Role of gasdermin D in bone resorption

对罕见疾病的研究通常会告知更常见的病理。我们广泛研究了NLRP 3 炎性体，其在自身炎性疾病如cryopyrin相关的周期性 综合征（CAPS）。这些病症的一个主要特征是IL-1β的过度产生，其也是高度 由放疗和化疗等程序引起，通常用于杀死恶性细胞或作为 预处理方案用于骨髓移植（BMT）。IL-1β有效促进骨吸收， 同时抑制骨形成，但IL-1阻断剂在治疗骨形成中的功效有限。 综合征相关的骨病变。这表明除了IL-1β以外， 加工，有助于在疾病中对骨骼的不利影响。炎性小体负责 IL-1β和IL-18的成熟。最近的研究已经确定GSDMD作为一种额外的关键底物， 炎性小体活化的GSDMD易位到质膜，在那里它形成孔， 分泌IL-1β和IL-18。然而，过度的孔形成损害膜的完整性，释放 促炎细胞质内容物进入细胞外环境。这种形式的细胞死亡，称为 焦亡是炎性的因此，虽然GSDMD是免疫应答和组织修复的正常参与者， 它的慢性激活促进炎症。我们推测，伴随的多种炎症反应的释放， 焦亡过程中的因素导致病理性骨丢失。因此，抑制GSDMD可以提供 与IL-1阻断剂相比具有上级功效，不仅在CAPS的情况下，而且在放射和化学疗法的情况下。 最近的药物发现工作已经确定双硫仑作为GSDMD孔形成活性的拮抗剂。 双硫仑是FDA批准的治疗酒精成瘾的药物。我们发现， 双硫仑抑制LPS刺激的IL-1β产生。双硫仑还抑制IL-1β分泌， 体外细胞凋亡和破骨细胞（OC）分化。为了进一步研究GSDMD在骨吸收中的作用， 我们在小鼠模型中确定了GSDMD功能丧失对骨骼的影响。初步结果表明 Gsdmd-/-小鼠的基线骨量高于WT小鼠。此外，旺盛的OC形成， 发生在CAPS小鼠中的Gsdmd在Gsdmd消融后正常化。Gsdmd基因敲除小鼠也对 辐射/BMT诱导的骨丢失。体外实验数据进一步证明，GSDMD的表达上调。 在OC分化过程中受到调节，这种蛋白质的遗传消除减少了OC的形成。这些结果 提示GSDMD在OC中起作用而不影响其存活，并调节骨吸收。 该建议的中心假设是GSDMD在病理条件下调节骨吸收 通过涉及其在炎症细胞和OC谱系中的作用的机制。我们将测试这个假设， 两个目的：目的1：确定GSDMD在病理条件下骨吸收中的作用。目标2：定义 GSDMD在OC谱系中的作用，并阐明其在这些细胞中的活化机制。

The study of rare diseases often informs more common pathologies. We have extensively studied the NLRP3 inflammasome, which is mutated in autoinflammatory disorders such as cryopyrin-associated periodic syndromes (CAPS). A major feature of these conditions is excessive production of IL-1β, which is also highly induced by procedures such as radiotherapy and chemotherapy, commonly used to kill malignant cells or as a conditioning regimen for bone marrow transplantation (BMT). IL-1β potently promotes bone resorption while simultaneously inhibiting bone formation, but IL-1 blocking agents have limited efficacy in the treatment of syndrome-associated bone pathologies. This suggests that other actions of the inflammasomes beyond IL-1β processing, contribute to adverse skeletal effects in diseases. The inflammasomes are responsible for the maturation of IL-1β and IL-18. Recent studies have identified GSDMD as an additional critical substrate of the inflammasomes. Activated GSDMD translocates to the plasma membrane where it forms pores through which IL-1β and IL-18 are secreted. However, excessive pore formation compromises membrane integrity, releasing pro-inflammatory cytoplasmic contents into the extracellular environment. This form of cell death, termed pyroptosis is inflammatory. Thus, while GSDMD is a normal participant in immune responses and tissue repair, its chronic activation promotes inflammation. We surmise that the concomitant release of multiple inflammatory factors during pyroptosis causes pathological bone loss. Therefore, inhibition of GSDMD could provide superior efficacy over IL-1 blockade, not only in the context of CAPS, but also radiation and chemotherapy. Recent drug discovery efforts have identified disulfiram as an antagonist of GSDMD-pore forming activity. Disulfiram is an FDA-approved drug for the treatment of alcohol addiction. We found that administration of disulfiram to mice inhibited LPS-stimulated IL-1β production. Disulfiram also inhibited IL-1β secretion, pyroptosis and osteoclast (OC) differentiation in vitro. To further study the role of GSDMD in bone resorption, we determined skeletal impact of GSDMD loss-of-function in mouse models. Preliminary results indicate that baseline bone mass was higher in Gsdmd-/- compared to WT mice. Moreover, the exuberant OC formation that occurred in CAPS mice was normalized upon Gsdmd ablation. Gsdmd null mice were also resistant to radiation/BMT-induced bone loss. In vitro data further demonstrated that expression of GSDMD was up- regulated during OC differentiation, and genetic ablation of this protein decreased OC formation. These results suggest that GSDMD is functional in OC without compromising their survival, and regulates bone resorption. The central hypothesis of this proposal is that GSDMD regulates bone resorption in pathological conditions through mechanisms involving its actions in inflammatory cells and OC lineage. We will test this hypothesis in two Aims: Aim 1: Determine the role of GSDMD in bone resorption in pathological conditions. Aim 2: Define the role of GSDMD in the OC lineage and elucidate the mechanisms of its activation in these cells.