Dissecting the heterogeneity and function of myeloid cells in lupus nephritis

项目摘要 狼疮性肾炎（LN）是系统性红斑狼疮的常见表现，可导致 不可逆肾损害。目前的免疫抑制疗法在超过一半的人中无法逆转疾病。 治疗的患者，只有2种新药已被批准的治疗，这两种药物都只提供适度的 改进.在人类和动物模型中，肾髓样细胞参与LN的肾损伤。 巨噬细胞具有高度可塑性，可以介导促炎和修复功能。因为 骨髓细胞有很多亚群，其中一些随着时间的推移而改变功能， 单细胞对于这些细胞的研究至关重要。第1阶段和第2阶段早期的初步研究 加速药品伙伴关系-SLE（AMP-SLE），其中我们都是成员，已经表明， LN肾脏中的巨噬细胞的多个亚群类似于小鼠中的髓样亚群，但其功能 这些亚群以及它们是致病的还是保护性的仍然不清楚。肾组织的研究 LN患者的肾脏活检体积小， 患者在其疾病过程中。因此，动物模型，当仔细和严格 使用，是特别有用的解决通过检查人类数据产生的假设。 我们的总体假设是，我们将在LN模型中鉴定与人类相关的髓系细胞亚群， 反映了疾病病理生理学、疾病阶段和对治疗的反应性的差异， 信息将有助于确定治疗干预和直接个性化治疗的新途径。我们 方法的重点是识别和测试人类相关细胞亚群和途径的功能， 体外和体内生理条件下。在目标1中，我们将完成单细胞RNASeq的整合， 分析来自5种不同LN模型的髓样细胞与来自AMP-SLE 2期的髓样细胞， 和独特的巨噬细胞亚群。我们将跟踪外周髓系细胞在转化过程中的命运 从血液进入肾脏，以确定每个子集的肾炎特异性特征以及它们如何 相互关联然后，我们将确定是否有一个特定的骨髓细胞谱， LN患者对标准治疗有反应或无反应。在目标2中，我们将确定如何 在体外和体内，肾髓样细胞与肾基质细胞相互作用。我们将分析肾脏的命运 缓解治疗的人相关小鼠模型中的骨髓细胞（应答和非应答小鼠中） 诱导治疗，以确定修复子集和途径。我们在这里的重点将是两个标准， 治疗和贝利木单抗（抗BAFF），因为这是迄今为止唯一获批用于治疗 LN和预防进一步的肾耀斑。在目标3中，我们将使用CRISPR文库用于体内靶向13种 我们已经在初步研究中确定了转录因子，以筛选新的治疗方法， 保护LN肾脏免于疾病进展、纤维化和终末期肾病。

PROJECT SUMMARY Lupus nephritis (LN) is a common manifestation of systemic lupus erythematosus that can lead to irreversible renal impairment. Current immunosuppressive therapies fail to reverse disease in more than half of treated patients and only 2 new drugs have been approved treatment, both of which confer only modest improvement. Renal myeloid cells are involved in renal injury in LN, both in humans and animal models. Macrophages are highly plastic and can mediate both pro-inflammatory and reparative functions. Because there are so many subsets of myeloid cells and some of them change their function over time, analysis of single cells is crucial to the study of these cells. Initial studies in Phase 1 and early Phase 2 of the Accelerating Medicines Partnerships-SLE (AMP-SLE), of which we are all members, have shown that there are multiple subsets of macrophages in LN kidneys that are analogous to myeloid subsets in mice but the function of these subsets and whether they are pathogenic, or protective is still not known. Study of kidney tissues in LN patients is hampered by the small size of the kidney biopsy and the infrequency of these biopsies in individual patients over the course of their disease. Therefore, animal models, when carefully and rigorously employed, are especially useful for addressing hypotheses generated by examining human data. Our overall hypothesis is that we will identify human-relevant myeloid cell subsets in LN models that reflect differences in disease pathophysiology, disease stage and responsiveness to treatments and that this information will help identify new pathways for therapeutic intervention and direct personalized treatment. Our approach is focused on identifying and testing the function of human-relevant cell subsets and pathways in vitro and under physiologic conditions in vivo. In Aim 1 we will complete the integration of single cell RNASeq analysis of myeloid cells from 5 different models of LN with that from AMP-SLE Phase 2 to map both shared and unique macrophage sub-populations. We will follow the fate of peripheral myeloid cells as they transition from the blood into the kidneys to define how the nephritis-specific profile of each subset and how they are related to each other. We will then determine whether there is a particular myeloid cell profile that is associated with response or non-response of LN patients to standard of care therapy. In Aim 2 we will determine how renal myeloid cells interact with renal stromal cells both in vitro and in vivo. We will analyze the fate of renal myeloid cells (in responder and non-responder mice) in human-relevant mouse models treated with remission induction therapies to identify reparative subsets and pathways. Our focus here will be on both standard of care therapy and on belimumab (anti-BAFF) since this is the only biologic drug so far approved for treatment of LN and for prevention of further renal flares. In Aim 3 we will use CRISPR libraries for in vivo targeting of 13 transcription factors we have identified in preliminary studies to screen for new therapeutic approaches to protect LN kidneys from disease progression, fibrosis, and end stage renal disease.