Mechanisms by which TLR9-deficiency and FasL Promote Cutaneous Lupus

摘要 常见的病理生理机制被认为促进了皮肤和全身的表现。 狼疮的症状。因此，更好地理解促进CLE的因素可能会提供重要的见解，如 关于系统性红斑狼疮的发病机制。然而，也可能是组织特异性效应机制解释了 对于SLE患者群体表现出的多样化的临床表现。由于75%的SLE患者表现出 某种皮肤损伤，而皮肤暴露在紫外线下往往与狼疮红斑有关，令人惊讶的是 有关CLE的发病、进展和复发的机械性研究相对较少。 造成这种差距的一个原因是，可用于CLE研究的小鼠模型有限--尽管 SLE小鼠模型众多，准确反映CLE中心特征的模型更多 有限的。我们现在已经建立了一种由T细胞引发的狼疮样皮肤炎症(LLSI)的可诱导模型 传输，这概括了CLE的许多功能。其中包括皮肤渗透的突出作用 产生干扰素γ的Th1细胞，角质形成细胞过度死亡，真皮/表皮自身抗体沉积 边界，CxCL9、CxCL10、CxCL11、CCL8表达增加，皮肤内pDC积聚。那里 我们的LLSI模型与其他诱因模型和遗传模型在机制上也有相似之处 程序性小鼠系统性红斑狼疮模型；它们都依赖于TLR7的表达，并因 TLR9缺失。因此，我们的LLSI小鼠提供了一种新颖、快速和可重复性的系统来探索 诱导和调节皮肤狼疮的效应机制。此应用程序将 关注TLR9和FasL。如前所述，TLR9对皮肤和皮肤的发育都有负面调节作用 系统性狼疮，但TLR9是否通过简单地与TLR7竞争访问 内体转运伴侣Unc93B1，或通过诱导依赖于TLR9信号的分子而主动 限制炎症的级联反应尚未得到解决。我们最近还表明， 皮损的发生完全依赖于T细胞FasL的表达，但FasL是否间接促进疾病 通过诱导细胞死亡和产生细胞碎片和/或直接通过诱导促炎因子的产生 细胞因子尚未解决。TLR9和FasL之间的相互作用可能是LLSI中一个重要的扩增环- TLR配体诱导Fas表达上调，FasL产生细胞碎片，激活内体TLRs。我们 建议通过使用基因靶向的小鼠来解决这些问题，这些小鼠在体外都有区分两者的突变 和体内(LLSI)研究。在目标1中，我们将使用表达正常水平的TLR9的小鼠，这种TLR9不能 在目标2中，我们将使用表达Caspase 8突变的小鼠，该突变可以移除 Caspase8自动裂解位点，从而阻止FasL诱导的细胞凋亡，但不能阻止趋化因子的产生。 总之，这些研究应该有助于确定针对TLR9和TLR9的最有效的治疗策略 FasL途径预防或改善皮肤性狼疮的发展。

ABSTRACT Common pathophysiological mechanisms are thought to promote the cutaneous and systemic manifestations of lupus. Thus a better understanding of the factors that promote CLE are likely to provide important insights as far as the pathogenesis of SLE. Nevertheless, it is also likely that tissue specific effector mechanisms account for the diverse clinical presentations exhibited by SLE patient populations. Since 75% of SLE patients exhibit skin lesions of some sort, and UV exposure of the skin is often associated with lupus flares, it is surprising that there have been relatively few mechanistic studies that address initiation, progression and recurrence of CLE. One reason for this gap is that murine models available for the study of CLE have been limited – despite the numerous murine models of SLE, models that accurately reflect the central features of CLE are much more limited. We have now develop an inducible model of lupus like skin inflammation (LLSI), initiated by T cell transfer, that recapitulates many of the features of CLE. These include a prominent role for skin-infiltrating IFNγ-producing Th1 cells, excessive keratinocyte death, autoantibody deposition at the dermal/epidermal border, increased expression of CxCL9, CxCL10, CxCL11, CCL8, and accumulation of pDCs in the skin. There are also mechanistic similarities between our LLSI model and other inducible as well as genetically programmed murine models of SLE; they all depend on the expression of TLR7 and are exacerbated by the absence of TLR9. Therefore our LLSI mice provide a novel, rapid and reproducible system for exploring the effector mechanisms responsible for the induction and regulation of cutaneous lupus. This application will focus on TLR9 and FasL. As mentioned, TLR9 negatively regulates the development of both cutaneous and systemic lupus, but whether TLR9 works passively by simply competing with TLR7 for access to the endosomal trafficking chaperone Unc93B1, or actively by inducing molecules dependent on a TLR9 signaling cascade that limit inflammation, has not been addressed. We have also recently shown that the development of skin lesions is completely dependent T cell FasL expression, but whether FasL promotes disease indirectly by inducing cell death and creating cell debris and/or directly by inducing the production of pro-inflammatory cytokines is unresolved. Interplay between TLR9 and FasL may be an important amplification loop in LLSI - TLR ligands induce upregulation of Fas and FasL generates cell debris that can activate endosomal TLRs. We propose to address the questions by using gene-targeted mice with discriminating mutations for both in vitro and in vivo (LLSI) studies. In Aim 1, we will use mice that express normal levels of a form of TLR9 that cannot engage MyD88, and in Aim 2, we will use mice that express a Caspase 8 mutation which removes the Caspase 8 autocleavage site and thereby prevents FasL-induced apoptosis but not chemokine production. Together, these studies should help identify the most effective therapeutic strategies for targeting TLR9 and FasL pathways to prevent or ameliorate the development of cutaneous lupus.