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.