COCHLEAR MACROPHAGES AND EPITHELIAL REPAIR

描述（由申请人提供）：虽然免疫系统在几乎所有器官系统中的存在已被广泛认识，但免疫对内耳的影响却很少受到关注。对人类受试者的研究表明，在一些患者中，皮质类固醇可以成功地治疗听力损失，这可能是由于它们的抗炎和免疫抑制作用。不幸的是，我们对影响炎症过程和内耳正常功能之间相互作用的细胞机制了解有限。由于类固醇治疗为成功治疗听力损失提供了这样的希望，炎症如何影响听力的具体机制值得进一步研究。我们研究的长期目标是确定炎症在耳蜗维持和损伤中的作用。在之前的工作中，我们已经证明毛细胞损伤引起了单核细胞和巨噬细胞形式的专业吞噬细胞强烈、快速地招募到小鼠耳蜗。本研究将使用新的药理学方法和敲除小鼠品系来研究巨噬细胞在耳蜗病理和损伤恢复中的作用。在第一组实验中，我们使用脂质体包裹氯膦酸钠在体内消耗耳蜗巨噬细胞，以确定招募的巨噬细胞对耳蜗损伤是保护还是有害。接下来，我们将研究在耳蜗单核细胞和巨噬细胞中表达的趋化因子受体CX3CR1在耳毒性损伤中的作用。我们的研究表明，CX3CR1基因缺失导致巨噬细胞进入耳蜗增加和耳毒性损伤增强。拟议的实验将确定抑制cx3cr1敲除小鼠中的巨噬细胞是否可以逆转这种作用。进一步的研究将检查主要清道夫受体CD36在受损毛细胞吞噬中的作用。我们已经证明CD36在耳蜗巨噬细胞和耳蜗支持细胞中都有表达，这表明这两种细胞类型都可能参与毛细胞碎片的吞噬。我们将使用CD36敲除小鼠来确定CD36是否对Corti器官中凋亡毛细胞的识别和去除至关重要。我们还将使用放射骨髓嵌合体分离CD36表达对白细胞和耳蜗支持细胞的影响。最后的实验将通过培养活体小鼠耳蜗的延时共聚焦成像来研究巨噬细胞在耳毒性中的动态作用。利用这项技术，我们可以跟踪巨噬细胞的运动和活动，以确定它们在耳毒性损伤中如何与毛细胞相互作用。这些实验将广泛研究毛细胞损伤后白细胞在内耳中的作用，并使我们开始了解免疫抑制如何影响内耳的正常过程和病理过程。

DESCRIPTION (provided by applicant): While the presence of the immune system in virtually all organ systems is widely recognized, little attention has been paid to the influences of immunity on the inner ear. Research in human subjects demonstrates that hearing loss is successfully treated with corticosteroids in some patients, presumably due to their anti-inflammatory and immunosuppressive effects. Unfortunately, we possess limited knowledge of the cellular mechanisms that influence interactions between inflammatory processes and normal function of the inner ear. Because steroid therapy provides such hope for successful treatment of hearing loss, the specific mechanisms of how inflammation affects hearing warrant further study. The long-term goal of our research is to identify the role of inflammation in cochlear maintenance and injury. In prior work, we have demonstrated that hair cell injury evokes a strong, rapid recruitment of professional phagocytes in the form of monocytes and macrophages into the mouse cochlea. The present studies will use novel pharmacological methods and knockout mouse strains to examine the role of macrophages in cochlear pathology and recovery from injury. In the first set of experiments, we use liposomally-encapsulated clodronate to deplete cochlear macrophages in vivo, in order to determine whether recruited macrophages are protective or detrimental in cochlear injury. Next, we will examine the role of the chemokine receptor CX3CR1, expressed in cochlear monocytes and macrophages, in ototoxic injury. Our studies have shown that genetic deletion of CX3CR1 leads to both increased macrophage entry into the cochlea and enhancement of ototoxic injury. Proposed experiments will determine whether suppressing macrophages in CX3CR1-knockout mice can reverse this effect. Additional studies will examine the involvement of the cardinal scavenger receptor, CD36, in the phagocytosis of injured hair cells. We have shown that CD36 is expressed by both cochlear macrophages and cochlear supporting cells, suggesting that both cell types may be involved in phagocytosis of hair cell debris. We will use CD36 knockout mice to determine whether CD36 is essential for the recognition and removal of apoptotic hair cells from the organ of Corti. We will also use radiation bone marrow chimeras to isolate the effects of CD36 expression on leukocytes versus cochlear supporting cells. A final experiment will examine the dynamic role of macrophages in ototoxicity by time lapse confocal imaging of the live mouse cochlea in culture. Using this technique, we can follow the movement and activity of macrophages to determine how they interact with hair cells in ototoxic injury. These experiments will broadly study the role of leukocytes in the inner ear after hair cell injury and will allow us to begin understanding how immunosuppression affects normal processes as well as pathological processes in the inner ear.