Anti-inflammatory role of MerTK in the RPE independent of diurnal outer segment phagocytosis

MerTK 在 RPE 中的抗炎作用不依赖于昼夜外节吞噬作用

基本信息

批准号：
10317323
负责人：
SILVIA C FINNEMANN
金额：
$ 23.91万
依托单位：
FORDHAM UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10317323
关键词：
Acute Adult Affect Age Animal Model Animals Anti-Inflammatory Agents Apoptosis Blindness Cell Communication Cell model Cells Cessation of life Childhood Complex Data Disease Distal Distress Embryonic Development Extracellular Protein Eye Family Functional disorder Genetic Transcription Hand Human IL18 gene Inflammation Inflammation Mediators Inflammatory Inherited Knock-out Ligands Ligation Link MERTK gene Mediating Mediator of activation protein Microglia Modeling Muller&apos s cell Mus Mutant Strains Mice Mutation Neurons Patients Phagocytes Phagocytosis Phosphatidylserines Phosphotransferases Photoreceptors Pilot Projects Plasma Cells Process Proteins Rattus Receptor Protein-Tyrosine Kinases Reporting Retina Retinal Degeneration Retinal Diseases Retinitis Pigmentosa Role Signal Transduction Surface Surgeon Teenagers Testing Translations cell type cohort college cytokine early onset experimental study follow-up in vivo inhibitor/antagonist mutant neuroinflammation novel phagocytosis receptor postnatal preservation prevent programs promoter receptor

项目摘要

Diurnal RPE phagocytosis is an essential aspect of lifelong continuous renewal of photoreceptor outer segments. Phagocytosis of distal photoreceptor outer segment tips requires activity of RPE surface receptors of the TAM (Tyro3/Axl/MerTK) receptor tyrosine kinase family. In human patients, mutations in the mertk gene cause an unusually severe form of retinitis pigmentosa with blindness in teenage years. The retina in mice and rats lacking MerTK develops normally but rapidly degenerates postnatally, with onset of photoreceptor death before 4 weeks of age. Neuroinflammation and specifically activation and redistribution of retinal microglia has been reported in adult MerTK-deficient animals and has been assumed to be a consequence of photoreceptor distress and debris accumulation caused by lack of daily clearance phagocytosis of spent outer segment tips by the RPE. However, we find that, surprisingly, robust increase in pro-inflammatory cytokines and microglia activation in MerTK-deficient rat and mouse retina precede postnatal onset of photoreceptor outer segment renewal and daily RPE phagocytosis. Moreover, our results show that anti-inflammatory treatment is effective in preventing microglia activation and extending photoreceptor survival and function only if applied at early postnatal age several days prior to the onset of diurnal outer segment renewal and RPE phagocytosis. In wild- type retina, MerTK is expressed early postnatally and mainly by the RPE (and not detected in microglia). This implies that loss of MerTK affects the RPE specifically, directly and before outer segment renewal begins. Thus, all available evidence supports microglia stimulation by MerTK-deficient RPE independently of RPE phagocytic activity and outer segment renewal. Here, we propose highly focused, key proof-of-principle pilot studies to rigorously scrutinize our completely novel concept that lack of MerTK in itself and unrelated to RPE phagocytosis causes RPE cells to release a pro-inflammatory cytokine, which elicits early onset, harmful neuroinflammation that in turn accelerates photoreceptor dysfunction and demise. We will test our hypothesis in 3 independent specific aims. Aim 1 will explore double mutant mice we just generated to test if cytokine is required for neuroinflammation in mertk-/- retina. Aim 2 will explore if inducing cytokine loss in the RPE alone is sufficient to eliminate early postnatal harmful neuroinflammation and whether cytokine mediator must be expressed early postnatally to induce neuroinflammation in mertk-/- retina. Aim 3 will explore polarized, differentiated RPE cell models (primary, unpassaged wild-type and MerTK-deficient RPE and passage 1 human RPE cells) to determine whether MerTK expression and its kinase signaling activity are directly linked mechanistically to cytokine transcription, translation, and/or release by RPE cells. Upon completion, this pilot study will have yielded unambiguous confirmation or disproof of our novel hypothesis and generated in vivo and culture models for large scope mechanistic and pre-translational follow-up.

昼夜 RPE 吞噬作用是光感受器外层终身持续更新的重要方面段。远端光感受器外节尖端的吞噬作用需要 RPE 表面受体的活性 TAM (Tyro3/Axl/MerTK) 受体酪氨酸激酶家族的成员。在人类患者中，突变在默特克基因导致异常严重的色素性视网膜炎并导致青少年失明。小鼠的视网膜和缺乏 MerTK 的大鼠发育正常，但出生后迅速退化，并伴有光感受器死亡 4周龄前。神经炎症以及视网膜小胶质细胞的具体激活和重新分布在成年 MerTK 缺陷动物中已有报道，并被认为是光感受器的结果由于缺乏每日清除消耗的外段尖端的吞噬作用而导致痛苦和碎片堆积通过 RPE。然而，令人惊讶的是，我们发现促炎细胞因子和小胶质细胞的强劲增加 MerTK 缺陷的大鼠和小鼠视网膜的激活先于出生后光感受器外节的出现更新和每日 RPE 吞噬作用。此外，我们的结果表明抗炎治疗是有效的仅在早期应用才能防止小胶质细胞激活并延长光感受器的存活和功能昼夜外节更新和 RPE 吞噬作用开始前几天的出生后年龄。在野外—— 视网膜型，MerTK 在出生后早期表达，主要由 RPE 表达（在小胶质细胞中未检测到）。这意味着 MerTK 的丢失会在外节更新开始之前直接、具体地影响 RPE。因此，所有可用的证据都支持 MerTK 缺陷的 RPE 独立于 RPE 刺激小胶质细胞吞噬细胞活性和外节更新。在这里，我们提出高度集中、关键的原理验证试点研究严格审查我们全新的概念，即本身缺乏 MerTK 并且与 RPE 无关吞噬作用导致 RPE 细胞释放促炎细胞因子，从而引发早期发病、有害的炎症神经炎症反过来会加速感光器功能障碍和死亡。我们将检验我们的假设 3个独立的具体目标。目标 1 将探索我们刚刚生成的双突变小鼠，以测试细胞因子是否 mertk-/- 视网膜神经炎症所需。目标 2 将探讨仅在 RPE 中诱导细胞因子损失是否有效足以消除产后早期有害的神经炎症以及是否必须使用细胞因子介质出生后早期表达，诱导 merk-/- 视网膜神经炎症。目标 3 将探索极化、分化的 RPE 细胞模型（原代、未传代的野生型和 MerTK 缺陷的 RPE 和第 1 代人 RPE 细胞）以确定 MerTK 表达及其激酶信号传导活性是否直接相关机械地影响 RPE 细胞的细胞因子转录、翻译和/或释放。该试点项目完成后研究将对我们的新假设产生明确的证实或反驳，并在体内产生以及用于大范围机械和翻译前随访的文化模型。