROM patients following treatment with OMrelevant cytokines and pathogens. 37 We speculate that OME ME cells may exhibit heightened sensitivity to pepsin as well and work is underway in our laboratory to test this hypothesis. In addition, the time and pH conditions for in vitro experiments were intended to simulate EER: a chronic condition, typically persisting for months to years, characterized by recurring, transient, and weakly-acid proximal reflux episodes.…”

“…This study was approved by Children's Wisconsin Institutional Review Board (protocol 369996-9). As previously, 37,38 informed consent was obtained prior to surgery from patients aged 6 months to 12 years with diagnosis of OME (unremitting MEF for >3 months) or ROM (≥3 episodes of OM over 6 months with resolution between) undergoing tympanostomy tube insertion (TTI) at Children's Wisconsin. MEF was collected using Juhn Tym Tap MEF collectors and surgeon ranked viscosity (0 = absent; 1 = thin serous; 2 = thick serous; 3 = mucoid; 4 = thick mucoid; and 5 = extremely thick mucoid).…”

“…Laryngoscope 132: February 2022 Samuels et al: Pepsin in Otitis Mediatranscribed using Superscript IV VILO (ThermoFisher Scientific, Waltham, MA). IL6, IL8, and TNF expression were analyzed using Taqman gene expression assays as previously 37.…”

Development of a physiological model of human middle ear epithelium

“… 9 When compared to primary cells these show marked differences both in baseline expression and stimuli‐induced expression of a range of inflammatory mediators. 10 The HMEEC‐1 cell line also lacks important phenotypic features, such as cilia, which have known functional importance, such as mucociliary transport around the Eustachian tube orifice.…”