Virus-infection in cochlear supporting cells induces audiosensory receptor hair cell death by TRAIL-induced necroptosis.

Although sensorineural hearing loss (SHL) is relatively common, its cause has not been identified in most cases. Previous studies have suggested that viral infection is a major cause of SHL, especially sudden SHL, but the system that protects against pathogens in the inner ear, which is isolated by the blood-labyrinthine barrier, remains poorly understood. We recently showed that, as audiosensory receptor cells, cochlear hair cells (HCs) are protected by surrounding accessory supporting cells (SCs) and greater epithelial ridge (GER or Kölliker's organ) cells (GERCs) against viral infections. Here, we found that virus-infected SCs and GERCs induce HC death via production of the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Notably, the HCs expressed the TRAIL death receptors (DR) DR4 and DR5, and virus-induced HC death was suppressed by TRAIL-neutralizing antibodies. TRAIL-induced HC death was not caused by apoptosis, and was inhibited by necroptosis inhibitors. Moreover, corticosteroids, the only effective drug for SHL, inhibited the virus-induced transformation of SCs and GERCs into macrophage-like cells and HC death, while macrophage depletion also inhibited virus-induced HC death. These results reveal a novel mechanism underlying virus-induced HC death in the cochlear sensory epithelium and suggest a possible target for preventing virus-induced SHL.

Engraftment of Human Stem Cell-Derived Otic Progenitors in the Damaged Cochlea.

Most cases of sensorineural deafness are caused by degeneration of hair cells. Although stem/progenitor cell therapy is becoming a promising treatment strategy in a variety of organ systems, cell engraftment in the adult mammalian cochlea has not yet been demonstrated. In this study, we generated human otic progenitor cells (hOPCs) from induced pluripotent stem cells (iPSCs) in vitro and identified these cells by the expression of known otic markers. We showed successful cell transplantation of iPSC-derived-hOPCs in an in vivo adult guinea pig model of ototoxicity. The delivered hOPCs migrated throughout the cochlea, engrafted in non-sensory regions, and survived up to 4 weeks post-transplantation. Some of the engrafted hOPCs responded to environmental cues within the cochlear sensory epithelium and displayed molecular features of early sensory differentiation. We confirmed these results with hair cell progenitors derived from Atoh1-GFP mice as donor cells. These mouse otic progenitors transplanted using the same in vivo delivery system migrated into damaged cochlear sensory epithelium and adopted a partial sensory cell fate. This is the first report of the survival and differentiation of hOPCs in ototoxic-injured mature cochlear epithelium, and it should stimulate further research into cell-based therapies for treatment of deafness.

Macrophages in the Human Cochlea: Saviors or Predators-A Study Using Super-Resolution Immunohistochemistry.

The human inner ear, which is segregated by a blood/labyrinth barrier, contains resident macrophages [CD163, ionized calcium-binding adaptor molecule 1 (IBA1)-, and CD68-positive cells] within the connective tissue, neurons, and supporting cells. In the lateral wall of the cochlea, these cells frequently lie close to blood vessels as perivascular macrophages. Macrophages are also shown to be recruited from blood-borne monocytes to damaged and dying hair cells induced by noise, ototoxic drugs, aging, and diphtheria toxin-induced hair cell degeneration. Precise monitoring may be crucial to avoid self-targeting. Macrophage biology has recently shown that populations of resident tissue macrophages may be fundamentally different from circulating macrophages. We removed uniquely preserved human cochleae during surgery for treating petroclival meningioma compressing the brain stem, after ethical consent. Molecular and cellular characterization using immunofluorescence with antibodies against IBA1, TUJ1, CX3CL1, and type IV collagen, and super-resolution structured illumination microscopy (SR-SIM) were made together with transmission electron microscopy. The super-resolution microscopy disclosed remarkable phenotypic variants of IBA1 cells closely associated with the spiral ganglion cells. Monitoring cells adhered to neurons with "synapse-like" specializations and protrusions. Active macrophages migrated occasionally nearby damaged hair cells. Results suggest that the human auditory nerve is under the surveillance and possible neurotrophic stimulation of a well-developed resident macrophage system. It may be alleviated by the non-myelinated nerve soma partly explaining why, in contrary to most mammals, the human's auditory nerve is conserved following deafferentiation. It makes cochlear implantation possible, for the advantage of the profoundly deaf. The IBA1 cells may serve additional purposes such as immune modulation, waste disposal, and nerve regeneration. Their role in future stem cell-based therapy needs further exploration.

Dynamic activation of basilar membrane macrophages in response to chronic sensory cell degeneration in aging mouse cochleae.

In the sensory epithelium, macrophages have been identified on the scala tympani side of the basilar membrane. These basilar membrane macrophages are the spatially closest immune cells to sensory cells and are able to directly respond to and influence sensory cell pathogenesis. While basilar membrane macrophages have been studied in acute cochlear stresses, their behavior in response to chronic sensory cell degeneration is largely unknown. Here we report a systematic observation of the variance in phenotypes, the changes in morphology and distribution of basilar membrane tissue macrophages in different age groups of C57BL/6J mice, a mouse model of age-related sensory cell degeneration. This study reveals that mature, fully differentiated tissue macrophages, not recently infiltrated monocytes, are the major macrophage population for immune responses to chronic sensory cell death. These macrophages display dynamic changes in their numbers and morphologies as age increases, and the changes are related to the phases of sensory cell degeneration. Notably, macrophage activation precedes sensory cell pathogenesis, and strong macrophage activity is maintained until sensory cell degradation is complete. Collectively, these findings suggest that mature tissue macrophages on the basilar membrane are a dynamic group of cells that are capable of vigorous adaptation to changes in the local sensory epithelium environment influenced by sensory cell status.

Trichostatin A reduces cisplatin-induced ototoxicity through the STAT6 signaling pathway.

Cisplatin-induced ototoxicity limits its wide application in the treatment of cancer. A number of pro-inflammatory factors have been shown to be involved in cisplatin-induced ototoxicity. Trichostatin A (TSA) is an anti-inflammatory agent that has been shown to exert protective effects against cisplatin-induced ototoxicity. In the present study, we hypothesized that TSA may protect cochlear hair cells from cisplatin-induced damage by regulating the interleukin (IL)-4/signal transducer and activator of transcription (STAT)6 signaling pathway. Wistar rat cochlear explants were cultured in DMEM. The differentially expressed genes of the basilar membrane were identified by microarray analysis of global expression profiles. Hair cells were stained with rhodamine phalloidin and observed under a scanning electron microscope to evaluate the protective effects of TSA against cisplatin-induced cochlear hair cell damage. The levels of cytokines in the supernatant of the cultured basilar membranes was measured using ELISA. STAT6 and phosphorylated (p-)STAT6 expression was measured by western blot analysis. Morphological observation revealed that cisplatin induced the disarrangement of the cochlear hair cells, as well as the fusion and detachment of the cilia, while these aberrant alterations were inhibited by TSA, suggesting that TSA exerts a protective effect against cisplatin-induced damage to hair cells. Furthermore, the increase in the expression of STAT6 and p-STAT6 induced by cisplatin was reversed by treatment with TSA, accompanied by the decreased expression of IL-1ß, IL-4 and IL-6. Therefore, our data demonstrate that TSA reduces cisplatin-induced ototoxicity by inhibiting pro-inflammatory factor-mediated STAT6 signaling. Thus, TSA may be used to prevent the side-effects associated with the use of cisplatin in cancer treatment.

Vestibular hair cell regeneration and restoration of balance function induced by math1 gene transfer.

HYPOTHESIS: Delivery of math1 using an adenovector (Admath1.11D) results in vestibular hair cell regeneration and recovery of balance function in ototoxin-treated adult mice. BACKGROUND: Loss of peripheral vestibular function is associated with disease processes such as vestibular neuronitis, aminoglycoside ototoxicity, and aging. Loss of vestibular hair cells is one of the mechanisms underlying balance dysfunction in all of these disorders. Currently, recovery from these diseases relies on central vestibular compensation rather than on local tissue recovery. Overexpression of the mammalian atonal homologue math1 has been demonstrated to induce generation of hair cells in neonatal organ of Corti cultures and in the guinea pig cochlea in vivo and could thus provide an approach to local tissue recovery. METHODS: Admath1.11D was applied to cultures of aminoglycoside-treated macular organs or in vivo in a mouse aminoglycoside ototoxicity model. Outcome measures included histologic examination, immunohistochemistry, swim testing, and evaluation of the horizontal vestibulo-ocular reflex. RESULTS: Delivery of math1 resulted in the generation of vestibular hair cells in vitro after aminoglycoside-mediated loss of hair cells. Math1-treated mice showed recovery of the vestibular neuroepithelium within 8 weeks after Admath1.11D treatment. Assessment of animals after vector infusion demonstrated a recovery of vestibular function compared with aminoglycoside-only-treated mice. CONCLUSION: Molecular replacement of math1 may provide a therapeutic means of restoring vestibular function related to vestibular hair cell loss.

Mononuclear phagocytes migrate into the murine cochlea after acoustic trauma.

Acoustic injury results in destruction of hair cells and numerous nonsensory cells of the cochlea. How these injured structures undergo repair is not well understood. This study was designed to examine the cochlea for the presence of mononuclear phagocytes after tissue injury caused by noise damage. We used octave band noise (8--16 kHz) at three levels (106, 112, and 120 dB) for 2 hours and studied the mice at 1, 3, 7, and 14 days after noise exposure to determine how noise affected hearing thresholds, hair cell number, and tissue injury in the cochlea. Furthermore, we assessed the cochlea for presence of inflammation by performing immunohistochemistry for CD45, common leukocyte antigen. We counted the number of CD45(+) cells that were present in the cochlea at the above-mentioned time points after noise. CD45 is present on all bone marrow-derived white blood cells and is not otherwise expressed in the inner ear. We found that, after noise exposure, there is a large increase in CD45(+) cells. These marrow-derived cells are concentrated in the spiral ligament and spiral limbus, areas that are known to be susceptible to acoustic injury. It is possible that this inflammatory response plays a role in propagating cellular damage in these areas. Immunohistochemistry demonstrates that these cochlear cells are derived from the monocyte/macrophage lineage and serve a phagocytic function in the inner ear.

Nestin expression in the developing rat cochlea sensory epithelia.

An intermediate filament (IF), nestin, is used as an immature cell marker because nestin occurs in neural progenitors during early development. Recent cell culture studies have indicated that proliferating otic progenitor cells express nestin in vitro. However, localization of nestin in the developing inner ear has not yet been clarified. In this study, the ontogenetical expression of nestin epitopes in the rat cochlea was examined immunohistochemically. Sensory epithelial cells in the rat Corti organ (e.g. hair cells and support cells) transiently demonstrated immunoreactivity for nestin during the late embryonic period. After birth, nestin expression in the sensory epithelia disappeared gradually. The findings of this study indicate that the expression of nestin epitopes in the developing cochlea is linked with the plasticities of sensory epithelial cells, such as proliferation or differentiation.

Evidence for a possible NOS back-up system in the organ of Corti of the guinea pig.

Recently, the two Ca(2+)/calmodulin-regulated nitric oxide synthase isoforms, nNOS and eNOS, and NO itself have been identified in the cochlea of vertebrates using specific antibodies and a new fluorescence indicator. In order to acquire more information about the quantitative and spatial distribution of these two constitutively expressed NOS isoforms (cNOS) in the organ of Corti at the cellular and subcelluar levels, ultrathin sections of London resin (LR) White-embedded cochleae of the guinea pig were incubated with various concentrations of commercially available antibodies to nNOS and eNOS. The immunoreactivity was visualized by a gold-labeled secondary antibody and the amount of the immunoreactions/microm(2) was quantified for different cell types and subcellular regions. Both NOS isoforms were identified to varying degrees in the same cell types and subcellular regions. A prominent eNOS immunoreactivity was identified in nearly every cell type. In all analyzed animals the highest number of gold-coupled anti-eNOS antibodies was always seen in the cells of the reticular lamina, especially in the cuticular structures of outer and inner hair cells, pillar cells and apical Deiters' cells. Also the microtubuli-containing cytoplasmic regions of Deiters' cells were scattered with gold-coupled anti-eNOS antibodies. A clear eNOS immunoreaction was also found in the remaining cytoplasm of inner and outer hair cells and in the apical Deiters' cells. Numerous anti-nNOS antibodies were located in the outer hair cells and in the cuticular structures of the apical Deiters' cells. The amount of the gold-labeled anti-nNOS antibodies in the cuticular plates of the pillar cells and outer hair cells and in the cytoplasm of inner hair cells and apical Deiters' cells were clearly less but still above unspecific background labeling. The spatial co-localization of the two NOS isotypes in the same cell regions was proven in double-labeling experiments. The spatial distribution of the two cNOS isoforms confirmed recent findings of other authors who localized NO distribution and production sites. The cNOS co-expression with similar function in the same cell type and subcellular regions may represent a functional "back-up system" in which one NOS isoform can replace the other in case of pathophysiological malfunction.

Inflammatory signals increase Fas ligand expression by inner ear cells.

There is considerable evidence that hearing and vestibular function can be influenced by immune processes. The inner ear has evolved mechanisms, such as the blood-labyrinthine barrier that limit immune responses and autoimmune processes to reduce the potential for damage to cochlear cells. Recently, expression of Fas ligand (FasL) in some non-lymphoid tissue, as in the anterior chamber of the eye, has been hypothesized to play a role in protection of sensitive organs from activated T-cells. We show that under resting conditions, cochlear cells express little or no FasL. However, after exposure to interferon-gamma in vitro, FasL is induced in many neonatal cochlear cells. In addition, we show that FasL is upregulated in adult cochlear cells after induction of a sterile labyrinthitis in vivo. The induction of FasL by inflammation may serve to limit cochlear immune responses, and to protect sensorineural tissue from immune and autoimmune damage.

Macrophage activity in organ cultures of the avian cochlea: demonstration of a resident population and recruitment to sites of hair cell lesions.

The factors that regulate the repair and regeneration of the sensory hair cells of the inner ear are not understood. Previous studies of hair cell injury in the lateral line sensory organs of amphibians and the cochleae of mammals have demonstrated that macrophages and other leukocytes are recruited to sites of hair cell lesions. The present study examined the distribution and activity of macrophages in organ cultures of the avian cochlea, a system whose regenerative abilities have been widely studied. Cochleae were removed from chicks and placed in organ culture, and precise hair cell lesions were created using a laser microbeam. Macrophages in the cultures were identified using histochemical, immunocytochemical, and morphologic criteria. It was found that (a) cultured cochleae contained a resident population of macrophages, and (b) increased numbers of macrophages were recruited to the sites of hair cell lesions. Furthermore, the latency of macrophage recruitment to lesions is consistent with a suggested role for macrophages in the initiation of hair cell regeneration.

Monoclonal antibody induced hearing loss.

Monoclonal antibodies KHRI-3 and KHRI-5 identify antigens expressed on inner ear supporting cells and auditory hair cells respectively. To determine if these antibodies affect inner ear function groups of syngeneic Balb/c mice were inoculated with hybridomas KHRI-3, KHRI-5 and other Ig-secreting hybridomas. Hybridomas UM-A9, UM-7F11, the non-secreting SP2/0 myeloma and mice with no hybridoma were used as controls. Animals were tested for auditory brainstem responses (ABR) for frequencies of 4, 8, 16 and 24 kHz, before the inoculation of the hybridomas and at intervals of 6 to 10 days thereafter or daily once tumors became palpable. In normal mice there were no changes in ABR thresholds over the course of the experiment. Other control animals showed little change in ABR even when the growth of the hybridoma or myeloma tumors were far advanced. Of the KHRI-5 hybridoma bearing animals only one of seven animals exhibited threshold shifts greater than 15 dB. In contrast, most mice bearing the KHRI-3 hybridoma exhibited high frequency threshold shifts of 40-50 dB that coincided temporally with the growth of the hybridoma, the presence of circulating KHRI-3 antibody, and greatly increased immunoglobulin titers. Ears from KHRI-3-bearing mice that developed high frequency hearing loss also had a novel type of lesion in the basal turn of the cochlea that was characterized by loss of outer hair cells and absence of typical supporting cell scars. Such changes were not found in control hybridoma-bearing mice. These findings suggest that KHRI-3 antibody has an effect on hearing that is secondary to damage to the organ of Corti and loss of outer hair cells. Our results have important implications for antibody-mediated mechanisms of hearing loss and provide an animal model in which to study this phenomenon.

Neuron-specific enolase-like immunoreactivity in inner hair cells but not outer hair cells in the guinea pig organ of Corti.

Neuron-specific enolase (NSE) has been localized only in neurons and cells with characteristics of neurons. The immunocytochemical localization of NSE was examined in guinea pig cochleae to determine if hair cells, which have some neuronal characteristics, would show NSE-like immunoreactive labeling. NSE-like immunoreactivity was seen in inner hair cells but not in outer hair cells. This is the first report of NSE-like immunoreactivity in a receptor cell. NSE-like immunoreactivity was also seen in efferent fibers and terminals and in both type I and type II spiral ganglion cells. The finding of NSE-like immunoreactivity in inner but not outer cells adds to the number of differences found between them and may be related to differences in function and action.