Supplementary Material for: Overexpression of CX3CL1-CX3CR1 Signaling in the Stria Vascularis Rather Than the Basilar Membrane may be the trigger for inflammaging in the Cochlea

Introduction: Inflammaging is a key mechanism in presbycusis. CX3CL1-CX3CR1 pathway is critical for cochlear macrophage-hair cell cross-talk. However, its role in Inflammaging remains unclear. Methods: To investigate the role of CX3CL1-CX3CR1 signaling in cochlear inflammaging, Single-cell RNA sequencing (scRNA-seq) data from young and aged mouse cochleae were analyzed to map CX3CL1-CX3CR1 distribution and aging-related trends. Findings were validated with immunofluorescence, RT-qPCR, and Western blot. A migration assay assessed CX3CL1-CX3CR1's influence on macrophage migration and inflammation. Results: scRNA-seq analysis showed CX3CL1 mainly located in the basal cells of stria vascularis, while CX3CR1 and TNF-a mainly located in macrophages.The mRNA levels of CX3CL1, CX3CR1, and TNF-a in the stria vascularis significantly upregulated in aged mice. The Western blot showed similar trends, but only the upregulation of soluble CX3CL1 was statistically significant. Exogenous CX3CL1 significantly promoted BV2 cell migration and TNF-a secretion induced by LPS, while such effects were canceled in BV2 cells with CX3CR1 interfered. Conclusion: The overexpression of CX3CL1 in the basal cells of the stria vascularis with aging may be a trigger point for activating the local inflammatory microenvironment in age-related hearing loss, but it still requires further in vivo intervention experiments for validation.

引言：衰老性炎症（Inflammaging）是老年性耳聋（presbycusis）的关键致病机制。CX3CL1-CX3CR1通路（CX3CL1-CX3CR1 pathway）对耳蜗巨噬细胞-毛细胞串扰至关重要，但其在衰老性炎症中的作用仍不明确。方法：为探究CX3CL1-CX3CR1信号通路在耳蜗衰老性炎症中的作用，本研究分析了年轻与衰老小鼠耳蜗的单细胞RNA测序（Single-cell RNA sequencing, scRNA-seq）数据，以绘制CX3CL1-CX3CR1的分布特征及衰老相关表达趋势。并通过免疫荧光（immunofluorescence）、实时定量聚合酶链反应（RT-qPCR）及蛋白质印迹法（Western blot）对分析结果进行验证。此外，采用迁移实验评估CX3CL1-CX3CR1通路对巨噬细胞迁移与炎症反应的影响。结果：单细胞RNA测序分析显示，CX3CL1主要表达于血管纹（stria vascularis）基底细胞，而CX3CR1与肿瘤坏死因子-α（TNF-α）则主要表达于巨噬细胞。衰老小鼠血管纹中CX3CL1、CX3CR1及TNF-α的mRNA水平显著上调；蛋白质印迹实验结果与之趋势一致，但仅可溶性CX3CL1的上调具有统计学显著性。外源性CX3CL1可显著促进脂多糖（LPS）诱导的BV2细胞迁移与TNF-α分泌，而该效应在CX3CR1干扰的BV2细胞中被阻断。结论：衰老状态下血管纹基底细胞中CX3CL1的过表达，可能是激活年龄相关性听力损失局部炎症微环境的触发点，但该结论仍需进一步通过体内干预实验加以验证。