Regulation Of Immune Responses In Humans and in Experimental Animals

我们对LRRK 2功能的研究集中在LRRK 2的“肠道限制性”LRRK 2抑制剂上，据推测其肺或肾毒性比以前的抑制剂小，同时保留LRRK 2抑制效力和特异性。这些抑制剂，合成的合作研究人员在山。Sinai Medical Center的LRRK 2激酶抑制剂（例如CZC-54252）是已知的（已经开发的）LRRK 2激酶抑制剂的类似物，其先前已经被合成用于治疗帕金森病。本质上，已知抑制剂的修饰涉及添加化学基团（称为运动载体），这些化学基团先前已显示出增加化合物的血液清除率，同时增强肠道滞留。修改后的抑制剂将受到密集的测试，由山。西奈半岛的研究人员确定他们的药代动力学特性，他们的激酶特异性和他们的毒性，最终使用它们来治疗克罗恩病的意图。 我们已经对修饰的抑制剂（CS-82）在无毒浓度下抑制TLR 4（LPS）、TLR 2（酵母聚糖）和Dectin-1（酵母聚糖耗尽的酿酒酵母，ZymD）刺激的细胞因子产生的比较能力进行了广泛的研究。 我们首先发现，虽然CS-82对LRRK 2表达影响很小或没有影响，但它极大地抑制了LRRK 2磷酸化以及磷酸化已知LRRK 2靶点Rab-10和Rab-12的能力;因此，它被证明是LRRK 2激酶功能的有效抑制剂。 关于对巨噬细胞细胞因子产生的影响，CS-82表现出与市售抑制剂相比相等或略微更大的抑制TLR诱导的TNF-α和IL-6产生的能力。 相反，其抑制IL-10产生的程度低于市售抑制剂。 这些抑制性质在体内表现为向患有DSS-结肠炎的小鼠施用CS-82证明能够改善结肠炎并减少发炎结肠中IL-12和TNF-α mRNA固有层产生。 在LRRK 2与NLRC 4炎性体活性的关系的研究中，我们在PA（炭疽芽孢杆菌保护性抗原）存在下用LPS加LFn-Fla或LFn-针（融合至嗜肺军团菌鞭毛蛋白或针蛋白的致死因子的N末端）刺激人DC，PA是进入细胞并通过NAIP相互作用激活NLRC 4的两种细菌物质。我们发现两种刺激都诱导IL-1b和IL-18的分泌，即，NLRC 4炎性体的产物。此外，我们发现两种修饰的抑制剂（CS-82和CS-190）抑制NLRC 4诱导的IL-1b分泌，而母体抑制剂具有更边际的抑制作用。修饰的抑制剂也抑制NLRC 4诱导的IL-18分泌，但程度较低。 对照研究表明，抑制剂对NLRP 3诱导的IL-1b分泌几乎没有影响，因此它们对NLRC 4炎性小体的抑制是炎性小体特异性作用。 最后，通过蛋白质印迹研究，我们表明，在所有抑制剂（亲本和修饰的）存在下，NLRC 4对细胞的刺激导致丝氨酸533处的NLRC 4磷酸化以及LRRK 2磷酸化的丧失。 这些研究表明，通过LRRK 2激酶在S533处的NLRC 4磷酸化对于NLRC 4诱导IL-1b确实重要，但对于诱导IL-18则不那么重要。然而，应该注意的是，在NLRC 4磷酸化受到抑制的情况下观察到IL-1b的产生（尽管水平降低），这表明炎性小体活性可以在没有磷酸化的情况下以一定水平进行。在抑制剂对来自克罗恩病患者的细胞的作用的有限研究中，我们发现用鞭毛蛋白刺激患者细胞导致比对照细胞更高的炎性小体刺激，这可能反映了更高的LRRK 2水平。这伴随着更大的IL-1b产生，但不是更大的IL-18产生。

Our studies of LRRK2 function has centered around "gut-restricted" LRRK2 inhibitors of LRRK2 that are presumed to have less lung or kidney toxicity than previous inhibitors while retaining LRRK2 inhibitory potency and specificity. These inhibitors, synthesized by collaborating investigators at Mt. Sinai Medical Center, are analogs of known (already developed) LRRK2 kinase inhibitors (such as CZC-54252) that have previously been synthesized for the treatment of Parkinson's Disease. In essence, the modifications of known inhibitor involves the addition of chemical groups (called kinetophores)that have previously been shown to increase blood clearance of a compound while its enhancing intestinal retention. The modified inhibitors will be subjected to intensive testing by the Mt. Sinai investigators to define their pharmacokinetic properties, their kinase specificity and their toxicity with the intention of ultimately using them to treat Crohn's disease. We have conducted extensive studies of the comparative capacity of modified inhibitor (CS-82) to suppress TLR4 (LPS), TLR2 (Zymosan) and Dectin-1 (Zymosan-depleted S.cerevisciae, ZymD) stimulated cytokine production at a non-toxic concentration. We found first that whereas CS-82 had little or no effect on LRRK2 expression it greatly inhibited LRRK2 phosphorylation and ability to phosphorylate known LRRK2 targets, Rab-10 and Rab-12; it was thus shown to be a potent inhibitor of LRRK2 kinase function. With respect to effect on macrophage cytokine production, CS-82 manifested equal or marginally greater ability to suppress TLR-induced TNF-a and IL-6 production than commercially available inhibitor. In contrast, it inhibited IL-10 production to a lesser extent than commercially available inhibitor. These inhibitory qualities were manifest in vivo in that administration of CS-82 to mice subjected to DSS-colitis proved able to ameliorate colitis and to reduce both IL-12 and TNF-a mRNA lamina propria production in the inflamed colon. In studies of the relation of LRRK2 to NLRC4 inflammasome activity we stimulated human DCs with LPS plus LFn-Fla or LFn-needle (N terminus of lethal factor fused to L.pneumophila flagellin or Needle protein) in the presence of PA (Bacillus anthracis protective antigen), two bacterial substances that enter cells and activate NLRC4 via NAIP interaction. We found that both stimuli induced secretion of IL-1b and IL-18, i,e., products of the NLRC4 inflammasome. In addition, we found that two modified inhibitors (CS-82 and CS-190)suppressed NLRC4-induced IL-1b secretion whereas the parent inhibitor had a much more marginal inhibitory effect. The modified inhibitor also inhibited NLRC4-induced IL-18 secretion but to a lesser extent. Control studies showed that the inhibitors had little effect on NLRP3-induced IL-1b secretion and therefore their inhibition of the NLRC4 inflammasome was an inflammasome-specific effect. Finally, with Western blot studies we showed that NLRC4 stimulation of cells in the presence of all inhibitors (both parent and modified) led to loss of NLRC4 phosphorylation at Serine 533 as well as LRRK2 phosphorylation. These studies imply that NLRC4 phosphorylation at S533 by LRRK2 kinase is indeed important for NLRC4 induction of IL-1b but less so for induction of IL-18. However, it should be noted that IL-1b production was observed (albeit at a reduced level) in the face of suppressed NLRC4 phosphorylation suggesting that inflammasome activity can proceed at some level without phosphorylation. In the limited studies of the effect of inhibitors on cells from patients with Crohn;s disease we found that stimulation of patient cells with flagellin led to higher inflammasome stimulation than in control cells, probably reflecting higher LRRK2 levels. This was accompanied by greater IL-1b production, but not greater IL-18 production.