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.

Introduction

The World Health Organization (WHO) has reported that 360 million people, more than 5% of the world’s population, suffer from disabling hearing loss [1]. Hearing loss is classified into two types; namely, conductive hearing loss and sensorineural hearing loss (SHL), the latter of which is the main type of hearing disability [2]. SHL is mainly caused by damage to cochlear hair cells (HCs), which function as audiosensory receptors [3]. Although the aetiology of SHL has not been identified, it has been suggested that viral infections such as cytomegalovirus (CMV), rubella, mumps, measles and herpes simplex virus (HSV) can cause it, especially with sudden SHL (SSHL), which usually develops in one ear within 72 h of infection [4,5]. Systemic corticosteroid administration is the primary treatment of choice for SSHL [6]. However, after corticosteroid treatment, hearing improvement is achieved in only 50% of patients, with 20% of them showing no change in hearing ability [7]. Because very little is known about the mechanisms underlying this disease and the anti-infection protection system in the inner ear, corticosteroids are still used despite their limited efficacies.

The inner ear is considered an immune-privileged site because the blood-labyrinthine barrier prevents access to it by the peripheral immune system [8]. The central nervous system, sensory organs, and gonads are separated from peripheral blood containing immune cells by a physical barrier to minimize any collateral tissue damage caused by an immune reaction [9]. It has been shown that lymphocytes reside only in the endolymphatic sac and are unlikely to participate in responses to pathogens [10], suggesting the innate immune system might be involved in the HC defence mechanism against pathogens. One of the earliest innate antiviral defence mechanisms is the type I interferon (IFN) system, and we have previously found that viral infection results in IFN-α/β production using the cochlear sensory epithelium isolated from newborn mice [11]. More recently, we have found that cochlear supporting cells (SCs), consisting of Hensen’s cells, Claudius’ cells, and greater epithelial ridge (GER or Kölliker’s organ) cells (GERCs) in the neonatal immature inner ear, function as macrophage-like cells and protect adjacent HCs from pathogens [12].

Macrophages are found in all tissues and have roles in development, homeostasis, tissue repair and immunity, and disruption of their repair and homeostatic functions can cause many disease states including metabolic disease and cancer [13]. Moreover, blocking agents, such as monoclonal antibodies or cytokine antagonists of cytokine activity, especially those produced early in the inflammatory cascade such as tumour necrosis factor (TNF)-α and interleukin (IL)-6, are used for treating several inflammatory diseases, including rheumatoid arthritis and inflammatory bowel disease [14]. In the central nervous system, tissue resident macrophages (microglial cells) are considered to be a causative factor in brain diseases, including inflammatory diseases such as multiple sclerosis, degenerative diseases such as Alzheimer’s disease (AD) or Parkinson’s disease (PD), and psychiatric disorders such as depression or schizophrenia, and as such they are a potential target for the treatment of these diseases [15,16]. In relation to these findings, we found that cochlear SCs function similarly to tissue resident macrophages that protect HCs from pathogens [12]. Therefore, understanding the role(s) played by macrophages in infection and inflammation and their causative role(s) in disorder of their associated tissues is considered to be important for the successful treatment of various diseases.

In the present study, we investigated the effects of viral infection in the isolated murine newborn cochlear sensory epithelium, and found that virus-infected SCs and GERCs produced TNF-related apoptosis-inducing ligand (TRAIL), and TRAIL induced HC death by necroptosis. In addition, a necroptosis inhibitor efficiently suppressed the virus-induced HC death. These results suggest the mechanism underlying virus-induced SHL, and provide a potential target for treatment strategies against SHL.

Materials and methods

Experimental animals

Postnatal day 2 (sex: unknown) ICR mice (SLC), interleukin 6 (Il6) null mice (Jackson Laboratory) and interferon (alpha and beta) receptor 1 (Ifnar1) null mice (B&K Universal) were used in this study. The animal experiment protocol was approved by the Ethics Committee on Animal Experiments of Nippon Medical School. It was carried out in accordance with the guidelines for Animal Experiments of Nippon Medical School and the guidelines of The Law and Notification of the Government of Japan, as well as the ARRIVE guidelines. Mice were maintained 12 hour light/12 hour dark cycle at 20–24°C with 40–70% humidity. They were allowed to have free access to standard laboratory mouse chow, MF (Oriental Yeast Co., ltd. Tokyo, Japan), and free access to drinking water. They were housed at a maximum number of five. All mice were checked for stress each day. Mice were euthanized by cervical dislocation for further experiments.

Preparation and treatment of cochlear sensory epithelium explant cultures

Cochlear sensory epithelia were resected and cultured as previously described [11]. All experiments began after an overnight incubation of each cochlea with 300 μl medium [Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich Inc., St. Louis, MO, USA) supplemented with D-glucose (6 g/l) and penicillin G (150 μg/ml)] for explant cultures. Each cochlea was then transferred to 400 μl medium containing 3.0 × 107 pfu/ml TMEV, which is an RNA virus. TMEV (GDVII strain) was propagated from viral cDNA and BHK21 cells [17]. Cultures were maintained for 9–12 h until TMEV began to infect the SCs. At 16–21 h, TMEV began to infect the GERCs and HC death was observed. HC death was almost completed by 24 h. To analyze the paracrine influence of SC/GERC-secreted tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), the TMEV-infected cochleae were incubated in rat anti-TRAIL antibody (0.01 mg/ml, GeneTex, clone N2B2) for 24 h. Phosphorylated mixed lineage kinase domain-like (p-Mlkl) expression in HCs suggested necroptosis, therefore necroptosis inhibitors such as necrostatin-1 (Abcam) or ponatinib (Selleckchem) were used at the specified concentrations for up to 24 h. Steroids such as dexamethasone are primarily used to treat SSHL in the clinic, although its effectiveness is limited and the mechanisms of action remain poorly understood. To compare the effectiveness of steroids with necrostatin-1 and to determine the unidentified mechanisms of steroids, 1.0 μM dexamethasone (Wako) was added to medium containing TMEV and cultivated for up to 24 h. Clophosome™ (91 μg/ml for up to 24 h; Funakoshi), which is a liposome-clodronate reagent, was used to deplete activated SCs and GERCs as macrophages to determine whether these macrophage-like cells injured HCs.

Immunohistochemistry

For whole mount immunohistochemistry, samples were fixed at room temperature (RT) for 15 min in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and then rinsed with PBS. All specimens were incubated in blocking solution at RT for 30 min in 10% goat serum with 0.2% Triton X-100 for all antibodies except the anti-cleaved caspase 3 antibody or for 15 min in 0.2% Triton X-100 and 15 min in 1% BSA in 0.2% Triton X-100 when colabelled with cleaved caspase 3. The primary antibodies used in this study were as follows: rabbit polyclonal anti-Myosin VIIA (Myo7a) (25–6790; 1:1000, Proteus Biosciences), mouse monoclonal anti-double-stranded RNA (dsRNA) (J2; 1:800, K1; 1:2000, English & Scientific Consulting), rabbit polyclonal anti-cleaved caspase 3 (#9664; 1:100, Cell Signaling), rabbit polyclonal anti-DR4 (GTX28414; 1:1000, GeneTex), rabbit polyclonal anti-DR5 (ab8416; 5 μg/ml, Abcam), and rabbit monoclonal anti-p-Mlkl (phospho S345) [EPR9515(2); 1:1000, Abcam]. Actin filaments were visualized with Alexa 594- or 633-labelled phalloidin (1:100, Invitrogen). The primary antibodies were visualised with Alexa 488- or 546-conjugated anti-rabbit or anti-mouse IgGs (1:1000, Invitrogen). Samples with nuclear staining were incubated in PBS containing 1 μg/ml DAPI (Invitrogen). Fluorescence images were captured under an FV1200 confocal microscope (Olympus). Computational section images were reconstructed by FV10-ASW (Olympus) after capturing images every 0.2–0.5 μm. Whole mount images of HCs were obtained by superposing images from the bottom to the top of HCs after capturing images every 0.5 μm. Whole mount images of virally infected, activated SCs and GERCs were obtained from single slices or several slices overlapped at the SC or GERC level after capturing images every 0.5 μm.

Cell counts

The numbers of inner and outer HCs in the sensory epithelium were counted along a 100-μm longitudinal distance in the basal- to mid-turn of each explant. As described above, we estimated from the basal- to mid-turn of the cochlea, because Hensen’s cells and Claudius’ cells in the apical turn overlapped with the inner side of the basal-turn. HCs with remaining stereocilia were counted as live HCs, and stereocilia were identified with phalloidin, which detects actin filaments.

Electron microscopy

Electron microscopy analysis was performed as described previously [18,19], with slight modification. Mouse inner ears were observed by transmission electron microscopy (TEM). The specimens were fixed with 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 60 min, washed five times in 0.1 M phosphate buffer, and post-fixed with 1% osmium tetroxide for 60 min at 4°C. The fixed inner ears were dehydrated with a graded ethanol series, and embedded in EponA2 according to the conventional method. Thin sections were cut with a diamond knife, stained with uranyl acetate and lead citrate, and examined with a JEM-1010 transmission electron microscope (JEOL) at an accelerating voltage of 80 kV.

RNA extraction, qRT-PCR, and microarray

For qRT-PCR, an RNeasy Micro Kit (Qiagen) was used to extract total RNA from three cochlear sensory epithelia that were cultivated under the following conditions for 16 h: DMEM alone; DMEM with TMEV; DMEM with TMEV and dexamethasone; DMEM with TMEV and Clophosome™; DMEM with LPS. cDNA was synthesized from DNase-treated total RNA using a PrimeScript RT reagent Kit (Takara Bio). Synthesized cDNA was subsequently mixed with TaqMan Universal PCR Master Mix (Applied Biosystems) in the presence of commercial TaqMan primer-probe sets of interest (Applied Biosystems). Real-time PCR quantification was performed using the ABI StepOnePlus Real-Time PCR System (Applied Biosystems). All reactions were performed in triplicate. Relative mRNA levels were calculated using the ΔΔCt method. For the invariant control, we used actin beta (Actb). For microarray, total RNA was extracted from nine mock explants, nine explants treated with 1000 ng/ml LPS for 9 or 16 h, and 14 explants infected with TMEV for 9 or 16 h using the RNeasy Micro Kit. Filgen (a biological technical service company) performed the microarray analysis using a GeneChip Mouse Gene 2.0 ST Array (Affymetrix). These data have been uploaded to the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) under accession code GEO: GSE89556.

Statistical analysis

The data are expressed as the mean ± standard error. One-way ANOVA was used for the HC counts in necrostatin-1 and ponatinib assays or Ifnar1 KO, Il6 KO, and LPS assays. P-values < 0.05 were considered statistically significant. Post hoc tests were performed using the Bonferroni method. P-values < 0.005 (necrostatin-1 assay) 0.0167 (ponatinib assay) or 0.0033 (Ifnar1 KO, Il6 KO, and LPS assays) were considered statistically significant. In the other experiments for which HC counts were needed, statistical analyses were performed by unpaired t-tests, in which P-values < 0.05 were considered statistically significant. In all qRT-PCR analyses, unpaired t-tests were used, in which P-values < 0.05 were considered statistically significant.

Results

Virus-infected SCs and GERCs induce HC death

We previously found that Theiler’s murine encephalomyelitis virus (TMEV) infection in the isolated murine newborn cochlear sensory epithelium induces IFN-α/β production [11]. TMEV is a small RNA picornavirus commonly used as an experimental model system for blood-brain barrier disruption [20]. Recently, we observed that TMEV infection is mainly established in SCs and HC infection is rarely observed in the presence of IFN-α/β produced by SCs that function as macrophage-like cells [12]. We also observed that SC infection is established in the early stage of TMEV infection (9 h after virus infection) and GERC infection is established in the later stage (16 h after virus infection) [12]. To understand the influence of virus infection in SCs on HCs, we analyzed the cell status of HCs using the same experimental system. It has been reported that TMEV infects macrophages [21]. Indeed, we have observed that TMEV infected almost all SCs and GERCs in our experimental system [12]. Therefore, although TMEV is not a virus that causes SHL in humans such as CMV, we used this experimental system to investigate the effects of infection with cochlear SCs that are protective cells against virus infection in mice. As a result, we found that HC death occurred at 16 h after a virus infection, despite very few virus-infected HCs being present (Fig 1A). Most HCs died within 24 h of infection (Fig 1B). Next, we analysed the effects of virus-inducible cytokines IFN-α/β [22], which induce cell death in virus-infected cells, and IL6 that has been reported to regulate apoptosis of TMEV-infected cells [23]. As shown in Fig 1C–1E, TMEV-induced HC death was not affected in IFN-α/β receptor 1 (Ifnar1) and IL6 (Il6) sensory epithelium. Moreover, signals of Toll-like receptors (TLRs), which recognize microbial components, induce apoptosis [24], but lipopolysaccharide (LPS) that activates TLR4 did not induce HC death (Fig 1C–1E). Although the time of infection establishment was different between SCs and GERCs, there were no significant differences between loss of inner HCs (IHCs) and outer HCs (OHCs) (Fig 1E). These results suggest that signal(s) other than virus components and virus-inducible cytokines IFN-α/β and IL6 induce HC death.

Fig 1

Temporal analysis of HC death following viral infection.

(A) At 16 h after TMEV infection of the cochlear sensory epithelium, HC death was initiated in spite of the presence of very few virus-infected HCs [*P < 0.05, t-test, Mock: n = 5, TMEV: n = 4, outer HCs (OHCs), inner HCs (IHCs)]. Even at 18 h after TMEV infection when HC death was progressing, very few dsRNA-positive HCs were observed. At this time point (16 h after TMEV infection), the majority of SCs were lost by TMEV infection, which was confirmed by the computational section (see the uninfected sample). (B) Severe HC damage was observed at 24 h after TMEV infection with migration of dsRNA-positive SCs and GERCs. Most dying HCs were still negative for dsRNA. Migration of virus-infected SCs and GERCs has been reported previously [12]. (C) Ifnar1 KO and Il6 KO mice experienced SC and GERC migration to the HC layer with severe HC damage during the virus infection. (D) LPS treatment did not induce HC death with no migration of SCs and GERCs. (E) IHC and OHC numbers (counted by phalloidin staining) at 24 h of incubation with TMEV or LPS (IHC and OHC: P < 0.0001, ANOVA; *P < 0.0001, Bonferroni). Both IHCs and OHCs were reduced significantly in TMEV-infected WT cochleae (n = 3) compared with WT with mock treatment (n = 6). Even when Ifnar1 (n = 4) or Il6 (n = 3) was deficient in cochlear sensory epithelia, HC death occurred with TMEV infection similarly to that seen in WT mice, which suggested that these cytotoxic cytokines do not induce HC death. Almost all HCs survived when treated with LPS (100 ng/ml: n = 4, 1000 ng/ml: n = 4). HC death during TMEV infection was also confirmed by HC counting based on Myo7a staining (*P < 0.0001, t-test, WT mock: n = 6, WT TMEV: n = 3). These observations indicate that some kinds of secreted proteins, which were secreted when infected with TMEV, except for IFNs and IL6, but not secreted when incubated with LPS, are the cause of HC death. (F) HC stereocilia (red arrowheads) degenerated over time after activation of SCs (black arrowhead) and GERCs as macrophages (TEM; OHC1: First row of OHCs, OHC2: Second row of OHCs, OHC3: Third row of OHCs). Cytoplasmic vacuolisation has been found in many virus-infected cells [25]. (G) qRT-PCR analysis revealed that virus infection (n = 5) was more effective to upregulate Trail expression compared with LPS treatment (n = 3) or mock treatment (n = 6) at 16 h (*P < 0.05, **P < 0.01, ***P < 0.0001, t-test). Scale bars: Immunostaining, 20 μm; TEM, 10 μm. Error bars, standard errors.

To clarify this death signal, we performed cDNA microarray analysis and found that expression of the cell death-inducing ligand Trail was induced by the viral infection, but not LPS (Fig 1G). TRAIL, a TNF superfamily protein, mediates killing of virus-infected cells and is involved in the pathogenesis of multiple virus-induced disorders [26]. It has also been shown that virus infection and IFN-α/β stimulation of immune cells induce expression of TRAIL [27].

TRAIL produced by virus infection induces HC death

TRAIL, a potent stimulator of apoptosis, works by binding to DR4 (also known as TRAILR1) and DR5 (also known as TRAILR2) death receptors [26,28]. Expression of DR4 and DR5 was found in HCs, but rarely in SCs (Fig 2A). We previously found that TMEV-infected SCs and GERCs express macrophage marker proteins and perform phagocytosis, which indicate that SCs and GERCs are macrophage-like cells [12]. It has been shown that TRAIL is induced in virus-infected macrophages [27], and that Trail is a transcriptional target of virus-induced transcription factor interferon regulatory factor 3 [29]. Indeed, in SHIELD (Shared Harvard Inner-ear Laboratory Database [30]), macrophage marker F4/80 and SC marker Sox2 were expressed in SC fractions [GFP(-)] and HC markers Myo7a, prestin, and Pou4f3 were expressed in the HC fraction [GFP(+); S1 Fig]. Moreover, Trail was expressed in SC fractions that were higher than HC fractions [especially at embryonic day 16 and postnatal day 0; S1 Fig]. Additionally, TMEV infections were mainly established in SCs and infections of HCs were rarely observed (Fig 1A). These findings suggest that TRAIL was produced by SCs, which function as macrophages, after TMEV infection. However, it cannot be ruled out that factor(s) produced by virus-infected SCs act on HCs to induce TRAIL. Moreover, The SC fraction is a cell population other than the HC fraction of sensory epithelial cells, which was thought to be absent of immune cells, but it cannot be completely ruled out that this population contains small numbers of macrophages and lymphocytes. However, these findings suggest that TRAIL was produced by SCs that function as macrophages after TEMV infection.

Fig 2

TMEV infection of SCs and GERCs results in TRAIL-mediated HC death.

(A) HC expression of TRAIL receptors DR4 (green) and DR5 (green). (B) TRAIL-neutralizing antibody (5 μg/ml) attenuated HC damage (*P < 0.01, **P < 0.001, ***P < 0.0001, t-test, Mock: n = 6, TMEV: n = 4, + anti-TRAIL antibody: n = 3). While HCs were protected by anti-TRAIL antibody during TMEV infection, the death of SCs was not attenuated, indicating that the anti-TRAIL antibody has its effect directly on HCs. (C) Stereocilia of HCs was almost intact when treated with recombinant TRAIL protein (6 μg/ml) for 24 h, but long term exposure to recombinant TRAIL protein (48 h) disorganised and deformed the stereocilia. Scale bars, 20 μm. Error bars, standard errors.

As shown in Fig 2B, HC death was efficiently suppressed by a TRAIL-neutralizing antibody. The specificity and neutralizing activity of the used antibody against TRAIL (monoclonal N2B2 antibody) have been shown previously [31,32]. Although it is possible that loss of SCs leads to HC death, loss of IHCs and OHCs was effectively blocked in the presence of the TRAIL-neutralizing antibody in spite of SC loss. In this result, the decrease of IHCs was almost suppressed by the TRAIL-neutralizing antibody, but a decrease of OHCs was slightly observed (Fig 2B). Regarding this difference, it is possible that there was a difference in the local TRAIL concentration and the effect of SC loss, but this has not been clarified at this time. These results indicated that TRAIL was the effector for virus-infected SC- and GERC-induced HC death. However, because four TRAIL receptors, which include DR4 and DR5, have been identified in humans [33], it is not to be elucidated whether only the binding of DR4 and DR5 expressed in HCs is important for HC death by TRAIL. However, recombinant TRAIL protein itself was not sufficient to induce HC death (Fig 2C). Therefore, these results indicated that HC death was triggered by TRAIL and suggested that other factor(s) are required to induce full death. Considering human diseases, it has been shown that the loss of both HCs and SCs occurs in many human SSHL cases [34], which suggests that virus-infected SCs induce HC death in SSHL.

Virus-induced HC death is not caused by apoptosis

Apoptosis is the most commonly observed cell death during a viral infection, and it is considered that host cells eliminate virus-infected cells via apoptosis, which aborts further viral infection [35]. During SC- and GERC-induced HC death, the activation of caspase 3, a mediator of apoptosis, was observed in the SCs and GERCs, but not in the HCs (Fig 3A). This suggests that the viral infection induced apoptosis in the virus-infected SCs, but that SC-mediated HC death is not caused by apoptosis. It has been shown that the effect of aminoglycoside, a drug known to be ototoxic to HCs, can be suppressed by a caspase inhibitor [36], indicating that this type of cell death is caused by apoptosis. However, the mechanism underlying this phenomenon is not fully understood [37]. Therefore, we next analysed the expression of inflammation and macrophage markers in the presence of aminoglycoside; however, the expression of these markers in SCs was not induced by gentamicin, an aminoglycoside antibiotic (Fig 3B). These results suggest that the virus-induced HC death mechanism differs from that induced by ototoxic drugs.

Fig 3

Virus-infected SC- and GERC-induced HC death is not mediated by apoptosis.

(A) Cleaved caspase 3 (green) was expressed in TMEV-infected SCs and GERCs (arrowheads in sections), but not in HCs, which indicated that apoptosis did not occur in HCs. At 20 h after infection (right panel), most SCs had died by apoptosis and infected GERCs had migrated onto the HC layer, as described previously [12]. (B) Tao et al. deposited RNA sequence datasets for sorted HCs (GFP positive) and surrounding cells including SCs (GFP-negative non-HCs) from explant cultures of an Atoh1-GFP organ of Corti treated with gentamicin for 3 h in the NCBI GEO database (GSE66775) [38]. In this database, we focused on macrophage- and inflammation-correlated gene expression changes in a non-HC population including SCs to compare virus infection with aminoglycoside-related injury. Here, we extracted inflammation markers Il6 and Il1b, and macrophage markers F4/80, Mac-1, and Iba1. No significant difference was observed in the expression levels of these genes in the non-HC population, which included SCs, between the control (n = 3) and gentamicin-treated group (n = 3). Scale bars, 20 μm.

Virus-induced death of HCs is mediated by TRAIL-induced necroptosis

Programmed cell death plays a fundamental role in animal development and tissue homeostasis, and is regulated by a variety of mechanisms such as apoptosis, necroptosis, ferroptosis and pyroptosis, among others [39]. Among these cell deaths, we found that addition of necrostatin-1 [40] and ponatinib [41] necroptosis inhibitors efficiently supressed SC- and GERC-induced HC death (Fig 4A and 4B). Indeed, in addition to expression of apoptosis-related genes, expression of necroptosis-related genes was induced in TMEV-infected cochlear sensory epithelium (S2 Fig). Moreover, although necrostatin-1 effectively suppressed HC loss, there was no suppression of SC loss (Fig 4A). This result suggests that the necroptosis inhibitor affected HC death, but did not inhibit SC death, which was caused by apoptosis (Fig 3A). Moreover, it has been suggested that reactive oxygen species (ROS) induce HC death [42]. However, genes involved in ROS production, such as Nox1-4, Tp53, Ptgs2 (prostaglandin-endoperoxide synthase 2), and Tnfa (tumour necrosis factor-α), showed no marked induction similar to apoptosis- and necroptosis-related genes by virus infection. Furthermore, the SC- and GERC-induced phosphorylation of the necroptosis-regulator (mixed lineage kinase domain-like, Mlkl) [43], which was also observed in the HCs, was blocked by necrostatin-1 treatment (Fig 4C). Moreover, it has been shown that TRAIL induces necroptosis under some conditions [44]. Therefore, these results suggest that SC- and GERC-induced HC death was caused by necroptosis [40]. We quantified HC death by the number of HCs along a longitudinal distance of 100 μm. However, TMEV treatment induced SC death, which resulted in disorganization of cochlear tissues. Therefore, it is possible that virus-induced disorganization led to the appearance of lower numbers of hair cells when counted in the same width. However, as shown in Fig 4A, necrostatin treatment, especially 400 mm treatment, significantly inhibited loss of HC numbers (HC number was not markedly reduced compared with that observed without virus infection shown in Fig 2B), whereas most SCs were lost. To more accurately assess HC death, we believe that the effects of disorganization in cochlear tissues need further analysis.

Fig 4

Necroptosis inhibitors supress HC damage.

(A, B) Necroptosis inhibitors necrostatin-1 (A) (IHC and OHC: P < 0.0001, ANOVA, 400 μM: n = 3, 200 μM: n = 3, 20 μM: n = 3, 0 μM: n = 4) and ponatinib (B) (IHC: P < 0.0001, OHC: P = 0.0021, ANOVA, 5 μM: n = 3, 0.5 μM: n = 4, 0 μM: n = 4) both attenuated HC death (*P < 0.001, **P < 0.0001, Bonferroni). While HCs were protected by Necrostatin-1 during TMEV infection, the death of SCs was not diminished, demonstrating that Necrostatin-1 functions directly to HCs as well as the TRAIL-neutralizing antibody. (C) p-Mlkl (green) expression in HCs induced by TMEV infection was inhibited by necrostatin-1. Thus, SCs and GERCs both induced HC necroptosis via the TRAIL-death receptor-signalling cascade. *P < 0.001, t-test, mock: n = 3, TMEV: n = 3. Scale bars, 20 μm. Error bars, standard errors.

Prevention of viral infection-induced HC death by targeting the macrophage functions of SCs and GERCs

Currently, corticosteroids are the primary effective therapeutic agents for SSHL [45]. To understand the role played by corticosteroids in virus infection-induced HC death, we investigated whether dexamethasone administration would result in more numbers of HCs in TMEV infections. In fact, dexamethasone suppressed virus infection-induced HC death (Fig 5A). Macrophage marker expression was also inhibited in the SCs and GERCs (Fig 5B), suggesting that corticosteroids inhibit virus-induced HC death by inhibiting virus-induced macrophage changes in SCs and GERCs. Conversely, IFN-α/β expression was unaffected, indicating the therapeutic benefit of corticosteroids in virus-induced SHL without suppressing the antiviral effect of IFN-α/β (Fig 5C). The same effect was also observed with Clophosome™ (Clopho), which contains macrophage-depleting anionic lipids [46] (Fig 5D and 5E). Therefore, these results suggest that in addition to necroptosis, macrophages are a candidate target for the prevention of virus-induced SSHL.

Fig 5

Targeting the macrophage functions of SCs and GERCs supresses HC damage.

(A–C) Corticosteroid dexamethasone (Dex) inhibited HC damage. (A) (*P < 0.05, **P < 0.0001, t-test; TMEV + Nec (necrostatin-1): n = 3, TMEV + Dex: n = 3, TMEV: n = 4). Dex downregulated macrophage marker expression (B) (Mock: n = 3, TMEV: n = 4, TMEV + Dex: n = 5), but did not downregulate type I IFN expression at 16 h (C) (Mock: n = 5, TMEV: n = 5, TMEV + Dex: n = 5) (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, t-test). The mechanism underlying the ability of Dex to treat SHL involves downregulation of SC and GERC macrophage functions in spite of type I IFN expression not being suppressed, which subsequently protects HCs from viral infection. (D, E) SC depletion following Clopho treatment leads to HC survival during virus infection. Clopho administration removed activated SCs as macrophages, thereby attenuating HC damage during TMEV infection (D) (*P < 0.01, **P < 0.0001, t-test, TMEV: n = 4, TMEV + Clopho: n = 3). Clopho administration to TMEV-infected explants downregulated macrophage marker expression (E) (16 h; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, t-test, Mock: n = 3, TMEV: n = 4, TMEV + Clopho: n = 6). (F) A diagram indicating the mechanism of HC death by SCs activated as macrophage-like cells and HC protection by the glucocorticoid, macrophage-depleting agent, and necroptosis inhibitor. Scale bars = 20 μm. Error bars, standard errors.

Discussion

We recently investigated the defence mechanism used by audiosensory receptor HCs against viral and bacterial infections, and found that the SCs and GERCs surrounding the HCs function like macrophages and protect these cells [12]. In the present study, we found that when excess virus infected the SCs and GERCs, the SCs changed from “guardians” to “aggressors” and caused HC death. It has been widely shown that inflammatory macrophages are initially beneficial to the body because they facilitate the clearance of invading organisms; however, they also trigger substantial collateral tissue damage, resulting in various diseases [13]. At present, we still do not know whether the same phenomena also occur in adult humans in response to a viral infection. Because it has been reported that both HCs and SCs are absent in many human SSHL cases [34], it is conceivable that in response to a viral infection virus-induced apoptosis of SCs and TRAIL-induced necroptosis of HCs concurrently occur in the inner ear, a process that may occur in SSHL. It has been shown that macrophages play roles in development, homeostasis, tissue repair and host defences against infection, and also play pathophysiological roles that result in chronic inflammation [13]. Furthermore, it is now well appreciated that many major diseases and conditions (e.g., atherosclerosis, obesity, diabetes and cancer) are associated with chronic inflammation, and that macrophages play a role in them [13,47]. Therefore, macrophage-targeting therapies are in development against such diseases [48,49].

TRAIL, a TNF superfamily member protein, induces apoptosis through the caspase activation pathway [33]. This protein is also known to induce necroptosis under certain conditions, such as acidic pH, depletion of the cellular inhibitor of apoptosis (cIAP) or TNF receptor-associated factor 2 (TRAF2), via the receptor-interacting serine/threonine protein kinase (RIPK) 1 and RIPK3 [33]. It has been shown that necroptosis promotes further cell death and neuroinflammation during the pathogenic processes of several neurodegenerative diseases, including multiple sclerosis, amyotrophic lateral sclerosis (ALS), AD and PD, through the death receptors of TNF superfamily members [50]. In living slices of human brain tissue, TRAIL was found to induce cell death in neurons and glias, suggesting that TRAIL acts as a destructive effector molecule in the human brain [51]. Furthermore, TRAIL is expressed in the brains of patients with AD and is completely absent from the brains of patients without AD [52]. In human multiple sclerosis lesions, and in mouse brains after the induction of experimental autoimmune encephalomyelitis, TRAIL is upregulated, predominantly by activated microglia and invading immune cells [53]. This suggests that TRAIL contributes to pathogenesis in neurological disorders. Our present results show that, as well as in the central nervous system, the TRAIL produced by activated macrophage-like cells is involved in sensory receptor disorders. Additionally, necrostatin-1, a necroptosis inhibitor, has shown efficacy in improving tissue injuries in animal models of diseases ranging from ischemic brain, kidney and heart injuries, to multiple sclerosis, ALS, and AD [54]. However, although several necroptosis inhibitors are effective against inflammation-mediated disorders [40,41], at present, only a few have passed to the clinical testing stage [55]. Further development of clinically useful necrosis inhibitors for the treatment of such diseases is expected.

Although it has been observed rarely, SSHL may occur in patients with COVID-19 (coronavirus disease 2019) [56]. In relation to this, it has been reported that respiratory tract infection by coronavirus, especially SARS (severe acute respiratory syndrome)-coronavirus, causes marked elevation of TRAIL production [57]. At present, corticosteroids are the primary effective therapeutic agents for SSHL [45]. However, the use of corticosteroids for treatment of viral infections delays virus clearance. In the present study, a potential role of macrophage activation in virus-induced SHL was supported by our results showing that corticosteroids inhibited virus-induced activation of macrophage functions in SCs and GERCs. Therefore, it is possible that this effect may be one of the pharmacological actions of corticosteroids in SHL. Our results also suggest that targeting macrophages or necroptosis may be effective for treatment of virus-induced SHL. This notion is supported by the results showing that macrophage depletion by Clopho or necroptosis inhibitors effectively suppressed HC death induced by virus infection. It is now considered that targeting macrophages is a potential therapy for many diseases including inflammatory diseases, metabolic diseases, and cancer [13,58]. Several methods have been developed to induce depletion, reprogramming, or repolarization of macrophages. Among them, nanotechnology-based systems (e.g., liposomes, dendrimers, gold nanoparticles, and polymeric nanoparticles) have been developed as specific delivery systems to the disease site [58]. Therefore, in patients with SHL, it will be important to develop a system that effectively delivers drugs to the inner ear through the blood-labyrinthine barrier. Moreover, it has been considered that necroptosis plays a crucial role in the regulation of various physiological processes and mediates various diseases such as ischemic brain injury, immunological disorders, and cancers. Therefore, development of therapeutic drugs targeting necroptosis has been carried out for various diseases [40,41]. In addition to these studies, our results suggest that necroptosis inhibitors may be effective therapeutic agents for virus-induced SSHL.

In conclusion, our results revealed novel TRAIL-mediated HC death induced by virus infection in cochlear sensory epithelium. Moreover, our results have shown that macrophage-targeting drugs and necroptosis inhibitors effectively protected HCs against virus infection in our ex vivo experimental system using cochlear sensory epithelia isolated from newborn mice. The weakness of our analysis is that it remains unclear whether this mechanism is also mediated by viruses that cause SHL other than TMEV, whether these treatments are also effective for SHL in mouse models, and the detailed induction mechanism of HC death. Moreover, it is unknown whether the same phenomenon seen in the inner ear of newborn mice applies to that of adult mice. However, we believe that our findings may shed new light on therapeutic paradigms for SSHL.

Expression changes of genes correlated with HCs and SCs during development.

Expression changes of genes correlated with HCs and SCs during development. (A–D) Shared Harvard Inner-ear Laboratory Database (SHIELD; https://shield.hms.harvard.edu/index.html) is a resource for RNAseq datasets from HCs (GFP-positive cells) and their surrounding cells including SCs (GFP-negative cells) at E16, P0, P4, and P7. Here, we extracted Trail (A), F4/80 (B), Sox2 (C), and Myo7a, prestin, and Pou4f3 (D) from this database. (A) Trail was expressed in SC fractions higher than HC fractions, especially at E16 and P0. (B) Macrophage marker F4/80 was expressed in SC fractions. (C) SC marker Sox2 was expressed in not only SC fractions, but also HC fractions during the embryonic stage, but the SC fractions maintained Sox2 expression, whereas the HC fractions did not after the postnatal stage. (D) HC markers Myo7a, prestin, and Pou4f3 were expressed in HC fractions.

(TIF)

Click here for additional data file.

Gene Ontology analysis of microarray data showing upregulation of necroptosis- and apoptosis-related genes by TMEV infection.

Gene Ontology analysis of microarray data showing upregulation of necroptosis- and apoptosis-related genes by TMEV infection. We performed microarray analysis of mock cochlear sensory epithelia, LPS-treated cochlear sensory epithelia (9 and 16 h), and TMEV-infected cochlear sensory epithelia (9 and 16 h) and then examined necroptosis-, apoptosis- and ROS-related genes by Gene Ontology analysis. Among necroptosis-related genes, Trail, Tlr3, and Mlkl were upregulated in TMEV-infected cochlear sensory epithelia at 16 h compared with mock- and LPS-treated cochlear sensory epithelia. Apoptosis-related genes, such as Stat1 and Jun, were upregulated in TMEV-infected cochlear sensory epithelia, especially at 16 h compared with mock- and LPS-treated cochlear sensory epithelia. However, ROS-related genes were not upregulated, except for Nox2, in TMEV-infected cochlear sensory epithelia compared with mock- and LPS-treated cochlear sensory epithelia.

(TIF)

Click here for additional data file.

29 Jun 2021

PONE-D-21-18184

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

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Reviewer #1: Sudden hearing loss is very common in clinic and Systemic corticosteroid administration is the primary treatment currently, although not all patients well respond to it, partly due to the poorly understood mechanisms underlying it. To obtain insights toward Sudden hearing loss, Hayashi et al in this study focused on characterizing the mechanisms underlying TMEV infection-caused hair cell death in cochlea. Briefly, each cochlear sample is incubated in medium for overnight before exposing to medium containing TMEV, which is an RNA virus. Three key findings are reported: 1) TRAIL expression in SCs is the key after TMEV infection; 2) Transformation of SCs (or GER/LER cells) into macrophages is another key step for HC death; 3) HC death occurs by necrosis, but not by apoptosis. The study is well-designed and clearly presented.

3 main comments are raised:

1) For the immunostaining images, the key single pannel is necessary. For instance, please show DR4 and DR5 single channel to better visualize their expression. In addition, a section view can be better demonstrate DR4 and 5’s expression in HCs. Furthermore, are DR4 and DR5 expressed in SCs?

2) How about the antibody specificity against TRAIL? Please provide evidence if possible. If the antibody is not specific, neutralizing experiments might have other explainations.

3) I also have a concern regarding whether TRAIL is critical in the process of HC death. In other words, as the authors speculate binding of TRAIL to DR4/5 is the key step, will HCs die in DR4 -/- or DR5 -/-, following TMEV infection? If answer is “ no”, more concerns are raised regarding the importance of binding of TRAIL with DR4 or DR5. The author should alert readers of this or provide further evidence to prove the direct involvement of TRAIL in TMEV induced HC death. Because it is one of the key findings in this study, it cannot be ignored.

Reviewer #2: The manuscript is interesting and presents data that suggest that certain viral infections may affect cochlear supporting cells in such a way that they respond via upregulation of Tnf-related apoptosis inducing ligand (TRAIL) which then binds to TRAIL receptors expressed by cochlear hair cells causing stereocilia degeneration and hair cell death. Some evidence is also presented to suggest that the hair cell death occurs via necroptosis and can be mitigated by treatment with anti-necroptosis agents or by inhibitors of macrophage activity. While the manuscript is largely novel and many of the findings appear sound, there are several aspects of the manuscript which need to be addressed before it can be made suitable for publication.

Specifically:

Major comments:

There are a lot of negative data, which may be fine, but the authors seem to interpret the lack of findings with more confidence than may be warranted without giving proper discussion to potential caveats such as insufficient doses, later timepoints, etc.

All of the hair cell counts have some issues. First, the TMEV treatment predominantly affects supporting cells which leads to disorganization. Such disorganization could lead to the appearance of lower numbers of hair cells when counted in windows that are 100um wide as the density of hair cells might be more severely affected than total hair cell number. Add this to the lack of cleaved caspase staining, and it becomes unclear the extent to which hair cells are dying. Though the MLKL staining counters this concern somewhat, all of these limitations should be discussed.

Second, the EM images suggest loss of bundles. If phalloidin was used to count surviving hair cells (as noted in the methods) then it is possible that loss of bundles would make the supposed loss of hair cells appear more exaggerated. It would be ideal if Myo7a counts could also be carried out and then there would not be ambiguity as a result of the stereocilia phenotype.

To the first point above, SC loss leading to disorganization, the authors have a note at one point about how the TRAIL neutralizing antibody treatment led to better hair cell survival even though SCs were still lost. However, there is no quantification of SCs in the paper. Actual quantification of the SCs would bolster this claim and help rebut the concern that disorganization is a main contributor to the observed phenotype rather than cell death.

Overall, much is left to be desired from the discussion. While the external information pertaining to necroptosis, macrophages, and TRAIL signaling is interesting, the manuscript would benefit greatly from added discussion that focuses specifically on the methodology and results of the studies therein, specifically consideration of what the strong conclusions are as well as any weaknesses, limitations, or other considerations. As noted above and below, this should include, but is not limited to, the choice of the virus used in this model, the methodology for counting of hair cells, and negative results.

No plan is mentioned for public access of the microarray data upon publication, rather the authors claim all data is in the manuscript, but given that a whole transcript GeneChip array was used, there should be a much larger dataset than what is included in supplemental figure 2. The data from this experiment should be uploaded to a publicly accessible database.

Minor comments:

The disorganization of bundles claimed to be elicited by recombinant TRAIL protein is not clear from the image (Fig 2c), single channel images of the phalloidin only should be shown, and ideally at higher magnification. Also, it is unclear whether the control is from a condition of 24 or 48 hours, but if only one control rather than 2 is to be presented, it should be from 48 hours.

Page 10, 2nd to last paragraph of the intro, it would be good to continue to make clear in this paragraph that the SCs are being likened to macrophages. Otherwise it is a bit unclear whether the authors are talking about possible roles for SCs or for actual macrophages which have been shown to reside in the inner ear and migrate to the sensory epithelium during times of stress or injury. While it becomes clear through the rest of the paper that the target is the SCs, making this more clear in this portion of the introduction will help limit confusion.

It would be preferable if the composition of the media (and possibly other details) of the explant procedure were outlined here rather than referring back to a reference from 2013.

TMEV (Theiler’s murine encephalitis virus) should be defined at its first use. Also, it could be helpful to justify the choice of this virus rather than murine CMV or others that might be more closely related to viruses known to cause hearing loss in humans.

p.11 line 86-87, what are the specified concentrations? These should be defined and justified, particularly for any that yielded negative results.

p. 15, TMEV is referred to as TEMV in multiple places. This persists through later portions of the manuscript. The abbreviation should be corrected to be consistent throughout.

p.15 line 188-192, wording is confusing, and should be framed more speculatively as these experiments are in vitro and hearing function was therefore not assessed. The authors can state that they speculate or hypothesize that TMEV infection might cause hearing loss prior to hair cell death since there is some evidence in vitro of bundle degeneration prior to hair cell death, but this would ultimately need to be validated in vivo.

Lines 198-211, this speculation about supporting cells producing TRAIL rather than hair cells should be moved to the discussion and presented as speculative with an acknowledgement that, in the absence of direct evidence, the authors do not know whether the increased TRAIL that was detected by qPCR is made by the SCs or not (no matter how reasonable such speculation may be).

Lines 234-235 this statement is an overreach… SCs could still be involved in hair cell death in response to aminoglycosides even if the specific transcripts that were examined in this study did not differ.

Lines 239- 240 should be ferroptosis and pyroptosis

In referencing the SHIELD data, the authors should be cautious in referring to “SC” expression as the dataset they are referring to only used a hair cell specific GFP, so the data they are referring to as “SC” gene expression is likely to include cell types other than just SCs, including macrophages or other immune cells.

Reviewer #3: This manuscript entitled virus-infection in cochlear supporting cells induces audiosensory receptor hair cell death by TRAIL-induced necroptosis, suggests a possible new target for preventing virus-induced sensorineural hearing loss. In this study, the authors show that supporting cells and GERCs induce hair cell death. This likely occurs via production of TRAIL as hair cell death is suppressed by TRAIL-neutralizing antibodies. Rather than through apoptotic mechanisms, this death occurs via necroptosis as it is inhibited by necroptosis inhibitors. Interestingly, corticosteroids also inhibited hair cell death via the inhibition of supporting cell/GERC transformation into macrophage-like cells. Hair cell death is also inhibited with macrophage depletion.

Overall, this is a well-written study. The authors do a nice job at systematically investigating the mechanism of hair cell death after viral infection. However, there are a few concerns that would need to be addressed prior to publication. Otherwise, this manuscript appears to make a significant contribution to the field.

In Figure 1F, the authors use TEM to show degeneration of hair cell stereocilia over time. Whereas the first panel shows stereocilia present at 16 hours, the second and third panels show loss of bundles by 21 hours prior to the loss of hair cells. They then claim that these data suggest hearing impairment is induced without hair cell death. However, the authors previously concluded in Figure 1A that hair cell death occurred at 16 hours post-viral infection, and that most hair cells died within 24 hours of infection. This leads me to think that these TEM scans may not be representative of the hair cell death process as proposed by the authors, especially if the numbers of hair cells at 24 hours as quantified in 1E are as low as ~1-2 IHC and 0 OHC in WT TMEV tissue.

In Figure 2, the authors report that hair cell death was suppressed by a TRAIL-neutralizing antibody. Whereas IHC numbers do not significantly differ between the Mock and the anti-TRAIL antibody, there is a statistically significant loss of OHC with the TRAIL-neutralizing antibody as compared to the Mock. This difference is not addressed by the authors.

The authors use gentamicin in Figure 3 as an ototoxic drug and analyze expression of markers in supporting cells in the presence of gentamicin. However, the figure legend only reports duration of gentamicin treatment and not dosage. The negative result of seeing no change in expression of these markers could certainly be due to a dose of gentamicin that is too low and/or incubation for too short a period of time. Further explanation and/or control experiments confirming appropriate ototoxic doses of gentamicin would be needed before supporting the conclusions made by the authors for this figure.

In Figure 4, the authors show that addition of necroptosis inhibitors necrostatin-1 and ponatinib suppress hair cell death. In Figure S2, they show that TEMV infection induces the expression of both apoptotic and necroptotic genes in the cochlear sensory epithelium. Expression of three necroptosis-related genes (Trail, Tlr3, Mlk1) increases in TMEV 16 hours, as does expression of numerous apoptosis-related genes. It would be interesting to note the changes to these genes in the presence of TMEV and necrostatin-1 or ponatinib. One would expect to see suppression of the necroptotic genes but not the apoptotic genes if these inhibitors had no effect on apoptosis.

The authors use dexamethasone in Figure 5 to suppress virus-induced hair cell death since steroids are the primary therapies for sudden sensorineural hearing loss. It would be interesting to see if prednisone, an alternative steroid used to treat SSHL with a much shorter half-life, has a similar effect. The authors note that dexamethasone inhibited hair cell damage, but not as strongly as necrostatin-1. Quantification of hair cell numbers comparing dexamethasone treatment versus necrostatin-1 treatment may be revealing to see whether this is truly the case. Since dexamethasone downregulates expression of macrophage markers in SCs and GERCs whereas necrostatin-1 inhibits necroptosis, it appears that the downstream signal—namely inhibition of necroptosis—contributes more significantly to the prevention of hair cell death than the upstream signal of suppressing macrophage expression in SCs and GERCs. Does this suggest that suppression of macrophage expression allows for activation of alternative mechanisms that still ultimately result in hair cell death via necroptosis? Answering these questions would be important to better elucidate whether targeting macrophages and/or necroptosis would be possible therapeutic avenues for the treatment of virus-induced SHL as described in lines 318-319.

Finally, a visual abstract or summary figure documenting the proposed mechanism of supporting cell-induced hair cell death would strongly enhance this paper.

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13 Oct 2021

Point-by-point responses to the reviewers’ comments

Ms No.: PONE-D-21-18184

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

We are grateful for the invaluable comments and suggestions made by the referees. In accordance with their suggestions, we have added some new results to adequately address the raised issues and have amended the manuscript. Please find below our point-by-point responses to each of their comments.

Reviewer #1

Sudden hearing loss is very common in clinic and Systemic corticosteroid administration is the primary treatment currently, although not all patients well respond to it, partly due to the poorly understood mechanisms underlying it. To obtain insights toward Sudden hearing loss, Hayashi et al in this study focused on characterizing the mechanisms underlying TMEV infection-caused hair cell death in cochlea. Briefly, each cochlear sample is incubated in medium for overnight before exposing to medium containing TMEV, which is an RNA virus. Three key findings are reported: 1) TRAIL expression in SCs is the key after TMEV infection; 2) Transformation of SCs (or GER/LER cells) into macrophages is another key step for HC death; 3) HC death occurs by necrosis, but not by apoptosis. The study is well-designed and clearly presented.

3 main comments are raised:

1) For the immunostaining images, the key single pannel is necessary. For instance, please show DR4 and DR5 single channel to better visualize their expression. In addition, a section view can be better demonstrate DR4 and 5’s expression in HCs. Furthermore, are DR4 and DR5 expressed in SCs?

In accordance with this comment, we have added a single channel image of DR4 or DR5 and a section view image of DR4 or DR5 in Fig. 2A. We have also shown the wide range of images that include SCs to present their expression in SCs clearer. In these images, significant expression of DR4 and DR5 was found in HCs, but rarely in SCs. Therefore, we have added the following sentence in the text: “Expression of DR4 and DR5 was found in HCs, but rarely in SCs (Fig. 2A)” (lines 209 to 210).

2) How about the antibody specificity against TRAIL? Please provide evidence if possible. If the antibody is not specific, neutralizing experiments might have other explainations.

In accordance with this comment, we have cited studies of the production of the N2B2 monoclonal antibody (Ref. 30) and its neutralization activity (Ref. 31), and added the following sentence in the text: “The specificity and neutralizing activity of the used antibody against TRAIL (monoclonal N2B2 antibody) have been shown previously [30, 31].” (lines 229 to 230).

3) I also have a concern regarding whether TRAIL is critical in the process of HC death. In other words, as the authors speculate binding of TRAIL to DR4/5 is the key step, will HCs die in DR4 -/- or DR5 -/-, following TMEV infection? If answer is “ no”, more concerns are raised regarding the importance of binding of TRAIL with DR4 or DR5. The author should alert readers of this or provide further evidence to prove the direct involvement of TRAIL in TMEV induced HC death. Because it is one of the key findings in this study, it cannot be ignored.

We agree with this comment. Indeed, we have not analyzed HC death in DR4-/- or DR5-/- after TMEV infection. Therefore, we revised the text as follows: “However, because four TRAIL receptors, which include DR4 and DR5, have been identified in humans [32], it is not to be elucidated whether only the binding of DR4 and DR5 expressed in HCs is important for HC death by TRAIL.” (lines 237 to 239).

Reviewer #2

The manuscript is interesting and presents data that suggest that certain viral infections may affect cochlear supporting cells in such a way that they respond via upregulation of Tnf-related apoptosis inducing ligand (TRAIL) which then binds to TRAIL receptors expressed by cochlear hair cells causing stereocilia degeneration and hair cell death. Some evidence is also presented to suggest that the hair cell death occurs via necroptosis and can be mitigated by treatment with anti-necroptosis agents or by inhibitors of macrophage activity. While the manuscript is largely novel and many of the findings appear sound, there are several aspects of the manuscript which need to be addressed before it can be made suitable for publication.

Specifically:

Major comments:

There are a lot of negative data, which may be fine, but the authors seem to interpret the lack of findings with more confidence than may be warranted without giving proper discussion to potential caveats such as insufficient doses, later timepoints, etc.

All of the hair cell counts have some issues. First, the TMEV treatment predominantly affects supporting cells which leads to disorganization. Such disorganization could lead to the appearance of lower numbers of hair cells when counted in windows that are 100um wide as the density of hair cells might be more severely affected than total hair cell number. Add this to the lack of cleaved caspase staining, and it becomes unclear the extent to which hair cells are dying. Though the MLKL staining counters this concern somewhat, all of these limitations should be discussed.

We agree with this comment. Therefore, in accordance with this comment, we have added the following sentences in the text: “We quantified HC death by the number of HCs along a longitudinal distance of 100 µm. However, TMEV treatment induced SC death, which resulted in disorganization of cochlear tissues. Therefore, it is possible that virus-induced disorganization led to the appearance of lower numbers of hair cells when counted in the same width. However, as shown in Fig. 4A, necrostatin treatment, especially 400 mm treatment, significantly inhibited loss of HC numbers (HC number was not markedly reduced compared with that observed without virus infection shown in Fig. 2B), whereas most SCs were lost. To more accurately assess HC death, we believe that the effects of disorganization in cochlear tissues need further analysis.” (lines 279 to 287).

Second, the EM images suggest loss of bundles. If phalloidin was used to count surviving hair cells (as noted in the methods) then it is possible that loss of bundles would make the supposed loss of hair cells appear more exaggerated. It would be ideal if Myo7a counts could also be carried out and then there would not be ambiguity as a result of the stereocilia phenotype.

To the first point above, SC loss leading to disorganization, the authors have a note at one point about how the TRAIL neutralizing antibody treatment led to better hair cell survival even though SCs were still lost. However, there is no quantification of SCs in the paper. Actual quantification of the SCs would bolster this claim and help rebut the concern that disorganization is a main contributor to the observed phenotype rather than cell death.

Owing to our mistake, the initial Fig. 2B was confusing because it only showed HCs. As presented in our revised Fig. 2B that shows a wider range, almost all SCs were lost after virus infection for 24 hours as determined by loss of DAPI-stained cells. However, HCs were still observed in the presence of the TRAIL-neutralizing antibody. We believe that the revised Fig. 2B will help readers to understand this phenomenon.

Overall, much is left to be desired from the discussion. While the external information pertaining to necroptosis, macrophages, and TRAIL signaling is interesting, the manuscript would benefit greatly from added discussion that focuses specifically on the methodology and results of the studies therein, specifically consideration of what the strong conclusions are as well as any weaknesses, limitations, or other considerations. As noted above and below, this should include, but is not limited to, the choice of the virus used in this model, the methodology for counting of hair cells, and negative results.

In accordance with this comment, we have added the following description in the Discussion: “In conclusion, our results revealed novel TRAIL-mediated HC death induced by virus infection in cochlear sensory epithelium. Moreover, our results have shown that macrophage-targeting drugs and necroptosis inhibitors effectively protected HCs against virus infection in our ex vivo experimental system using cochlear sensory epithelia isolated from newborn mice. The weakness of our analysis is that it remains unclear whether this mechanism is also mediated by viruses that cause SHL other than TMEV, whether these treatments are also effective for SHL in mouse models, and the detailed induction mechanism of HC death. Moreover, it is unknown whether the same phenomenon seen in the inner ear of newborn mice applies to that of adult mice. However, we believe that our findings may shed new light on therapeutic paradigms for SSHL.” (lines 372 to 380).

No plan is mentioned for public access of the microarray data upon publication, rather the authors claim all data is in the manuscript, but given that a whole transcript GeneChip array was used, there should be a much larger dataset than what is included in supplemental figure 2. The data from this experiment should be uploaded to a publicly accessible database.

In accordance with this comment, we have added the following description to the text: “These data have been uploaded to the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) under accession code GEO: GSE89556” (lines 157 to 159).

Minor comments:

The disorganization of bundles claimed to be elicited by recombinant TRAIL protein is not clear from the image (Fig 2c), single channel images of the phalloidin only should be shown, and ideally at higher magnification. Also, it is unclear whether the control is from a condition of 24 or 48 hours, but if only one control rather than 2 is to be presented, it should be from 48 hours.

In accordance with this comment, we have added a single channel image of phalloidin for each condition. The control is from the condition of 48 hours. We have added this information in Fig. 2C.

Page 10, 2nd to last paragraph of the intro, it would be good to continue to make clear in this paragraph that the SCs are being likened to macrophages. Otherwise it is a bit unclear whether the authors are talking about possible roles for SCs or for actual macrophages which have been shown to reside in the inner ear and migrate to the sensory epithelium during times of stress or injury. While it becomes clear through the rest of the paper that the target is the SCs, making this more clear in this portion of the introduction will help limit confusion.

In accordance with this comment, we have added the following description to the second to last paragraph of the Introduction: “In relation to these findings, we found that cochlear SCs function similarly to tissue resident macrophages that protect HCs from pathogens [12].” (lines 59 to 60).

It would be preferable if the composition of the media (and possibly other details) of the explant procedure were outlined here rather than referring back to a reference from 2013.

We prepared Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich Inc., St. Louis, MO, USA) supplemented with D-glucose (6 g/l) and penicillin G (150 �g/ml) for explant cultures. We have added this information to the Materials and Methods (lines 82 to 83).

TMEV (Theiler’s murine encephalitis virus) should be defined at its first use. Also, it could be helpful to justify the choice of this virus rather than murine CMV or others that might be more closely related to viruses known to cause hearing loss in humans.

In accordance with this comment, we have added the following description to the introduction of TMEV: “We previously found that Theiler’s murine encephalomyelitis virus (TMEV) infection in isolated murine newborn cochlear sensory epithelium induces IFN-α/β production [11]. TMEV is a small RNA picornavirus commonly used as an experimental model system for blood-brain barrier disruption [20]. Recently, we observed that TMEV infection is mainly established in SCs and HC infection is rarely observed in the presence of IFN-α/β produced by SCs that function as macrophage-like cells [12]. We also observed that SC infection is established in the early stage of TMEV infection (9 h after virus infection) and GERC infection is established in the later stage (16 h after virus infection) [12]. To understand the influence of virus infection in SCs on HCs, we analyzed the cell status of HCs using the same experimental system. It has been reported that TMEV infects macrophages [21]. Indeed, we have observed that TMEV infected almost all SCs and GERCs in our experimental system [12]. Therefore, although TMEV is not a virus that causes SHL in humans such as CMV, we used this experimental system to investigate the effects of infection with cochlear SCs that are protective cells against virus infection in mice.” (underlined text is newly written; lines 174 to 187).

p.11 line 86-87, what are the specified concentrations? These should be defined and justified, particularly for any that yielded negative results.

The concentration of the TRAIL-neutralizing antibody was 0.01 mg/ml. We have added this information to the Materials and Methods (line 90).

p. 15, TMEV is referred to as TEMV in multiple places. This persists through later portions of the manuscript. The abbreviation should be corrected to be consistent throughout.

This was a mistake. In accordance with this comment, we have carefully checked and rewritten the text.

p.15 line 188-192, wording is confusing, and should be framed more speculatively as these experiments are in vitro and hearing function was therefore not assessed. The authors can state that they speculate or hypothesize that TMEV infection might cause hearing loss prior to hair cell death since there is some evidence in vitro of bundle degeneration prior to hair cell death, but this would ultimately need to be validated in vivo.

We agree with this comment. Therefore, we have deleted the sentence “which suggested that this HC death signal induced hearing impairment without HC death” from the text.

Lines 198-211, this speculation about supporting cells producing TRAIL rather than hair cells should be moved to the discussion and presented as speculative with an acknowledgement that, in the absence of direct evidence, the authors do not know whether the increased TRAIL that was detected by qPCR is made by the SCs or not (no matter how reasonable such speculation may be).

In accordance with this comment, we have added the following description to the text: “These findings suggest that TRAIL was produced by SCs, which function as macrophages, after TEMV infection. However, it cannot be ruled out that factor(s) produced by virus-infected SCs act on HCs to induce TRAIL.” (lines 225 to 227).

Lines 234-235 this statement is an overreach… SCs could still be involved in hair cell death in response to aminoglycosides even if the specific transcripts that were examined in this study did not differ.

We agree with this comment and have changed the following description from “These results suggest that virus-induced HC death differs from that induced by ototoxic drugs, and SCs and GERCs are not involved in aminoglycoside-induced HC death” to “These results suggest that the virus-induced HC death mechanism differs from that induced by ototoxic drugs” (lines 258 to 259).

Lines 239- 240 should be ferroptosis and pyroptosis

This was a mistake and we have carefully checked and rewritten the text.

In referencing the SHIELD data, the authors should be cautious in referring to “SC” expression as the dataset they are referring to only used a hair cell specific GFP, so the data they are referring to as “SC” gene expression is likely to include cell types other than just SCs, including macrophages or other immune cells.

In accordance with this comment, we have added the following description to the text: “The SC fraction is a cell population other than the HC fraction of sensory epithelial cells, which was thought to be absent of immune cells, but it cannot be completely ruled out this population contains small numbers of macrophages and lymphocytes. However, these findings suggest that TRAIL was produced by SCs that function as macrophages after TEMV infection.” (lines 221 to 225).

Reviewer #3

This manuscript entitled virus-infection in cochlear supporting cells induces audiosensory receptor hair cell death by TRAIL-induced necroptosis, suggests a possible new target for preventing virus-induced sensorineural hearing loss. In this study, the authors show that supporting cells and GERCs induce hair cell death. This likely occurs via production of TRAIL as hair cell death is suppressed by TRAIL-neutralizing antibodies. Rather than through apoptotic mechanisms, this death occurs via necroptosis as it is inhibited by necroptosis inhibitors. Interestingly, corticosteroids also inhibited hair cell death via the inhibition of supporting cell/GERC transformation into macrophage-like cells. Hair cell death is also inhibited with macrophage depletion.

Overall, this is a well-written study. The authors do a nice job at systematically investigating the mechanism of hair cell death after viral infection. However, there are a few concerns that would need to be addressed prior to publication. Otherwise, this manuscript appears to make a significant contribution to the field.

In Figure 1F, the authors use TEM to show degeneration of hair cell stereocilia over time. Whereas the first panel shows stereocilia present at 16 hours, the second and third panels show loss of bundles by 21 hours prior to the loss of hair cells. They then claim that these data suggest hearing impairment is induced without hair cell death. However, the authors previously concluded in Figure 1A that hair cell death occurred at 16 hours post-viral infection, and that most hair cells died within 24 hours of infection. This leads me to think that these TEM scans may not be representative of the hair cell death process as proposed by the authors, especially if the numbers of hair cells at 24 hours as quantified in 1E are as low as ~1-2 IHC and 0 OHC in WT TMEV tissue.

As the reviewer commented, our study did not provide sufficient results to consider the association between loss of stereocilia and HC death. Therefore, we have deleted the following comment in the text: “Interestingly, prior to cell death, there was loss of the critical structures involved in sound signal transduction, namely sensory hairs (Fig. 1F), which suggested that this HC death signal induced hearing impairment without HC death”.

In Figure 2, the authors report that hair cell death was suppressed by a TRAIL-neutralizing antibody. Whereas IHC numbers do not significantly differ between the Mock and the anti-TRAIL antibody, there is a statistically significant loss of OHC with the TRAIL-neutralizing antibody as compared to the Mock. This difference is not addressed by the authors.

In accordance with this comment, we have added the following description to the text: “In this result, the decrease of IHCs was almost suppressed by the TRAIL-neutralizing antibody, but a decrease of OHCs was slightly observed (Fig. 2B). Regarding this difference, it is possible that there was a difference in the local TRAIL concentration and the effect of SC loss, but this has not been clarified at this time.” (lines 232 to 236).

The authors use gentamicin in Figure 3 as an ototoxic drug and analyze expression of markers in supporting cells in the presence of gentamicin. However, the figure legend only reports duration of gentamicin treatment and not dosage. The negative result of seeing no change in expression of these markers could certainly be due to a dose of gentamicin that is too low and/or incubation for too short a period of time. Further explanation and/or control experiments confirming appropriate ototoxic doses of gentamicin would be needed before supporting the conclusions made by the authors for this figure.

Tao et al. mentioned in their study that cochlear sensory epithelia were treated with 0.5 mM gentamicin for 3 h and then gentamicin was washed out and replaced with fresh medium. While no detectable hair cell loss was observed at 3 h, severe hair cell damage was caused by gentamicin at 24 h. We believe that these results demonstrate the validity of this model of explant culture to compare with that of virus infection.

In Figure 4, the authors show that addition of necroptosis inhibitors necrostatin-1 and ponatinib suppress hair cell death. In Figure S2, they show that TEMV infection induces the expression of both apoptotic and necroptotic genes in the cochlear sensory epithelium. Expression of three necroptosis-related genes (Trail, Tlr3, Mlk1) increases in TMEV 16 hours, as does expression of numerous apoptosis-related genes. It would be interesting to note the changes to these genes in the presence of TMEV and necrostatin-1 or ponatinib. One would expect to see suppression of the necroptotic genes but not the apoptotic genes if these inhibitors had no effect on apoptosis.

To show the effect of the necroptosis inhibitor on SC death, we changed Fig. 4A to a wide range of images that included SCs. As shown in this revised figure, although necrostatin-1 effectively suppressed HC loss, there was no suppression of SC loss. This result suggested that the necroptosis inhibitor affected HC death, but did not inhibit SC death, which was caused by apoptosis (Fig. 3A). We have not investigated the effect of the necroptosis inhibitor on the expression of apoptosis-related genes, but believe that this result shows that it does not affect induction of apoptosis. We have added the following comment to the text: “Moreover, although necrostatin-1 effectively suppressed HC loss, there was no suppression of SC loss (Fig. 4A). This result suggests that the necroptosis inhibitor affected HC death, but did not inhibit SC death, which was caused by apoptosis (Fig. 3A).” (lines 268 to 271).

The authors use dexamethasone in Figure 5 to suppress virus-induced hair cell death since steroids are the primary therapies for sudden sensorineural hearing loss. It would be interesting to see if prednisone, an alternative steroid used to treat SSHL with a much shorter half-life, has a similar effect. The authors note that dexamethasone inhibited hair cell damage, but not as strongly as necrostatin-1. Quantification of hair cell numbers comparing dexamethasone treatment versus necrostatin-1 treatment may be revealing to see whether this is truly the case. Since dexamethasone downregulates expression of macrophage markers in SCs and GERCs whereas necrostatin-1 inhibits necroptosis, it appears that the downstream signal—namely inhibition of necroptosis—contributes more significantly to the prevention of hair cell death than the upstream signal of suppressing macrophage expression in SCs and GERCs. Does this suggest that suppression of macrophage expression allows for activation of alternative mechanisms that still ultimately result in hair cell death via necroptosis? Answering these questions would be important to better elucidate whether targeting macrophages and/or necroptosis would be possible therapeutic avenues for the treatment of virus-induced SHL as described in lines 318-319.

This is an error in our description. In fact, the effectiveness of necroststin-1 and dexamethasone cannot be compared in this experiment. Indeed, there are no results to deny that suppression of macrophages allows activation of alternative mechanisms that still ultimately result in hair cell death. We have described this in the Discussion (third paragraph) that both macrophage-targeting therapy and a necroptosis inhibitor may be candidates for SHL treatment without superiority and we believe that these statements will not mislead readers. We have changed the description in the Figure 5 legend from “Corticosteroid dexamethasone (Dex) also inhibited HC damage, but not as strongly as necrostatin-1 (Nec)” to “Corticosteroid dexamethasone (Dex) inhibited HC damage”.

Finally, a visual abstract or summary figure documenting the proposed mechanism of supporting cell-induced hair cell death would strongly enhance this paper.

In accordance with this comment, we have added a visual abstract.

Submitted filename: Hayashi.Responses.docx

Click here for additional data file.

10 Nov 2021

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

PONE-D-21-18184R1

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Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

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Reviewer #1: My concerns are addressed in a satisfied way. It is an important topic in Sudden hearing loss filed, but whether the DR4 and DR5 (or other receptors) are indeed critical for HC death remains unclear in vivo, because solid genetic evidence are missed in this study. But I am ok to publish this initial study, and more thorough studies could be followed.

Reviewer #3: (No Response)

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16 Nov 2021

PONE-D-21-18184R1

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

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