Complement as a modulator of immunosuppression and progression in Polycystic Kidney Disease

项目摘要 常染色体显性多囊肾病（ADPKD）是最常见的单等位基因肾病 肾囊肿是一种常见的肾病，其特征是肾囊肿持续生长，导致终末期肾病。一个FDA 批准的治疗是可用的，但它只是减缓囊肿生长和影响生活质量，突出了紧迫的 需要新的治疗方案。ADPKD表现为高表型变异，这表明ADPKD的机制 除了基因突变之外，PKD 1或PKD 2也会影响疾病的严重程度。最近的数据显示， 免疫细胞作为疾病严重程度的调节剂。多篇出版物强调M2样肾巨噬细胞 促进囊性进展。相反，来自我的K 01的数据，使用缓慢渐进的正交Pkd 1 p.R3277C（Pkd 1 RC/RC）ADPKD模型，显示CD 8 + T细胞可以抑制肾囊肿的生长。我们进一步发现， 肾脏囊性微环境显示出免疫抑制的多种特征 CD 4+调节性T细胞，代谢重编程和免疫检查点的参与。事实上，免疫 在Pkd 1 RC/RC模型中，检查点抑制剂导致CD 8 + T细胞的再活化，减轻囊性疾病。 目前尚不清楚的是驱动免疫抑制的机制，这可能是重要的/新的 治疗目标在癌症中，一种在细胞和分子水平上与PKD平行的疾病， 补体途径已显示促进免疫抑制微环境。补体 级联反应是调节适应性免疫的先天免疫系统的中心部分。补充已经 显示在PKD细胞、患者肾脏/囊液和鼠模型中上调，包括Pkd 1 RC/RC 正如我们的初步数据所示。此外，补体的遗传丢失或非选择性抑制 蛋白质C3减缓鼠PKD模型中的囊肿生长。我们假设补体信号驱动PKD， 部分，通过创造一个免疫抑制微环境，损害适应性免疫系统停止 囊肿生长该项目有两个目标。在目标1中，我们将利用荧光激活细胞分选，流式细胞术， 和多光谱免疫荧光成像，以确定哪些细胞是关键的生产者和反应者， Pkd 1 RC/RC肾脏中的补体信号传导。我们将进一步将这些发现与疾病的严重程度联系起来。在Aim中 2，我们将测试是否有针对性的补体抑制剂导致囊性疾病。这将是此类研究中的第一个 在与ADPKD正交的模型Pkd 1 RC/RC小鼠中使用临床相关的特异性补体抑制剂。 我们将进一步利用复杂的64-荧光团能力的流式细胞术方法来研究如何 补体抑制改变了囊性免疫微环境，特别关注免疫抑制 功能.这些调查直接扩展了我的NIDDK K 01工作，并将提供关键的初步数据 竞争R 01应用程序。获得这个R 03将进一步促进我过渡到一个独立的 一个创新的，临床前和转化研究计划的研究者，专注于研究的作用， 囊状微环境作为PKD的调节剂和治疗靶点。

PROJECT SUMMARY Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common, monoallelic nephropathy worldwide, characterized by continuous renal cysts growth leading to end stage kidney disease. A single FDA approved therapy is available, but it merely slows cyst growth and impacts quality of life, highlighting the urgent need for new treatment options. ADPKD presents with high phenotypic variability, suggesting that mechanisms beyond the genetic mutation to either PKD1 or PKD2 influence disease severity. Recent data have implicated immune cells as modulators of disease severity. Multiple publications highlight that M2-like renal macrophages promote cystic progression. Conversely, data from my K01, using the slowly progressive, orthologous Pkd1 p.R3277C (Pkd1RC/RC) ADPKD model, show that CD8+ T cells can inhibit renal cyst growth. We further found that the renal cystic microenvironment displays multiple features of immunosuppression such as increased numbers of CD4+ regulatory T cells, metabolic reprogramming, and engagement of immune checkpoints. Indeed, immune checkpoint inhibitors, which lead to reactivation of CD8+ T cells, alleviate cystic disease in the Pkd1RC/RC model. What remains unknown are the mechanisms that drive immunosuppression, which may present important/novel therapeutic targets. In cancer, a disease that parallels PKD at the cellular and molecular level, activation of the complement pathway has been shown to promote an immunosuppressive microenvironment. The complement cascade is a central part of the innate immune system that regulates adaptive immunity. Complement has been shown to be upregulated in PKD cells, patient kidneys/cyst fluid, and murine models, including the Pkd1RC/RC mouse as shown by our preliminary data. Further, genetic loss or non-selective inhibition of the complement protein C3 slows cyst growth in murine PKD models. We hypothesize that complement signaling drives PKD, in part, by creating an immunosuppressive microenvironment which impairs the adaptive immune system to halt cyst growth. This project has two aims. In Aim 1, we will utilize fluorescent-activated cell sorting, flow cytometry, and multispectral immunofluorescent imaging to determine which cells are key producers and responders of complement signaling in the Pkd1RC/RC kidney. We will further correlate these findings to disease severity. In Aim 2, we will test if targeted complement inhibition alleviates cystic disease. This will be the first of such studies using a clinically relevant, specific complement inhibitor in a model orthologous to ADPKD, the Pkd1RC/RC mouse. We will further utilize sophisticated 64-fluorophore capable flow cytometry methods to investigate how complement inhibition alters the cystic immune microenvironment with specific focus on immunosuppressive features. These investigations directly expand upon my NIDDK K01 work and will provide key preliminary data for a competitive R01 application. Obtaining this R03 will further facilitate my transition into an independent investigator with an innovative, preclinical, and translational research program that focuses on the role of the cystic microenvironment as modulator of and therapeutic target for PKD.