Role of Flip Macrophages

描述（由申请人提供）： 关节内衬中的巨噬细胞和滑膜成纤维细胞是类风湿性关节炎（RA）中观察到的破坏的核心。虽然Fas和Fas配体（FasL）表达的细胞在滑膜衬里，在体内很少观察到细胞凋亡。我们已经证明Flice样抑制蛋白（Flip）在RA关节中高度表达。Flip表达的抑制导致Fas介导的正常巨噬细胞和RA关节巨噬细胞的凋亡。我们的初步数据表明，在巨噬细胞上的Fas/FasL相互作用中断后，通过Toll样受体（TLR）和IL-1受体1（IL-1 R1）途径介导的NF-κ B的激活和IL-6的表达受到抑制。此外，在RAW 264.7巨噬细胞样细胞中的实验证明，Flip或FADD的异位表达（其在Fas连接后被募集到Fas死亡诱导信号复合物（DISC））抑制LPS诱导的NF-κ B和IL-6启动子报告子的活化。在缺乏TLR的293细胞中，Flip或FADD都不能抑制TNF α诱导的NF-κ B或IL-6启动子激活。这些观察结果表明，在Fas/FasL相互作用后，如发生在RA关节衬里中，Flip和FADD被募集到Fas DISC，通过TLR/IL-1 R1途径促进增强的活化。尽管Flip的两种同种型（FlipL和FlipS）都能够防止细胞凋亡并调节TLR/IL-1 R1通路，但与其他细胞类型如滑膜成纤维细胞相比，Flips在巨噬细胞中独特地表达。我们的初步数据表明，FlipL和FlipS在转录后水平上以细胞类型特异性方式受到差异调节。我们在目标1中提出，检查负责通过TLR/IL-1 R1途径调节激活的机制。目的2提出表征负责调节Flip亚型的细胞类型特异性表达的机制。由于尚未检查flip缺失的体内效应，因此目的3提出检查小鼠巨噬细胞中flip的细胞类型特异性缺失的效应。这些研究将为RA的发病机制提供深入的见解，并可能开发新的治疗策略。

DESCRIPTION (provided by applicant): Macrophages and synovial fibroblasts in the joint lining are central to the destruction observed in rheumatoid arthritis (RA). Although Fas and Fas ligand (FasL) are expressed on cells in the synovial lining, in vivo apoptosis is rarely observed. We have demonstrated that Flice Like Inhibitory Protein (Flip) is highly expressed in the RA joint. Suppression of the expression of Flip resulted in Fas-mediated apoptosis of normal macrophages and those from the RA joint. Our preliminary data demonstrates that following the interruption of Fas/FasL interactions on macrophages, activation of NF-kappaB and the expression of IL-6, mediated through the toll like receptor (TLR) and IL-1 receptor 1 (IL-1R1) pathways, was suppressed. Further, experiments in RAW 264.7 macrophage-like cells demonstrate that the ectopic expression of either Flip or FADD, which are recruited to the Fas Death Inducing Signal Complex (DISC) following Fas ligation, suppressed the LPS-induced activation of NF-kappaB and IL-6 promoter reporters. In 293 cells, which lack TLRs, neither Flip or FADD suppressed TNFalpha-induced NF-kappaB or IL-6 promoter activation. These observations suggest that following Fas/FasL interaction, as occurs in the lining in the RA joint, Flip and FADD are recruited to the Fas DISC, promoting enhanced activation through the TLR/IL-1 R1 pathway. Although both isoforms of Flip (FlipL and FlipS) are capable of protecting against apoptosis and modulating the TLR/IL-1R1 pathway, Flips is uniquely expressed in macrophagess, compared to other cell types such as synovial fibroblasts. Our preliminary data suggests that FlipL and FlipS are differentially regulated at the post-transcriptional level in a cell type-specific fashion. We propose in aim 1 to examine the mechanisms responsible for the modulation of activation through the TLR/IL-1 R1 pathway. Aim 2 proposes to characterize the mechanisms responsible for regulating the cell type-specific expression of the Flip isoforms. Since the in vivo effects of deletion of flip have not been examined, aim 3 proposes to examine the effects of the cell type-specific deletion of flip in macrophages in mice. These studies will provide insights into mechanisms that contribute to the pathogenesis of RA and potentially the development of new therapeutic strategies.