The immune response is an important component of the cochlear response to stress. As an important player in the cochlear immune system, the basilar membrane immune cells reside on the surface of the scala tympani side of the basilar membrane. At present, the immune cell properties in this region and their responses to stress are not well understood. Here, we investigated the functional role of these immune cells in the immune response to acoustic overstimulation. This study reveals that tissue macrophages are present in the entire length of the basilar membrane under steady-state conditions. Notably, these cells in the apical and the basal sections of the basilar membrane display distinct morphologies and immune protein expression patterns. Following acoustic trauma, monocytes infiltrate into the region of the basilar membrane, and the infiltrated cells transform into macrophages. While monocyte infiltration and transformation occur in both the apical and the basal sections of the basilar membrane, only the basal monocytes and macrophages display a marked increase in the expression of MHC II and CIITA, a MHC II production cofactor, suggesting the site-dependent activation of antigen-presenting function. Consistent with the increased expression of the antigen-presenting proteins, CD4+ T cells, the antigen-presenting partner, infiltrate into the region of the basilar membrane where antigen-presenting proteins are upregulated. Further pathological analyses revealed that the basal section of the cochlea displays a greater level of sensory cell damage, which is spatially correlated with the region of antigen-presenting activity. Together, these results suggest that the antigen-presenting function of the mononuclear phagocyte population is activated in response to acoustic trauma, which could bridge the innate immune response to adaptive immunity.

BackgroundThe cochlea is the sensory organ of hearing. In the cochlea, the organ of Corti houses sensory cells that are susceptible to pathological insults. While the organ of Corti lacks immune cells, it does have the capacity for immune activity. We hypothesized that resident cells in the organ of Corti were responsible for the stress-induced immune response of the organ of Corti. This study profiled the molecular composition of the immune system in the organ of Corti and examined the immune response of non-immune epithelial cells to acoustic overstimulation.MethodsUsing high-throughput RNA-sequencing and qRT-PCR arrays, we identified immune- and inflammation-related genes in both the cochlear sensory epithelium and the organ of Corti. Using bioinformatics analyses, we cataloged the immune genes expressed. We then examined the response of these genes to acoustic overstimulation and determined how changes in immune gene expression were related to sensory cell damage.ResultsThe RNA-sequencing analysis reveals robust expression of immune-related genes in the cochlear sensory epithelium. The qRT-PCR array analysis confirms that many of these genes are constitutively expressed in the resident cells of the organ of Corti. Bioinformatics analyses reveal that the genes expressed are linked to the Toll-like receptor signaling pathway. We demonstrate that expression of Toll-like receptor signaling genes is predominantly from the supporting cells in the organ of Corti cells. Importantly, our data demonstrate that these Toll-like receptor pathway genes are able to respond to acoustic trauma and that their expression changes are associated with sensory cell damage.ConclusionThe cochlear resident cells in the organ of Corti have immune capacity and participate in the cochlear immune response to acoustic overstimulation.

Exposure to intense noise induces apoptosis in hair cells in the cochlea. To identify the molecular changes associated with noise-induced apoptosis, we used quantitative real-time PCR to evaluate the changes in 84 apoptosis related genes in cochlear samples from the sensory epithelium and lateral wall. Sprague Dawley rats exposed to a continuous noise at 115 dB SPL for 2 h. The exposure caused a 40-60 dB threshold shift 4 h post-exposure that decreased to 20-30 dB 7 days post-exposure. These functional changes were associated with apoptotic markers including nuclear condensation and fragmentation and TUNEL staining. Immediately after the noise exposure, 12 genes were downregulated, whereas only one gene (Traf4) was upregulated. At 4 h post-exposure, 8 genes were upregulated; 3 (Tnrsf1a, Tnfrsf1b, Tnfrst5) belonged to the Tnfrsf family, 3 (Bir3, Mcl1 and Prok2) have anti-apoptotic properties and 1 (Gadd45a) is a target of p53. At 7 d post-exposure, all the upregulated genes returned to pre-noise levels. Interestingly, the normal control cochlea had high constitutive levels of several apoptosis-related genes. These constitutively expressed genes, together with the inducible genes, may participate in the induction of cochlear apoptotic activity.

Our previous RNA-sequencing analysis of the rat cochlear genes identified multiple biological processes and molecular pathways in the cochlear response to acoustic overstimulation. However, the biological processes and molecular pathways that are common to other species have not been documented. The identification of these common stress processes is pivotal for a better understanding of the essential response of the cochlea to acoustic injury. Here, we compared the RNA-sequencing data collected from mice and rats that sustained a similar, but not identical, acoustic injury. The transcriptome analysis of cochlear genes identified the differentially expressed genes in the mouse and rat samples. Bioinformatics analysis revealed a marked similarity in the changes in the biological processes between the two species, although the differentially expressed genes did not overlap well. The common processes associated with the differentially expressed genes are primarily associated with immunity and inflammation, which include the immune response, response to wounding, the defense response, chemotaxis and inflammatory responses. Moreover, analysis of the molecular pathways showed considerable overlap between the two species. The common pathways include cytokine-cytokine receptor interactions, the chemokine signaling pathway, the Toll-like receptor signaling pathway, and the NOD-like receptor signaling pathway. Further analysis of the transcriptional regulators revealed common upstream regulators of the differentially expressed genes, and these upstream regulators are also functionally related to the immune and inflammatory responses. These results suggest that the immune and inflammatory responses are the essential responses to acoustic overstimulation in the cochlea.

Acoustic trauma, a leading cause of sensorineural hearing loss in adults, induces a complex degenerative process in the cochlea. Although previous investigations have identified multiple stress pathways, a comprehensive analysis of cochlear responses to acoustic injury is still lacking. In the current study, we used the next-generation RNA-sequencing (RNA-seq) technique to sequence the whole transcriptome of the normal and noise-traumatized cochlear sensory epithelia (CSE). CSE tissues were collected from rat inner ears 1 d after the rats were exposed to a 120-dB (sound pressure level) noise for 2 h. The RNA-seq generated over 176 million sequence reads for the normal CSE and over 164 million reads for the noise-traumatized CSE. Alignment of these sequences with the rat Rn4 genome revealed the expression of over 17000 gene transcripts in the CSE, over 2000 of which were exclusively expressed in either the normal or noise-traumatized CSE. Seventy-eight gene transcripts were differentially expressed (70 upregulated and 8 downregulated) after acoustic trauma. Many of the differentially expressed genes are related to the innate immune system. Further expression analyses using qRT-PCR confirmed the constitutive expression of multiple complement genes in the normal organ of Corti and the changes in the expression levels of the complement factor I (Cfi) and complement component 1, s subcomponent (C1s) after acoustic trauma. Moreover, protein expression analysis revealed strong expression of Cfi and C1s proteins in the organ of Corti. Importantly, these proteins exhibited expression changes following acoustic trauma. Collectively, the results of the current investigation suggest the involvement of the complement components in cochlear responses to acoustic trauma.