Murali Sree Varshini, Stothert Andrew R, Pereyra Elyssa, Batalkina Lyudmila V, Kaur Tejbeer
Department of Otolaryngology and Brain Health Institute, Rutgers University, Robert Wood Johnson Medical School, Piscataway, New Jersey, U.S.A., 08854 (present address).
Department of Biomedical Sciences, Creighton University, School of Medicine, Omaha, Nebraska, U.S.A., 68178.
bioRxiv. 2025 Aug 13:2025.08.04.668473. doi: 10.1101/2025.08.04.668473.
Cochlear injury activates the resident macrophages (RM) and recruits the blood-circulating monocytes and monocyte-derived macrophages (Mo/Mo-M), but their specific functions in the injured cochlea are unknown. It is well established that the chemokine fractalkine receptor (CXCR1), expressed by cochlear macrophages, influences the density of those macrophages and promotes synaptic repair and spiral ganglion neuron survival in the injured cochlea. As CXCR1 is expressed on both RM and Mo/Mo-M, it remains unclear if CXCR1-expressing RM and Mo/Mo-M are distinct and differentially promote SGN survival after cochlear injury. Here, we demonstrate the use of fate mapping via a tamoxifen-inducible CXCR1 mouse model (CXCR1:R26RFP) wherein CXCR1-expressing RM and Mo/Mo-M are endogenously labeled with different fluorescent reporters to define the heterogeneity in cochlear macrophages regarding their origin, turnover, spatiotemporal distribution, morphology, and fate following a loud acoustic trauma. After 60 days of tamoxifen injections at 4 weeks of age, long-lived cochlear RM were YFP+ RFP+ with 98.0 ± 1.7% recombinant efficiency, and short-lived blood-circulating CXCR1 lineage (Mo/Mo-M) were YFP+ RFP- with 2.5 ± 1.1% recombinant efficiency. Following an acoustic trauma of 112 dB SPL at 8-16 kHz octave band for 2 hours, morphologically similar RM and Mo/Mo-M were observed in the spiral ganglion, lamina, ligament, and around the sensory epithelium. Quantification of RM and Mo/Mo-M in the spiral lamina and ganglion revealed distinct spatial and temporal distribution patterns. Furthermore, recruited Mo/Mo-M expressed classical monocyte markers such as Ly6C and CCR2. Both RM and Mo/Mo-M were positive for proliferation marker, Ki67, and negative for apoptotic marker, cleaved caspase-3, suggesting that the overall increase in macrophage numbers in the noise-injured cochlea is a contribution of both the proliferation of RM and recruitment of Mo/Mo-M. Probing for blood-clotting protein, fibrinogen, showed its presence in the cochlea after acoustic trauma, suggesting vascular damage that positively and strongly correlated with the time course of recruitment of blood-circulating Mo/Mo-M in the noise-injured cochlea. These data imply that macrophages in the noise-injured cochlea are heterogeneous regarding their ontogeny, distribution, and fate. They offer a robust tool to study the precise roles of resident and recruited macrophages in healthy and pathological ears.
耳蜗损伤会激活驻留巨噬细胞（RM），并募集血液循环中的单核细胞和单核细胞衍生的巨噬细胞（Mo/Mo-M），但其在受损耳蜗中的具体功能尚不清楚。众所周知，耳蜗巨噬细胞表达的趋化因子fractalkine受体（CXCR1）会影响这些巨噬细胞的密度，并促进受损耳蜗中的突触修复和螺旋神经节神经元存活。由于CXCR1在RM和Mo/Mo-M上均有表达，目前尚不清楚表达CXCR1的RM和Mo/Mo-M是否不同，以及在耳蜗损伤后是否会差异促进螺旋神经节神经元存活。在此，我们展示了通过他莫昔芬诱导的CXCR1小鼠模型（CXCR1:R26RFP）进行命运图谱分析的应用，其中表达CXCR1的RM和Mo/Mo-M通过不同的荧光报告基因进行内源性标记，以确定耳蜗巨噬细胞在大声创伤后的起源、更新、时空分布、形态和命运方面的异质性。在4周龄时注射他莫昔芬60天后，长寿的耳蜗RM为YFP+RFP+，重组效率为98.0±1.7%，而短命的血液循环CXCR1谱系（Mo/Mo-M）为YFP+RFP-，重组效率为2.5±1.1%。在8-16kHz倍频程带以112dB SPL的强度进行2小时的声创伤后，在螺旋神经节、板层、韧带和感觉上皮周围观察到形态相似的RM和Mo/Mo-M。对螺旋板层和神经节中的RM和Mo/Mo-M进行定量分析，揭示了不同的时空分布模式。此外，募集的Mo/Mo-M表达经典的单核细胞标志物，如Ly6C和CCR2。RM和Mo/Mo-M的增殖标志物Ki67均为阳性，凋亡标志物裂解的半胱天冬酶-3均为阴性，这表明噪声损伤耳蜗中巨噬细胞数量的总体增加是RM增殖和Mo/Mo-M募集共同作用的结果。对凝血蛋白纤维蛋白原的检测显示，声创伤后其在耳蜗中存在，这表明血管损伤与噪声损伤耳蜗中血液循环的Mo/Mo-M募集的时间进程呈正相关且相关性很强。这些数据表明，噪声损伤耳蜗中的巨噬细胞在其个体发生、分布和命运方面存在异质性。它们为研究驻留和募集的巨噬细胞在健康和患病耳朵中的精确作用提供了一个强大的工具。
