Regulation Of Immune Responses In Humans and in Experimental Animals

在最初的研究中，我们通过Western印迹研究证实，LRRK2基因多态与LRRK2表达增加相关，因此是一种功能突变的获得。这一发现与LRRK2过度表达的影响有关，因为这种异常的小鼠表现出更严重的DSS-结肠炎。此外，LRRK2转基因小鼠的树突状细胞显示Dectin-1介导的TNF-α和IL-23的诱导与MAPK、NF-kappaB和NFAT信号成分的磷酸化、阳性的NF-kappaB和NFAT报告分析以及NFAT的核移位相关。 在进一步的研究中，我们证实了Dectin-1刺激树突状细胞导致K-63泛素化LRRK2，并且这是由TAB2和TRAF6介导的。此外，LRRK2与TRAF6联合诱导NEMO的K-63多泛素化，在报告实验中LRRK2与TAB2或LRRK2与TRAF6在报告实验中均诱导NK-kappaB。因此，LRRK2成为核因子-kappaB活化的主要促进器。 在LRRK2与自噬关系的研究中，我们用LRRK2KOxLC3gfp转基因小鼠和LRRK2Tgxgfp转基因小鼠表明，LRRK2的缺失和过度表达分别与自噬增加和自噬减少有关。这一结果得到了LC3转化的蛋白质印迹研究的证实。关于这些影响自噬的机制，我们在Western印迹和Duolink研究中发现LRRK2与BEKIN-1结合，LRRK2增强BEKIN-1与TAB2的相互作用，LRRK2与TAB2结合，LRRK2诱导BEKIN-1降解。因此，LRRK2结合到吞噬体膜上，并通过其促进Beclin-1的降解来抑制自噬。在初步研究中，我们有证据表明抑制自噬导致LRRK2表达增强；因此，LRRK2基因多态患者的LRRK2表达增加可能表现出对先天刺激的促炎反应增强，因为这种表达增加导致抑制自噬和反馈LRRK2介导的对核因子-kappaB或NFAT激活的刺激。 在最后一轮研究中，我们证实了LRRK2抑制剂逆转了LRRK2对自噬和核因子-kappaB激活的影响，并改善了DSS-结肠炎。因此，这种抑制剂成为克罗恩病患者的一种可能的治疗方法，特别是那些具有LRRK2基因多态的患者。

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.