Suppressor of Cytokine Signaling and Control of Inflammation in Lyme Disease

描述（由申请人提供）：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。螺旋体侵入多个器官并诱发急性和慢性炎症。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控制莱姆病炎症所需的机制。最近我们观察到伯氏疏螺旋体单独或与IL-10共同诱导J774巨噬细胞中细胞因子信号传导抑制因子（SOCS） 1和3的表达。这与il -10介导的促炎细胞因子IL-6、IL-12p40、IL-18、TNF？和il - 1 ?。我们的假设是，单独或联合IL-10诱导巨噬细胞中SOCS1/SOCS3的表达在莱姆病过程中的炎症控制中具有重要的功能；SOCS1/SOCS3可能作为IL-10抗炎活性的介质或炎症细胞因子信号传导的直接调节剂。具体目标是：1。利用RNA干扰（RNAi）沉默J774巨噬细胞中socs1/socs3基因表达(a)对伯氏疏螺旋体诱导的il -10介导的炎症细胞因子抑制和(b)对炎症细胞因子信号传导的影响。我们将通过将小干扰RNA （siRNA）双链体传递到巨噬细胞中，然后在IL-10存在和不存在的情况下用活螺旋体或螺旋体脂蛋白刺激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？（ii） SOCS通过抑制STAT蛋白（响应IL-6、IL-12、IFN?）和NF?的激活和DNA结合活性，对炎症细胞因子信号传导的影响。B（响应IL-1¿，IL-12， TNF?）使用基因功能测定。2. 比较莱姆病耐药C57BL/6J和疾病易感C3H/HeJ小鼠在感染伯氏伯氏杆菌前和感染过程中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抗炎活性和疾病的相关性。我们将使用来自小鼠骨髓、淋巴结和脾脏的巨噬细胞（并刺激它们，如Aim 1），通过qPCR、Western blot和共聚焦显微镜评估SOCS的表达水平。我们将把巨噬细胞中的SOCS表达与il -10介导的促炎细胞因子抑制（如Aim 1）和关节组织中的SOCS表达与疾病严重程度联系起来。如果SOCS（或其下游介质）在这两种菌株的小鼠细胞和组织中表达差异，并且SOCS沉默导致巨噬细胞中莱姆病炎症反应失调，那么SOCS途径可能决定了对莱姆病的易感和抗性，正如小鼠模型所示，也许也适用于人类。

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.