Search

Inflammatory reaction plays a crucial role in the pathophysiology of acquired hearing loss such as ototoxicity and labyrinthitis. In our earlier work, we showed the pivotal role of otic fibrocytes in cochlear inflammation and the critical involvement of proinflammatory cytokines in cisplatin ototoxicity. We also demonstrated that otic fibrocytes inhibit monocyte chemoattractant protein 1 (CCL2) upregulation in response to interleukin-10 (IL-10) via heme oxygenase 1 (HMOX1) signaling, resulting in suppression of cochlear inflammation. However, it is still unclear how IL-10 affects inflammation-mediated cochlear injury. Here we aim to determine how hypochlorous acid, a model inflammation mediator affects cochlear cell viability and how IL-10 affects hypochlorous acid-mediated cochlear cell injury. NaOCl, a sodium salt of hypochlorous acid (HOCl) was found to induce cytotoxicity of HEI-OC1 cells in a dose-dependent manner. Combination of hydrogen peroxide and myeloperoxidase augmented cisplatin cytotoxicity, and this synergism was inhibited by N-Acetyl-L-cysteine and ML-171. The rat spiral ligament cell line (RSL) appeared to upregulate the antioxidant response element (ARE) activities upon exposure to IL-10. RSL cells upregulated the expression of NRF2 (an ARE ligand) and NR0B2 in response to CoPP (a HMOX1 inducer), but not to ZnPP (a HMOX1 inhibitor). Adenovirus-mediated overexpression of NR0B2 was found to suppress CCL2 upregulation. IL-10-positive cells appeared in the mouse stria vascularis 1 day after intraperitoneal injection of lipopolysaccharide (LPS). Five days after injection, IL-10-positive cells were observed in the spiral ligament, spiral limbus, spiral ganglia, and suprastrial area, but not in the stria vascularis. IL-10R1 appeared to be expressed in the mouse organ of Corti as well as HEI-OC1 cells. HEI-OC1 cells upregulated Bcl-xL expression in response to IL-10, and IL-10 was shown to attenuate NaOCl-induced cytotoxicity. In addition, HEI-OC1 cells upregulated IL-22RA upon exposure to cisplatin, and NaOCl cytotoxicity was inhibited by IL-22. Taken together, our findings suggest that hypochlorous acid is involved in cochlear injury and that IL-10 potentially reduces cochlear injury through not only inhibition of inflammation but also enhancement of cochlear cell viability. Further studies are needed to determine immunological characteristics of intracochlear IL-10-positive cells and elucidate molecular mechanisms involved in the otoprotective activity of IL-10.

Cochlear inflammatory diseases, such as tympanogenic labyrinthitis, are associated with acquired sensorineural hearing loss. Although otitis media is extremely frequent in children, tympanogenic labyrinthitis is not commonly observed, which suggests the existence of a potent anti-inflammatory mechanism modulating cochlear inflammation. In this study, we aimed to determine the molecular mechanism involved in cochlear protection from inflammation-mediated tissue damage, focusing on IL-10 and hemoxygenase-1 (HMOX1) signaling. We demonstrated that IL-10Rs are expressed in the cochlear lateral wall of mice and rats, particularly in the spiral ligament fibrocytes (SLFs). The rat SLF cell line was found to inhibit nontypeable Haemophilus influenzae (NTHi)-induced upregulation of monocyte chemotactic protein-1 (MCP-1; CCL2) in response to IL-10. This inhibition was suppressed by silencing IL-10R1 and was mimicked by cobalt Protoporphyrin IX and CO-releasing molecule-2. In addition, IL-10 appeared to suppress monocyte recruitment through reduction of NTHi-induced rat SLF cell line-derived chemoattractants. Silencing of HMOX1 was found to attenuate the inhibitory effect of IL-10 on NTHi-induced MCP-1/CCL2 upregulation. Chromatin immunoprecipitation assays showed that IL-10 inhibits NTHi-induced binding of p65 NF-κB to the distal motif in the promoter region of MCP-1/CCL2, resulting in suppression of NTHi-induced NF-κB activation. Furthermore, IL-10 deficiency appeared to significantly affect cochlear inflammation induced by intratympanic injections of NTHi. Taken together, our results suggest that IL-10/HMOX1 signaling is involved in modulation of cochlear inflammation through inhibition of MCP-1/CCL2 regulation in SLFs, implying a therapeutic potential for a CO-based approach for inflammation-associated cochlear diseases.

Bucillamine is used for the treatment of rheumatoid arthritis. This study investigated the protective effects of bucillamine against cisplatin-induced damage in auditory cells, the organ of Corti from postnatal rats (P2) and adult Balb/C mice. Cisplatin increases the catalytic activity of caspase-3 and caspase-8 proteases and the production of free radicals, which were significantly suppressed by pretreatment with bucillamine. Bucillamine induces the intranuclear translocation of Nrf2 and thereby increases the expression of γ-glutamylcysteine synthetase (γ-GCS) and glutathione synthetase (GSS), which further induces intracellular antioxidant glutathione (GSH), heme oxygenase 1 (HO-1) and superoxide dismutase 2 (SOD2). However, knockdown studies of HO-1 and SOD2 suggest that the protective effect of bucillamine against cisplatin is independent of the enzymatic activity of HO-1 and SOD. Furthermore, pretreatment with bucillamine protects sensory hair cells on organ of Corti explants from cisplatin-induced cytotoxicity concomitantly with inhibition of caspase-3 activation. The auditory-brainstem-evoked response of cisplatin-injected mice shows marked increases in hearing threshold shifts, which was markedly suppressed by pretreatment with bucillamine in vivo. Taken together, bucillamine protects sensory hair cells from cisplatin through a scavenging effect on itself, as well as the induction of intracellular GSH.

BackgroundWe recently showed that beta-defensins have antimicrobial activity against nontypeable Haemophilus influenzae (NTHi) and that interleukin 1 alpha (IL-1 alpha) up-regulates the transcription of beta-defensin 2 (DEFB4 according to new nomenclature of the Human Genome Organization) in human middle ear epithelial cells via a Src-dependent Raf-MEK1/2-ERK signaling pathway. Based on these observations, we investigated if human middle ear epithelial cells could release IL-1 alpha upon exposure to a lysate of NTHi and if this cytokine could have a synergistic effect on beta-defensin 2 up-regulation by the bacterial components.MethodsThe studies described herein were carried out using epithelial cell lines as well as a murine model of acute otitis media (OM). Human cytokine macroarray analysis was performed to detect the released cytokines in response to NTHi exposure. Real time quantitative PCR was done to compare the induction of IL-1 alpha or beta-defensin 2 mRNAs and to identify the signaling pathways involved. Direct activation of the beta-defensin 2 promoter was monitored using a beta-defensin 2 promoter-Luciferase construct. An IL-1 alpha blocking antibody was used to demonstrate the direct involvement of this cytokine on DEFB4 induction.ResultsMiddle ear epithelial cells released IL-1 alpha when stimulated by NTHi components and this cytokine acted in an autocrine/paracrine synergistic manner with NTHi to up-regulate beta-defensin 2. This synergistic effect of IL-1 alpha on NTHi-induced beta-defensin 2 up-regulation appeared to be mediated by the p38 MAP kinase pathway.ConclusionWe demonstrate that IL-1 alpha is secreted by middle ear epithelial cells upon exposure to NTHi components and that it can synergistically act with certain of these molecules to up-regulate beta-defensin 2 via the p38 MAP kinase pathway.