COCHLEAR MACROPHAGES AND EPITHELIAL REPAIR

描述(申请人提供)：虽然免疫系统在几乎所有器官系统中的存在被广泛认识，但很少有人注意到免疫对内耳的影响。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知之甚少。由于类固醇治疗为成功治疗听力损失提供了希望，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护和损伤中的作用。在之前的工作中，我们已经证明了毛细胞损伤会引起专业吞噬细胞以单核细胞和巨噬细胞的形式强烈、快速地募集到小鼠耳蜗里。本研究将使用新的药理学方法和基因敲除小鼠品系来研究巨噬细胞在耳蜗病理学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的氯屈膦酸盐在体内耗尽耳蜗巨噬细胞，以确定招募的巨噬细胞在耳蜗损伤中是保护还是有害的。接下来，我们将研究在耳蜗单核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的基因缺失会导致巨噬细胞进入耳蜗的数量增加，并加强耳毒性损伤。拟议中的实验将确定抑制CX3CR1基因敲除小鼠的巨噬细胞能否逆转这一效应。其他研究将检查主要清道夫受体CD36在受损毛细胞吞噬过程中的作用。我们发现CD36在耳蜗单核巨噬细胞和支持细胞上均有表达，提示这两种细胞可能参与了毛细胞碎片的吞噬作用。我们将使用CD36基因敲除小鼠来确定CD36是否是识别和去除Corti器官中的凋亡毛细胞所必需的。我们还将使用放射骨髓嵌合体来分离CD36表达对白细胞和耳蜗支持细胞的影响。最后一项实验将通过对培养的小鼠耳蜗活组织进行延时共聚焦成像，来检验巨噬细胞在耳毒性中的动态作用。使用这项技术，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。这些实验将广泛地研究毛细胞损伤后白细胞在内耳中的作用，并将使我们开始了解免疫抑制如何影响内耳的正常过程以及病理过程。 公共卫生相关性：在各种形式的损伤后，内耳退化，炎症细胞离开血管进入耳蜗组织。这些炎性细胞在多大程度上有助于修复或加剧损伤尚不确定。我们将使用抑制炎症的重要细胞成分--单核细胞和巨噬细胞的方法，来确定这些细胞如何影响暴露于耳毒性药物后的内耳损伤。

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. PUBLIC HEALTH RELEVANCE: After various forms of injury, the inner ear degenerates and inflammatory cells exit the blood vessels and enter the cochlea. To what degree these inflammatory cells could be helpful for repair or could serve to exacerbate injury is uncertain. We will use methods to suppress monocytes and macrophages, important cellular components of inflammation, to determine how these cells affect injury in the inner ear after exposure to ototoxic medications.