Activated tissue-resident macrophages contribute to hair cell insults in noise-induced hearing loss

doi:10.21203/rs.3.rs-3789678/v1

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Activated tissue-resident macrophages contribute to hair cell insults in noise-induced hearing loss

https://doi.org/10.21203/rs.3.rs-3789678/v1

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Background

Macrophages serve as the primary immune cell population and assume a pivotal role in the immune response within the damaged cochleae, yet the origin and role of macrophages in response to noise exposure remains controversial.

Methods

We generated Ccr2^RFP/+ Cx3cr1^GFP/+ dual-reporter mice to identify the infiltrated and tissue-resident macrophages in the noise-exposed cochlea.

Results

Under steady-state, tissue-resident macrophages present in the Rosenthal's canal (SGNs), spiral ligament, and basilar membrane of the adult cochlea. After noise exposure, activated resident macrophages change in morphology, increase in abundance, and migrate to the region of hair cells, leading to the loss of outer hair cells and the damage of ribbon synapses. Meanwhile, peripheral monocytes are not implicated in the noise-induced hair cell insults. Furthermore, the noise-induced activity and redistribution of macrophages were abolished by the inhibition of TLR4 signaling, resulting in alleviated insults of hair cells and partial recovery of hearing function.

Conclusions

Our findings point to cochlea resident macrophages as pro-inflammatory and detrimental players in acoustic trauma and introduce a potential therapeutic target in noise-induced hearing loss.

Macrophages

Acoustic trauma

Hair cells

Ribbon synapse

Inflammatory response

The cochlea was long considered immune privileged due to the presence of blood-labyrinth barriers (BLB). However, the activities of Immune cells and inflammation have been described in acute and chronic insults to the cochlea, such as noise, ototoxic injury, aging, or a cochlear implant insertion [1–8]. In response to these insults, macrophages are the primary immune cell population in the cochlea and play a major role in immune activity [9]. High-level noise exposure (NE) induces the damage of cochlear sensory cells, resulting in permanent hearing loss. Numerous of researches have shown dramatic alteration in the abundance, morphology, and mediator expression of cochlear macrophages after NE, indicating the activity of macrophages in response to acoustic trauma. However, before we conclude that cochlear macrophages would be a therapeutic target in noise-induced hearing loss (NIHL), pivotal questions need to be clarified. What is the origin of activated macrophages in the cochlea, i.e., are they cochlear tissue-resident or infiltrated macrophages? Whether the activated cochlear macrophages are injurious or protective to cochlear function?

Tissue-resident macrophages, with specific expression signatures of CX3CR1, CD163, and CD11b, distribute across modiolus, spiral ganglion neurons (SGNs), and spiral ligament of the cochlea. Under physiological conditions, they contribute to the maintenance of homeostasis and maintain their population with a turnover time of several months [10]. Infiltrated macrophages, with specific expression signatures of CCR2 and Ly6C, are recruited from the bone marrow monocytes. They infiltrate into tissues and differentiate into macrophages almost instantly following acute insults. Generally, both activated resident and infiltrated macrophages contribute to the immune response to insults by producing pro-inflammatory and anti-inflammatory mediators, phagocytosis, and antigen presentation. In NIHL, the evidence regarding the origin of activated macrophages in the cochlea is conflicting. It is unclear which macrophage population is activated and initiates the response to NE.

The role of activated macrophages in NIHL also remains to be determined. Evidenced by the increasing pro-inflammatory cytokines and chemokines in the cochlea following acoustic trauma, macrophages are proposed to initiate the inflammation and may result in the subsequent loss of cochlear sensory cells. Meanwhile, macrophages are involved in reparative response by producing anti-inflammatory mediators, clearing cellular debris and pathogens from tissues. These reparative functions are evidenced by the activity of macrophages in tissue repair after NE and cochlear implantation [1, 5, 7]. Whether and when macrophages are protective/ injurious is critical to understanding the role of macrophages in NIHL.

To address the above questions, in the present study, we employed the CX3CR1 and CCR2 fluorescence labeling mice to identify the tissue-resident and infiltrated macrophages in the cochlea. We determined the changes in the abundance, distribution, and morphology of these macrophages across different regions of the cochlea after NE. The roles of macrophage activity in NIHL and the relevance of the TLR4-NLRP3 pathway were also examined.

All animal experiments conducted in this study are approved by the Institutional Animal Care and Use Committee (IACUC) of Southern Medical University. The principles established by the Animal Care Committee of Southern Medical University were adhered to throughout the experiments.

Subjects

C57BL/6 wild-type (WT) female mice aged 1.5-2 months and weighting 15-20g were used in our experiments. B6.129(Cg)-Cx3cr1^tm1Litt Ccr2^tm2.1Ifc/JernJ mice (Stock number: 032127) and B6.129P2(Cg)Cx3Cr^1tm1Litt/J mice (Stock number: 005582) (The Jackson Laboratory, Bar Harbor, ME, USA) at the same age were also used in our study. B6.129(Cg)-Cx3cr1^tm1Litt Ccr2^tm2.1Ifc/JernJ mice, also known as Ccr2^RFPCx3cr1^GFP mice, express red fluorescent protein in CCR2-positive peripheral monocytes, T cells, and NK cells; and green fluorescent protein in monocytes, dendritic cells, NK cells, and brain microglia. B6.129P2(Cg)-Cx3Cr^1tm1Litt/J mice, also known as Cx3cr1^GFP mice, express green fluorescent protein in the place of Cx3cr1. Heterozygous mice (Ccr2^RFP/+Cx3cr1^GFP/+ and Cx3cr1^GFP/+) were created by cross-breeding corresponding homozygous with C57BL/6J mice. For genotyping, using One Step Mouse Genotyping Kit (Vazyme, Nanjing, China), genomic DNA was extracted from a small piece of mice tail and was used as template for PCR with specific primers. The primers used in the genotyping protocols are listed in Additional file 1: Table S1. Mice were housed in a room maintained at 22 (± 2) °C and subjected to a 12:12 light/dark cycle with lights on at 8:00 AM.

Auditory brainstem response (ABR) measurements

ABR measurements were performed to evaluate the auditory function of the mice as previously described [11]. Animals were anesthetized with an intraperitoneal (i.p.) injection of sodium pentobarbital (80 mg/kg) and were maintained on 0.5% inhaled isoflurane, and placed on the center of an anti-vibration table in a soundproof room. A subdermal needle electrode (recording electrode) was located over the skull vertex, and the reference and ground electrodes were placed ventrolaterally to the external pinna. For close-field ABR recording, each ear was individually stimulated using the tube of a close-field speaker (MF1, Tucker-Davis Technology, Alachua, FL, USA). For open-field ABR recording, the speaker was positioned 10 cm away toward the head of the animal. Software SigGenRZ (Tucker-Davis Technology, Alachua, FL, USA) was used to generate click and 5 ms tone bursts with various frequencies and intensities. Click and tone burst stimulus intensities were gradually decreased by 5 dB from 95 dB sound pressure level (SPL). The ABR waveforms were amplified, recorded, averaged, and stored by the software BioSigRZ. Real-time averages of the amplified evoked responses from 256 sweeps were made. The ABR thresholds were defined as the minimum sound intensity at which averaged waveforms could be distinguished. An SPL threshold of 95 dB was assigned when there was no significant ABR wave at maximum strength.

Noise exposure

Animals were placed in a vertical acrylic cylinder (10 cm in diameter, 20 cm high) to receive noise exposure. Continuous Gaussian white noise at a level of 110 dB SPL for 3 hours was generated by the software SigGenRZ. The signal was routed through a power supply (P350, Breeze Audio, China) and a power amplifier (TAS5630, Breeze Audio, China) to a loudspeaker positioned above the acrylic cylinder. The noise level was calibrated using a sound level meter (2610, Brüel & Kjær, Denmark).

Trans-tympanic Administration

Animals were anesthetized with pentobarbital sodium (80mg/kg, i.p.). A heating pad was used to maintain their body temperature during administration. Specific TLR4 inhibitor TAK-242 (0.1mg/kg, HY-11109, MedChemExpress Company, New Jersey, USA) was dissolved in a mixture of a vehicle containing 10% DMSO, 5% Tween-80, 40% PEG300, and 45% saline. Utilizing a 33-G cannula, 5 µl of either TAK-242 or the vehicle alone was gently injected through the tympanic membrane under a surgical stereomicroscope. Animals were allowed to recover on the heating pad before being returned to the animal housing facility. To evaluate the efficacy of trans-tympanic drug delivery, membrane dye FM1-43 (Invitrogen, USA) was trans-tympanically injected and the dye uptake was examined in hair cells (Additional file 1: Fig. S2A).

Endocochlear potential (EP) recording

EP was recorded according to the previously described method [12], with a detailed protocol described as follows. Briefly, after anesthesia, a tracheotomy was performed to exposure the cochlea under a surgical stereomicroscope. Using a fine dental drill, a small hole was opened on the lateral wall of the cochlear basal turn. Controlled by a micromanipulator, a recording microelectrode (pulled by a glass pipet and filled with 150 mM KCl) was inserted into the scala media, while the ground electrode was placed on the dorsal neck muscles. The response from the microelectrode was amplified using the Multiclamp700B amplifier (Molecular Devices, Foster City, CA, USA) and acquired using the software pClamp 10 (Molecular Devices, Foster City, CA, USA). The voltage changes during the microelectrode penetration were continuously recorded. Bilateral ears were analyzed for each animal.

Histology and Immunostaining

Histology and Immunostaining procedure as described in our previous study [11, 13]. In brief, the mice were deeply anesthetized and killed by decapitation. The cochleae were dissected from the temporal bone, and perfused with 4% paraformaldehyde overnight at 4°C. After 5 days of decalcification, the organs of Corti were isolated and divided into three segments evenly (apical, middle, and basal) for subsequent immunostaining. For quantifying CX3CR1 and CCR2 positive cells, the cochlea was serially sectioned in the axial plane at a thickness of 10 µm. Cochlea tissues were permeabilized by 0.3% Triton X-100 in PBS for 30 min followed by blocking with 10% normal goat serum for 1h at room temperature. Tissues were incubated overnight at 4°C with primary antibodies as followed: rabbit anti-myosin 7a (1:1,000, 25-6790, Protein Tech, Chicago, IL, United States), rat anti-F4/80 (1:1,000, MF48000, Invitrogen, Carlsbad, CA, United States), rabbit anti-TLR4 (1:1,000, ab13556, Abcam, United States), rabbit anti- F4/80 (1:1,000, 30325, Cell Signaling Technology, United States), rat anti-NLRP3 (1:1,000, Invitrogen, Carlsbad, CA, United States), mouse anti-CtBP2 (1:1,000, 612044, BD Biosciences, United States), mouse anti-GluR2 (1:1,000, MAB397, Millipore, United States). After incubation with the primary antibodies, the tissues were rinsed with PBS, and incubated in the dark with secondary antibodies for 1 h at room temperature. The secondary antibodies (Invitrogen, Carlsbad, CA, United States) were as follows: Myosin 7a staining: Alexa Fluor 568 goat anti-rabbit IgG, Alexa Fluor 647 goat anti-rabbit IgG or Alexa Fluor 405 goat anti-rabbit IgG; F4/80 and TLR4 staining: Alexa Fluor 488 goat anti-rat IgG and Alexa Fluor 405 donkey anti-rabbit IgG; F4/80 and NLRP3 staining: Alexa Fluor 488 goat antirabbit IgG and Alexa Fluor 405 goat anti-rat IgG; Myosin 7a, CtBP2 and GluR2 staining: Alexa Fluor 405 donkey anti-rabbit IgG, Alexa Fluor 488 goat anti-mouse IgG2a and Alexa Fluor 568 goat anti-mouse IgG1. The fluorescence images were acquired using a confocal laser scanning microscopy (Olympus, Japan).

Immunoblot Analysis

Whole cochleae were homogenized with a glass pestle in ice-cold RIPA lysis buffer (Byotime Biotechnology, China) containing 1% PMSF (Fdbio science, China). Following a 30-minute incubation on ice, the lysate underwent centrifugation at 12000× g at 4°C for 15 minutes. Protein concentration was determined using the Bio-Rad Protein Assay BCA kit. Subsequently, 30 µg total protein from each sample underwent separation via SDS-PAGE and transfer to a PVDF membrane (Millipore, Bedford, MA, USA). The blots were blocked with 5% non-fat dry milk in TBST and subjected to an overnight incubation at 4°C with primary antibodies targeting TLR4 (ab13556, Abcam, USA), NLRP3 (ab4207, Abcam, USA), NF-κB (8242, Cell Signaling Technology, USA), MyD88 (4283, Cell Signaling Technology, USA), ASC (67824, Cell Signaling Technology, USA), IL-1β (ab9722, Abcam, USA) and GAPHD (AG019, Byotime Biotechnology, China) diluted in primary antibody dilution buffer (Byotime Biotechnology, China). Following a PBS wash, the PVDF membrane underwent incubation with secondary antibodies conjugated to HRP (SA00001-1, SA00001-2, Proteintech, China) for 2 hours. The chemiluminescence detection system (Image 600, GE Healthcare, Chicago, IL, USA) was employed to detect protein bands. Subsequently, protein grayscale analysis was conducted using the Image Quant TL software (GE Healthcare, Chicago, IL, USA).

Statistical analysis

Statistical analysis was conducted using GraphPad Prism 8.4.2 (GraphPad Software). The data are presented as the mean ± SEM or SD, derived from a minimum of three independent experiments. Student’s t-test was utilized for comparing two groups, while analysis of variance (ANOVA) was employed for comparisons among multiple groups. Post hoc analysis using Tukey’s test was performed after ANOVA. Experiments with two factors underwent analysis with a two-way ANOVA. A significance threshold of P < 0.05 was applied for all tests.

Noise exposure induces permanent hearing loss and insults to hair cells.

In the present study, WT, Cx3cr1^GFP/+, and Ccr2^RFP/+ Cx3cr1^GFP/+ mice were exposed to white noise at 110 dB SPL for 3h. Such high-level noise exposure was reported to induce a permanent threshold shift in CBA/CaJ and C57BL/6 mice [1, 10, 14–16]. As shown in Fig. 1A, an average of 40 dB of the free-field ABR threshold shift in 16 kHz was observed at 1 day (D1) and 7 days (D7) post-NE for animals, suggesting severe hearing loss in both WT and reporter mice. The monaural ABR measurements in WT mice showed that the threshold lifting lasted for at least 14 days after exposure at all frequencies and clicks examined (Fig. 1B), indicating it should be a permanent hearing loss in our experimental design. More evidence comes from the morphological examination of cochlear sensory hair cells. Compared with the control group, the mice in D14 showed a significant loss of outer hair cells (OHCs) at apical, middle, and basal turns of the cochlea, whereas the inner hair cells (IHCs) showed no significant decrease (Fig. 1C and D). However, the severe hearing loss in our experiment may not be attributed to such mild loss in OHCs completely. Therefore, we also examined the ribbon synapses that transfer signals from IHCs to SGNs. As shown by the immunostaining of pre- (CtBP2) and post-synaptic (GluR2) puncta of ribbon synapses, the density of ribbon synapses was significantly decreased at all cochlear locations compared with the controls (Fig. 1E and F). These data indicate that insults of hair cells, especially the loss of ribbon synapses, are responsible for the NIHL in our experiment.

No infiltration of peripheral monocytes into the cochlea after noise exposure.

To distinguish the origin of cochlear macrophage populations after noise exposure, we used the Ccr2 ^RFP/+Cx3cr1^GFP/+ dual-reporter mice in our experiment. In this mouse model, the CCR2-positive peripheral monocytes are labeled with red fluoresce (CCR2-RFP), while the CX3CR1-positive tissue-resident macrophages are labeled with green fluoresce (CX3CR1-GFP). This mouse model has been widely used for tracking monocytes recruitment in the inflammation response in brain injury [9, 17, 18]. Under control conditions, as shown by the cochlear cross-section, CX3CR1-GFP cells were found in multiple regions, including the Rosenthal's canal (SGNs), basilar membrane (BM), spiral ligament (SL), and stria vascularis (SV). CCR2-RFP cells were only found sporadically among SGNs and SL (Fig. 2A). As shown by the representative images of a cochlea at D7, NE increased the number of CX3CR1-GFP cells. However, CCR2-RFP cells were still rarely observed after NE (Fig. 2A). The dynamics of the CX3CR1-GFP and CCR2-RFP cell abundance in SGNs, SV, and SL regions are shown in Fig. 2B. As CX3CR1-GFP cells were increased in all regions, no significant increase of CCR2-RFP cells was detected at different times after NE. To examine the integrity of the intrastrial fluid–blood barrier, the EP was measured at different times after NE. A ~ 40% reduction in EP value was recorded immediately after NE, and then fully recovered to control value within 24 hours (Fig. 2C). This drop in EP indicates the temporary breakdown of the intrastrial fluid–blood barrier, which is essential for hearing function and cochlear immune homeostasis. The loss of the integrity of the intrastrial fluid–blood barrier may result in the leakage of molecules and cells from the capillaries. Interestingly, our data showed that no peripheral monocytes were allowed to infiltrate into the cochlear at any time after NE, even during this period.

The macrophages were further examined in the whole mounts of the BM, where pathologies were found in hair cells after NE. As shown by the representative images in Fig. 2D, compared with the control condition, abundant CX3CR1-GFP cells were observed after NE, especially in the region of hair cells where CX3CR1-GFP cells were rarely present in a steady state. Consistent with other regions, little CCR2-RFP cells under control conditions and no increase of their abundance after NE were found in our experiments (data of D3 and D7 were not shown). These results confirmed that peripheral monocytes were not infiltrated into the cochlear, suggesting that infiltrated macrophages were not implicated in the response to noise trauma.

Resident macrophages are activated after noise exposure.

A previous study showed that depletion of CCR2 may promote the loss of hair cells after NE in the CX3CR1^GFP/+ mouse strain [16]. Although our experiments have shown no difference in the noise-induced ABR shift between the Ccr2 ^RFP/+Cx3cr1^GFP/+ mice and the WT controls (Fig. 1A), the impact of CCR2 gene manipulation on the CX3CR1 positive macrophages is uncertain. Therefore, instead, the activity of tissue-resident macrophages in the BM was examined in the CX3CR1^GFP/+ mouse strain, in which the tissue-resident macrophages are labeled with green fluorescence of the CX3CR1 biomarker.

Under control conditions, CX3CR1-GFP cells were distributed near the modiolus side along the entire BM, while only a few CX3CR1-GFP cells were located in the region of hair cells (Fig. 3A). These CX3CR1-GFP cells were stellate in shape with a slender body and multiple long dendritic processes. After NE, these cells showed amoeboid morphology with shorter and less ramified processes. Such change in morphology implies immune activation of CX3CR1-GFP macrophages after NE. To quantify the noise-induced changes in abundance and distribution, the numbers of CX3CR1-GFP cells in the different regions of the BM are shown in Fig. 3B. A dramatic increase of CX3CR1-GFP cells was observed in the region of hair cells (-100 ~ 0 µm and 0 ~ 100 µm from the IHCs) at all cochlear locations, while a significant increase was also found in most other regions of the BM.

Toll-like receptor 4 (TLR4) is a cellular receptor of inflammatory factors and endogenous molecules of damaged cellular debris. Upon the binding of these ligands, TLR4 activates macrophages and the innate immune system through the NF-κB signaling pathway [19–21]. In activated macrophages, the NLRP3 inflammasome pathway is usually associated with damaging inflammatory processes and apoptosis. Both signaling pathways have been documented to modulate the insults of cochlear sensory cells [22, 23]. However, the interaction of these signaling pathways and their roles remained unknown in macrophages after NE. In agreement with previous studies [24, 25], we found that TLR4/NF-κB and NLRP3 inflammasome pathways were expressed in cochlear macrophages and activated by NE (Additional file 1: Fig. S1). The up-regulation of NF-κB and expression of IL-1β suggests that cochlear macrophages were activated and may play a pro-inflammatory role after NE. To further confirm the effect of these pathways on the activation of cochlear macrophages, TAK-242, the TLR4 inhibitor was introduced into the cochlea through the trans-tympanic route without detriment to hearing function (Additional file 1: Fig. S2). Under the administration of TAK-242, the expression levels of TLR4, MyD88, and NF-κB p65 were decreased significantly, indicating that the noise-up-regulated TLR4 pathway was inhibited. Accompanied, the activation of the NLRP3 pathway was also inhibited as reflected by a remarkable reduction of NLRP3, ASC, and IL-1β (Fig. 4A). Interestingly, the increase of CX3CR1-GFP cells and their redistribution in the BM were also abolished by TAK-242 (Fig. 4B and C). These results, together with the changes in the morphology, abundance, and distribution, indicated that cochlear resident macrophages were activated and switched to a pro-inflammatory phenotype by NE.

TAK-242 attenuates ribbon synapse reduction and hearing loss in noise trauma.

To determine the roles of activated macrophages in NIHL, TAK-242 was applied to inhibit the activation of cochlear macrophages via the TLR4-NLRP3 pathway. As shown in Fig. 5, the noise-induced loss of CtBP2 puncta and paired ribbon synapses were significantly reduced in the IHCs treated with TAK-242. Considering the insult of ribbon synapse was the major pathology of noise trauma for hair cells in our experiment, this result suggests that the inhibition of macrophage activation may have a protective effect in noise-exposed cochlea. This conclusion was further confirmed by the in vivo measurements of hearing function. Immediately after NE, one ear of the animal was trans-tympanic treated with TAK-242 while the other ear was treated with the vector (Fig. 6A). For the vector controls, monaural ABR thresholds were elevated significantly one day after NE (D1). Such NIHL was persistent, without any recovery observed at D7 and D14 (Fig. 6B). For those ears treated with TAK-242, although NE induced comparative acute hearing loss at D1, the ABR thresholds were recovered partially at most frequencies tested at D7 and D14 (Fig. 6B and 6C). The recovery of hearing function was accompanied by our morphological finding, indicating that activated macrophages promote the insults of hair cells and contribute to permanent hearing loss in noise trauma.

Our experiments show that the abundance, distribution, and morphology of cochlear resident macrophages are dramatically changed by high-level NE. Together with these changes, the noise-induced macrophage activation was further confirmed by the up-regulated TLR4-NLRP3 pathway. Meanwhile, no conspicuous presence of infiltrated macrophages under control conditions or distinct changes in this population was observed after NE in the cochlea. Through the TLR4-NLRP3 pathway, the inhibition of resident macrophage activity alleviates the trauma of cochlear hair cells and the accompanying hearing loss after NE. Our finding in this study reveals that the activity of tissue-resident macrophages, but not infiltrated macrophages, contributes to the insults of hair cells in NIHL.

Macrophages are found present in the cochlea of humans and mice [9, 26, 27]. Derived from the yolk sac and the fetal liver, these macrophages are long persisting in the cochlea from the embryonic stage [28, 29]. Consistent with existing reports [9], our results reveal that macrophages reside in the Rosenthal's canal (SGN), organ of Corti, and lateral wall of the adult cochlea (Figs. 2 and 3). In the stria vascularis (SV), processes of perivascular-resident macrophage-like melanocytes (PVM/Ms) contact with endothelial cells and ensheath strial capillaries, which is essential for forming of BLB and maintaining the EP [30]. The integrity of the intrastrial fluid–blood barrier prevents the leakage of molecules and cells from the blood vessels, implying that tissue-resident macrophages are the major population in healthy cochlea. This opinion is supported by our result that most cochlear macrophages are CX3CR1 positive before NE (Fig. 2). Under steady state, resident macrophages contribute to the maintenance of immune homeostasis with a relatively slow renewal through the infiltration of bone marrow-derived monocytes to the cochlea [3, 31, 32]. This may explain the origin of sporadic CCR2-positive macrophages observed in the cochlea under control conditions (Fig. 2).

NE was widely reported to cause temporary (reversible) or permanent threshold shifts of hearing, depending on the intensity and duration of NE used in different studies. In the present study, high-level noise exposure was designed to induce acute and irreversible hearing loss, evidenced by the loss of hair cells and the trauma of ribbon synapse (Fig. 1). Our data show that noise increased the number of macrophages in the regions of SGN, basilar membrane, SV, and SL (Fig. 2). The change in macrophage number is accompanied by its re-distribution in the basilar membrane, particularly in the region adjacent to sensory hair cells (Fig. 3). Interestingly, the changes in the distribution and number of cochlear macrophages were carried out by CX3CR1 labeled cells, indicating that cochlear resident macrophages were the major population in response to noise trauma. One possible approach to of circulation macrophages infiltrated in the cochlea is through the insulted intrastrial fluid–blood barrier after NE. Activated by NE, PVM/Ms detached from capillary walls, resulting in the increased permeability of the blood-labyrinth barrier and the drop in EP [33]. Our experiments show that EP decreased immediately and recovered fully one day after NE, suggesting that the loss of integrity of the blood-labyrinth barrier is transient. During this short period, no sign of CCR2 labeled macrophage was detected in the cochlea, and circulation macrophages were not likely infiltrated in the cochlea through the integral BLB thereafter.

However, a transient increase in the permeability of molecules from the blood vessels is possible to initiate the immune response of cochlear macrophages. Besides the changes in distribution and abundance, the distinction between macrophage morphology before and after NE provides more evidence for the activation of cochlear macrophages. As previously reported, our data show that under homeostatic conditions, cochlear macrophages have branched morphology with ramified long processes [9]. However, after NE, macrophages acquired a small and rounded amoeboid shape with less ramified extensions (Fig. 3). As an indicator of activated immune state, this change in morphology indicates the activation of macrophages after NE [5, 27, 34, 35]. This conclusion is also supported by the up-regulation of TLR4 signaling, which has been reported to modulate the cochlear immune response after NE [22].

TLR4 has been known expressed in cochlear macrophages and implicated in acoustic trauma [22]. Interacting with its ligands leaked from the temporally break-down BLB, or endogenous molecules of damaged cellular debris, TLR4 can activate cochlear macrophages through the NF-κB signaling pathway [19–21]. Although the TLR4 signaling was confirmed to be associated with the activation of macrophages in our experiment, the role of activated cochlear macrophages in acoustic injury still needs to be determined. By producing inflammatory molecules and recruiting more immune cells, TLR4 signaling-activated macrophages have been documented to be implicated in the loss of hearing and hair cells after acoustic injury [22]. In a lower-level NE mouse model, however, the hearing was reported to fully recover without any hair cell loss, even though cochlear macrophages were activated comparable [35]. Meanwhile, activated macrophages are also linked to the protective and reparative processes after acoustic injury [16, 36]. These contradictory results may be due to the difference in the experimental design and animal models used in these studies. Our experiments show the up-regulation of NLRP3 signaling, which mediates the pro-inflammatory responses in activated macrophages [37, 38], is associated with the noise-induced TLR4 activity (Fig. 4, Supplementary Fig. 1). Blocking of the TLR4 activity inhibited the activation and re-distribution of cochlear resident macrophages, resulting in less trauma in hair cells and a partial reversal of hearing loss (Figs. 5 and 6, Supplementary Fig. 2). Our results confirm that the activity of cochlear macrophages plays a pro-inflammatory role and aggravates the insults of hair cells in acoustic injury. Beyond the inflammation-induced cell damage, another possible mechanism of the activated macrophages aggravating cell damage is the phagocytic function. For example, serving as macrophage in the central neural system, microglia in the brain have been found to phagocytose cell debris and redundant synapses in Alzheimer's disease [39, 40]. Activated cochlear macrophages may play a similar role in noise-induced ribbon synapse loss. The effects of NE on the cochlear macrophage and their impacts on tissue pathogenesis are summarized in Fig. 7.

The portion of hearing loss cannot be eliminated by the inhibition of macrophage activity revealing that other mechanisms are involved in the noise-induced trauma. For example, the violent mechanical force of vibration peels hair bundles off from the hair cells [41]. This cellular debris would initiate the activation of cochlear macrophage via the TLR4- NLRP3 pathway. Meanwhile, without the implication of macrophage activity, loss of hair bundles leads to the dysfunction of hair cells immediately and apoptosis of insulted cells in two weeks [42]. Other effectors besides cochlear immune responses, including ROS-mediated cochlear cell apoptosis and autophagy, are well-documented to contribute the acute insults to the cochlea after NE [43–45]. In conclusion, our results indicate that cochlear resident macrophages are activated and contribute to the insults of hair cells in NIHL.

Our findings reveal that cochlear tissue-resident macrophages, but not infiltrated peripheral monocytes, are implicated in the responses to NE. Cochlear macrophages are activated by NE through the TLR4-NLRP3 signaling pathway, and promote the insults to cochlear hair cells in NIHL.

BLB: Blood-labyrinth barriers; NE: Noise exposure; NIHL: Noise-induced hearing loss; SGNs: Spiral ganglion neurons; ABR: Auditory brainstem response; SPL: Sound pressure level; EP: Endocochlear potential; OHCs: Outer hair cells; IHCs: Inner hair cells; BM: Basilar membrane; SL: Spiral ligament; SV: Stria vascularis; TLR4: Toll-like receptor 4; NLRP3: NOD-, LRR- and pyrin domain-containing 3; PVM/Ms: perivascular-resident macrophage-like melanocytes.

Acknowledgments

We thank Dr. Fengxian Li for generous help in mouse models and in reviewing the manuscript.

Author contributions

HZ and JT designed experiments and revised the manuscript. JP conducted the experiments, analyzed the data, and wrote the manuscript. KW, JQ, DC, AC, and YY conducted the experiments. All authors read and approved the final manuscript.

Funding

This study was supported by the "Technology Innovation 2030-Major Projects" on brain science and brain-like computing of the Ministry of Science and Technology of China (2021ZD0202603), the National Key Research and Development Program of China (2022YFC2402700), the National Natural Science Foundation of China (82271156), the Guangdong Basic and Applied Basic Research Foundation (2022A1515012036, 2023A1515012557).

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Ethics approval and consent to participate

Consent for publication

Not applicable.

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Activated tissue-resident macrophages contribute to hair cell insults in noise-induced hearing loss

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Abstract

Background

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Introduction

Materials and Methods

Subjects

Auditory brainstem response (ABR) measurements

Noise exposure

Trans-tympanic Administration

Endocochlear potential (EP) recording

Histology and Immunostaining

Immunoblot Analysis

Statistical analysis

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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