Characterizing the negative signaling in dendritic cells and macrophages to attenuate inflammation and bone destruction in rheumatoid arthritis

这项提议的目的是了解负性内源性信号传导的潜在机制， 减少类风湿性关节炎（RA）的慢性炎症，这可以为治疗类风湿性关节炎提供新的治疗靶点。 与炎症和组织破坏有关的疾病。目前的RA治疗有严重的负面影响 对患者的免疫系统有影响，并可能导致恶性肿瘤、肺炎和肺结核。正调节子 RA炎症反应的负性内源性因子（如NF-κB）已经得到很好的表征，然而， 调节RA诱导的炎症和骨破坏，特别是在巨噬细胞和树突状细胞中， 类风湿关节炎的病理状态仍然难以捉摸。拟议的研究将描述负内源性 巨噬细胞和树突状细胞（DC）的分化和活性调节因子， 炎症和组织破坏。我们注意到巨噬细胞或树突状细胞中Gα13缺乏， 加重小鼠RA模型的炎症反应。AAV介导的局部Gna 13组成型过表达 活性形式（Gna 13 CA）不仅显著减少骨破坏，而且显著抑制 结果表明，Gna 13 CA可以在RA小鼠模型中预防炎症。我们 RNA测序（RNA-seq）分析显示Gα13可能负性调节巨噬细胞和DC 分化和活化以及ChIP测序分析显示NF-κB基序富集 Gα13缺陷巨噬细胞炎症相关基因全基因组启动子的位点结合 用TNFα或LPS刺激。我们的初步数据表明，Gα13缺陷促进Akt活性，STAT 3 在树突状细胞和巨噬细胞中的磷酸化和NF-κB信号通路活化。基于 在我们的初步研究中，我们假设Gα13是一个主要的负调节因子， 通过Gα13/RhoA/AKT/IKK/NF-κB途径与类风湿关节炎相关的骨破坏 巨噬细胞和树突细胞。提出了三个具体目标来检验我们的假设。在目标1中，我们 确定Gα13在类风湿性关节炎（RA）炎症和骨破坏中的作用， 通过功能丧失建立条件性基因敲除（CKO）小鼠模型的表型和病理机制 在RA的方法。目的2：明确Gα 13 CA在炎症和骨组织中的作用及治疗作用 通过表征条件性转基因的表型和病理机制， 在RA中通过功能获得方法在过表达（OE）小鼠模型中进行。我们将描述 Gα13通过Gα13/ 巨噬细胞和树突状细胞中的RhoA/AKT/IKK/NF-κB信号传导、STAT 3信号传导和TLR信号传导途径 目标3中的细胞。这项拟议的研究将提供重要的见解，细胞的负调控， 免疫系统有效地靶向RA中的炎症和骨破坏。从本研究中获得的知识 可能为治疗RA和其它炎性骨疾病带来潜在的上级治疗手段。

The goal of this proposal is to understand the mechanisms underlying how negative endogenous signaling reduces chronic inflammation in rheumatoid arthritis (RA), which can provide novel therapeutic targets for diseases related to inflammation and tissue destruction. Current therapies for RA have serious negative effects on the patients’ immune system, and can cause malignancies, pneumonia, and tuberculosis. Positive regulators of inflammation in RA (such as NF-κB) have been well characterized, however, the negative endogenous regulators of RA-induced inflammation and bone destruction, especially in macrophages and dendritic cells in RA pathological condition remain elusive. The proposed study will characterize the negative endogenous regulators of differentiation and activity in macrophages and dendritic cells (DCs) that attenuate RA-induced inflammation and tissue destruction. We noted that Gα13 deficiency in macrophages or dendritic cells aggravated inflammation in mouse RA model. AAV mediated overexpression of local Gna13 constitutively active form (Gna13CA) not only significantly reduced bone destruction, but also dramatically inhibited inflammation in RA, indicating that Gna13CA could protect against inflammation in a mouse model of RA. Our RNA sequencing (RNA-seq) analysis showed that Gα13 may negatively regulate macrophage and DC differentiation and activation and ChIP sequencing (ChIP-seq) analysis showed the enrichment of NF-κB motif site binding on the genome-wide promoters of the genes related to inflammation in Gα13 deficient macrophages with TNFα or LPS stimulations. Our preliminary data showed that Gα13 deficiency promotes Akt activity, STAT3 phosphorylation and NF-κB signaling pathway activation in dendritic cells and macrophages. Based on our preliminary studies, we hypothesize that Gα13 is a master negative regulator that attenuates inflammation and bone destruction associated with rheumatoid arthritis through the Gα13/RhoA/AKT/IKK/NF-κB pathway in macrophages and dendritic cells. Three specific aims are proposed to test our hypothesis. In Aim 1, we will determine the role of Gα13 in inflammation and bone destruction in rheumatoid arthritis (RA) by characterizing the phenotypes and pathomechanism of conditional knockout (CKO) mouse models via loss-of-function approach in RA. In Aim 2, we will define the roles and therapeutic effects of Gα13CA in inflammation and bone destruction in RA by characterizing the phenotypes and pathomechanism of conditional transgenic overexpression (OE) mouse models via gain-of-function approach in RA. We will characterize the molecular mechanism by which Gα13 regulates inflammation and bone destruction in RA through Gα13/ RhoA/AKT/IKK/NF-κB signaling, STAT3 signaling, and TLRs signaling pathways in macrophages and dendritic cells in Aim 3. The proposed study will provide important insights into the negative regulation of the cells of the immune system to effectively target inflammation and bone destruction in RA. Knowledge gained from this study may bring forth potential superior therapeutic means for treating RA and other inflammatory bone diseases.