Regulatory Role of Toll-like receptor signaling in lupus development

系统性红斑狼疮（SLE）是一种病因不明的慢性、危及生命的自身免疫性疾病，可导致多器官病变和肾脏破坏。自身耐受性的丧失伴随着产生抗DNA和其他抗自身抗体的自身反应性B细胞克隆的出现，随后形成致病性免疫复合物（IC）。这些IC可以通过内体核酸感应Toll样受体（TLR）、TLR 3、TLR 7和TLR 9激活免疫细胞以产生I型干扰素。依赖于I型干扰素和TLR信号传导的鼠模型是所谓的降植烷诱导的全身性自身免疫模型。我们最近已经从这些小鼠的细胞和组织的特点，并确定了干扰素刺激的基因（ISG）签名相媲美的ISG签名在人类狼疮患者中发现的。由于TLR 9是细菌和哺乳动物自身DNA的重要内体核酸传感器，因此认为TLR 9的缺乏在鼠狼疮模型中具有保护性。然而，令人惊讶的是，TLR 9 KO小鼠显示出恶化的狼疮病理和肾脏疾病，表明TLR 9信号传导在狼疮和可能的其他系统性自身免疫疾病中的保护作用。人们提出了不同的假设来解释这种保护，但确切的机制尚不清楚。使用在实验室中建立的降植烷诱导的自身免疫性疾病的模型，我们建议更详细地研究TLR 9缺乏在疾病的开始和后期阶段的影响。 具体地说，我们想研究潜在的致病性炎症单核细胞在TLR 9缺陷的降植烷注射小鼠中增加募集的潜在机制。此外，我们希望了解在该模型中缺乏TLR 9的情况下导致加速和更严重肾脏疾病的因素。我们计划通过用同种异型和CD 45不同的TLR 7 KO和TLR 9 KO小鼠重建正常小鼠来产生混合骨髓嵌合体。然后用降植烷攻击这些细胞，并测定源自2种亲本菌株的B细胞、DC、巨噬细胞和嗜中性粒细胞对疾病过程各个方面的贡献。此外，我们希望鉴定一个狼疮患者亚群，其中与TLR 9诱导的浆细胞样树突状细胞中干扰素α产生相比，TLR 7向增加的转变伴随着HEp-2细胞上的细胞质染色模式和IgM同种型的保护性抗DNA抗体的丧失。我们希望在这个子集中，我们可以丰富患者的TLR 9信号级联缺陷，这使他们容易患狼疮。最后，我们提出用转染到胞质溶胶中的核酸刺激患者来源的单核细胞，并希望建立一种测定法来鉴定在胞质溶胶核酸降解/传感途径中具有缺陷的患者。

Systemic lupus erythematosus (SLE) is a chronic, life threatening autoimmune disorder of unknown etiology, leading to multiple organ pathologies and kidney destruction. Loss of self tolerance is accompanied by the emergence of autoreactive B-cell clones producing anti-DNA and other anti-self antibodies with subsequent formation of pathogenic immune complexes (IC). These ICs can activate immune cells via endosomal nucleic acid sensing Toll-like receptors (TLRs), TLR3, TLR7 and TLR9 to produce type I interferons. A murine model which is dependent on type I Interferon and TLR signaling is the so-called pristane-induced model of systemic autoimmunity. We have recently characterized cells and tissues from these mice and identified an interferon stimulated gene (ISG) signature comparable to the ISG signature found in human lupus patients. Since TLR9 is an important endosomal nucleic acid sensor of bacterial but also mammalian self-DNA, it was thought, that deficiency of TLR9 would be protective in murine lupus models. Surprisingly, however TLR9 KO mice showed exacerbated lupus pathology and kidney disease, indicating a protective role for TLR9 signaling in lupus and likely other systemic autoimmune diseases. Different hypotheses have been proposed to explain this protection, however the exact mechanism is not understood. Using the well-established model of pristane-induced autoimmune disease in the lab we propose to study the effect of TLR9 deficiency in the initiation and the later stages of the disease in more detail. Specifically we want to study the underlying mechanism of the increased recruitment of potentially pathogenic inflammatory monocytes in TLR9 deficient pristane injected mice. Further, we want to understand the factors that cause accelerated and more severe kidney disease in the absence of TLR9 in this model. We plan to generate mixed bone marrow chimeras, produced by reconstituting normal mice with allotype- and CD45-disparate TLR7 KO and TLR9 KO mice. These will then be challenged with pristane and the contribution of B cells, DCs, macrophages and neutrophils, derived from the 2 parental strains to various aspects of the disease process will be determined. Furthermore, we wish to identify a subset of lupus patients in which a shift towards increased TLR7 compared to TLR9 induced Interferon alpha production in plasmacytoid dendritic cells is accompanied by a cytoplasmic staining pattern on HEp-2 cells and a loss of protective anti-DNA antibodies of IgM isotype. We hope that in this subset we might enrich patients with defects in the TLR9 signaling cascade, which predispose them to lupus development. Lastly, we propose to stimulate patient derived monocytes with nucleic acids transfected into the cytosol and wish to establish an assay to identify patients with defects in the cytosolic nucleic acid degradation/sensing pathways.