Suppressor of Cytokine Signaling and Control of Inflammation in Lyme Disease

描述（由申请方提供）：莱姆病由伯氏疏螺旋体螺旋体引起，是一种新兴的传染病，因此具有重大的公共卫生意义。螺旋体侵入多个器官并引起急性和慢性炎症。由螺旋体诱导的炎症在疾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B.单独或与IL-10一起加和诱导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-12 p40，IL-18，TNF？IL-1？我们推测B.在莱姆病过程中，巨噬细胞中的伯氏螺旋体单独或与IL-10一起在控制炎症方面具有重要的功能; SOCS 1/SOCS 3将作为IL-10抗炎活性的介质或作为炎性细胞因子信号传导的直接调节剂。具体目标是：1.确定使用RNA干扰（RNAi）沉默J774巨噬细胞中socs 1/socs 3基因表达对IL-10介导的由B诱导的炎性细胞因子抑制的影响（a）。burgdorferi和（B）对炎性细胞因子信号传导的影响。我们将通过将小干扰RNA（SiRNA）双链体递送到巨噬细胞中，然后在存在和不存在IL-10的情况下用活螺旋体或螺旋体脂蛋白刺激来沉默socs基因。以这种方式，我们将评估（i）IL-10抑制促炎细胞因子（IL-6、IL-12 p40、IL-18、TNF β、TNF α）的能力，IL-1？）使用定量实时PCR（qPCR）和ELISA，以及（ii）SOCS通过抑制STAT蛋白的活化和DNA结合活性（响应IL-6、IL-12、IFN？）在NF？B（对IL-1、IL-12、TNF？的反应）使用基因功能分析。2.比较B治疗前和治疗过程中来自莱姆病抗性C57 BL/6 J和疾病易感性C3 H/HeJ小鼠的原代巨噬细胞和关节组织中SOCS 1/SOCS 3的表达。Burgdorferi感染，并将SOCS 1/SOCS 3表达与IL-10抗炎活性和疾病相关联。我们将采用源自小鼠骨髓、淋巴结和脾脏的巨噬细胞（并如目的1中所述刺激它们），通过qPCR、蛋白质印迹和共聚焦显微镜评估SOCS的表达水平。我们将（i）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（ii）关节组织中的SOCS表达与疾病严重程度相关联。如果SOCS（或其下游介质）在来自这两种菌株的小鼠的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，则SOCS途径可能决定对莱姆病的易感性和抗性，如在小鼠中建模的那样，并且可能也在人类中。 莱姆病是由伯氏疏螺旋体引起的一种新出现的传染病，因此具有重大的公共卫生意义。由螺旋体在多个器官中诱导的炎症在疾病发病机制中起主要作用，但其调节尚不清楚。实现这一提议的目标将解释炎症是如何调节的，并有助于理解莱姆病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.