Therapeutic modulation of calcium- and calciprotein particle-induced NLRP3 inflammasome activation in rheumatoid arthritis

NLRP3炎性小体的激活和随后的IL-1β释放是先天免疫系统最强大的促炎机制，并已报道在许多病理情况下驱动炎症。钙敏感受体在甲状旁腺和肾脏中监测全身钙稳态，但它也在免疫细胞中强烈表达，并被发现是炎症性疾病中重要的促炎介质。我们之前报道过，钙诱导的信号通过钙感知受体触发单核细胞NLRP3炎性体激活，导致大量IL-1β和其他caspase1依赖性细胞因子的释放，以及TNF和IL-6的释放。最近，我们可以进一步阐明潜在的机制，表明在钙浓度足够的情况下，含有磷酸盐和胎儿蛋白A的钙蛋白颗粒以CaSR信号依赖的方式形成并被单核细胞巨噬症吸收。类风湿关节炎是最常见的自身免疫性疾病之一，其特征是中小型关节的破坏性炎症，往往使人衰弱，有些患者也会出现内脏器官的炎症。该疾病的长期后果，特别是对疾病活动控制不佳的患者，包括主要由于心血管事件频率增加而导致的预期寿命显著降低。在一项临床研究中，我们的研究小组可以证明，离子化钙诱导类风湿关节炎患者单核细胞IL-1β反应增加，类风湿关节炎的单核细胞比对照单核细胞吸收钙蛋白颗粒的速度要高得多。在这个项目中，我们将测试各种方法来干扰治疗这一途径，以抑制RA中钙诱导的炎症。首先，我们将使用药理学和遗传学方法来抑制CaSR信号，并研究在这些条件下自身免疫性关节炎的病程。在第二种方法中，患关节炎的小鼠将接受巨噬细胞抑制剂的治疗，以调节关节炎的进程。第三，用组织蛋白酶抑制剂和组织蛋白酶b缺陷小鼠研究关节炎中钙蛋白颗粒溶酶体的分解。此外，我们将进一步研究从CPP降解到NLRP3激活和IL-1β释放的细胞内途径。我们将使用关节炎的遗传小鼠模型以及胶原蛋白和胶原抗体诱导的关节炎来测试这一途径中每个步骤与炎症发生和延续的相关性。在项目结束时，我们将确定最有希望的治疗方法来抑制人类类风湿关节炎中钙诱导的NLRP3炎性体激活。

Activation of the NLRP3 inflammasome and subsequent IL-1β release is the most powerful pro-inflammatory mechanism of the innate immune system and has been reported to drive inflammation in numerous pathological scenarios. The calcium sensing receptor is monitoring systemic calcium homeostasis in parathyroid gland and kidney, but it is also strongly expressed in immune cells and has been found to be an important pro-inflammatory mediator in inflammatory diseases. We have reported previously, that calcium-induced signaling via the calcium sensing receptor triggers NLRP3 inflammasome activation in monocytes resulting in the release of high amounts of IL-1β and other Caspase1-dependent cytokines, but also of TNF and IL-6. Recently, we could further elucidate the underlying mechanism by showing, that in the presence of sufficient calcium concentration, calciprotein particles containing phosphate and fetuin A are formed und taken up by monocyte macropinocytosis in a CaSR signaling dependent fashion. Rheumatoid arthritis is one of the most common autoimmune diseases and characterized by a destructive and often debilitating inflammation of small and medium sized joints, and in some patients also of internal organs. Long-term consequences of the disease, in particular in patients with poorly controlled disease activity, include a significantly decreased life expectancy primarily due to an increased frequency of cardiovascular events. In a clinical study, our group could show that ionized calcium induces increased IL-1β responses in monocytes from patients with rheumatoid arthritis, and that RA monocytes take up calciprotein particles at a much higher rate than control monocytes. In this project, we will test various approaches to interfere therapeutically with this pathway, in order to inhibit calcium induced inflammation in RA. First, we will use pharmacologic and genetic methods to inhibit CaSR signaling and investigate the course of autoimmune arthritis under those conditions. In the second approach, arthritic mice will be treated with inhibitors of macropinocytosis in order to modulate the course of arthritis. And thirdly, Cathepsin inhibitors and cathepsin B-deficient mice will be used to investigate lysosomal breakdown of calciprotein particles in arthritis. In addition, we will further investigate the intracellular pathway leading from CPP degradation to NLRP3 activation and IL-1β release. The relevance of each step of this pathway for the initiation and perpetuation of inflammation will be tested using genetic mouse model of arthritis as well as collagen- and collagen-antibody-induced arthritis. At the conclusion of the project, we will have identified the most promising therapeutic approach to the inhibition of calcium induced NLRP3 inflammasome activation in human rheumatoid arthritis.