Otitis media (OM) is a common disease in young children, accounting for more office visits and surgeries than any other pediatric condition. It is associated with an estimated cost of five billion dollars annually in the USA. Moreover, chronic and recurrent middle ear (ME) disease leads to hearing loss during critical periods of language acquisition and learning leading to delays in reaching developmental milestones and risking permanent damage to the ME and inner ear in severe cases. Therefore, research to understand the disease pathogenesis and identify new therapeutics is important. Although OM is a multifactorial disease, targeting the molecular mechanisms that drive inflammation and OM resolution is critical. In this review, we discuss the current evidence suggesting that innate immune receptors and effectors play key roles in OM by mediating both the ME inflammatory responses and recovery.

BACKGROUND: A number of drugs are toxic to the cochlear sensory cells known as hair cells (HCs), resulting in hearing loss. Treatment with survival-promoting growth factors, antioxidants, and inhibitors of cell death pathways or proteinases have been shown to reduce HC damage in in vivo and/or in vitro animal models. Conversely, translation to humans has often been disappointing. This may be due to the complexity of intracellular damage processes. We hypothesized that combining treatments targeting different cellular processes would be more effective. METHODS: Using an in vitro model of gentamicin ototoxicity for murine cochlear hair cells, we screened all 56 possible combinations of inhibitors targeting five different cell damage mechanisms, plus the activator of one cell survival pathway, each of which have been shown to be singly effective in preventing HC loss in experimental studies. A high dose of gentamicin (200 μM) was used over three days in culture. All compounds were added at a dosage below that required for significant protection in the assay, and only this single dose was then employed. This was done so that we could more easily detect interactive, as opposed to additive, effects. RESULTS: Increasing protection of hair cells was observed as combinations of compounds were increased from two to four factors, although not all combinations were equally protective. The optimal combination of four compounds consisted of an anti-oxidant, an apoptosis inhibitor, an autophagy inhibitor and a protective growth factor. Increasing the number of factors to five or six resulted in decreased protection. CONCLUSION: The results support the hypothesis that targeting multiple cellular damage or survival pathways provides more an effective hair cell protection approach. The results help to identify critical interactions among the cellular processes that operate in gentamicin ototoxicity. They also suggest that inhibiting too many biological processes impairs functions critical to HC survival, resulting in decreased protection.

Viral infections have already been implicated with otitis media and sudden sensorineural hearing loss. However, the pathophysiology of COVID-19 as it relates to otologic disorders is not well-defined. With the spread of SARS-CoV-2, it is important to evaluate its colonization of middle ear mucosa. Middle ear and nasal tissue samples for quantitative RT-PCR and histologic evaluations were obtained from post-mortem COVID-19 patients and non-diseased control patients. Here we present evidence that SARS-CoV-2 colonizes the middle ear epithelium and co-localizes with the primary viral receptor, angiotensin-converting enzyme 2 (ACE2). Both middle ear and nasal epithelial cells show relatively high expression of ACE2, required for SARS-CoV-2 entry. The epithelial cell adhesion molecule (EpCAM) was use as a biomarker of epithelia. Furthermore, we found that the viral load in the middle ear is lower than that present in the nasal cavity.

Otitis media (OM) is a common pediatric disease for which systemic antibiotics are often prescribed. While local treatment would avoid the systemic treatment side-effects, the tympanic membrane (TM) represents an impenetrable barrier unless surgically breached. We hypothesized that the TM might harbor innate biological mechanisms that could mediate trans-TM transport. We used two M13-bacteriophage display biopanning strategies to search for mediators of trans-TM transport. First, aliquots of linear phage library displaying 10(10th) 12mer peptides were applied on the TM of rats with active bacterial OM. The middle ear (ME) contents were then harvested, amplified and the preparation re-applied for additional rounds. Second, the same naïve library was sequentially screened for phage exhibiting TM binding, internalization and then transit. Results revealed a novel set of peptides that transit across the TM to the ME in a time and temperature dependent manner. The peptides with highest transport capacities shared sequence similarities. Historically, the TM was viewed as an impermeable barrier. However, our studies reveal that it is possible to translocate peptide-linked small particles across the TM. This is the first comprehensive biopanning for the isolation of TM transiting peptidic ligands. The identified mechanism offers a new drug delivery platform into the ME.

A major aspect of pathology in otitis media (OM), the most common childhood bacterial disease, is hyperplasia of the middle ear mucosa. Activation of innate immune receptors during OM leads to the activation of NF-κB, a pleiotropic transcription factor involved both in inflammation and tissue growth. To explore the role of NF-κB in mucosal hyperplasia during OM, we evaluated the expression of genes involved in two modes of NF-κB activation during a complete episode of acute, bacterial OM in mice. We also determined the effects of inhibitors of each pathway on infection-stimulated mucosal growth in vitro. A majority of the genes that mediate both the canonical and the non-canonical pathways of NF-κB activation were regulated during OM, many with kinetics related to the time course of mucosal hyperplasia. Inhibition of either pathway reduced the growth of cultured mucosal explants in a dose-dependent manner. However, inhibition of the canonical pathway produced a greater effect, suggesting that this mode of NF-κB activation dominates mucosal hyperplasia during OM.

Pathogen recognition following infection in mammals depends mainly on TLRs and NLRs. Herein, we evaluate the role of NOD1 and NOD2 signaling in the inflammatory responses of the middle ear (ME) mucosa and leukocytes recruitment to infection site during otitis media (OM). OM is a common pediatric disease with prevalent repercussions on hearing health. While many risk factors have been implicated to OM proneness, immunity and the triggering of inflammation are central to OM pathology. We observed that many genes encoding members of the NOD leucine-rich repeat and their downstream adaptor/effector molecules were strongly regulated during the course of OM. When compared to wild type C57BL/6 mice, NOD1- and NOD2-deficient mice were susceptible to prolonged OM infection by non-typeable Haemophilus influenza. NOD1-deficient mice appeared to have reduced macrophage enlistment with a delayed inflammatory response by neutrophils and prolonged mucosal hyperplasia, whereas NOD2 knockouts exhibited an overall reduction in the number of leukocytes recruited to the ME, leading to delayed bacterial clearance. Altogether, these data show that the NODs play a role in the pathogenesis and recovery of OM and reinforce the importance of innate immune signaling in the protective host response.

Single-cell transcriptomics was used to profile cells of the normal murine middle ear. Clustering analysis of 6770 transcriptomes identified 17 cell clusters corresponding to distinct cell types: five epithelial, three stromal, three lymphocyte, two monocyte, two endothelial, one pericyte and one melanocyte cluster. Within some clusters, cell subtypes were identified. While many corresponded to those cell types known from prior studies, several novel types or subtypes were noted. The results indicate unexpected cellular diversity within the resting middle ear mucosa. The resolution of uncomplicated, acute, otitis media is too rapid for cognate immunity to play a major role. Thus innate immunity is likely responsible for normal recovery from middle ear infection. The need for rapid response to pathogens suggests that innate immune genes may be constitutively expressed by middle ear cells. We therefore assessed expression of innate immune genes across all cell types, to evaluate potential for rapid responses to middle ear infection. Resident monocytes/macrophages expressed the most such genes, including pathogen receptors, cytokines, chemokines and chemokine receptors. Other cell types displayed distinct innate immune gene profiles. Epithelial cells preferentially expressed pathogen receptors, bactericidal peptides and mucins. Stromal and endothelial cells expressed pathogen receptors. Pericytes expressed pro-inflammatory cytokines. Lymphocytes expressed chemokine receptors and antimicrobials. The results suggest that tissue monocytes, including macrophages, are the master regulators of the immediate middle ear response to infection, but that virtually all cell types act in concert to mount a defense against pathogens.

Objective and design

The human c2orf40 gene encodes a tumor suppressor gene called esophageal cancer-related gene-4 (ECRG4) with pro- and anti-inflammatory activities that depend on cell surface processing. Here, we investigated its physical and functional association with the innate immunity receptor complex.

Methods

Interactions between ECRG4 and the innate immunity receptor complex were assessed by flow cytometry, immunohistochemistry, confocal microscopy, and co-immunoprecipitation. Phage display was used for ligand targeting to cells that overexpress the TLR4-MD2-CD14.

Results

Immunoprecipitation and immunohistochemical studies demonstrate a physical interaction between ECRG4 and TLR4-MD2-CD14 on human granulocytes. Flow cytometry shows ECRG4 on the cell surface of a subset of CD14(+) and CD16(+) leukocytes. In a cohort of trauma patients, the C-terminal 16 amino acid domain of ECRG4 (ECRG4(133-148)) appears to be processed and shed, presumably at a thrombin-like consensus sequence. Phage targeting this putative ligand shows that this peptide sequence internalizes into cells through the TLR4/CD14/MD2 complex, but modulates inflammation through non-canonical, NFκB signal transduction.

Conclusions

ECRG4 is present on the surface of human monocytes and granulocytes. Its interaction with the human innate immunity receptor complex supports a role for cell surface activation of ECRG4 during inflammation and implicates this receptor in its mechanism of action.

Introduction

Patients with otitis media (OM) encounter significant functional hearing impairment with conductive, or a combined hearing loss and long-term sequelae involving impaired speech/language development in children, reduced academic achievement and irreversible disorders of middle and inner ear requiring a long time therapy and/or multiple surgeries. In its persistent chronic form, Otitis media (COM) can often only be treated by undergoing ear surgery for hearing restoration. The persistent inflammatory reaction plays a major role, often caused by multi-resistant pathogens in the ear. Herein, we present outcomes of patients implanted with currently the only FDA approved active Middle Ear Implant Vibrant Soundbridge (VSB), suffering from persistent COM.

Methods

The study enrolled 42 patients, treated by performing middle ear (ME) surgery to different extents and implanted with the VSB to various structures in the ME. Included were 17 children and 25 adults that had recurrent and/or persisting OM and significant hearing loss. Preoperative and postoperative patients' audiometric data were evaluated and the benefit with VSB assessed using the Glasgow Benefit Inventory for adults and pediatric cohorts. The microbial spectrum of pathogens was assessed before and after surgery, exploring the colonization of the otopathogens, as well as the intestinal microbiome from individually burdened patients.

Results

The mean functional gain is 29.7 dB HL (range from 10 to 56.2 dB HL) with a significant improvement in speech intelligibility in quiet. Following VSB implantation, no significant differences in coupling were observed at low complication rates. Postoperatively patients showed significantly increased benefit with VSB compared to the untreated situation, including less otorrhea, pain, medical visits, and medication intake, with no recurrent OM and significant bacterial shift in otopathogens. The analysis of the intestinal microbiome displayed a high abundance of bacterial strains that might be linked to chronic and persistent inflammation.

Conclusions

Functional ear surgery including rehabilitation with a VSB in patients suffering from COM present to be safe and effective. The successful acceptance accompanied by the improved audiological performance resulted in significant benefit with VSB, with a shift in the ear pathogens and altered microbiome and thus is a great opportunity to be treated.

In humans, esophageal cancer-related gene 4 (ECRG4) is encoded by four exons in the c2orf40 locus of chromosome 2. Translation of ECRG4 messenger ribonucleic acid produces a 148 amino acid-secreted 17 KDa protein that is then processed to 14, ten, eight, six, four, and two KDa peptides, depending on the cell in which the gene is expressed. As hypermethylation at the c2orf40 locus inhibits ECRG4 gene expression in many epithelial cancers, several investigators have speculated that ECRG4 is a candidate tumor suppressor. Indeed, overexpression of ECRG4 inhibits cell proliferation in vitro, but it also has a wide range of effects in vivo beyond its antitumor activity. ECRG4 overexpression affects apoptosis, senescence, cell migration, inflammation, injury, and infection responsiveness. ECRG4 activities also depend on its cellular localization, secretion, and post-translational processing. These cytokine/chemokine-like characteristics argue that ECRG4 is not a traditional candidate tumor suppressor gene, as originally predicted by its downregulation in cancer. We review how insights into the regulation of ECRG4 gene expression, knowledge of its primary structure, and the study of its emerging physiological functions come together to support a much more complex role for ECRG4 at the interface of inflammation, infection, and malignancy.