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

系统性红斑狼疮（SLE）的DNA和染色质抗体驱动自身免疫。SLE发病机制的主要问题与自身反应性B细胞能够识别的DNA的物理形式以及防止DNA自我耐受性破坏的机制有关。分泌的脱氧核糖核酸酶DNASE1L3的零突变和半胚变异分别与家族性和散发性SLE有关。Boris Reizis及其同事为这一观察提供了机制上的解释：他们在实验性SLE中发现来自微粒的染色质是自身反应性B细胞的潜在自身抗原。微粒含有凋亡细胞的基因组DNA。显然，循环的DNASE1L3具有独特的消化微粒DNA的能力，从而限制了其抗原性。重要的是，该模型描述了一种涉及分泌酶的细胞外源性自我耐受机制，因此可以开发用于治疗目的。此外，相同的亚型DNASE1L3变异也与系统性硬化症（SSc）有关，相似类型的dna驱动免疫复合物是SLE和SSc的特征。因此，本项目的总体目标是系统地测试DNASE1L3及其内源性DNA底物在SLE和SSc中的作用。这一目标将通过两个研究目标来实现。目的1探讨微粒DNA作为抗原在人类SLE中的作用。DNASE1L3活性、抗体结合微粒DNA和微粒DNA含量将在400多名特征明确的SLE患者和匹配的健康供者中进行测量。研究人群将允许比较临床和临床前患者的样本。以下假设将被探讨：1)IgG抗体与微粒DNA结合发生在很大一部分SLE患者中，但在健康个体中没有发生；2)循环微粒中的DNA含量在SLE患者中增加，并与抗体与微粒DNA结合和DNASE1L3活性降低相关；3)微粒相关DNA和染色质抗体在疾病早期出现，与疾病严重程度相关，甚至预测其严重程度。目的2探讨DNASE1L3在小鼠SSc模型中的作用和治疗价值。我们将在博莱霉素诱导小鼠模型和紧致皮肤小鼠遗传模型中评估dnase1l3缺乏对临床和免疫学参数的影响。为了测试DNASE1L3的治疗效用，将给小鼠注射一种编码人类DNASE1L3的腺病毒载体。详细探讨以下假设：i) DNASE1L3缺陷小鼠比野生型小鼠发展出更严重的实验性SSc形式，ii)微颗粒DNA含量增加，抗体与微颗粒DNA结合和DNASE1L3活性降低与实验性SSc疾病严重程度相关，iii) DNASE1L3递送预防和恢复实验性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.