Suppressor of Cytokine Signaling and Control of Inflammation in Lyme Disease

描述(申请人提供)：莱姆病，由伯氏疏螺旋体引起，是一种新出现的传染病，因此，具有重大的公共卫生意义。螺旋体侵袭多个器官，引起急性和慢性炎症。由螺旋体引起的炎症在该病的发病机制中起主要作用。我们广泛的长期目标是确定莱姆病炎症控制所需的机制(S)。最近，我们观察到伯氏杆菌单独或与IL-10共同诱导J774巨噬细胞表达细胞因子信号转导抑制因子(SOCS)1和3。这与IL-10介导的抑制促炎细胞因子IL-6、IL-12p40、IL-18、TNF？和IL-1？我们的假设是，伯氏杆菌单独或与IL-10共同诱导巨噬细胞SOCS1/SOCS3的表达在莱姆病病程中的炎症控制中具有重要的功能；SOCS1/SOCS3可能作为IL-10抗炎活性的介质或作为炎症细胞因子信号的直接调节因子。1.应用RNA干扰技术(RNAi)研究沉默J774巨噬细胞中Socs1/SOCS3基因表达的作用：(A)抑制IL-10介导的伯氏杆菌诱导的炎性细胞因子；(B)对炎性细胞因子信号转导的影响。我们将通过向巨噬细胞递送小干扰RNA(SiRNA)双链，然后在存在和不存在IL-10的情况下用活的螺旋体或螺旋体脂蛋白刺激来沉默SOCS基因。通过这种方式，我们将评估(I)IL-10抑制促炎细胞因子(IL-6、IL-12p40、IL-18、TNF？、IL-1？)的能力。使用实时定量聚合酶链式反应(QPCR)和ELISAs，以及(Ii)SOCs通过抑制STAT蛋白的激活和DNA结合活性(对IL-6、IL-12、干扰素？)的激活和DNA结合活性而对炎性细胞因子信号转导的影响。核因子？B(对IL-1β、IL-12、肿瘤坏死因子？)使用基因功能分析。2.比较莱姆病抗性C57BL/6J和易感C3H/HeJ小鼠感染伯氏杆菌前后原代巨噬细胞和关节组织中SOCS1/SOCS3的表达，并探讨SOCS1/SOCS3表达与IL-10抗炎活性及疾病的关系。我们将使用来自小鼠骨髓、淋巴结和脾的巨噬细胞(并像目标1那样刺激它们)通过qPCR、Western印迹和共聚焦显微镜来评估SoCs的表达水平。我们将把巨噬细胞中SOCS的表达(I)与IL-10介导的对关节组织中促炎症细胞因子的抑制(如目标1)和(II)与疾病的严重程度相关联。如果SOCs(或其下游介质)在这两个菌株的小鼠细胞和组织中差异表达，并且SOCS沉默导致巨噬细胞中莱姆病炎症反应的失调，那么SOCS途径可能决定了对莱姆病的易感性和抵抗力，就像在小鼠身上建模的那样，也许在人类中也是如此。 莱姆病是由伯氏疏螺旋体引起的一种新发传染病，具有重大的公共卫生意义。炎症是由螺旋体在多个器官中引起的，在疾病的发病机制中起着重要作用，但其调节机制尚不清楚。实现这一建议的目标将解释炎症是如何调节的，并有助于理解莱姆病的易感性和抵抗力的基础。

DESCRIPTION (provided by applicant): Lyme disease, caused by the spirochete Borrelia burgdorferi, is an emerging infectious disease, and therefore, of major public health significance. The spirochete invades multiple organs and induces acute and chronic inflammatory conditions. Inflammation, as induced by the spirochete, plays a primary role in the pathogenesis of the disease. Our broad long-term goal is to identify the mechanism(s) required for the control of inflammation in Lyme disease. Recently we observed that B. burgdorferi alone or together with IL-10 additively induced the expression of the suppressor of cytokine signaling (SOCS) 1 and 3 in J774 macrophages. This correlated with the IL-10-mediated inhibition of the pro-inflammatory cytokines IL-6, IL-12p40, IL-18, TNF? and IL-1?. Our hypothesis is that expression of SOCS1/SOCS3 induced by B. burgdorferi alone or together with IL-10 in macrophages is functionally important in the control of inflammation during the course of Lyme disease; SOCS1/SOCS3 would act either as mediators of the IL-10 anti-inflammatory activity or as direct modulators of inflammatory cytokine signaling. The specific aims are to: 1. Determine the effect of silencing socs1/socs3 gene expression in J774 macrophages using RNA interference (RNAi) (a) on the IL-10-mediated inhibition of inflammatory cytokines induced by B. burgdorferi and (b) on inflammatory cytokine signaling. We will silence socs genes by delivering small interfering RNA (siRNA) duplexes into macrophages, followed by stimulation with live spirochetes or spirochetal lipoproteins in the presence and absence of IL-10. In this fashion we will assess (i) the ability of IL-10 to inhibit pro-inflammatory cytokines (IL-6, IL-12p40, IL-18, TNF?, IL-1?) using quantitative real-time PCR (qPCR) and ELISAs and (ii) the effect of SOCS on inflammatory cytokine signaling by their ability to inhibit the activation and DNA binding activities of STAT proteins (in response to IL-6, IL-12, IFN?) and of NF?B (in response to IL-1¿, IL-12, TNF?) using gene function assays. 2. Compare the expression of SOCS1/SOCS3 in primary macrophages and joint tissues from Lyme disease-resistant C57BL/6J and disease- susceptible C3H/HeJ mice before and during the course of a B. burgdorferi infection, and to correlate SOCS1/SOCS3 expression with IL-10 anti-inflammatory activity and with disease. We will employ macrophages derived from the bone marrow, lymph nodes and spleen from mice (and stimulate them as in Aim 1) to assess the expression levels of SOCS by qPCR, Western blot and confocal microscopy. We will correlate SOCS expression (i) in macrophages with the IL-10-mediated inhibition of pro-inflammatory cytokines (as in Aim 1) and (ii) in joint tissues with disease severity. If SOCS (or their downstream mediators) are differentially expressed in cells and tissues of mice from those two strains, and SOCS silencing results in the dysregulation of Lyme disease inflammatory responses in macrophages, then the SOCS pathway may dictate susceptibility and resistance to Lyme disease, as modeled in mice, and perhaps also in humans. Lyme disease, caused by the spirochete Borrelia burgdorferi, is an emerging infectious disease, and therefore, of major public health significance. Inflammation, as induced by the spirochete in multiple organs, plays a major role in disease pathogenesis, but its regulation is not understood. Achieving this proposal's goal will explain how inflammation is regulated, and help understand the basis of susceptibility and resistance to Lyme disease.