The role of DNASE1L3 and its endogenous substrate, chromatin in apoptotic cell microparticles, in human systemic lupus erythematosus and experimental systemic sclerosis

DNA和染色质抗体驱动系统性红斑狼疮（SLE）的自身免疫。SLE发病机制的主要问题是自身反应性B细胞识别的DNA的物理形式和防止对DNA的自身耐受性破坏的机制。分泌型脱氧核糖核酸酶DNASE 1 L3的突变和亚纯型变体分别与家族性和散发性SLE有关。Boris Reizis及其同事为这一观察结果提供了一种机制解释：他们将微粒中的染色质鉴定为实验性SLE中自身反应性B细胞的潜在自身抗原。微粒含有来自凋亡细胞的基因组DNA。显然，循环DNASE 1 L3是唯一能够消化微粒中的DNA，从而限制其抗原性。重要的是，该模型描述了一种涉及分泌酶的自身耐受性的细胞外在机制，因此可以开发用于治疗目的。同样的亚型DNASE 1 L3变异体也与系统性硬化症（SSc）和类似类型的DNA-因此，本项目的总体目标是系统地检测DNASE 1 L3及其内源性DNA底物在SLE和SSc中的参与。本研究将通过两个研究目标来实现这一目标：第一个目标是探讨微粒DNA作为抗原在人类SLE中的作用。将在400多名具有良好特征的SLE患者和匹配的健康供体中测量DNASE 1 L3活性、与微粒DNA结合的抗体和微粒DNA含量。研究人群将允许比较临床与临床前患者的样本。将探讨以下假设：i）IgG抗体与微粒DNA的结合发生在大部分SLE患者中，但不发生在健康个体中，ii）SLE患者中循环微粒中的DNA含量增加，并且与抗体与微粒DNA的结合和降低的DNASE 1 L3活性相关，目的2探讨DNA酶1 L3在SSc小鼠模型中的作用和治疗效用。将在博来霉素诱导的小鼠模型中评估DNA酶1 L3缺陷对临床和免疫学参数的影响，以及在一个皮肤紧绷的遗传模型中。为了测试DNASE 1 L3的治疗效用，将用编码人DNASE 1 L3的腺病毒载体注射小鼠。i）DNASE 1 L3缺陷型小鼠比野生型小鼠发展出更严重形式的实验性SSc，ii）微粒DNA含量增加、抗体与微粒DNA的结合和DNASE 1 L3活性降低与实验性SSc中的疾病严重程度相关，和iii）DNASE 1 L3递送预防和逆转实验性SSc。

Antibodies to DNA and chromatin drive autoimmunity in systemic lupus erythematosus (SLE). Major questions of SLE pathogenesis concern the physical form of DNA that can be recognized by autoreactive B cells and the mechanisms that prevent the breakdown of self-tolerance to DNA.Null mutations and hypomorphic variants of the secreted deoxyribonuclease DNASE1L3 are linked to familial and sporadic SLE, respectively. Boris Reizis and colleagues provided a mechanistic explanation for this observation:They identified chromatin from microparticles as latent self-antigen for autoreactive B cells in experimental SLE. Microparticles contain genomic DNA from apoptotic cells. Apparently, circulating DNASE1L3 is uniquely capable of digesting the DNA in microparticles, thereby restricting its antigenicity.Importantly, this model describes a cell-extrinsic mechanism of self-tolerance that involves a secreted enzyme and therefore can be developed for therapeutic purposes.Moreover, the same hypomorphic DNASE1L3 variant is also linked to systemic sclerosis (SSc) and similar types of DNA-driven immune complexes are characteristic for both, SLE and SSc.Thus, the overall goal of this project is to systematically test the involvement of DNASE1L3 and its endogenous DNA substrate in SLE and SSc. This goal will be pursued through two research objectives.Objective 1 explores the role of microparticle DNA as an antigen in human SLE.DNASE1L3 activity, antibody binding to microparticle DNA and microparticle DNA content will be measured in more than 400 well-characterized SLE patients and matched healthy donors. The study-population will allow comparing samples from clinical versus preclinical patients.Following hypotheses will be explored: i) Binding of IgG antibodies to microparticle DNA occurs in a substantial fraction of SLE patients, but not in healthy individuals, ii) DNA content in circulating microparticles is increased in SLE patients and correlates with binding of antibodies to microparticle DNA and reduced DNASE1L3 activity, and iii) Antibodies to microparticles-associated DNA and chromatin develop early in the disease and correlate with or even predict its severity.Objective 2 explores the role and therapeutic utility of DNASE1L3 in murine models of SSc.The effect of DNASE1L3-deficiency on clinical and immunological parameters will be assessed in the bleomycin-induced mouse model, and in a genetic model with tight skin1 mice. To test the therapeutic utility of DNASE1L3, mice will be injected with an adenoviral vector encoding human DNASE1L3.In detail following hypotheses will be explored: i) DNASE1L3 deficient mice develop a more aggravated form of experimental SSc than wild type mice, ii) Increased microparticle DNA content, binding of antibodies to microparticle DNA and reduced DNASE1L3 activity correlate with diseases severity in experimental SSc, and iii) DNASE1L3 delivery prevents and reverts experimental SSc.