The transcriptional landscape of the cultured murine middle ear epithelium in vitro.

Otitis media (OM) is the most common paediatric disease and leads to significant morbidity. Although understanding of underlying disease mechanisms is hampered by complex pathophysiology, it is clear that epithelial abnormalities underpin the disease. The mechanisms underpinning epithelial remodelling in OM remain unclear. We recently described a novel in vitro model of mouse middle ear epithelial cells (mMEECs) that undergoes mucociliary differentiation into the varied epithelial cell populations seen in the middle ear cavity. We now describe genome wide gene expression profiles of mMEECs as they undergo differentiation. We compared the gene expression profiles of original (uncultured) middle ear cells, confluent cultures of undifferentiated cells and cells that had been differentiated for 7 days at an air liquid interface (ALI). >5000 genes were differentially expressed among the three groups of cells. Approximately 4000 genes were differentially expressed between the original cells and day 0 of ALI culture. The original cell population was shown to contain a mix of cell types, including contaminating inflammatory cells that were lost on culture. Approximately 500 genes were upregulated during ALI induced differentiation. These included some secretory genes and some enzymes but most were associated with the process of ciliogenesis. The data suggest that the in vitro model of differentiated murine middle ear epithelium exhibits a transcriptional profile consistent with the mucociliary epithelium seen within the middle ear. Knowledge of the transcriptional landscape of this epithelium will provide a basis for understanding the phenotypic changes seen in murine models of OM.

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

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)-1ß, 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.

Transcriptomic analysis of tobacco-flavored E-cigarette and menthol-flavored E-cigarette exposure in the human middle ear.

Electronic cigarettes (e-cigarettes) are the most widely used electronic nicotine delivery systems and are designed to imitate smoking and aid in smoking cessation. Although the number of e-cigarette users is increasing rapidly, especially among young adults and adolescents, the potential health impacts and biologic effects of e-cigarettes still need to be elucidated. Our previous study demonstrated the cytotoxic effects of electronic liquids (e-liquids) in a human middle ear epithelial cell (HMEEC-1) line, which were affected by the manufacturer and flavoring agents regardless of the presence of nicotine. In this study, we aimed to evaluate the gene expression profile and identify potential molecular modulator genes and pathways in HMEEC-1 exposed to two different e-liquids (tobacco- and menthol-flavored). HMEEC-1 was exposed to e-liquids, and RNA sequencing, functional analysis, and pathway analysis were conducted to identify the resultant transcriptomic changes. A total of 843 genes were differentially expressed following exposure to the tobacco-flavored e-liquid, among which 262 genes were upregulated and 581 were downregulated. Upon exposure to the menthol-flavored e-liquid, a total of 589 genes were differentially expressed, among which 228 genes were upregulated and 361 were downregulated. Among the signaling pathways associated with the differentially expressed genes mediated by tobacco-flavored e-liquid exposure, several key molecular genes were identified, including IL6 (interleukin 6), PTGS2 (prostaglandin-endoperoxide synthase 2), CXCL8 (C-X-C motif chemokine ligand 8), JUN (Jun proto-oncogene), FOS (Fos proto-oncogene), and TP53 (tumor protein 53). Under menthol-flavored e-liquid treatment, MMP9 (matrix metallopeptidase 9), PTGS2 (prostaglandin-endoperoxide synthase 2), MYC (MYC proto-oncogene, bHLH transcription factor), HMOX1 (heme oxygenase 1), NOS3 (nitric oxide synthase 3), and CAV1 (caveolin 1) were predicted as key genes. In addition, we identified related cellular processes, including inflammatory responses, oxidative stress and carcinogenesis, under exposure to tobacco- and menthol-flavored e-liquids. We identified differentially expressed genes and related cellular processes and gene signaling pathways after e-cigarette exposure in human middle ear cells. These findings may provide useful evidence for understanding the effect of e-cigarette exposure.

Cytological and microbiological characteristics of middle ear effusions in brachycephalic dogs.

BACKGROUND: Middle ear effusion is common in brachycephalic dogs with similarities to otitis media with effusion in children. Association with the cranial and eustachian tube morphology and bacterial infection is suspected in both species. HYPOTHESIS/OBJECTIVES: To determine cytological and bacteriological features of middle ear effusions in dogs, provide information on histological features, and further assess the dog as a model of the human disease. ANIMALS: Sixteen live dogs, 3 postmortem cases of middle ear effusion, and 2 postmortem controls. METHODS: Prospective; clinical investigation using computed tomography, magnetic resonance imaging, video-otoscopy, myringotomy; cytological assessment of 30 and bacteriology of 28 effusions; histology and immunohistochemistry (CD3 for T-lymphocytes, Pax5 for B lymphocytes and MAC387 for macrophages) of 10 middle ear sections. RESULTS: Effusions were associated with neurological deficits in 6/16 (38%) and concurrent atopic dermatitis and otitis externa in 9/16 (56%) of live cases. Neutrophils and macrophages predominated on cytology (median 60 [range 2%-95.5%] and 27 [2%-96.5%]) whether culture of effusions was positive or not. In histology sections, the mucosa was thickened in affected dogs but submucosal gland dilatation occurred in affected and unaffected dogs. There was no bacterial growth from 22/28 (79%) of effusions. Bacteria isolated from the other 6 (21%) were predominantly Staphylococcus pseudintermedius (4/6, 67%). CONCLUSIONS AND CLINICAL IMPORTANCE: Clinical, morphological, and cytological findings in middle ear effusions of dogs and people suggest similar pathogeneses. Middle ear effusion of dogs could be a useful model of human otitis media with effusion. Such comparisons can improve understanding and management across species.

CXCL12 is required for stirrup-shaped stapes formation during mammalian middle ear development.

BACKGROUND: The mammalian middle ear comprises a chain of three ossicles-the malleus, incus, and stapes-each of which has a unique morphology for efficiently transmitting sound information. In particular, the stapes, which is attached to the inner ear, is stirrup-shaped with a head and base connected by two crural arches, forming the stapedial foramen, through which the stapedial artery passes. However, how the stapes acquires this critical stirrup shape for association with the stapedial artery during development is not clear. RESULTS: C-X-C motif chemokine ligand 12 (CXCL12) is a chemoattractant essential for cellular movement and angiogenesis. In Cxcl12 -/- embryos, migration of neural crest cells into the prospective middle ear regions and their mesenchymal condensation to form the three ossicles proceed normally in correct alignment with each other and the inner ear. However, in the absence of CXCL12, the stapes loses its stirrup shape and instead exhibits a columnar shape lacking the crural arches and central hole. In addition, although the stapedial artery initially forms during early mesenchymal condensation of the stapes, it degenerates without CXCL12 function. CONCLUSION: CXCL12 plays an essential role in establishing the stirrup-shaped architecture of the stapes, possibly by maintaining the stapedial foramen and stapedial artery throughout development.

Comparison between in vitro toxicities of tobacco- and menthol-flavored electronic cigarette liquids on human middle ear epithelial cells.

Since electronic-cigarettes (e-cigarettes) are considered less toxic than conventional tobacco smoking, the use of e-cigarettes has increased, and the market for e-cigarette liquids (e-liquids) is continuously increasing. However, many studies showed that e-cigarettes may cause various harmful effects in lung, oral and heart. In this study, we investigated the effects of e-liquids on otitis media (OM) using human middle ear epithelial cells (HMEECs). Menthol-flavored e-liquid induced significant cell death in HMEECs (IC50: 1.45 ± 0.14%) and tobacco-flavored e-liquid led to increase in inflammatory cytokine levels and higher mucin production. Flavored e-liquids decreased the mRNA levels of genes encoding epithelial sodium channels (ENaCs) in HMEECs. Apoptosis and autophagy reactions were induced by exposure of HMEECs to menthol- and tobacco-flavored e-liquids. Tobacco-flavored e-liquids caused a greater increase in the levels of autophagosome marker, LC3-II, compared to menthol-flavored e-liquids, which was followed by cell death. These results demonstrate that flavored e-liquids cause cytotoxicity via apoptosis, autophagy, inflammatory response, and mucin production in HMEECs. The flavors present in e-liquids might be a risk factor for the development of otitis media.

Vinpocetine Suppresses Streptococcus pneumoniae-Induced Inflammation via Inhibition of ERK1 by CYLD.

Otitis media (OM) is the most common bacterial infection in children. It remains a major health problem and a substantial socioeconomic burden. Streptococcus pneumoniae (S. pneumoniae) is one of the most common bacterial pathogens causing OM. Innate inflammatory response plays a critical role in host defense against bacterial pathogens. However, if excessive, it has a detrimental impact on the middle ear, leading to middle ear inflammation, a hallmark of OM. Currently, there has been limited success in developing effective therapeutic agents to suppress inflammation without serious side effects. In this study, we show that vinpocetine, an antistroke drug, suppressed S. pneumoniae-induced inflammatory response in cultured middle ear epithelial cells as well as in the middle ear of mice. Interestingly, vinpocetine inhibited S. pneumoniae-induced inflammation via upregulating a key negative regulator cylindromatosis (CYLD). Moreover, CYLD suppressed S. pneumoniae-induced inflammation via inhibiting the activation of ERK. Importantly, the postinfection administration of vinpocetine markedly inhibited middle ear inflammation induced by S. pneumoniae in a well-established mouse OM model. These studies provide insights into the molecular mechanisms underlying the tight regulation of inflammation via inhibition of ERK by CYLD and identified vinpocetine as a potential therapeutic agent for suppressing the inflammatory response in the pathogenesis of OM via upregulating negative regulator CYLD expression.

Keratinocyte growth factor (KGF) induces stem/progenitor cell growth in middle ear mucosa.

OBJECTIVE: The middle ear epithelium is derived from the neural crest and endoderm, which line distinct regions of the middle ear cavity. In this study, we investigated the localization of stem/progenitor cells in the middle ear mucosa of adult mice and the effects of keratinocyte growth factor (KGF) on the cell kinetics of stem/progenitor cells in vivo. METHODS: In this study, after KGF-expression vector was transfected in the ear, two kinds of thymidine analogues, BrdU and EdU, were transferred at different time points. BrdU was detected by immunohistochemistry and EdU was detected by click chemistry. We also performed immunohistochemistry using anti-Keratin14 (K14) antibody (an undifferentiated epithelial cell marker), anti-p63 antibody (a stem/progenitor cell marker) and anti-acetylated α-tubulin antibody (a ciliated epithelial cell marker). RESULTS: A large number of EdU-positive cells were detected in the thickened mucosal epithelium of the pars flaccida and attic region at Day 1 after KGF transfection. Interestingly, in the mucosal epithelium overlying the promontory of the cochlea, many EdU-positive cells were detected. These cells were also positive for K14 and p63. The acetylated α-tubulin positive cells were reduced in the attic region at Day 1 after KGF transfection. CONCLUSION: These findings indicate that KGF over-expression may increase stem/progenitor cell proliferation in the mucosal epithelium not only within the attic which is typical in middle ear cholesteatoma, but also overlying the promontory of the cochlea.

Single origin of the epithelium of the human middle ear.

OBJECTIVE: The epithelium lining the human middle ear and adjacent temporal bone cavity shows a varying morphological appearance throughout these cavities. Its embryologic origin has long been debated and recently got attention in a newly proposed theory of a dual embryologic origin. The epithelial morphology and its differentiating capabilities are of significance in future mucosa-targeted therapeutic agents and could affect surgical approaches of the temporal bone. This study aims to analyze reported murine histological findings that led to the theory of a dual epithelial embryological origin and immunohistochemically investigate whether such an epithelial embryological origin in the human fetal middle ear could be true. METHODS: By combining a sagittal sectioning technique and immuno-histochemical staining, a comprehensive immuno-histological overview of the fetal human middle ear during a critical stage of tympanic cavitation was provided. A critical analysis of previously reported findings leading to the theory of a dual epithelial embryological origin and a comparison of these findings to the findings in the human fetal middle ear was performed. RESULTS: The reported findings and critical analysis provide multiple arguments for an entirely endodermal embryonic origin of the epithelium lining the tympanic cavity. CONCLUSION: Different morphological epithelial appearances throughout the tympanic and temporal bone cavities could be explained by different stages of epithelial differentiation rather than different embryologic origin and endodermal rupture does not seem to be a necessity for these cavities to form.

Isolation and Culture of Primary Mouse Middle Ear Epithelial Cells.

Epithelial abnormalities underpin the development of the middle ear disease, otitis media (OM). Until now, a well-characterized in vitro model of the middle ear (ME) epithelium that replicates the complex cellular composition of the middle ear has not been available. This chapter describes the development of a novel in vitro model of mouse middle ear epithelial cells (mMECs), cultured at the air-liquid interface (ALI). This system enables recapitulation of the characteristics of the native murine ME epithelium. We demonstrate that mMECs undergo differentiation into the varied cell populations seen within the native middle ear. Overall, our mMEC culture system can help better understand the cell biology of the middle ear and improve our understanding of the pathophysiology of OM. The model also has the potential to serve as a platform for validation of treatments designed to reverse aspects of epithelial remodeling underpinning OM development.

Effect of electronic cigarettes on human middle ear.

OBJECTIVE: Electronic cigarettes (e-cigarettes) are the most commonly used electronic nicotine delivery systems and are a relatively new product designed for smoking cessation. The market scale of electronic cigarettes is growing rapidly, but the potential impact of e-cigarettes on public health has not yet been verified. In this study, we examined the effect of e-liquids on a human middle ear epithelial cell (HMEEC) line. MATERIAL AND METHODS: The main components of e-liquids are propylene glycol, vegetable glycerin and flavoring agents with or without nicotine. We analyzed 73 bottles of e-liquids from 12 different manufacturers, evaluated the trace elements in e-liquids, and identified the cytotoxicity of e-liquids on HMEECs in the presence or absence of nicotine. RESULTS: In the trace elements analysis, nickel, arsenic, cadmium, and lead were detected in the e-liquids. E-liquids without nicotine decreased cell viability, and the average IC 50 value of total e-liquids (n = 73) was 2.48 ±â€¯0.93%. Among the different flavors, menthol-flavored e-liquids significantly reduced cell viability, and their average IC 50 value (n = 28) was 1.85 ±â€¯0.80%. The average IC 50 values were distinct among manufacturers and the proportion of the solvents. CONCLUSION: The present study provides evidence that e-cigarettes influence and reduce human middle ear cell viability even without the application of nicotine. Additionally, the cytotoxicity of e-liquids was affected by the flavoring agents.

Stem cells in middle ear cholesteatoma contribute to its pathogenesis.

Cholesteatoma is a potentially life-threatening middle ear lesion due to the formation of an inflamed ectopic mass of keratinizing squamous epithelium. Surgical removal remains the only treatment option, emphasizing the need to gain a better understanding of this severe disease. We show for the first time that stem cells residing in cholesteatoma tissue contribute to disease progression. Cells expressing the "stemness" markers Nestin and S100B were detected in middle ear cholesteatoma and auditory canal skin. Isolated Nestin + /S100B + -cells showed the capability for self-renewal, neurosphere formation and differentiation into mesodermal and ectodermal cell types. Compared to auditory canal skin stem cells middle ear cholesteatoma-derived stem cells displayed an enhanced susceptibility to inflammatory stimuli, and this suggested a possible contribution to the inflammatory environment in cholesteatoma tissue. Cholesteatoma derived stem cells were able to differentiate into keratinocyte-like cells using factors mimicking the microenvironment of cholesteatoma. Our findings demonstrate a new perspective on the pathogenesis of cholesteatoma and may lead to new treatment strategies for this severe middle ear lesion.

Evidence against the mucosal traction theory in cholesteatoma.

OBJECTIVES: To investigate the distribution of ciliated epithelium in the human middle ear and its potential role in the formation of cholesteatoma. STUDY DESIGN: Comparative human temporal bone study. METHODS: We selected temporal bones from 14 donors with a diagnosis of cholesteatoma, 15 with chronic otitis media without retraction pockets, 14 with chronic otitis media with retraction pockets, 14 with cystic fibrosis (CF), and 16 controls. We mapped the distribution of the ciliated cells in the mucosal lining of the middle ear and tympanic membrane using three-dimensional reconstruction analysis, and counted the number of ciliated cells in the middle ear mucosa. RESULTS: Ciliated cells are extremely sparse in the epithelial lining of the lateral surface of the ossicles in the epitympanum and the medial surface of the tympanic membrane. Furthermore, there is a significant decrease in the number of ciliated cells in these areas in temporal bones with cholesteatoma, chronic otitis media, chronic otitis media with retraction pockets, and CF compared to controls. Ciliated cells most commonly are located at the hypotympanum and the Eustachian tube opening but not the tympanic membrane or epitympanum. CONCLUSION: The paucity of ciliated epithelial cells on the medial side of the tympanic membrane and the lateral surface of the ossicles in the epitympanum in cases with cholesteatoma and/or chronic otitis media do not support the mucosal migration theory of cholesteatoma formation. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:1663-1667, 2018.

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

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.

Temporal and spatial expression patterns of Hedgehog receptors in the developing inner and middle ear.

The mammalian inner ear is a complex organ responsible for balance and hearing. Sonic hedgehog (Shh), a member of the Hedgehog (Hh) family of secreted proteins, has been shown to play important roles in several aspects of inner ear development, including dorsoventral axial specification, cochlear elongation, tonotopic patterning, and hair cell differentiation. Hh proteins initiate a downstream signaling cascade by binding to the Patched 1 (Ptch1) receptor. Recent studies have revealed that other types of co-receptors can also mediate Hh signaling, including growth arrest-specific 1 (Gas1), cell-adhesion molecules-related/down-regulated by oncogenes (Cdon), and biregional Cdon binding protein (Boc). However, little is known about the role of these Hh co-receptors in inner ear development. In this study, we examined the expression patterns of Gas1, Cdon, and Boc, as well as that of Ptch1, in the developing mouse inner ear from otocyst (embryonic day (E) 9.5) until birth and in the developing middle ear at E15.5. Ptch1, a readout of Hh signaling, was expressed in a graded pattern in response to Shh signaling throughout development. Expression patterns of Gas1, Cdon, and Boc differed from that of Ptch1, and each Hh co-receptor was expressed in specific cells and domains in the developing inner and middle ear. These unique and differential expression patterns of Hh co-receptors suggest their roles in mediating various time- and space-specific functions of Shh during ear development.

Cilia distribution and polarity in the epithelial lining of the mouse middle ear cavity.

The middle ear conducts sound to the cochlea for hearing. Otitis media (OM) is the most common illness in childhood. Moreover, chronic OM with effusion (COME) is the leading cause of conductive hearing loss. Clinically, COME is highly associated with Primary Ciliary Dyskinesia, implicating significant contributions of cilia dysfunction to COME. The understanding of middle ear cilia properties that are critical to OM susceptibility, however, is limited. Here, we confirmed the presence of a ciliated region near the Eustachian tube orifice at the ventral region of the middle ear cavity, consisting mostly of a lumen layer of multi-ciliated and a layer of Keratin-5-positive basal cells. We also found that the motile cilia are polarized coordinately and display a planar cell polarity. Surprisingly, we also found a region of multi-ciliated cells that line the posterior dorsal pole of the middle ear cavity which was previously thought to contain only non-ciliated cells. Our study provided a more complete understanding of cilia distribution and revealed for the first time coordinated polarity of cilia in the epithelium of the mammalian middle ear, thus illustrating novel structural features that are likely critical for middle ear functions and related to OM susceptibility.

Morphology and Ciliary Motion of the Middle Ear Mucosa of Neonatal and Adult Gerbils.

BACKGROUND/AIMS: Since the middle ear cavity plays an important role in middle ear effusion, the goal of this study is to examine the morphological and functional development of the middle ear mucosa in neonatal and adult gerbils. METHODS: Gerbils aged between postnatal day 1 (P1) and P26 were used for the experiments. The morphological development of the middle ear cavity was measured by using the celloidin embedding method and scanning electronic microscope. Ciliary motion was determined and calibrated by a photodiode-based measurement system. RESULTS: The area of the middle ear increased significantly from 0.42 ± 0.02 mm2 (n = 8) at P1 to 2.6 ± 0.03 mm2 (n = 8) at P26. At P1, all regions were covered by densely populated ciliated cells. A significant reduction of the density of ciliated cells was found between P8 and P18 (p < 0.05). The beat frequency at P1 is not statistically different from that of P8 (p > 0.05). However, the beat frequency increased significantly between P8 and P18 (p < 0.05). CONCLUSION: Middle ear mucosa underwent a significant change between P1 and P18. The cilia morphology and function (ciliary motion) are fully mature at P18. This may explain the high incidence of middle ear effusion in the neonatal period in humans.

The suppressive effects of metformin on inflammatory response of otitis media model in human middle ear epithelial cells.

OBJECTIVE: Metformin is a well-known anti-diabetic agent, but its mechanism is unclear. Recently, many reports have described the anti-inflammatory effects of metformin on various cell types, including human vascular smooth muscle cells and endothelial cells. This study was designed to investigate the anti-inflammatory effect of metformin on lipopolysaccharide (LPS) induced inflammation in human middle ear epithelial cell lines (HMEECs). METHODS: The effect of pretreatment by metformin (0, 1, 2, 4 mM) was evaluated by the inflammatory response in the HMEECs exposed to LPS (10 ng/ml). For verifying the suppression effect of metformin on the inflammatory cytokines, tumor necrosis factor-alpha (TNF-α) was evaluated by real-time polymerase chain reaction, and COX-2 protein was assessed by western blotting. Intracellular reactive oxygen species (ROS) was measured using 2', 7'-dichlorofluorescein diacetate (DCFHDA) fluorocytometer. RESULTS: Stimulation by LPS 10 ng/ml concentration showed 12.4 folds increase the expression of TNF-α mRNA compared to control on HMEECs. Pretreatment of metformin dose dependently suppressed the expression of TNF-α mRNA induced by LPS (2 mM, p = 0.03). The amount of COX-2 protein production was significantly decreased by metformin pretreatment (4 mM, p = 0.01). The production of ROS was decreased significantly by pretreatment of metformin (p = 0.03). CONCLUSIONS: These findings suggest that the inflammatory response and oxidative stress induced by LPS could be suppressed by metformin in HMEECs. Therefore, metformin may have a therapeutic potential for the treatment of the otitis media.

Association of microRNA 146 with middle ear hyperplasia in pediatric otitis media.

OBJECTIVE: Toll-like receptor signaling activated by bacterial otitis media pathogens in the middle ear has been shown to play a key role in OM susceptibility, pathogenesis and recovery. Recent studies implicate microRNA 146 (miR-146) in regulation of inflammation via negative feedback of toll-like receptor signaling (TLR) in a wide variety of tissues, however its involvement in otitis media is unknown. METHODS: Human middle ear epithelial cells were stimulated with proinflammatory cytokines, interleukin 1 beta or tumor necrosis factor alpha, for two to twenty-four hours. Middle ear biopsies were collected from children with otitis media with effusion (n = 20), recurrent otitis media (n = 9), and control subjects undergoing cochlear implantation (n = 10). miR-146a, miR-146b expression was assayed by quantitative PCR (qPCR). Expression of miR-146 targets involved in TLR signaling, IRAK1 and TRAF6, was assayed by qPCR in middle ear biopsies. Middle ear biopsies were cryosectioned and epithelial thickness measured by a certified pathologist. RESULTS: Proinflammatory cytokines induced expression of miR-146 in middle ear epithelial cells in vitro. Middle ear miR-146a and miR-146b expression was elevated in otitis media patients relative to control subjects and correlated with middle ear epithelial thickness. A trend towards inverse correlation was observed between miR-146 and TRAF6 expression in the clinical population. CONCLUSIONS: This report is the first to assess miRNA expression in a clinical population with OM. Findings herein suggest miR-146 may play a role in OM.

Middle Ear Response of Muc5ac and Muc5b Mucins to Nontypeable Haemophilus influenzae.

IMPORTANCE: Chronic otitis media with effusion is characterized by middle ear secretion of mucin glycoproteins, predominantly MUC5B; MUC5AC, the other secretory mucin studied frequently, has also been identified in the middle ear. Emerging evidence suggests a dichotomous role for these mucins in innate immune responses. We hypothesized that MUC5AC is an acute responder and MUC5B is expressed at later time points, reflecting a chronic situation. OBJECTIVE: To determine middle ear regulation of MUC5B and MUC5AC following in vitro bacterial and cytokine exposure. DESIGN, SETTING, AND SAMPLES: An in vitro cell-based model of mucin gene regulation was conducted in a basic science laboratory at a tertiary pediatric hospital. The study was conducted from July 1, 2014, to June 30, 2015; data analysis was performed in July 2015. INTERVENTIONS: Nontypeable Haemophilus influenzae (NTHi) lysates were generated and used to stimulate mouse middle ear epithelial cells (mMEECs) for 2 hours during 3 weeks. MAIN OUTCOMES AND MEASURES: Real-time quantitative polymerase chain reaction, luciferase assays, Western blot assay, and immunofluorescence techniques were performed to determine Muc5ac and Muc5b expression over time, Cxcl2 chemokine response, and nuclear factor-κB activation. Luciferase reporter assays were performed to evaluate specific promoter responses after NTHi exposure. RESULTS: Nontypeable H influenzae lysates (200 µg/mL) drove differential mucin gene activation, with Muc5ac being induced up to 2.04 fold at 24 hours and 2.79 fold at 96 hours (P < .05) and Muc5b being induced only at more long-term points: 1.61 fold at 96 hours, 1.41 fold at 1 week, and 1.53 fold at 3 weeks (P < .05). Although NTHi lysates induced robust, early nuclear factor-κB nuclear translocation with nuclear factor-κB-dependent induction of Cxlc2 expression, the lysates had minimal to no effect on Muc5ac and Muc5b promoter activity. However, in contrast to NTHi lysates, CXCL2 induced significant transcription of both Muc5b and Muc5ac as early as 24 hours. CONCLUSIONS AND RELEVANCE: Nontypeable H influenzae lysates activate differential mucin gene activation in mMEECs. Although Muc5ac is an early response mucin gene, Muc5b appears to react as a chronic response mucin.