Pharmacological Inhibition of Cochlear Mitochondrial Respiratory Chain Induces Secondary Inflammation in the Lateral Wall: A Potential Therapeutic Target for Sensorineural Hearing Loss

<div><p>Cochlear lateral wall has recently been reported as a common site of inflammation, yet precise molecular mechanisms of the inflammatory responses remain elucidated. The present study examined the inflammatory responses in the lateral wall following acute mitochondrial dysfunction induced by a mitochondrial toxin, 3-nitropropionic acid (3-NP). Reverse-transcription (RT)-PCR revealed increases in the expression of the proinflammatory cytokines interleukin (IL)-1β and IL-6. Immunohistochemistry showed an increase in the number of activated cochlear macrophages in the lateral wall, which were in close proximity to IL-6-expressing cells. A genome-wide DNA microarray analysis of the lateral wall revealed that 35% and 60% of the genes showing >2-fold upregulation at 1 d and 3 d post-3-NP administration, respectively, were inflammatory genes, including CC- and CXC-type chemokine genes. High expression of CCL-1, 2, and 3 at 1 d, and of CCL-1, 2, 3, 4, and 5, CCR-2 and 5, and CX3CR1 at 3 d post-3-NP administration, coupled with no change in the level of CX3CL1 expression suggested that macrophages and monocytes may be involved in the inflammatory response to 3-NP-mediated injury. Quantitative (q)RT-PCR showed a transient induction of IL-1β and IL-6 expression within 24 h of 3-NP-mediated injury, followed by sustained expression of the chemoattractants, CCL-2, 4 and 5, up until 7 d after injury. The expression of CCL-2 and IL-6 was higher in animals showing permanent hearing impairment than in those showing temporary hearing impairment, suggesting that these inflammatory responses may be detrimental to hearing recovery. The present findings suggest that acute mitochondrial dysfunction induces secondary inflammatory responses in the lateral wall of the cochlear and that the IL-6/CCL-2 inflammatory pathway is involved in monocyte activation. Therefore, these secondary inflammatory responses may be a potential post-insult therapeutic target for treatments aimed at preventing the damage caused by acute mitochondrial dysfunction in the cochlear lateral wall.</p></div

Expression of proinflammatory cytokines in 3-NP-injured cochlea.

<p>RT-PCR data for three major proinflammatory cytokines, IL-6, IL-1β, and TNF-α, and the house-keeping gene GAPDH. There was a significant difference in IL-6 expression in the 3-NP-treated cochleae compared with the saline-treated controls. Whole cochleae were harvested and used for the assay.</p

Time-dependent expression of IL-6 and chemokines in the 3-NP-injured cochlea lateral wall.

<p>Quantitative RT-PCR to examine the expression of IL-6 and chemokines, including CCL2 (MCP-1), CCL5 (RANTES) and CCL4 (MIP-1β), in the injured cochlear lateral wall. The expression of IL-6 was induced 3 h after 3-NP administration. IL-6 expression was higher in the PTS model (blue lines) than in the temporal threshold shift model (pink lines) at Day 1 (<b>a</b>; p<0.01; purple), suggesting that IL-6 was a detrimental factor. IL-6 expression was high in the PTS model at Day 1 post-injury but was quickly downregulated. Chemokines were induced in the 3-NP-injured lateral wall from 6 h to 7 d post 3-NP administration. Chemokine expression was induced more slowly than that of proinflammatory cytokines, but it was sustained (<b>a</b>–<b>d</b>). The level of CCL2 expression in the PTS and TTS models was significantly different, being greater in the PTS model than in the TTS model (<b>b</b>; p<0.05 (green) and p<0.01 (purple)), suggesting that CCL2 was also a detrimental factor. * indicates p<0.05, ** indicates p<0.01.</p

IL-6-expressing cells in 3-NP-injured cochlea were accompanied by macrophages.

<p>Cells expressing IL-6 (<b>a</b> and <b>c</b>) or Iba-1 (<b>b</b> and <b>d</b>) in the temporal threshold shift (TTS) model were examined by immunohistochemistry at Day 1 by using neighboring sections. IL-6 was expressed in type III fibrocytes (<b>a</b> and <b>c</b>; blue arrows) and in the mesenchyme cells beneath the basement membrane (<b>c</b>; black arrows). Cochlear macrophages expressing Iba-1 were identified adjacent to cells expressing IL-6, including in the lateral portion of the type II fibrocyte region that contacts type III cells (<b>b</b> and <b>d</b>; orange arrows), and were seen infiltrating the cells beneath the mesenchyme of the basilar membrane (<b>d</b>; black arrows). Iba-1-expressing cells were not observed in the type III area in which IL-6 was expressed (blue arrows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090089#pone-0090089-g003" target="_blank">Fig. 3<b>a</b> and <b>b</b></a>).</p

IL-6 expression in 3-NP-injured cochlea.

<p>Expression of IL-6 mRNA in whole cochlea. The temporal threshold shift (TTS) model (induced with 300 mM 3-NP) showed a significant induction of IL-6 mRNA from Day 1, which was sustained up until Day 3. The expression of IL-6 mRNA at Day 3 was also significantly higher in the permanent threshold shift (PTS) model (induced by 500 mM 3-NP) than in the saline-treated controls (*p<0.01, **p<0.05; N.D: not detected).</p

Upregulated CC-type chemokine genes in the lateral wall of 3-NP-injured cochlea.

<p>The values displayed refer to the -fold changes compared with the saline-treated controls.</p><p>Genes showing >2-fold changes are listed.</p><p>NC: no change compared with control.</p

Upregulated CXC-type chemokine genes in the lateral wall of 3-NP-injured cochlea.

<p>The values displayed refer to the -fold changes compared with the saline-treated controls.</p><p>Genes showing >2-fold changes are listed.</p><p>NC: no change compared with control.</p

Quantification of photons in different groups of mice with TT injections.

<p>(A) Time course of photon bioluminescence for each group of mice. (B) Peak photon counts.</p

Photon bioluminescence in GFAP-luc mice.

<p>(A) Differences in photon bioluminescence of round windows with and without obstructions. The left panel shows photon bioluminescence in a mouse's left ear, in which the round window has been obstructed. The right panel shows the lack of bioluminescence in the mouse's right ear, in which the round window remains intact. (B) Time course showing photon bioluminescence in ears of mice with ip, with and without round window obstruction. ip;intraperitoneal injection, RWM; round window membrane.</p

Time course of photon bioluminescence.

<p>(A) Time course of photon bioluminescence in five groups of animals: Luc ip, luciferase-expressing mice receiving intraperitoneal injections; Luc TT, luciferase-expressing mice receiving transtympanic injections; Luc TT RW closure, luciferase-expressing mice that received transtympanic injections after round window membrane obstruction; WT ip, wild-type mice receiving intraperitoneal injections. Background, background (no photons). (B) Bar graphs showing the highest photon counts by region. ip, intraperitoneal injection; TT, transtympanic injection; RW cl/TT, transtympanic injection after round window membrane obstruction. (C) Delivery time into GFAP-expressing cells of inner ear. Average time when the signal was maximum in each group was shown (mean ± SEM). Same conventions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048480#pone-0048480-g002" target="_blank">Fig. 2</a>. *p<0.05.</p