Investigating the role of B cells in pulmonary fibrosis resulting from STING gain-of-function autoinflammation

项目摘要/摘要 肺纤维化是一个知之甚少的过程，被认为涉及免疫的前一阶段。 渗入和发炎。博莱霉素是一种DNA损伤诱导剂，常用于实验诱导 小鼠肺纤维化，表明核酸的免疫感应可能与肺纤维化的病因有关 纤维性肺病。CGAS-STING是一种胞质dsdna传感通路，受到严格的调控，以 通过区分对危险信号的免疫反应，如病原体或 遗传毒性应激和对惰性生理信号的免疫耐受性。这一规定被宪法所违反 活性刺痛突变，导致一种称为刺痛相关的自体炎症综合征 起病于婴儿期的血管病(SAVI)，患者出现免疫异常。与一致 核酸感应失调促进肺纤维化的观念；SAVI患者迅速死亡 以治疗难治性炎症性肺纤维化。为了解决SAVI肺部疾病治疗的迫切需要， 我们已经开发出表达SAVI突变STINGV154M(Vm)的基因靶向小鼠，并发现这些 小鼠概括了人类疾病的方方面面，包括炎症性肺纤维化。研究我们的 Savi小鼠模型不仅在于它对SAVI疾病研究的影响，而且在于它对SAVI疾病的广泛理解 导致肺纤维化的免疫缺陷，这个话题到目前为止一直受到以下限制 目前的纤维性肺病模型。我们的中心假设是SAVI纤维化肺病的获得性 是由自身反应性B细胞介导的。以下关键发现支持这一假说：(I)VM小鼠具有 以淋巴细胞减少伴高活化为特征的淋巴细胞严重免疫异常 在剩余的淋巴细胞中，这一特征也见于SAVI患者。(Ii)对B和T进行基因消融 淋巴细胞通过Rag1缺乏，我们发现在VM小鼠的肺部疾病中需要淋巴细胞； 然而，当我们在VM小鼠中通过TcRαβ缺乏特异性地消融βT细胞时，我们发现这些小鼠 坚持发展成暴发性肺部疾病。(Iii)此外，我们的初步数据表明，VM B细胞 在肺血管外空间蓄积，并独立于αβT细胞被激活。在这份提案中， 目标1将利用小鼠基因确定B细胞在VM SAVI纤维化肺部炎症中的作用 靶向抗体治疗B细胞缺陷及药物耗竭模型的研究。目标2将 使用小鼠模型确定VM SAVI肺B细胞是否通过自身反应促进疾病 通过对BCR谱系和抗体反应性的表征来限制BCR谱系对外来抗原的作用 在VM小鼠体内。该方法包括：生存研究，肺组织病理学分析，肺功能测试， 小鼠肺切片的流式细胞术、bcr谱带测序和抗体染色。我们的研究将 明确B细胞在VM SAVI肺纤维化中的作用，并将广泛提供对B细胞驱动的洞察 纤维性肺病的发病机制。

PROJECT SUMMARY/ABSTRACT Pulmonary fibrosis is a poorly understood process that is thought to involve a preceding stage of immune infiltration and inflammation. Bleomycin, a DNA damage inducing agent, is often used to experimentally induce pulmonary fibrosis in mice, indicating that immune sensing of nucleic acids may be tied to the etiology of fibrotic lung disease. cGAS-STING is a cytosolic dsDNA sensing pathway, which is tightly regulated to preserve immune homeostasis by discriminating immune response to danger signals like pathogens or genotoxic stress and immune tolerance to inert physiologic signals. This regulation is broken by constitutively active STING mutations, which cause an autoinflammatory syndrome known as STING Associated Vasculopathy with onset in Infancy (SAVI), in which patients develop immune abnormalities. Consistent with the notion that dysregulated nucleic acid sensing promotes pulmonary fibrosis; SAVI patients rapidly succumb to treatment resistant inflammatory lung fibrosis. To address the urgent need for SAVI lung disease therapy, we have developed gene-targeted mice that express the SAVI mutation STINGV154M(VM) and found that these mice recapitulate aspects of human disease including inflammatory lung fibrosis. The utility of studying our SAVI mouse model lies not only in its impact on studying SAVI disease, but broadly for understanding the immune defects that drive pulmonary fibrosis, a topic that has thus far been constrained by the limitations of current fibrotic lung disease models. Our central hypothesis is that the acquisition of SAVI fibrotic lung disease is mediated by autoreactive B cells. The following key findings support this hypothesis: (i) VM mice possess severe immune abnormalities of lymphocytes characterized by lymphopenia with concomitant hyperactivation of remaining lymphocytes, a feature also seen in SAVI patients. (ii) Using genetic ablation of B and T lymphocytes by Rag1 deficiency, we found that lymphocytes are required for lung disease in VM mice; however, when we specifically ablated αβ T cells by TCRβ deficiency in VM mice, we found that these mice persisted in developing fulminant lung disease. (iii) Additionally, our preliminary data indicates that VM B cells accumulate in the lung extravascular space and become activated independent of αβ T cells. In this proposal, Aim 1 will determine the contribution of B cells to VM SAVI fibrotic lung inflammation using mouse genetic models of B cell deficiency and pharmacologic depletion of B cells by targeted antibody treatment. Aim 2 will determine whether VM SAVI lung B cells promote disease through autoreactivity using mouse models that restrain the BCR repertoire to foreign antigens and by characterizing the BCR repertoire and antibody reactivity in VM mice. The approach includes: survival studies, lung histopathologic analysis, pulmonary function testing, flow cytometry, BCR repertoire sequencing, and antibody staining of mouse lung sections. Our studies will discern the role of B cells in VM SAVI lung fibrosis and will broadly provide insight into B cell driven mechanisms of fibrotic lung disorders.