Hair Cell Generation in Cochlear Culture Models Mediated by Novel γ-Secretase Inhibitors.

Sensorineural hearing loss is prevalent within society affecting the quality of life of 460 million worldwide. In the majority of cases, this is due to insult or degeneration of mechanosensory hair cells in the cochlea. In adult mammals, hair cell loss is irreversible as sensory cells are not replaced spontaneously. Genetic inhibition of Notch signaling had been shown to induce hair cell formation by transdifferentiation of supporting cells in young postnatal rodents and provided an impetus for targeting Notch pathway with small molecule inhibitors for hearing restoration. Here, the oto-regenerative potential of different γ-secretase inhibitors (GSIs) was evaluated in complementary assay models, including cell lines, organotypic cultures of the organ of Corti and cochlear organoids to characterize two novel GSIs (CPD3 and CPD8). GSI-treatment induced hair cell gene expression in all these models and was effective in increasing hair cell numbers, in particular outer hair cells, both in baseline conditions and in response to ototoxic damage. Hair cells were generated from transdifferentiation of supporting cells. Similar findings were obtained in cochlear organoid cultures, used for the first time to probe regeneration following sisomicin-induced damage. Finally, effective absorption of a novel GSI through the round window membrane and hair cell induction was attained in a whole cochlea culture model and in vivo pharmacokinetic comparisons of transtympanic delivery of GSIs and different vehicle formulations were successfully conducted in guinea pigs. This preclinical evaluation of targeting Notch signaling with novel GSIs illustrates methods of characterization for hearing restoration molecules, enabling translation to more complex animal studies and clinical research.

Anti-inflammatory and Oto-Protective Effect of the Small Heat Shock Protein Alpha B-Crystallin (HspB5) in Experimental Pneumococcal Meningitis.

Sensorineural hearing loss is the most common long-term deficit after pneumococcal meningitis (PM), occurring in up to 30% of surviving patients. The infection and the following overshooting inflammatory host response damage the vulnerable sensory cells of the inner ear, resulting in loss of hair cells and spiral ganglion neurons, ultimately leading to elevated hearing thresholds. Here, we tested the oto-protective properties of the small heat shock protein alpha B-crystallin (HspB5) with previously reported anti-inflammatory, anti-apoptotic and neuroprotective functions, in an experimental model of PM-induced hearing loss. We analyzed the effect of local and systemic delivery of HspB5 in an infant rat model of PM, as well as ex vivo, using whole mount cultures. Cytokine secretion profile, hearing thresholds and inner ear damage were assessed at predefined stages of the disease up to 1 month after infection. PM was accompanied by elevated pro-inflammatory cytokine concentrations in the cerebrospinal fluid (CSF), leukocyte and neutrophil infiltration in the perilymphatic spaces of the cochlea with neutrophils extracellular trap formation during the acute phase of the disease. Elevated hearing thresholds were measured after recovery from meningitis. Intracisternal but not intraperitoneal administration of HspB5 significantly reduced the levels of TNF-α, IL-6 IFN-γ and IL-10 in the acute phase of the disease. This resulted in a greater outer hair cell survival, as well as improved hearing thresholds at later stages. These results suggest that high local concentrations of HspB5 are needed to prevent inner ear damage in acute PM. HspB5 represents a promising therapeutic option to improve the auditory outcome and counteract hearing loss after PM.

A Triple Co-Culture Model of the Human Respiratory Tract to Study Immune-Modulatory Effects of Liposomes and Virosomes.

The respiratory tract with its ease of access, vast surface area and dense network of antigen-presenting cells (APCs) represents an ideal target for immune-modulation. Bio-mimetic nanocarriers such as virosomes may provide immunomodulatory properties to treat diseases such as allergic asthma. In our study we employed a triple co-culture model of epithelial cells, macrophages and dendritic cells to simulate the human airway barrier. The epithelial cell line 16HBE was grown on inserts and supplemented with human blood monocyte-derived macrophages (MDMs) and dendritic cells (MDDCs) for exposure to influenza virosomes and liposomes. Additionally, primary human nasal epithelial cells (PHNEC) and EpCAM+ epithelial progenitor cell mono-cultures were utilized to simulate epithelium from large and smaller airways, respectively. To assess particle uptake and phenotype change, cell cultures were analyzed by flow cytometry and pro-inflammatory cytokine concentrations were measured by ELISA. All cell types internalized virosomes more efficiently than liposomes in both mono- and co-cultures. APCs like MDMs and MDDCs showed the highest uptake capacity. Virosome and liposome treatment caused a moderate degree of activation in MDDCs from mono-cultures and induced an increased cytokine production in co-cultures. In epithelial cells, virosome uptake was increased compared to liposomes in both mono- and co-cultures with EpCAM+ epithelial progenitor cells showing highest uptake capacity. In conclusion, all cell types successfully internalized both nanocarriers with virosomes being taken up by a higher proportion of cells and at a higher rate inducing limited activation of MDDCs. Thus virosomes may represent ideal carrier antigen systems to modulate mucosal immune responses in the respiratory tract without causing excessive inflammatory changes.