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

昼夜RPE吞噬是光感受器外膜终生持续更新的重要方面 细分市场。远端光感受器外节末端的吞噬作用需要RPE表面受体的活性 (Tyro3/Ax1/MerTK)受体酪氨酸激酶家族成员。在人类患者中，突变 在MerTK中 基因 导致一种异常严重的色素性视网膜炎，并在青少年时期失明。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，出现光感受器死亡 4周龄前。神经炎症和视网膜小胶质细胞的特异性激活和重新分布 已报道在成年MerTK缺乏的动物中，并被认为是光感受器的结果 残存外段尖端缺乏每日清除吞噬作用引起的窘迫和碎屑堆积 在RPE旁边。然而，我们发现，令人惊讶的是，促炎细胞因子和小胶质细胞的强劲增长 MerTK缺陷大鼠和小鼠视网膜的激活先于光感受器外段的出生后开始 更新和每日RPE吞噬。此外，我们的结果表明，抗炎治疗是有效的。 只有在早期使用才能阻止小胶质细胞的激活和延长光感受器的存活和功能 出生后几天开始出现昼夜外段更新和RPE吞噬。在野外- 在视网膜类型中，MerTK在出生后早期表达，主要通过RPE(小胶质细胞未检测到)。这 意味着MerTK的丢失会在外部数据段更新开始之前对RPE产生特定的、直接的影响。 因此，所有可用的证据都支持MerTK缺乏的RPE对小胶质细胞的刺激不依赖于RPE 吞噬活性和外节更新。在这里，我们建议进行高度集中、关键的原则验证试点 研究严格审查我们的全新概念，即缺乏MerTK本身且与RPE无关 吞噬作用导致RPE细胞释放一种促炎细胞因子，这会导致早发、有害的 神经炎症，进而加速光感受器功能障碍和死亡。我们将检验我们的假设 在三个独立的具体目标中。Aim 1将探索我们刚刚培育的双突变小鼠，以测试细胞因子是否 MerTK-/-视网膜神经炎症所需。目标2将探索是否仅在RPE中诱导细胞因子丢失是 足以消除出生后早期有害的神经炎症以及细胞因子介质是否必须 在出生后早期表达以诱导MerTK-/-视网膜的神经炎症。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的表达和其激酶信号活性是否直接相关 由RPE细胞机械地转录、翻译和/或释放细胞因子。完成后，这一试点 研究将明确地证实或反证我们的新假设，并在体内产生 以及大范围机械性和翻译前跟进的文化模式。

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.