Immunoregulatory Defects In Inflammatory Bowel Disease

基于来自先前研究的证据，即通过胞壁酰二肽（MDP）激活NOD 2导致TLR 2应答的下调，并且这种调节的缺乏导致Th 1应答的增强，在初始研究中，我们用MDP处理患有葡聚糖硫酸盐诱导的结肠炎（DSS-结肠炎）或三硝基苯磺酸诱导的结肠炎（TNBS-结肠炎）的小鼠，以确定后者对体内炎症的影响。 事实上，我们发现MDP给药大大降低了这两种模型中的炎症。 这种下调作用的基础在随后的树突状细胞体外研究中变得明显，在这些研究中，我们表明，用MDP预刺激细胞使细胞在一系列炎性细胞因子的测定中对多种TLR刺激剂无反应。 旨在揭示这种效应机制的后续研究表明，MDP刺激NOD 2可诱导IRF-4的表达，IRF-4是一种先前被证明可抑制TLR反应的因子，并且IRF-4的基因沉默可导致反应的恢复。 有了这种见解，我们然后回到实验性结肠炎的体内研究，并表明通过施用包封在病毒包膜中以促进细胞进入的IRF-4特异性siRNA来体内下调IRF-4消除了MDP对TNBS-结肠炎的作用。 此外，它消除了MDP给药对TLR配体细胞因子诱导的下调作用。 此外，我们发现MDP给药不能影响IRF-4缺陷小鼠诱导的DSS-结肠炎。 在将这些发现与克罗恩病相关的最后一系列研究中，我们用表达野生型NOD 2或携带与克罗恩病中发现的相同的移码突变的NOD 2的质粒重建NOD 2缺陷小鼠。 然后，我们表明MDP给药预防了用野生型NOD 2重建的小鼠中的DSS结肠炎，但不能预防用突变（移码突变）NOD 2重建的小鼠。 因此，移码突变赋予的表型，其特征在于不能获得MDP诱导的无反应性。

Based on evidence from previous studies that activation of NOD2 by muramyl dipeptide (MDP) leads to the down-regulation of TLR2 responses and that the absence of such regulation leads to heightened Th1 responses, in initial studies we treated mice with either dextran-sulfate-induced colitis (DSS-colitis) or trinitrobenzene sulfonic acid-induced colitis (TNBS-colitis) with MDP to determine the latters effect on inflammation in vivo. Indeed, we found that MDP administration greatly decreased the inflammation in both of these models. The basis of this down-regulatory effect then became apparent in subsequent in vitro studies of dendritic cells in which we showed that pre-stimulation of the cells with MDP rendered the cells unresponsive to a wide variety of TLR stimulants in assays of a spectrum of inflammatory cytokines. Subsequent studies designed to reveal the mechanism of this effect showed that MDP stimulation of NOD2 induced the expression of IRF-4, a factor previously shown to inhibit TLR responses and that gene silencing of IRF-4 led to restoration of responses. With this insight in hand we then returned to in vivo studies of experimental colitis and showed that in vivo down-regulation of IRF-4 by administration of IRF-4-specific siRNA encapsulated in a viral envelope to facilitate cell entry abolished the effect of MDP on TNBS-colitis. In addition, it abolished the down-regulatory effect of MDP administration on TLR ligand cytokine induction. In addition, we showed that MDP administration was unable to affect DSS-colitis induced in IRF-4-deficient mice. In a final series of studies relating these findings to Crohns disease we re-constituted NOD2-deficient mice with plasmids expressing either wild type NOD2 or NOD2 bearing a frameshift mutation identical to that found in Crohns disease. We then showed that MDP administration prevented DSS-colitis in mice reconstituted with wild type NOD2 but not mice reconstituted with mutated (frameshift mutation) NOD2. Thus, the frameshift mutation conferred a phenotype characterized by an inability to acquire MDP-induced unresponsiveness.