【作者】 张伟娟；

【导师】 熊思东； 徐薇； 高波；

【作者基本信息】 复旦大学 ， 免疫学， 2010， 博士

【摘要】 活化淋巴细胞来源DNA (ALD-DNA)诱导系统性红斑狼疮(SLE)的新机制：巨噬细胞极化及其作用系统性红斑狼疮(systemic lupus erythematosus, SLE)是一个潜在的致死性疾病,以出现各种典型的自身免疫症状包括自身免疫反应、血管炎、关节炎和肾小球肾炎等为特征。在美国SLE病人总数已经超过二十五万,90%发生于育龄期妇女,严重影响人类健康。随着治疗手段的进步,狼疮性肾炎病人5年的生存率从二十世纪五十年代的44%提高到现在的82%,但是SLE病人的平均寿命只有44岁。SLE以其复杂多变的症状引起临床工作者的广泛关注,而SLE几乎累及免疫系统的各个成份,更是引起了免疫学工作者极大的兴趣,针对SLE的发病机制和SLE防治方法的研究一直是科学家关注的重大前沿课题。作为SLE病人血清学特征的抗双链DNA (double-stranded DNA, dsDNA)抗体,已经被证明是致病性的,可以引起免疫复合物沉积和组织损伤,与SLE疾病的严重程度密切相关。SLE病人遗传学的研究发现抗dsDNA自身抗体大多属于对dsDNA具有高亲和力的IgG亚类,不同于体细胞突变产生的抗体。研究显示自身DNA可以诱导抗dsDNA抗体产生。一般情况下,哺乳动物的DNA免疫原性较弱,不会引起免疫应答。寻找引起自身免疫反应和抗dsDNA抗体产生的驱动成份是免疫学家关注的热点。我们实验室在寻找SLE驱动原的过程中发现用活化淋巴细胞来源的DNA (activated lymphocyte-derived DNA, ALD-DNA)免疫同系的雌性BALB/c小鼠,可以产生一系列的SLE症状,包括高水平的抗dsDNA自身抗体、蛋白尿、免疫复合物沉积和肾小球肾炎,这些症状模拟病人体内由大量凋亡细胞来源的自身DNA引起的SLE症状,因此ALD-DNA免疫的小鼠可以被作为理想的小鼠狼疮模型进行探讨SLE疾病可能的细胞与分子免疫学机制。SLE通常被认为是由自身抗体介导的全身性炎症反应和由T／B细胞介导的适应性免疫应答所诱发的组织损伤,但是SLE发病和疾病进展的细胞与分子机制仍不清楚。有研究提示,在SLE小鼠中,显著激活的巨噬细胞和其它髓系细胞大量浸润到淋巴组织和肾脏中,启动和促进适应性免疫应答,从而导致SLE的发生。越来越多的证据显示F4／80+巨噬细胞是SLE肾炎中主要的浸润细胞,在SLE肾炎的发生过程中扮演重要角色,而浸润的巨噬细胞发挥保护性还是病理性作用有待阐明。功能上的可塑性和多样性是单核巨噬细胞的显著特点之一,巨噬细胞随着周围环境的变化,功能上会发生显著变化,这些功能上的变化,也称为功能上的巨噬细胞极化,可以产生有不同基因表达谱和不同功能的巨噬细胞亚群。目前认为M1和M2(包括M2a、M2b和M2c)是单核巨噬细胞功能上连续变化过程的两个极端。在SLE疾病过程中巨噬细胞是否发生极化、极化类型及其机制鲜有报道。本研究的目的在于：(1)探讨巨噬细胞在ALD-DNA诱导SLE发病中的作用；(2)通过表型分析和细胞因子表达谱鉴定,分析SLE模型鼠肾炎组织中巨噬细胞的活化和极化类型以及可能的分子机制；(3)研究导致自身DNA清除障碍和打破免疫耐受引起巨噬细胞产生免疫应答的机制；(4)设计体内外实验探索SLE疾病可能的防治方法。我们的研究分为以下四部分：1.巨噬细胞在ALD-DNA诱导SLE发病中的作用体内未被清除的凋亡细胞来源的自身DNA具有免疫原性,可以引发一系列的免疫应答,从而导致抗自身DNA的抗体产生和抗体介导的组织损伤,这在SLE病人体内非常普遍,但是巨噬细胞是否在SLE发病过程中发挥作用仍不清楚。在本课题中,我们在ALD-DNA免疫的SLE小鼠模型中,发现狼疮肾炎组织中有大量的活化巨噬细胞浸润。ALD-DNA可以在体内和体外诱导巨噬细胞分泌细胞因子TNF-α、IL-1β、IL-6和IL-10,并上调表达表面活化标志包括MHC class-Ⅱ、CD40、CD80和CD86,但是非活化淋巴细胞来源的DNA (unactivated lymphocyte-derived DNA, UnALD-DNA)并不能引起巨噬细胞的活化。我们进一步发现活化的巨噬细胞在体外可以促进T分泌IL-4和IL-10,促进B细胞产生抗dsDNA的自身抗体,从而参与ALD-DNA诱导的自身免疫反应。更重要的是去除SLE模型小鼠体内的巨噬细胞可以有效减轻尿蛋白水平、缓解狼疮性肾炎的症状。这些研究结果提示巨噬细胞在SLE发病过程中扮演重要角色,ALD-DNA通过诱导巨噬细胞活化进而启动针对自身抗原的固有免疫和适应性免疫应答,从而造成免疫复合物沉积和组织损伤。以上发现为SLE的发病机制提供了新视野,为临床SLE疾病的治疗提供了以控制巨噬细胞浸润和活化作为靶点的可能的新治疗策略。2. ALD-DNA诱导巨噬细胞极化的Notch途径在SLE病人体内存在固有免疫应答失调,包括异常巨噬细胞的活化。活化的巨噬细胞会发生功能上极化,但是在SLE疾病过程中活化巨噬细胞的极化类型和机制并不清楚。作为一个重要的决定细胞命运的局部细胞相互作用机制,Notch信号通路在调控各种免疫细胞的发育和分化过程中发挥关键作用,但是Notch通路是否在巨噬细胞极化过程中发挥作用并不清楚。本研究中,我们课题组用ALD-DNA免疫小鼠,建立了SLE模型。在SLE模型鼠中,我们发现狼疮肾炎组织中有M2b巨噬细胞的浸润。在体内M2b巨噬细胞以及体外ALD-DNA诱导的M2b巨噬细胞中,我们发现Notch1信号通路的活性增加。通过抑制Notch1信号通路以及高表达Notch1胞内活性片段的方法,我们发现ALD-DNA诱导的M2b极化依赖于Notch1信号通路的活化。Notch1信号通路活化后通过激活PI3K和MAPK信号通路促进NF-κB p50入核从而驱动ALD-DNA诱导的巨噬细胞M2b极化。进一步发现用Notch信号通路抑制剂Y-分泌酶抑制剂(γ-secretase inhibitor, GSI)处理小鼠,可以通过钝化巨噬细胞M2b极化从而缓解SLE症状。我们的研究结果显示Notch1途径依赖的巨噬细胞M2b极化可能在SLE疾病中发挥重要作用,提示阻断Notch1信号通路可以作为SLE的潜在治疗方法。3. Jagged1参与巨噬细胞M2极化的Notch途径巨噬细胞被微环境中不同的诱导因素活化后表现出不同的表型和功能特征,导致巨噬细胞极化为M1或者M2型巨噬细胞。虽然这些巨噬细胞亚群的分化和功能已经研究清楚,但是对于决定巨噬细胞极化事件的因素中除了可溶性的细胞因子(例如IFN-γ或者IL-4)之外的细胞表面受体的作用知之甚少。在本研究中我们提供直接证据表明Jagged1和巨噬细胞表面的受体Notch1相互作用后传导信号,促进巨噬细胞发生M2极化。Jagged1-Notch1信号通路的活化和效应细胞分化有剂量依赖关系,高水平的Jagged1刺激Notch1信号通路会导致巨噬细胞M2活化标志显著增加。我们的研究结果提示Jagged1活化的Notch1信号通路决定巨噬细胞M2极化而不发生M1极化,同时也提供一个巨噬细胞M2类型极化的可能机制。4.SAP通过诱导巨噬细胞极化类型转换缓解SLE发病作为正常存在的DNA结合蛋白,血清淀粉状蛋白P成份(Serum amyloid P component, SAP)可以激活巨噬细胞介导的细胞核成份包括dsDNA的吞噬。在我们前期研究中发现,ALD-DNA免疫小鼠可以诱导SLE的发生,有理由推测SAP在SLE疾病过程中发挥重要作用。在本课题中,我们发现ALD-DNA免疫的SLE小鼠血清中轻微升高的SAP浓度伴随着DNA浓度显著升高,导致SLE小鼠血清中SAP浓度与DNA浓度的比值显著低于正常小鼠,而且其比值和SLE小鼠血清中抗dsDNA的水平成负相关。进一步的研究发现SAP可以和DNA结合从而促进巨噬细胞介导的DNA吞噬。在体外,SAP和DNA结合后可以使巨噬细胞极化类型从DNA诱导type-Ⅱ型(M2b)转为SAP-DNA复合物诱导的替代性途径活化类型(alternatively activated macrophages, AAMΦs; M2a)。AAMΦs分泌大量的细胞因子IL-10一方面可以阻止DNA诱导巨噬细胞type-Ⅱ活化,另一方面可以促进AAMΦs对DNA的吞噬。更为重要的是我们用SAP处理小鼠,增加SLE小鼠体内的SAP水平,通过诱导巨噬细胞极化为AAMΦs,从而缓解肾小球肾炎的症状。以上研究结果显示SAP在SLE疾病过程中发挥保护性的作用,增加SAP水平可以通过诱导巨噬细胞极化类型从促炎性的type-Ⅱ类型转换为抗炎性的AAMΦs,从而缓解SLE。本研究提示SAP可能参与巨噬细胞极化类型转换和维持巨噬细胞对自身DNA的免疫耐受,可以作为SLE疾病治疗的有效途径之一。更多还原

【Abstract】 The study on the new mechanism for ALD-DNA induced SLE:macrophage polarization and its role in SLESystemic lupus erythematosus (SLE) is a potentially fatal disease characterized by the prototypic autoimmune syndrome with heterogeneous manifestations frequently including autoimmunity, vasculitis, arthritis, and glomerulonephritis. In the United States, the number of patients with SLE exceeds 250,000. The 90 percent of SLE patients are women of childbearing age. Recently, the 5-y survival rate of patients with lupus glomerulonephritis increased from 44%in the 1950s to 82%. Despite great advances in the treatment of this autoimmune disease, the mean age of death of patients dying from systemic lupus erythematosus is 44 y. To the clinician, SLE is important because it is a potentially fatal disease that is easily confused with many other disorders. To the immunologist, lupus is intriguing because all the key components of the immune system are involved in the underlying mechanisms of the SLE disease.The anti-dsDNA autoantibody, which is a serological hallmark of SLE, has been proved to be pathogenic and could cause subsequent tissue deposition of immune complexes (IC) and tissue damage. Anti-dsDNA antibodies are highly specific for SLE and levels of anti-dsDNA antibodies in serum tend to reflect disease activity. Genetic studies in SLE patients revealed that anti-dsDNA autoantibodies, which generally belong to IgG subtype with high-affinity binding to dsDNA, differ from the germ line due to somatic mutations. Generally, mammalian DNA gains poor immunogenicity and could not trigger the immune response. Accumulating data indicated that undigested DNA released from apoptotic cells could induce macrophage activation and trigger a set of immune response, thus producing autoantibodies to self-DNA, which occurs commonly in SLE patients. In this study, we utilized the SLE murine model established by our group previously through immunizing syngeneic female BALB/c mice with a self-DNA released from apoptotic lymphocytes which termed as activated lymphocyte-derived DNA. A series of SLE syndrome including highly anti-dsDNA antibodies, proteinuria, immune complex deposition, and glomerulonephritis were developed in our murine model, which resembles human SLE syndrome accompanied with abundant self-DNA released from unremoved apoptotic cells. Thereby the ALD-DNA immunized mice could be used as an ideal murine lupus model to explore the potential cellular and molecular immunological mechanisms responsible for SLE disease.SLE syndrome is generally considered to be autoantibody-mediated systemic inflammation and tissue damage triggered by aggressive T and B cell responses of the adaptive immune system. Yet, the underlying cellular and molecular mechanisms for onset and progression of SLE are still poorly understood. It was reported that markedly activated macrophages and other myeloid cells which infiltrated in lymphoid tissues and kidneys, mediated the onset and propagation of an aggressive adaptive immune response, thereby leading to SLE pathogenesis in mice. Accumulating data demonstrated that F4/80+macrophages represented the major inflammatory infiltrated cells and played a crucial pathogenic role in the development of SLE nephritis. However, It is still unclear whether the activated macrophages found in the kidneys have a pathogenic or protective role. Furthermore, macrophages display remarkable plasticity and can change their physiology in response to exposure to various microenvironmental signals. Functional macrophage polarization represents different extremes of a continuum ranging from M1, M2a (alternatively activated macrophages, AAMΦs), M2b (type-II), to M2c. The concrete phenotype and mechanism for functional macrophage polarization in SLE remains unclear.PART ONE:Induction of inflammatory and immune responses by macrophages stimulated with ALD-DNA:implications for the pathogenesis of SLEUndigested DNA released from apoptotic cells could trigger a set of immune response, thus producing autoantibodies to self-DNA, which occurs commonly in SLE patients. But the role of macrophages in the pathogenesis of SLE remains largely unknown. In this study, we report that in the SLE murine model generated by immunization with ALD-DNA, the nephritic tissues were found infiltrated with activated macrophages. ALD-DNA could induce the secretion of TNF-α, IL-1β, IL-6, and IL-10 and the expression of activation markers including MHC class-II, CD40, CD80, and CD86 in macrophages in vitro and in vivo. However, DNA derived from un-activated lymphocytes (UnALD-DNA) could not. Furthermore, activated macrophages were found to be involved in the ALD-DNA induced autoimmune response via promoting the cytokine production by T cells and autoantibody production by B cells when stimulated with ALD-DNA. More importantly, macrophage depletion could decrease the urine protein and induce the remission of established lupus nephritis in SLE murine model. Our findings suggest that ALD-DNA activates macrophages and, thereby, may crucially contribute to the pathogenesis of SLE.PART TWO:Blockade of Notchl signaling alleviates murine lupus via blunting macrophage activation and M2b polarizationSystemic lupus erythematosus (SLE) patients are found to be accompanied with innate immunity dysregulation including abnormally macrophage activation. But the concrete phenotype and the mechanism for functional polarization of the activated macrophages during pathogenesis of SLE remains unknown. As an important local cellular interaction mechanism responsible for cell fate determination, Notch signaling is reported to exert crucial functions in the development and differentiation of various immunocytes, whereas its role in macrophage polarization is not fully understood. Herein, in the SLE murine model generated by immunization with activated lymphocyte-derived DNA (ALD-DNA), the nephritic tissues were found infiltrated with M2b-polarized macrophages. Notchl signaling activity was significantly up-regulated in the ALD-DNA induced M2b macrophages in vitro and in vivo. Furthermore, ALD-DNA induced M2b polarization was found to be dependent on enhanced Notchl signaling through accelerating NF-κB p50 translocation into nucleus mediated by PI3K and MAPK pathways. Moreover, blockade of Notchl signaling with y-secretase inhibitor (GSI) treatment could ameliorate murine lupus through impeding macrophage M2b polarization. Our results implied that Notchl signaling dependent M2b-polarized macrophages might play a pivotal role in the pathogenesis of SLE, which could provide Notchl signaling blockade as a potential therapeutic approach for SLE disease.PART THREE:Notchl engagement by Jaggedl bias the M2 functional differentiation of activated macrophagesFollowing activation by antigen, macrophages execute distinct genetic programs that result in their differentiation toward the type 1 or type 2 macrophages (M1 or M2) phenotype. Although the differentiation and function of these macrophage subsets has been well studied, little is known about the contribution to these differentiation events of cell surface receptors other than those for soluble cytokines, such as IFN-y or IL-4. Here, we provide direct evidence that the Jaggedl interaction with Notchl on macrophages transduces signals, promoting development toward the M2 phenotype. The positive role of Notch signaling in effector cell differentiation was dose dependent, with high levels of Jaggedl stimulation resulting in increased macrophage M2 polarization. Our data revealed a clear contribution of Notchl pathways engaged by Jaggedl to M2 versus M1 fate decisions, while also providing insight into another mechanism for inhibition of macrophage M2 activation.PART FOUR:Serum amyloid P component ameliorates murine lupus via biasing macrophage activation to the alternative pathwaySerum amyloid P component (SAP) has been reported to activate macrophage-mediated phagocytosis of nuclear debris, whereas its role in the pathogenesis of systemic lupus erythematosus (SLE) is not fully understood. Herein, decreased serological ratios of SAP to DNA were found to be negatively correlated with the titers of anti-dsDNA antibodies in SLE patients and lupus murine model. SAP was shown to promote macrophage-mediated DNA uptake through binding to DNA. Furthermore, type-Ⅱpolarized macrophages induced by DNA could be switched to alternatively activated macrophages (AAMΦs) by SAP-DNA complex in vitro. IL-10 secreted by AAMΦs was found to predominantly impede DNA induced macrophage type-II activation and promote DNA phagocytosis. More importantly, reinforced SAP level in vivo could efficiently ameliorate glomerulonephritis through inducing renal macrophage alternative activation in lupus murine model. Taken together, our results reveal a protective role of SAP in lupus, which suggest that enhanced SAP level could alleviate SLE syndrome via switching the polarized phenotype of macrophages from pro-immune type-II activation to anti-immune alternative activation. This might provide SAP as a potential therapeutic approach for SLE disease.更多还原