Inner ear dysfunction secondary to chronic otitis media (OM), including high-frequency sensorineural hearing loss or vertigo, is not uncommon. Although chronic middle ear inflammation is believed to cause inner ear dysfunction by entry of OM pathogen components or cytokines from the middle ear into the inner ear, the underlying mechanisms are not well understood. Previously, we demonstrated that the spiral ligament fibrocyte (SLF) cell line up-regulates monocyte chemotactic protein 1 (MCP-1) expression after treatment with nontypeable Haemophilus influenzae (NTHI), one of the most common OM pathogens. We hypothesized that the SLF-derived MCP-1 plays a role in inner ear inflammation secondary to OM that is responsible for hearing loss and dizziness. Antibiotics have led to a dramatic decline in the incidence of life-threatening complications of otitis media (OM), such as meningitis or brain abscess (3). However, inner ear dysfunction secondary to chronic OM, including high-frequency sensorineural hearing loss or vertigo, is not uncommon (13,26,36,55,60). Although chronic middle ear inflammation is believed to cause inner ear dysfunction by entry of OM pathogen components or cytokines from the middle ear into the inner ear, the underlying mechanisms are not well understood (18,32,39,44,52,87).The inner ear is a sensory organ for hearing (cochlea) and equilibrium (vestibule). It consists of a variety of specialized cell types (50, 51), such as sensory hair cells, supporting cells, sulcus cells, and spiral ligament fibrocytes (SLFs), which are the most abundant cell types exposed to the perilymph. The type of inner ear cells that respond to proinflammatory signals entering the inner ear remain unknown. Considering that SLFs are one of the abundant cell types in the cochlea and that they secrete cytokines and chemokines after proinflammatory stimuli (72, 97), we hypothesized that the SLFs are major responders to such signals.Preliminary studies of human temporal bones with labyrinthitis showed the infiltration of lysozyme-positive round cells with a monomorphic nucleus into the spiral ligament (unpublished data). Also, SLF cell lines (96) showed an induction in monocyte chemotactic protein 1 (MCP-1) expression after treatment with lysate of nontypeable Haemophilus influenzae (NTHI), one of the most common OM pathogens (72). Moreover, it has previously been shown that monocytes can infiltrate cochlea exhibiting chronic middle ear inflammation or acoustic trauma (22,34,37). These results led us to focus on MCP-1 as an SLF-derived proinflammatory chemokine attracting effector cells and causing inner ear dysfunction.MCP-1, also known as the chemokine C-C motif ligand 2, is produced by various cells, including endothelial cells, smooth muscle cells, fibroblasts, and macrophages, in response to cytokines, growth factors, or bacterial components (9,46,78). It is encoded by an immediate-early gene (33) and is up-regulated by various stimuli such as bacterial lipopolysaccharide (LPS), interleukin-1 (IL-1), tumor necrosis fact...

Background: All mucosal epithelia, including those of the tubotympanium, are secreting a variety of antimicrobial innate immune molecules (AIIMs). In our previous study, we showed the bactericidal/bacteriostatic functions of AIIMs against various otitis media pathogens. Among the AIIMs, human β-defensin 2 is the most potent molecule and is inducible by exposure to inflammatory stimuli such as bacterial components or proinflammatory cytokines. Even though the β-defensin 2 is an important AIIM, the induction mechanism of this molecule has not been clearly

BackgroundOtitis media (OM), one of the most common pediatric infectious diseases, causes inner ear inflammation resulting in vertigo and sensorineural hearing loss. Previously, we showed that spiral ligament fibrocytes (SLFs) recognize OM pathogens and up-regulate chemokines. Here, we aim to determine a key molecule derived from SLFs, contributing to OM-induced inner ear inflammation.MethodsLive NTHI was injected into the murine middle ear through the tympanic membrane, and histological analysis was performed after harvesting the temporal bones. Migration assays were conducted using the conditioned medium of NTHI-exposed SLFs with and without inhibition of MCP-1/CCL2 and CCR2. qRT-PCR analysis was performed to demonstrate a compensatory up-regulation of alternative genes induced by the targeting of MCP-1/CCL2 or CCR2.ResultsTranstympanic inoculation of live NTHI developed serous and purulent labyrinthitis after clearance of OM. THP-1 cells actively migrated and invaded the extracellular matrix in response to the conditioned medium of NTHI-exposed SLFs. This migratory activity was markedly inhibited by the viral CC chemokine inhibitor and the deficiency of MCP-1/CCL2, indicating that MCP-1/CCL2 is a main attractant of THP-1 cells among the SLF-derived molecules. We further demonstrated that CCR2 deficiency inhibits migration of monocyte-like cells in response to NTHI-induced SLF-derived molecules. Immunolabeling showed an increase in MCP-1/CCL2 expression in the cochlear lateral wall of the NTHI-inoculated group. Contrary to the in vitro data, deficiency of MCP-1/CCL2 or CCR2 did not inhibit OM-induced inner ear inflammation in vivo. We demonstrated that targeting MCP-1/CCL2 enhances NTHI-induced up-regulation of MCP-2/CCL8 in SLFs and up-regulates the basal expression of CCR2 in the splenocytes. We also found that targeting CCR2 enhances NTHI-induced up-regulation of MCP-1/CCL2 in SLFs.ConclusionsTaken together, we suggest that NTHI-induced SLF-derived MCP-1/CCL2 is a key molecule contributing to inner ear inflammation through CCR2-mediated recruitment of monocytes. However, deficiency of MCP-1/CCL2 or CCR2 alone was limited to inhibit OM-induced inner ear inflammation due to compensation of alternative genes.

Background: Lysozyme is an antimicrobial innate immune molecule degrading peptidoglycan of the bacterial cell wall. Lysozyme shows the ubiquitous expression in wide varieties of species and tissues including the tubotympanum of mammals. We aim to investigate the effects of lysozyme depletion on pneumococcal clearance from the middle ear cavity.

The inner ear, composed of the cochlea and the vestibule, is a specialized sensory organ for hearing and balance. Although the inner ear has been known as an immune-privileged organ, there is emerging evidence indicating an active immune reaction of the inner ear. Inner ear inflammation can be induced by the entry of pro-inflammatory molecules derived from middle ear infection. Since middle ear infection is highly prevalent in children, middle ear infection-induced inner ear inflammation can impact the normal development of language and motor coordination. Previously, we have demonstrated that the inner ear fibrocytes (spiral ligament fibrocytes) are able to recognize nontypeable Haemophilus influenzae (NTHi), a major pathogen of middle ear infection, and up-regulate a monocyte-attracting chemokine through TLR2-dependent NF-κB activation. Here, we aimed to determine molecular mechanism involved in NTHi-induced cochlear infiltration of polymorphonuclear cells. The rat spiral ligament fibrocytes (SLFs) were found to release CXCL2 in response to NTHi via activation of c-Jun, leading to the recruitment of polymorphonuclear cells to the cochlea. We also demonstrated that MEK1/ERK2 signaling pathway is required for NTHi-induced CXCL2 up-regulation in the rat SLFs. Two AP-1 motifs in the 5’-flanking region of CXCL2 appeared to function as a NTHi-responsive element, and the proximal AP-1 motif was found to have a higher binding affinity to NTHi-activated c-Jun than the distal one. Our results will enable us to better understand the molecular pathogenesis of middle ear infection-induced inner ear inflammation.