Regulation Of Immune Responses In Humans and in Experimental Animals

在最初的研究中，我们用蛋白质印迹研究确定了LRRK 2多态性与LRRK 2表达增加相关，因此是功能突变的获得。 这一发现与LRRK 2过表达的影响相关，因为具有这种异常的小鼠表现出更严重的DSS-结肠炎。 此外，来自LRRK 2转基因小鼠的树突状细胞表现出增加的Dectin-1介导的TNF-α和IL-23诱导，其与MAPK、NF-κ B和NFAT信号传导组分的磷酸化、阳性NF-κ B和NFAT报告基因测定以及NFAT的核转位相关。 在进一步的研究中，我们确定了树突状细胞的Dectin-1刺激导致LRRK 2的K-63多聚泛素化，并且这是由TAB 2和TRAF 6介导的。此外，LRRK 2与TRAF 6联合诱导NEMO的K-63多聚泛素化，并且在报道基因测定中，LRRK 2和TAB 2或LRRK 2和TRAF 6两者在报道基因测定中诱导NK-κ B。因此，LRRK 2成为NF-κ B激活的主要促进剂。 在LRRK 2与自噬关系的研究中，我们用LRRK 2KOxLC 3gfp转基因小鼠和LRRK 2 Tgxgfp转基因小鼠表明，LRRK 2缺失和过表达分别与自噬增加和自噬减少相关。 该结果通过LC 3转化的蛋白质印迹研究证实。关于这些影响自噬的机制，我们在Western blot和duolink研究中发现LRRK 2与beclin-1结合，LRRK 2增强beclin-1与Rubicon的相互作用，LRRK 2与TAB 2和Rubicon联合诱导beclin-1降解。因此，LRRK 2与吞噬体膜结合并通过其增强beclin-1降解的能力来抑制自噬。在初步研究中，我们有证据表明自噬的抑制导致LRRK 2表达增强;因此，在具有LRRK 2多态性的患者中，LRRK 2表达的增加似乎可能表现出对先天刺激的促炎反应增强，因为这种表达的增加导致自噬的抑制和反馈LRRK 2介导的NF-κ B激活或NFAT激活的刺激。 在最后一轮研究中，我们确定了LRRK 2抑制剂逆转了LRRK 2对自噬和NF-κ B活化的作用，并改善了DSS结肠炎。 因此，这种抑制剂作为克罗恩病患者，特别是具有LRRK 2多态性的患者的可能治疗而出现。

In initial studies we established with Western blot studies that the LRRK2 polymorphism was associated with increased expression of LRRK2 and was thus a gain of function mutation. This finding correlated with the effect of LRRK2 over-expression since mice with this abnormality exhibited more severe DSS-colitis. In addition, dendritic cells from LRRK2 transgenic mice exhibited increased Dectin-1-mediated induction of TNF-alpha and IL-23 associated with phosphorylation of MAPK, NF-kappaB and NFAT signaling components, positive NF-kappaB and NFAT reporter assays and nuclear translocation of NFAT. In further studies we established that Dectin-1 stimulation of dendritic cells results in K-63 polyubiquitination of LRRK2 and that this is mediated by TAB2 and TRAF6. In addition, LRRK2 in association with TRAF6 induces K-63 polyubiquitination of NEMO and in a reporter assay both LRRK2 and TAB2 or LRRK2 and TRAF6 induces NK-kappaB in a reporter assay. Thus, LRRK2 emerges as a major facilitator of NF-kappaB activation. In studies of the relation of LRRK2 to autophagy we showed with LRRK2KOxLC3gfp transgenic mice and LRRK2Tgxgfp transgenic mice that LRRK2 deletion and over-expression was associated with increased autophagy and decreased autophagy respectively. This result was confirmed by studies of Western blot studies of LC3 conversion. As to the mechanism of these effects on autophagy, we showed that LRRK2 binds to beclin-1 in Western blot and duolink studies, LRRK2 enhances the interaction of beclin-1 with Rubicon and LRRK2 in association with TAB2 and Rubicon induces beclin-1 degradation. Thus, LRRK2 binds to the phagosomal membrane and inhibits autophagy via it ability to enhance degradation of beclin-1. In preliminary studies we have evidence that inhibition of autophagy leads to enhanced LRRK2 expression; thus, it appears likely that increased LRRK2 expression in patients with LRRK2 polymorphisms exhibit enhanced pro-inflammatory responses to innate stimuli because such increased expression causes inhibition of autophagy and feedback LRRK2-mediated stimulation of NF-kappaB activation or NFAT activation. In a final round of studies we established that LRRK2 inhibitors reverse the effects of LRRK2 on autophagy and NF-kappaB activation and ameliorate DSS-colitis. Thus, such inhibitors emerge as a possible treatment of patients with Crohn's disease, particularly those with LRRK2 polymorphisms.