Regulation Of Immune Responses In Humans and in Experimental Animals

在巨噬细胞/树突状细胞中LRRK2信号功能的初步研究中，我们发现携带LRRK2转基因小鼠的细胞在暴露于天然配体时表现出更多的细胞因子反应，天然配体通过激活SYK刺激细胞，SYK是一种细胞表面信号分子，参与Dectin受体信号转导，少量参与TLR信号转导。此外，在Western印迹研究中，我们确定LRRK2不仅与SYK相互作用，而且与下游SYK相关信号复合体的成分相互作用，Bcl10/MALT-1/CARD9为LRRK2作为SYK信号通路的组成部分提供了重要支持。 在更具体地讨论LRRK2如何影响自噬的研究中，我们发现，有些令我们惊讶的是，来自LRRK2转基因小鼠的巨噬细胞在Dectin途径的刺激下，表现出Lc3向Lc3 II的转化减少，后者是出现在激活的自噬途径远端的分子。对LRRK2缺陷小鼠巨噬细胞的补充研究表明，LRRK2缺乏与自噬增加有关，从而证实了转基因细胞的结果。 在进一步研究LRRK2介导的自噬抑制的分子机制时，我们发现LRRK2与自噬启动子Beclin相互作用，这种相互作用与Beclin二聚体的破坏有关。由于LRRK2也与Caspase8相互作用，而Caspase8是上述Bcl10信号复合体的组成部分，因此BECLIN的破坏可能由caspase8介导。 最近来自其他实验室的研究表明，自体修复会导致激活抑制，也会扰乱IL-1β信号。因此，目前的研究表明，LRRK2基因多态性在克罗恩病炎症中的作用机制之一是，这些基因多态性导致促炎性炎症小体/IL-1β分泌的正常调节中的自噬相关缺陷。

In initial studies of LRRK2 signaling function in macrophages/dendritic cells we established that cells from mice bearing an LRRK2 transgene manifest increased cytokine responses when exposed to innate ligands that stimulate cells by activating SYK, a cell surface signaling molecule involved in dectin receptor signaling and to a lesser extent in TLR signaling. This, plus the fact that in Western blot studies we determined that LRRK2 interacts with SYK as well as with components of the downstream SYK-associated signaling complex, BCL10/MALT-1/CARD9 provided important support for the idea that LRRK2 acts as component of the SYK signaling pathway. In studies more specifically addressing how LRRK2 affects autophagy we found, somewhat to our surprise, that macrophages from LRRK2 transgenic mice upon stimulation of the dectin pathway exhibit decreased conversion of LC3 to LC3 II, the latter a molecule that appears at the distal end of the activated autophagy pathway. Complementary studies of macrophages from LRRK2-deficient mice showed that LRRK2 deficiency was associated with increased autophagy, thus confirming the result obtained with transgenic cells. In additional studies to address the issue of the molecular mechanism of LRRK2-mediated autophagy inhibition we showed that LRRK2 interacts with the autophagy initiator, beclin, and such interaction is associatd with breakdown of beclin dimers. Beclin breakdown may be mediated by caspase8 since LRRK2 also interacts with caspase8 and the latter is component of the BCL10 signaling complex mentioned above. Recent studies from other laboratories have shown that autophaphy leads to inhibition of activation and also to disturbances in IL-1beta signaling. The present studies therefore suggest that one of the mechanisms by which LRRK2 polymorphisms contribute to the inflammation of Crohn's disease is that these polymorphisms result in autophagy-related defects in the normal regulation of pro-inflammatory inflammasome/IL-1beta secretiion.