Therapeutic Potential of 1‑(1,2,3,5,6,7-Hexahydro‑s‑indacen-4-yl)-3-[4-(2-hydroxypropan-2-yl)furan-2-yl]sulfonylurea (MCC950), a Potent Agent for Improving Autoinflammatory Hearing Loss in Mice with Myeloid Cell-Specific Conditional Expression of Mutant Nucleotide-Binding Oligomerization Domain, Leucine-Rich Repeat, and Pyrin Domain-Containing Protein 3 (Nlrp3)

Abstract

Autoinflammatory hearing loss due to NLRP3 inflammasome activation is poorly understood. We developed a novel myeloid-specific Nlrp3 mutant mouse model (Nlrp3 ^D301NneoR/+; LysM ^Cre/+) expressing the D301N Nlrp3 variant in macrophages and neutrophils, offering a platform for studying inflammasome-mediated cochlear inflammation and hearing loss. Quantitative PCR and MRI showed elevated Nlrp3 expression in the cochlea, with significant inflammatory changes in cochlear and middle ear regions. Histology revealed strial vascular degeneration, contributing to hearing loss. We tested MCC950, a selective NLRP3 inflammasome inhibitor, which significantly improved auditory thresholds, especially in the 8–32 kHz range, and reduced cochlear inflammation, as shown by decreased IL-1β levels. MCC950 also improved overall health, including weight gain and reduced alopecia. Its small size and selective inhibition make it a promising alternative to existing treatments like Anakinra and Canakinumab. These findings position MCC950 as a potential treatment for NLRP3 mutation-associated hearing loss.

Hearing is fundamental to human communication, cognitive integrity, and social engagement. In early life, it is critical for language acquisition, with even mild impairment disrupting phonological development, vocabulary growth, and literacy. In later life, hearing loss is a major modifiable risk factor for dementia, with severe deficits accelerating cognitive decline through sensory deprivation, social isolation, and increased cognitive load. Preserving hearing is therefore essential for both language development and long-term brain health.

Sensorineural hearing loss (SNHL) is largely irreversible, and current management focuses on rehabilitationthrough amplification or cochlear implantationrather than addressing underlying disease mechanisms. Among its diverse causes, gain-of-function mutations in the NLRP3 genebest known for their role in cryopyrin-associated periodic syndromes (CAPS)have been identified as a distinct cause of progressive bilateral SNHL, designated DFNA34 in nonsyndromic cases. , These mutations lead to constitutive NLRP3 inflammasome activation, excessive interleukin-1β (IL-1β) and IL-18 production, and localized cochlear autoinflammation, likely mediated by resident macrophages. −

Importantly, NLRP3-related hearing loss is one of the few genetic forms of deafness shown to respond to pharmacologic intervention, with IL-1 receptor antagonist therapy (anakinra) capable of stabilizing or even improving hearing in some patients. − Given that effective gene-based therapies are unavailable for most hereditary deafnessaside from experimental OTOF gene therapytargeting the NLRP3-IL-1β pathway represents a promising precision medicine approach for otherwise untreatable, progressive hearing loss. ,

Despite this therapeutic potential, the mechanisms linking NLRP3 activation to cochlear dysfunction remain poorly understood, largely due to the absence of reliable in vivo models. , Existing mouse models with pan-hematopoietic or hair cell-specific NLRP3 expression suffer from severe systemic toxicity and early mortality, precluding longitudinal studies and limiting insights into cell type-specific pathogenic processes.

To address this gap, we developed a conditional knock-in mouse model (Nlrp3 ^D301NneoR/+ ; LysM ^Cre/+ ) that selectively expresses mutant NLRP3 in myeloid cells, including macrophages and neutrophils, enabling targeted investigation of cochlear immune responses without systemic lethality. This platform provides an opportunity to evaluate disease mechanisms and test precision therapeutic strategies.

MCC950, a potent and selective small-molecule NLRP3 inhibitor, effectively blocks both canonical and noncanonical inflammasome activation in vitro and in vivo. However, its efficacy in treating autoinflammatory hearing loss has not yet been established. Here, we characterize the auditory phenotype, inflammatory profile, and survival of our conditional mutant mice and assess the therapeutic potential of MCC950. Our findings demonstrate the utility of this model, highlight the role of myeloid-derived inflammasome activity, and support MCC950 as a promising and effective precision medicine therapy for autoinflammatory hearing loss.

Results

Establishment of an Autoinflammatory Hearing Loss Model in Mice with Myeloid Cell-Specific Conditional Expression of Mutant Nlrp3

We successfully established a reliable autoinflammatory hearing loss model using the Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mouse, in which the Nlrp3 D301N variant is specifically expressed in myeloid cells (macrophages and neutrophils). This model enabled a focused investigation of NLRP3 inflammasome activation and its impact on cochlear inflammation and auditory function.

We assessed Nlrp3 expression in the brain, cochlea, and hippocampus of mutant (Nlrp3 ^D301NneoR/+; LysM ^Cre/+) mice in comparison to wild-type (Nlrp3 ^D301NneoR/+; LysM ^+/+) (Figure A). Quantitative real-time PCR analysis revealed significantly elevated Nlrp3 mRNA levels in mutants, with the cochlea showing the highest increase (16.5-fold), followed by the whole brain (8.9-fold) and hippocampus (7.1-fold). These results strongly suggest tissue-specific activation of the Nlrp3 inflammasome pathway, which may contribute to differential inflammatory responses and disease susceptibility across neural and auditory tissues.

1.

Increased Nlrp3 expression and cochlear inflammation inNlrp3 ^D301NneoR/+;LysM ^Cre/+ mice. (A) Comparison of Nlrp3 transcript expression in whole brain , cochlea, and hippocampus between mutant mice (Nlrp3 ^D301NneoR/+; LysM ^Cre/+) and wild-type mice (Nlrp3 ^D301NneoR/+; LysM ^+/+): quantitative real-time PCR results comparing relative Nlrp3 expression levels in the whole brain, cochlea, and hippocampus of Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice and Nlrp3 ^D301NneoR/+; LysM ^+/+ mice. Data are presented as mean ± SE (n = 3 per group). (B) Contrast-enhanced MRI images demonstrating cochlear fluid compartment enhancement. Pre- and postcontrast T1-weighted MRI scans of cochleae (yellow dashed circles) from Nlrp3 ^D301NneoR/+; LysM ^+/+ mice show no noticeable enhancement. In contrast, significant and more pronounced cochlear fluid compartment enhancement is observed in Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice. Middle ear inflammation is also evident in Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice (white arrowheads). The MRI images of Nlrp3 ^D301NneoR/+; LysM ^+/+ mice shown here are identical to those previously reported by Kim et al., as all genotypes were imaged concurrently in a single MRI session using the same wild-type mouse.

Acquisition of magnetic resonance images (MRI) scans further validated these findings (Figure B). Post-contrast imaging revealed no cochlear enhancement in Nlrp3 ^D301NneoR/+; LysM ^+/+ mice, indicating the absence of significant inflammation. In contrast, Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice displayed marked cochlear enhancement, suggesting active inflammation, alongside consistent middle ear inflammation. These MRI results emphasize the genotype-dependent inflammatory phenotype in both cochlear and middle ear structures.

Histological analysis of cochlear tissues showed no significant changes in the organ of Corti, spiral ganglion, or spiral limbus, with no differences in cell density between Nlrp3 ^D301NneoR/+; LysM ^+/+ and Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice (Figure A–C). Despite inflammation, these cochlear regions remained largely unaffected. However, significant inflammatory alterations were observed in other cochlear areas. Compared to Nlrp3 ^D301NneoR/+; LysM ^+/+ mice, Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice exhibited structural changes in the inner ear. The osseous spiral lamina showed increased vascular dilation, indicative of hyperemia, and the spiral ligament appeared paler and edematous, suggesting fluid accumulation and tissue swelling (Figure S1). Degeneration of the stria vascularis was noted, with thinning and disruption of cellular organization in both apical and middle cochlear turns. Quantitative analysis revealed significant reductions in strial vascular area and stria vascularis thickness in Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice, especially in the apex (Figure D–G). Vascular assessments also showed reduced vessel numbers in the stria vascularis, particularly in the cochlear apex, correlating with strial atrophy (Figure H,I). These findings support the MRI results of enhanced cochlear signal intensity. Further studies with larger cohorts are needed to fully evaluate vascular dilation and edema in the osseous spiral lamina and spiral ligament.

2.

Histological and quantitative assessment of cochlear morphology and vascularity in wild-type mice (Nlrp3 ^D301NneoR/+;LysM ^+/+) and mutant mice (Nlrp3 ^D301NneoR/+;LysM ^Cre/+). (A) Representative images of hematoxylin and eosin (H&E) staining of cochlear turns (yellow arrows indicate vascular areas). Scale bars represent 200 μm and zoom-in images 50 μm. Quantitative analyses of cell density revealed no significant difference between Nlrp3 ^D301NneoR/+; LysM ^+/+ mice and Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice in both apex (B) and middle turns (C). However, significant decreases were identified in stria vascularis area (D, E), thickness (F, G), and number of vessels (H, I) in Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice compared to Nlrp3 ^D301NneoR/+; LysM ^+/+ mice. Data represent mean ± SEM (n = 6 per group); *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

These results confirm the successful establishment of a reliable model of autoinflammatory hearing loss and highlight that NLRP3 inflammasome activation in myeloid cells induces significant cochlear inflammation and vascular degeneration, particularly in the stria vascularis, leading to hearing loss.

Therapeutic Potential of MCC950 in Preserving Auditory Function by Attenuating NLRP3-Mediated Cochlear Inflammation

To evaluate the therapeutic potential of MCC950, a selective NLRP3 inflammasome inhibitor, we monitored its effects on body weight, auditory function, and systemic inflammation in Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice.

Specifically, at 1 and 2 weeks, Nlrp3 ^D301NneoR/+; LysM ^+/+ mice had higher body weights (1 week: 2.74 g, 2 weeks: 5.78 g) than Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice treated with MCC950 (1 week: 2.44 g, 2 weeks: 5.28 g) and PBS (1 week: 2.83 g, 2 weeks: 4.16 g). By 3 weeks, MCC950-treated mice (6.94 g) and controls (7.06 g) had similar weights, while PBS-treated mice did not survive (Figure A). Due to the small sample size (limited to average group data without individual replicates), statistical comparisons using unpaired two-tailed t-tests were not feasible and did not yield valid results.

3.

Body weight trajectories and auditory function in wild-type mice (Nlrp3 ^D301NneoR/+;LysM ^+/+) and mutant mice (Nlrp3 ^D301NneoR/+;LysM ^Cre/+) following MCC950 or PBS treatment. (A) Body weight comparison over time in Nlrp3 ^D301NneoR/+; LysM ^+/+ mice and Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice treated with MCC950 or PBS. Body weights of mice were recorded at 1, 2, and 3 weeks posttreatment initiation. Data points represent mean values for each group. Statistical comparisons were not applicable due to data limitations. (B) ABR threshold comparisons between Nlrp3 ^D301NneoR/+; LysM ^+/+ mice, MCC950-treated Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice, and PBS-treated Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice at postnatal day 20 (P20). Data presented as mean ± SD Nlrp3 ^D301NneoR/+; LysM ^+/+ mice (n = 5), MCC950-treated Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice (n = 5), and PBS-treated Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice (n = 8). *p < 0.05, ***p < 0.001; ns, not significant.

Auditory brainstem responses (ABR) testing showed significant improvements in auditory thresholds at 4, 8, 16, and 32 kHz in MCC950-treated Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice compared to PBS-treated mice (Figure B and Figure S2). Notably, at higher frequencies, thresholds in MCC950-treated mice were similar to wild-type controls, indicating strong protection of cochlear function, especially in medial and basal regions. PBS-treated mice showed severe hearing impairment at all frequencies, highlighting MCC950’s protective effects.

In addition to auditory protection, MCC950 treatment also improved the overall health of the Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice. The treated mice exhibited reduced alopecia and increased body mass compared to the PBS-treated group, which demonstrated more severe systemic signs of inflammation, including significant alopecia and weight loss, with no survivors beyond 2 weeks of treatment (Figure A). These observations suggest that MCC950 not only alleviates cochlear inflammation but also improves the general condition of the mice. Additionally, images of Nlrp3 ^+/+; LysM ^Cre/+ and Nlrp3 ^D301NneoR/+; LysM ^+/+ mice at postnatal day 16 (P16), prior to any treatment exposure, are provided for genotype comparison (Figure S3).

4.

Effects of MCC950 treatment on physical appearance and longitudinal auditory function inNlrp3 ^D301NneoR/+;LysM ^Cre/+ mice. (A) Representative images showing improvements in physical appearance and reduced alopecia in MCC950-treated Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice at 0, 2, and 6 weeks postinjection, compared to PBS-treated Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice. (B) Longitudinal ABR threshold measurements indicating progressive auditory improvement at frequencies of 4, 8, 16, and 32 kHz over the 6-week period post-MCC950 injection.

Longitudinal ABR assessment of an additional Nlrp3 ^+/+; LysM ^Cre/+ mouse showed significant auditory recovery after MCC950 treatment. Initially, the mouse exhibited impaired thresholds at P16, but following 2 weeks of MCC950 treatment, auditory function improved, with further recovery at 6 weeks, highlighting the long-term therapeutic benefits of MCC950.

Finally, to assess the anti-inflammatory effects of MCC950, we measured IL-1β levels in cochlear tissues and serum (Figure A,B). In the PBS-treated Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice, elevated IL-1β levels were prominently observed both in cochlear tissues and serum, indicative of active inflammation. Conversely, MCC950 treatment significantly reduced IL-1β expression, aligning these levels more closely with those observed in Nlrp3 ^D301NneoR/+; LysM ^+/+ mice. This notable decrease suggests MCC950 effectively modulates inflammation, potentially fostering a favorable microenvironment conducive to preserving cochlear function and mitigating autoimmune damage within the inner ear.

5.

Effects of MCC950 treatment on IL-1β levels in cochlear tissues and serum. Cochlear tissue (A) and serum (B) levels of IL-1β were measured by ELISA in wild-type mice (Nlrp3 ^D301NneoR/+; LysM ^+/+) and mutant mice (Nlrp3 ^D301NneoR/+; LysM ^Cre/+) treated with MCC950 or PBS at 2 weeks posttreatment. Data are presented as mean ± SEM (n = 4 per group, 4 cochleae from 4 different animals); **p < 0.01, ***p < 0.001; ns, not significant.

Discussion

In this study, we successfully established a reliable and novel myeloid-specific Nlrp3 mutant mouse model of autoinflammatory hearing loss. The Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mutant mice express the Nlrp3 D301N variant specifically in myeloid cells, including macrophages and neutrophils. Notably, the severe phenotype observed in our myeloid-specific model aligns with previous findings in Nlrp3̂{D301N/+};LysM-Cre mice, which also exhibited early lethality and systemic inflammation. This confirms that NLRP3 hyperactivation in myeloid lineage cells (macrophages and neutrophils) is sufficient to drive the lethal pathology, marking them as the predominant effectors of the disease. This model provides a unique and valuable platform for investigating the role of NLRP3 inflammasome activation in cochlear inflammation and hearing loss. The successful establishment of this model represents a significant advancement in understanding the mechanisms underlying autoinflammatory hearing loss and offers an essential tool for investigating therapeutic interventions targeting NLRP3. Additionally, we demonstrate that MCC950, a selective NLRP3 inflammasome inhibitor, exhibits significant therapeutic potential in preserving auditory function and reducing cochlear inflammation in this model.

One of the key findings of this study is the tissue-specific activation of the NLRP3 inflammasome in the cochlea, as evidenced by significantly increased Nlrp3 expression in cochlear tissues compared to the brain and hippocampus. This highlights the cochlea as a major site of NLRP3-driven inflammation, which is crucial for the pathogenesis of hearing loss in this model. Our findings align with previous studies implicating the NLRP3 inflammasome in sensorineural hearing loss. , Gain-of-function mutations in NLRP3 typically affect the protein, but increased Nlrp3 transcripts in the cochlea and brain likely result from transcriptional priming, not genetic changes. Chronic inflammasome activation elevates IL-1β, activating NF-κB, which upregulates NLRP3 transcription, enhancing tissue vulnerability in immune-privileged areas like the brain and cochlea. , The observed inflammation, confirmed by MRI, emphasizes the genotype-dependent inflammatory phenotype within cochlear and middle ear structures. Histological analysis further supports these observations, revealing that the hearing loss observed was not due to damage to sensory hair cells or cochlear supporting structures. Instead, degeneration of the stria vascularisevidenced by reduced thickness, vascular area, and vessel numbersuggests vascular dysfunction as a key mechanism. These results corroborate findings from autoimmune inner ear disease models and support the hypothesis that strial vascular injury contributes to sensorineural hearing impairment. These findings underscore the importance of targeting NLRP3-mediated inflammation in the cochlea as a strategy to mitigate hearing loss.

A novel and significant aspect of this study is the demonstration of the therapeutic potential of MCC950, a selective NLRP3 inflammasome inhibitor, in preserving auditory function and reducing cochlear inflammation. Our results show that MCC950 treatment significantly improved ABR thresholds at higher frequencies, particularly at 8 kHz, 16 kHz, and 32 kHz, bringing the ABR thresholds of treated mutant mice closer to those of wild-type controls. This improvement is consistent with prior studies showing that immunosuppressive treatments can improve hearing in DFNA34-related autoinflammatory hearing loss. , This represents a substantial therapeutic benefit, suggesting that MCC950 effectively mitigates cochlear inflammation and protects against hearing loss caused by NLRP3 inflammasome activation. Notably, this is the first study demonstrating that targeting NLRP3 inflammasome activation can preserve cochlear function in a model of autoinflammatory hearing loss. The long-term improvements in ABR thresholds observed over a six-week period highlight the sustained therapeutic effects of MCC950. These findings provide new insights into the role of NLRP3 in cochlear inflammation and hearing loss, further establishing MCC950 as a promising therapeutic candidate for treating autoinflammatory hearing loss. Further supporting the therapeutic efficacy of MCC950, we observed a marked reduction in IL-1β levels in both cochlear tissues and serum following treatment. Elevated IL-1β levels in PBS-treated mutant mice indicated active inflammation, while MCC950 treatment normalized these levels, highlighting the anti-inflammatory effects of the drug. These findings align with previous reports demonstrating the reduction of inflammation by blocking IL-1β activity with immunosuppressive drugs such as Anakinra and Canakinumab. , These findings suggest that MCC950 exerts its protective effect through modulation of the inflammatory environment within the cochlea, contributing not only to the preservation of auditory function but also to improvements in systemic health, as evidenced by reduced alopecia and increased body mass in treated mice.

Several distinctive pharmacological attributes of MCC950 further enhance its therapeutic potential. MCC950s small molecular weight (<1 kDa) enhances its ability to penetrate the round window membrane, making intratympanic delivery feasible and reducing systemic toxicity risks. Unlike Anakinra and Canakinumab, MCC950 specifically inhibits the NLRP3 inflammasome, targeting an upstream pathway, increasing therapeutic specificity, and minimizing off-target effects.

This study has limitations, including a short observation period and limited mechanistic insights into hearing impairment. MCC950’s adverse effects and short half-life restrict its long-term use. Future research should evaluate its long-term efficacy, optimize delivery, clarify macrophage-mediated mechanisms to improve clinical outcomes, and include additional IL-1β production data from sham control groups.

In conclusion, this study not only confirms the successful establishment of a reliable myeloid-specific Nlrp3 mutant mouse model of autoinflammatory hearing loss but also provides new evidence for the therapeutic potential of MCC950 in preserving auditory function and reducing cochlear inflammation. These findings represent a crucial step forward in the development of targeted therapies for inflammatory forms of sensorineural hearing loss and pave the way for future studies aimed at optimizing the use of NLRP3 inhibitors in clinical settings.

Materials and Methods

Animals and Housing

Myeloid-specific Nlrp3 mutant mice (Nlrp3 ^D301NneoR/+; LysM ^Cre/+) and wild-type mice (Nlrp3 ^D301NneoR/+; LysM ^+/+) were generated by crossbreeding and treated with MCC950 from P3. Full details on animal generation, housing, genotyping, and treatment protocols are provided in the Supporting Information and Figure S4.

Evaluation of Expression of Mutant Nlrp3 in Several Tissues in the Nlrp3^D301NneoR/+; LysM^Cre/+ Mice

Nlrp3 expression in brain, cochlea, and hippocampus was assessed by qRT-PCR using TaqMan probes. Total RNA was isolated and normalized to Gapdh. Relative expression was calculated using the 2^–ΔΔCt method. Detailed protocols and primer/probe information are provided in the Supporting Information.

MCC950 Administration

MCC950 (20 mg/kg) was administered intraperitoneally twice weekly from P3 for 3 weeks. One mutant mouse (Nlrp3 ^D301NneoR/+; LysM ^Cre/+) received delayed treatment starting at P16. PBS was used for control injections. Complete dosing schedule and treatment rationale are available in the Supporting Information.

Auditory Brainstem Responses (ABRs)

ABRs were measured in Nlrp3 ^D301NneoR/+; LysM ^+/+ mice, MCC950-treated Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice, and PBS-treated Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice at P20. One mutant mouse (Nlrp3 ^D301NneoR/+; LysM ^Cre/+) was tested longitudinally from P16 to assess delayed treatment effects. Hearing thresholds at 4–32 kHz were determined under anesthesia using tone-burst stimuli. Mice without detectable responses were assigned a 110 dB threshold. Full ABR setup, anesthesia protocol, and electrode placement are detailed in the Supporting Information.

Enzyme-Linked Immunosorbent Assay (ELISA)

IL-1β levels in serum and cochlear tissue were quantified using standard ELISA. Blood was collected via cardiac puncture, and cochleae were harvested, lysed, and processed for protein extraction. Cytokine levels were normalized by protein content. Full anesthesia protocol, tissue preparation, and ELISA procedures are provided in the Supporting Information.

Acquisition of Magnetic Resonance Images (MRI)

To assess cochlear inflammation, pre- and postcontrast MRI was performed on Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mice and wild-type mice at P15 using a 7T scanner. Gadolinium contrast was administered via tail vein. T1-weighted images were acquired before and 30 min after injection. Detailed MRI parameters and animal handling protocols are provided in the Supporting Information.

Cochlear Extraction and Preparation of Histologic Sections

Hematoxylin–eosin (H&E) staining was performed on cochlear sections collected at P15 to assess structural abnormalities. The experimental groups comprised three animals per group, with six cochleae analyzed per group. Cochleae were fixed, embedded in paraffin, and sectioned at 5 μm. Detailed tissue preparation and staining protocols are available in the Supporting Information.

Quantification of Histological Features

Quantification of cell density, stria vascularis area and thickness, and vessel density within the stria vascularis was conducted using a systematic approach based on established stereological methods, as outlined in the Supporting Information.

Survival Analysis

Survival was monitored in MCC950-treated and untreated Nlrp3 ^D301NneoR/+; LysM ^Cre/+ mutant mice. Duration was recorded from treatment initiation to death or study end point. Full survival monitoring criteria and group details are described in the Supporting Information.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 9. Tests included t-tests and one-way ANOVA with Tukey’s post hoc comparisons. Significance was set at *p < 0.05, **p < 0.01, and ***p < 0.001. Complete statistical methods and analysis criteria are detailed in the Supporting Information.

All data generated or analyzed during this study are included in this submitted manuscript and its Supporting Information files.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.5c00695.

• The methods include animal housing, mutant Nlrp3 expression evaluation, MCC950 administration, ABR, ELISA, MRI acquisition, cochlear extraction, histology, histological quantification, survival, and statistical analysis. Supplementary figures show cochlear inflammation matching MRI (Figure S1), ABR thresholds post-PBS injection (Figure S2), physical traits comparison between wild-type and mutant mice (Figure S3), and Nlrp3D301NneoR allele generation and activation (Figure S4) (PDF)

∇.

N.-T.T. and B.J.K. contributed to the study conception and design, material preparation, data collection, and/or analyses. N.-T.T., B.J.K., M.K., P.G.J., H.J., J.Y., J.A.K., J.-Y.K., J.H.P., and Y.K. performed experiments and performed data analyses. B.J.K., Y.K., and B.Y.C. supervised the study. The first draft of the manuscript was written by N.-T.T., B.J.K., and B.Y.C. with all authors commenting on previous versions of the manuscript. All authors read and approved the final manuscript.

Ethics Approval and Consent to Participate: The animal study was approved by the Institutional Animal Care and Use Committee of Seoul National University Bundang Hospital (Seongnam, Republic of Korea, IACUC No.: BA-2004-294-038-09).

The authors declare no competing financial interest.