The immunological mechanism of PGRNs anti-inflammatory effect

性状（由申请方提供）：肿瘤坏死因子-a（TNF？）由于其位于促炎细胞因子级联反应的顶点，以及其在炎症发病机制中的主导地位，因此受到了最大的关注。抗TNF治疗已被接受为治疗炎性关节炎的有效方法。在对颗粒蛋白前体（PGRN）生长因子结合蛋白的全面筛选中，我们发现PGRN与TNFR 1和TNFR 2结合。随后的研究表明，PGRN保护调节性T细胞（Treg）免受TNF？的负调节; PGRN信号传导的丧失使得B6小鼠对胶原诱导的关节炎（CIA）高度敏感，而重组PGRN预防CIA模型中的炎症性关节炎;并且用PGRN的体内处理刺激Treg细胞中的IL-10产生，并且IL-10信号传导对于体外PGRN刺激的Treg功能是重要的。这些研究导致了中心假设，即PGRN通过与以下物质的相互作用发挥其保护作用： 炎症性关节炎过程中TNFR和IL-10产生Treg细胞的刺激具体目的：（1）研究PGRN在炎症性关节炎发病中的作用及其机制。我们将确定是否诱导敲除PGRN的小鼠与现有的炎症性关节炎的关节炎症状。我们假设PGRN缺陷小鼠中观察到的易感表型可能是由于Treg细胞的低频率。我们将研究PGRN在CIA发病机制中的免疫调节作用，重点关注PGRN在Treg发育中的作用及其潜在机制。(2)阐明TNFR在炎症性关节炎发病机制中PGRN介导的信号传导中的作用。我们将首先比较TNF？/ TNFR和PGRN/TNFR，使用串联亲和纯化，然后进行质谱分析。我们还将比较PGRN和TNF？的关键细胞内事件，包括信号传导、靶基因表达谱和NF？正常人和炎性关节炎患者Treg细胞中的B通路。此外，我们将阐明TNFR是重要的介导PGRN的保护作用，在CIA使用TNFR 1-/-和TNFR 2-/-小鼠。(3)确定IL-10在炎症性关节炎发病机制中介导PGRN作用的作用。为了深入了解已知的PGRN疗法的机制，我们将使用IL-10-GFP报告基因老虎小鼠和Foxp 3 RFP报告基因FIR小鼠来鉴定IL-10表达的非Treg细胞和响应于CIA中PGRN的获得或丧失的Treg细胞，这将提供关于为什么PGRN缺陷的B6小鼠变得对CIA高度易感以及IL-10是否是CIA中PGRN的靶标的更多了解。为了检验PGRN通过IL-10介导所观察到的对CIA的治疗效果的假设，我们将确定PGRN诱导的表达IL-10的TGFRP的过继转移是否可以抑制CIA，以及IL-10信号传导的阻断是否抑制PGRN对CIA的作用。提出的研究不仅将为CIA的发病机制提供新的见解，而且还将导致开发用于炎性关节炎和其他TNFR介导的病理和病症的新的治疗策略。

DESCRIPTION (provided by applicant): Tumor necrosis factor-a (TNF?) has received the greatest attention because of its position at the apex of the pro-inflammatory cytokine cascade, and its dominance in the pathogenesis of inflammation. Anti-TNF therapies have been accepted as the effective approach to treating inflammatory arthritis. In a global screen for the binding proteins of progranulin (PGRN) growth factor, we found that PGRN bound to TNFR1 and TNFR2. Subsequent studies demonstrated that PGRN protected regulatory T cells (Treg) from negative regulation by TNF?; Loss of PGRN signaling rendered B6 mice highly susceptible to collagen-induced arthritis (CIA), whereas re-combinant PGRN prevented inflammation arthritis in CIA models; and in vivo treatment with PGRN stimulated IL-10 production in Treg cells and IL-10 signaling was important for PGRN-stimulated Treg function in vitro. These studies have led to the central hypothesis, that PGRN exerts its protective effects through its interaction with TNFR and stimulation of IL-10 producing Treg cells in the course of inflammatory arthritis. The Specific Aims are: (1) To determine the roles of PGRN and underlying mechanisms in the pathogenesis of inflammatory arthritis. We will determine whether inducible knockout of PGRN worsens arthritic symptoms in mice with existing inflammatory arthritis. We hypothesize that the susceptible phenotype seen in PGRN deficient mice may be due to the low frequency of Treg cells. We will study the immune regulatory roles of PGRN in the pathogenesis of CIA, focusing on the role of PGRN and the underlying mechanisms in Treg development. (2) To elucidate TNFR's role in PGRN-mediated signaling in the pathogenesis of inflammatory arthritis. We will first compare the receptor complexes of TNF?/TNFR and PGRN/TNFR using tandem affinity purification, followed by mass spectrometry. We will also compare the key intracellular events of PGRN and TNF?, including signaling, target gene expression profiling, and activation of the NF?B pathway in human Treg cells isolated from normal and the patients with inflammatory arthritis. Furthermore, we will elucidate which TNFR is important for mediating PGRN's protective role in CIA by using TNFR1-/- and TNFR2-/- mice. (3) To define the role of IL-10 in mediating PGRN action in the pathogenesis of inflammatory arthritis. To gain insight into the mechanisms of already known therapies of PGRN, we will use IL-10-GFP reporter tiger mice and Foxp3 RFP reporter FIR mice to identify the IL-10 expressing non-Treg cells and Treg cells in response to gain- or loss-of- PGRN in CIA, which would provide more insight on why PGRN-deficient B6 mice become highly susceptible to CIA, and whether IL-10 is the target of PGRN in CIA. To test the hypothesis that PGRN via IL-10 mediates the observed therapeutic effect upon CIA, we will determine if adoptive transfer of PGRN-induced IL-10-expressing Tregs can suppress CIA, and whether blockage of IL-10 signaling inhibits PGRN's effects on CIA. The pro- posed studies will not only provide new insights into the pathogenesis of CIA, but also lead to the development of novel therapeutic strategies for inflammatory arthritis and other TNFR-mediated pathologies and conditions.