Hearing rehabilitation and microbial shift after middle ear surgery with Vibrant Soundbridge in patients with chronic otitis media.

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.

[Immunomodulation in Cholesteatoma]. / Immunmodulation im Cholesteatom.

INTRODUCTION: The etiopathogenesis of chronic otitis media epitympanalis/cholesteatoma and its proliferative destructive course with possible complications such as destruction of bony structures with hearing loss, vestibular dysfunction, facial nerve paralysis and intracranial complications are still unexplained. Surgery is still the way to go. New studies are increasingly looking at the innate immune system. METHODS: Our studies were carried out in a mouse model in WT mice and immundeficient KO-mice, as well as in cholesteatoma and healthy ear canal skin and middle ear tissue, which was removed during ear surgery. The expression analyses were carried out at the gene and protein level using TNF as the major target for therapy evaluation. By means of TUNEL staining and immunohistochemistry the level of apoptosis was evaluated. RESULTS: The uncontrolled undirected cholesteatoma growth shows an immunomodulatory profile with up and down-regulation of various gene networks, especially those involved in TNF downstream and upstream signaling pathways. TNF in cholesteatoma is modulated both inflammatorily and apoptotically and therefore is suitable as a possible therapeutic approach in various models. CONCLUSIONS: Cholesteatoma might be immunomodulatory regulated.

Macrophage Depletion in CCR2-/- Mice Delays Bacterial Clearance and Enhances Neutrophil Infiltration in an Acute Otitis Media Model.

BACKGROUND: Otitis media (OM) is a common and potentially serious disease of childhood. Although OM is multifactorial on origin, bacterial infection is a unifying component. Many studies have established a critical role for innate immunity in bacterial clearance and OM resolution. A key component of innate immunity is the recruitment of immune and inflammatory cells, including macrophages. METHODS: To explore the role of macrophages in OM, we evaluated the expression of genes related to macrophage function during a complete episode of acute OM in the mouse caused by middle ear (ME) inoculation with Haemophilus influenzae. We also combined CCR2 deficiency with chlodronate liposome toxicity to deplete macrophages during OM. RESULTS: Macrophage genes were robustly regulated during OM. Moreover, macrophage depletion enhanced and prolonged the infiltration of neutrophils into the infected ME and increased the persistence of bacterial infection. CONCLUSIONS: The results illustrate the critical role played by macrophages in OM resolution.

Role of the PI3K/AKT pathway and PTEN in otitis media.

Mucosal hyperplasia is common sequela of otitis media (OM), leading to the secretion of mucus and the recruitment of leukocytes. However, the pathogenic mechanisms underlying hyperplasia are not well defined. Here, we investigated the role of the AKT pathway in the development of middle mucosal hyperplasia using in vitro mucosal explants cultures and an in vivo rat model. The Akt inhibitor MK2206 treatment inhibited the growth of middle ear mucosal explants in a dose-dependent manner. In vivo, MK2206 also reduced mucosal hyperplasia. Unexpectedly, while PTEN is generally thought to act in opposition to AKT, the PTEN inhibitor BPV reduced mucosal explant growth in vitro. The results indicate that both AKT and PTEN are mediators of mucosal growth during OM, and could be potential therapeutic targets.

Lack of the hyaluronan receptor CD44 affects the course of bacterial otitis media and reduces leukocyte recruitment to the middle ear.

BACKGROUND: CD44 is a multifunctional molecule that plays major roles in both leukocyte recruitment and tissue proliferation. Since mucosal hyperplasia and leukocyte infiltration of the middle ear cavity are major features of otitis media, we evaluated the role of CD44 in the pathophysiology and course of this disease in a mouse model of middle ear infection. Expression of genes related to CD44 function were evaluated using gene arrays in wild-type mice. The middle ears of mice deficient in CD44 were inoculated with non-typeable Haemophilus influenzae. Histopathology and bacterial clearance were compared to that seen in wild-type controls. RESULTS: We observed strong up-regulation of CD44 and of genes related to its role in leukocyte extravasation into the middle ear, during the course of acute otitis media. Mice deficient in CD44 exhibited reduced early mucosal hyperplasia and leukocyte recruitment, followed by delayed resolution of infection and persistent inflammation. CONCLUSIONS: CD44 plays an important role in OM pathogenesis by altering the mucosal growth and neutrophil enlistment. Targeted therapies based on CD44 could be useful adjuncts to the treatment of middle ear infections.

Inflammation associated with noise-induced hearing loss.

Inflammation is a complex biological response to harmful stimuli including infection, tissue damage, and toxins. Thus, it is not surprising that cochlear damage by noise includes an inflammatory component. One mechanism by which inflammation is generated by tissue damage is the activation of damage-associated molecular patterns (DAMPs). Many of the cellular receptors for DAMPS, including Toll-like receptors, NOD-like receptors, and DNA receptors, are also receptors for pathogens, and function in the innate immune system. DAMP receptors are known to be expressed by cochlear cells, and binding of molecules released by damaged cells to these receptors result in the activation of cell stress pathways. This leads to the generation of pro-inflammatory cytokines and chemokines that recruit pro-inflammatory leukocytes. Extensive evidence indicates pro-inflammatory cytokines including TNF alpha and interleukin 1 beta, and chemokines including CCL2, are induced in the cochlea after noise exposure. The recruitment of macrophages into the cochlea has also been demonstrated. These provide substrates for noise damage to be enhanced by inflammation. Evidence is provided by the effectiveness of anti-inflammatory drugs in ameliorating noise-induced hearing loss. Involvement of inflammation provides a wide variety of additional anti-inflammatory and pro-resolution agents as potential pharmacological interventions in noise-induced hearing loss.

Otitis Media and Nasopharyngeal Colonization in ccl3-/- Mice.

We previously found CC chemokine ligand 3 (CCL3) to be a potent effector of inflammation during otitis media (OM): exogenous CCL3 rescues the OM phenotype of tumor necrosis factor-deficient mice and the function of macrophages deficient in several innate immune molecules. To further delineate the role of CCL3 in OM, we evaluated middle ear (ME) responses of ccl3-/-mice to nontypeable Haemophilus influenzae (NTHi). CCL chemokine gene expression was evaluated in wild-type (WT) mice during the complete course of acute OM. OM was induced in ccl3-/- and WT mice, and infection and inflammation were monitored for 21 days. Phagocytosis and killing of NTHi by macrophages were evaluated by an in vitro assay. The nasopharyngeal bacterial load was assessed in naive animals of both strains. Many CCL genes showed increased expression levels during acute OM, with CCL3 being the most upregulated, at levels 600-fold higher than the baseline. ccl3-/- deletion compromised ME bacterial clearance and prolonged mucosal hyperplasia. ME recruitment of leukocytes was delayed but persisted far longer than in WT mice. These events were linked to a decrease in the macrophage capacity for NTHi phagocytosis and increased nasopharyngeal bacterial loads in ccl3-/- mice. The generalized impairment in inflammatory cell recruitment was associated with compensatory changes in the expression profiles of CCL2, CCL7, and CCL12. CCL3 plays a significant role in the clearance of infection and resolution of inflammation and contributes to mucosal host defense of the nasopharyngeal niche, a reservoir for ME and upper respiratory infections. Therapies based on CCL3 could prove useful in treating or preventing persistent disease.

Innate Immunity: Orchestrating Inflammation and Resolution of Otitis Media.

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.

Esophageal cancer-related gene-4 (ECRG4) interactions with the innate immunity receptor complex.

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.

Combinatorial protection of cochlear hair cells: not too little but not too much.

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.

Anti-inflammatory response to 1,8-Cineol and associated microbial communities in Otitis media patients.

Chronic Otitis Media (COM) is defined as long term inflammation and colonization with pathogenic bacteria due to a defect or retraction of the tympanic membrane. Surgical interventions are often augmented by antibiotic resistance development and therefore, off-label treatment using the natural drug 1,8-Cineol was carried out. All COM patients underwent antibiotic therapy and middle ear surgery and developed antibiotic resistances. Microbiological investigations from the auditory canal and stool samples were performed in correlation with the clinical course. Therapy of COM patients with 1,8-Cineol revealed a clear reduction of inflammatory microbes P. aeruginosa and Proteus mirabilis in ear samples as well as intestinal Prevotella copri, which was associated with an improved clinical outcome in certain individuals. The present off-label study revealed manifold anti-inflammatory effects of the natural monoterpene 1,8-Cineol in Otitis media patients. A better understanding of the underlying mechanisms will improve the current treatment options and possible forms of application of this natural drug.

PANEL 3: Otitis media animal models, cell culture, tissue regeneration & pathophysiology.

OBJECTIVE: To review and summarize recently published key articles on the topics of animal models, cell culture studies, tissue biomedical engineering and regeneration, and new models in relation to otitis media (OM). DATA SOURCE: Electronic databases: PubMed, National Library of Medicine, Ovid Medline. REVIEW METHODS: Key topics were assigned to the panel participants for identification and detailed evaluation. The PubMed reviews were focused on the period from June 2019 to June 2023, in any of the objective subject(s) or keywords listed above, noting the relevant references relating to these advances with a global overview and noting areas of recommendation(s). The final manuscript was prepared with input from all panel members. CONCLUSIONS: In conclusion, ex vivo and in vivo OM research models have seen great advancements in the past 4 years. From the usage of novel genetic and molecular tools to the refinement of in vivo inducible and spontaneous mouse models, to the introduction of a wide array of reliable middle ear epithelium (MEE) cell culture systems, the next five years are likely to experience exponential growth in OM pathophysiology discoveries. Moreover, advances in these systems will predictably facilitate rapid means for novel molecular therapeutic studies.

Immunomodulatory Response of the Middle Ear Epithelial Cells in Otitis Media.

HYPOTHESIS: The middle ear (ME) epithelium transforms because of changed immunomodulation during infection. INTRODUCTION: The epithelial cells of the tympanic cavity represent the first line of defense in the context of otitis media. They can convert from a typical mucosal site into a respiratory epithelium and vice versa. Our goal is to depict the specific immune response of epithelial cells after infection at the molecular level. METHODS: The investigations were carried out on healthy and inflamed ME tissue, removed during surgical interventions in mouse and human models, and in a human in-vitro cell model in human ME epithelial cell line. We determined the epithelial localization of the protein expression of Toll- and NOD-like immune receptors and their associated signaling molecules using immunohistochemistry. In addition, we examined growth behavior and gene expression due to direct stimulation and inhibition. RESULTS: We found clinically and immunobiologically confirmed transformation of the inflamed ME epithelium depending on their origin, as well as differences in the distribution of Toll-like receptors and nucleotide-binding oligomerization domain-like receptors in the epithelial cell lining. Dysregulated gene and protein expression of the inflammatory and apoptotic genes could be modulated by stimulation and inhibition in the epithelial cells. CONCLUSIONS: The local ME mucosal tissue is believed to modulate downstream immune activity after pathogen invasion via intrinsic cellular mechanism. Using translation approaches to target these molecular pathways may offer more reliable clinical resolution of otitis media in the future.

A transcytotic transport mechanism across the tympanic membrane.

Drug treatments for middle ear diseases are currently delivered systemically, or locally after opening the impermeable tympanic membrane (TM). We previously used bacteriophage display to discover novel peptides that are actively transported across the intact TM, with a variety of transport rates. Peptide structures were analyzed for evidence regarding the mechanism for this unexpected transport, which was then tested by the application of chemical inhibitors. Primary sequences indicated that trans-TM peptides share one of two amino acid motifs. Secondary structures revealed that linear configurations associate with higher transport rates than coiled structures. Tertiary analysis indicated that the shared sequence motifs are prominently displayed at the free ends of rapidly transported peptide phage. The shared motifs were evaluated for similarity to known motifs. The highest probability matches were for protein motifs involved in transmembrane transport and exosomes. Overall, structural findings suggest that the shared motifs represent binding sequences. They also implicate transcytosis, a polarized cell transport mechanism consisting of endocytosis, transcellular transport, and exocytosis. Inhibitor studies indicated that macropinocytosis, retrograde transport through Golgi and exocytosis participate in transport across the TM, consistent with transcytosis. This process can be harnessed to noninvasively deliver therapeutics to the middle ear.

Essential Role of the Innate Immune Adaptor RIP2 in the Response to Otitis Media.

Intracellular nucleotide binding and oligomerization domain (NOD) and Toll-like (TLR) receptors have emerged as pivotal sensors of infection. Both Nod1 and Nod2 contain a caspase activation and recruitment domain (CARD) that interacts with the adaptor protein RIP2 (receptor-interaction protein-2). This leads to ubiquitination of RIP2 and in turn to the activation of NFκB and MAPK transcription factors, to command the host defensive response against pathogenic infections. RIP2 is also activated by TLRs 2 and 4, although the mechanism of this activation is less. The role of RIP2 in otitis media (OM) pathogenesis has yet to be examined. Herein, we used in vivo animal models including C57BL/6 wild-type (WT) and RIP2-/- knockout mice inoculated in the middle ear (ME) with non-typeable Haemophilus influenzae (NTHi), a common human OM pathogen, to evaluate the expression of RIP2 and its signaling genes at the cellular level to determine the role of RIP2 in OM pathogenesis and recovery. The Nod1, Nod2, and Ripk2 genes are minimally expressed in the normal ME. However, they are strongly upregulated during acute OM, as are many genes related to RIP2 signaling. However, while signaling genes were expressed by various ME cell types, only mucosal epithelial and stromal cells expressed the NODs, RIP2, and signaling genes required for the activation of the host defensive response. Whereas WT mice clear ME bacteria and recover from OM within 5 days after infection, RIP2-deficient mice show persistent ME bacterial carriage and inflammation to at least 15 days. This includes significantly prolonged mucosal hyperplasia and ME leukocytic infiltration. Recruitment of macrophages is also delayed in comparison to WT mice. Thus, RIP2 is required to elicit a robust innate immune response that promotes bacterial clearance and increases host innate resistance. The results also identify the structural cells of the ME mucosa, as opposed to leukocytes, as the primary sites of NOD/RIP2 activity in the infected ME.

HB-EGF Plays a Pivotal Role in Mucosal Hyperplasia During Otitis Media Induced by a Viral Analog.

Otitis media (OM), the most common childhood illness, can be caused by bacterial and/or viral infection. Hyperplasia of the middle ear (ME) mucosa is an important component of OM that contributes to its deleterious sequelae. Our previous research revealed that ME mucosal hyperplasia in bacterially induced OM was associated with expression of the heparin-binding epidermal growth factor (HB-EGF) gene, and that HB-EGF induced the proliferation of ME mucosal explants in culture. We used single-cell RNA-Seq to identify ME cells that express Hbegf and related genes involved in mediating responses to this factor. To determine the degree to which a viral infection might induce mucosal hyperplasia, and to assess the role of HB-EGF in hyperplasia in vivo, we used, Poly(I:C) to simulate a ME viral infection, Western blotting to confirm ME protein expression, and a specific inhibitor to block the effects of HB-EGF during OM. Genes for HB-EGF and its receptor were expressed in the ME primarily by epithelial, stromal and endothelial cells. Poly(I:C) induced prominent ME mucosal hyperplasia, peaking two days after ME injection. Immunostaining revealed that cleavage of proHB-EGF into its soluble form (sHB-EGF) was strongly induced in response to Poly(I:C). Inhibition of the sHB-EGF receptor dramatically reduced the hyperplastic response of the mucosa. The results demonstrate that a synthetic analog of viral double-stranded RNA interaction can induce OM including a strong proliferative response of the ME mucosa, independent of bacteria. They also indicate that HB-EGF is the dominant growth factor responsible for ME mucosal hyperplasia in vivo.

Resolution of otitis media in a humanized mouse model.

Otitis media (OM) is one of the largest public health problems of children and has devastating impacts in developing countries. The substantial medical and human costs involved have led to research to understand the disease and improve treatment. Animal models of OM have yielded critical information about the immune, inflammatory and genetic mechanisms of OM. However, it is important to link animal studies to human immune and inflammatory responses. In recent years, "humanized" mice have become a valuable tool to study the human immune system in an animal model. Here we describe the first use of humanized mice to study OM. We demonstrate that humanized mice with a sufficient degree of engraftment recapitulate a normal middle ear (ME) inflammatory response to bacterial infection, including the recruitment of human immune cells, and exhibit normal recovery. Moreover, these animals exhibit regulated expression of human-specific immune and inflammatory genes in the ME. In contrast, mice with insufficient engraftment fail to resolve OM. This model has many potential uses in OM research, including using hematopoietic stem cells from patients with differing degrees of OM susceptibility, to understand the role of human immune responses in proneness to this common childhood disease.

Immunohistochemical and qPCR Detection of SARS-CoV-2 in the Human Middle Ear Versus the Nasal Cavity: Case Series.

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.

The ECRG4 cleavage product augurin binds the endotoxin receptor and influences the innate immune response during otitis media.

Otitis media (OM), the most common disease of childhood, is typically characterized by bacterial infection of the middle ear (ME). Prominent features of OM include hyperplasia of the ME mucosa, which transforms from a monolayer of simple squamous epithelium with minimal stroma into a full-thickness respiratory epithelium in 2-3 days after infection. Analysis of the murine ME transcriptome during OM showed down-regulation of the tumor suppressor gene Ecrg4 that was temporally related to mucosal hyperplasia and identified stromal cells as the primary ECRG4 source. The reduction in Ecrg4 gene expression coincided with the cleavage of ECRG4 protein to release an extracellular fragment, augurin. The duration of mucosal hyperplasia during OM was greater in Ecrg4 -/- mice, the number of infiltrating macrophages was enhanced, and ME infection cleared more rapidly. ECRG4-null macrophages showed increased bacterial phagocytosis. Co-immunoprecipitation identified an association of augurin with TLR4, CD14 and MD2, the components of the lipopolysaccharide (LPS) receptor. The results suggest that full-length ECRG4 is a sentinel molecule that potentially inhibits growth of the ME stroma. Processing of ECRG4 protein during inflammation, coupled with a decline in Ecrg4 gene expression, also influences the behavior of cells that do not express the gene, limiting the production of growth factors by epithelial and endothelial cells, as well as the activity of macrophages.

Immunomodulation as a Protective Strategy in Chronic Otitis Media.

Introduction: Major features of the pathogenesis in otitis media, the most common disease in childhood, include hyperplasia of the middle ear mucosa and infiltration by leukocytes, both of which typically resolve upon bacterial clearance via apoptosis. Activation of innate immune receptors during the inflammatory process leads to the activation of intracellular transcription factors (such as NF-κB, AP-1), which regulate both the inflammatory response and tissue growth. We investigated these leading signaling pathways in otitis media using mouse models, human samples, and human middle ear epithelial cell (HMEEC) lines for therapeutic immunomodulation. Methods: A stable otitis media model in wild-type mice and immunodeficient KO-mice, as well as human tissue samples from chronic otitis media, skin from the external auditory canal and middle ear mucosa removed from patients undergoing ear surgery, were studied. Gene and protein expression of innate immune signaling molecules were evaluated using microarray, qPCR and IHC. In situ apoptosis detection determined the apoptotic rate. The influence of bacterial infection on immunomodulating molecules (TNFα, MDP, Tri-DAP, SB203580, Cycloheximide) in HMEEC was evaluated. HMEEC cells were examined after bacterial stimulation/inhibition for gene expression and cellular growth. Results: Persistent mucosal hyperplasia of the middle ear mucosa in chronic otitis media resulted from gene and protein expression of inflammatory and apoptotic genes, including NODs, TNFα, Casp3 and cleaved Casp3. In clinical chronic middle ear samples, these molecules were modulated after a specific stimulation. They also induced a hyposensitive response after bacterial/NOD-/TLR-pathway double stimulation of HMEEC cells in vitro. Hence, they might be suitable targets for immunological therapeutic approaches. Conclusion: Uncontrolled middle ear mucosal hyperplasia is triggered by TLRs/NLRs immunoreceptor activation of downstream inflammatory and apoptotic molecules.