Role of Flip Macrophages

描述(由申请人提供)： 关节衬里中的巨噬细胞和滑膜成纤维细胞是类风湿性关节炎(RA)破坏的中心。尽管Fas和Fas配体(FasL)表达在滑膜衬里的细胞上，但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关节中高表达。抑制Flip的表达导致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Fas/FasL相互作用被阻断后，通过Toll样受体(TLR)和IL-1受体1(IL-1R1)途径介导的NF-kappaB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表明，Fas连接后被招募到Fas死亡诱导信号复合体(DISC)的Flip或Fadd的异位表达抑制了内毒素诱导的NF-kappaB和IL-6启动子报告的激活。在缺乏TLRs的293细胞中，Flip或FADD都不能抑制TNFpha诱导的NF-kappaB或IL-6启动子的激活。这些观察表明，在Fas/FasL相互作用之后，就像在RA关节的衬里中发生的那样，Flip和FADD被招募到Fas盘，通过TLR/IL-1 R1途径促进激活。虽然Flip的两种异构体(FlipL和Flips)都能够保护细胞不发生凋亡并调节TLR/IL-1R1通路，但与滑膜成纤维细胞等其他类型的细胞相比，Flip在巨噬细胞中唯一表达。我们的初步数据表明，FlipL和Flip在转录后水平上以细胞类型特有的方式受到差异调控。我们在目标1中建议研究通过TLR/IL-1R1途径调节激活的机制。目的2建议表征负责调节翻转异构体的细胞类型特异性表达的机制。由于FliP的缺失在体内的影响尚未得到检验，Aim 3建议检测小鼠巨噬细胞中特定细胞类型的FliP缺失的影响。这些研究将为类风湿性关节炎的发病机制提供洞察力，并有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.