The Central Role of IL-33 in Immune-Mediated Scarring

描述(由申请人提供)： 夸张的疤痕或纤维化使一系列疾病复杂化，在所有器官中都会发生，并可能无情、虚弱和致命。它在经历过军事接触的老年患者中尤为普遍，给退伍军人带来了严重的问题。过度的先天和获得性免疫反应会导致纤维化，但确切的机制仍不完全清楚。最近的研究结果表明，白介素33在纤维化的发病机制中发挥了作用。IL-33以两种形式存在，并具有双重活性。核内IL-33(INIL33)与染色质结合，似乎调节基因表达。相比之下，细胞外IL-33(EXIL33)通过与细胞表面受体T1/ST2结合，促进Th2反应而发挥作用。EXIL33的活动正在积极调查中，而INIL33的活动一直被低估，没有详细研究或与疾病有关。我们的数据表明，纤维化主要(但不完全)是由INIL33驱动的。在肝纤维化患者的组织和博莱霉素损伤的小鼠模型中，INIL3的水平都升高，在这种模型中，INIL33的形式比流亡更占优势。在细胞培养和动物模型中，INIL33的基因传递导致胶原堆积，促纤维化细胞因子和基质金属蛋白酶的产生增加，以及Smad3的激活，所有这些都被认为是导致纤维化的原因。当与博莱霉素损伤联合使用时，INIL33可促进胶原堆积和促纤维化细胞因子的表达。如果有的话，INIL33到EXIL33的转化很少，没有Th2激活的迹象，T1/ST2基因缺陷对INIL33驱动的纤维化的减弱作用也很小。我们假设，INIL33是一种T1/ST2非依赖性、Th2非依赖性的多种促纤维化机制的上游激活剂，在成纤维细胞中INIL33的高表达通过直接和间接机制促进这些细胞的胶原沉积。将解决以下特定目标：1.对IL-33分子进行功能定位，以确定该蛋白中与培养的原代人成纤维细胞和小鼠体内的核定位、染色质结合和转录调控有关的区域。2.明确IL-33对成纤维细胞促纤维化作用的直接机制(结合胶原启动子和上调胶原基因转录)和间接机制(T1/ST2依赖和独立调节促纤维化细胞因子和基质金属蛋白酶)的相对作用。3.划定 通过确定成纤维细胞特异性IL-33缺陷、普遍存在的IL-33缺陷和普遍存在的T1/ST2缺陷在博莱霉素损伤模型中对炎症和纤维化的影响，体内成纤维细胞特异性表达IL-33和T1/ST2细胞表面受体的病理生理学作用。这些研究的成功完成将阐明INIL-33激活成纤维细胞的作用和机制，为未来开发合理的IL-33靶向抗纤维化策略奠定基础。

DESCRIPTION (provided by applicant): Exaggerated scarring, or fibrosis, complicates a spectrum of diseases, occurs in all organs, and can be relentless, debilitating, and deadly. It is particularly prevalent in older patients and thoe who have experienced military exposures, posing a serious problem for veterans. Exaggerated innate and adaptive immune responses drive fibrosis, yet the exact mechanisms remain incompletely understood. Recent findings suggest a role for interleukin (IL)-33 in the pathogenesis of fibrosis. IL-33 exists in two forms and has a dual activity. Intranuclear IL-33 (INIL33) is bound to chromatin and appears to regulate gene expression. By contrast, extracellular IL-33 (EXIL33) acts by binding to the cell-surface receptor T1/ST2 and facilitating Th2 responses. The activities of EXIL33 are under active investigation, whereas the activities of INIL33 have been underappreciated and not studied in detail or in association with disease. Our data suggest that fibrosis is driven primarily (yet not exclusively) by INIL33. The levels of INIL3 are elevated in tissues of patients with fibrosis as well as in the mouse model of bleomycin injury, in which the INIL33 form predominates over EXIL. Gene delivery of INIL33 in cell culture and in the animal model leads to collagen accumulation, increased production of profibrotic cytokines and matrix metalloproteinases, as well as Smad3 activation, all of which are known to contribute to fibrosis. When combined with bleomycin injury, INIL33 potentiates collagen accumulation and the expression of profibrotic cytokines. There is minimal, if any, conversion of INIL33 to EXIL33, no signs of Th2 activation, and gene deficiency of T1/ST2 has minimal, if any, attenuating effect on INIL33-driven fibrosis. We hypothesize that INIL33 is a T1/ST2-independent, Th2-independent, upstream activator of multiple profibrotic mechanisms and that elevated expression of INIL33 in fibroblasts contributes to collagen deposition by these cells through direct and indirect mechanisms. The following Specific Aims will be addressed: 1. Functionally map the IL-33 molecule to identify regions in this protein that confer nuclear localization, chromatin binding, and transcriptional regulation in cultured primary human fibroblasts and in mice in vivo. 2. Define the relative roles of direct mechanisms (binding to the collagen promoter and upregulating collagen gene transcription) and indirect mechanisms (both T1/ST2-dependent and -independent regulation of profibrotic cytokines and matrix metalloproteinases) of profibrotic regulation of fibroblasts by IL-33 in cell culture. 3. Delineate the pathophysiological roles of fibroblast-specific expression of IL-33 and of the T1/ST2 cell-surface receptor in vivo by determining the effects of fibroblast-specific IL-33 deficiency, ubiquitous IL-33 deficiency, and ubiquitous T1/ST2 deficiency on inflammation and fibrosis in the bleomycin injury model. Successful completion of these studies will clarify the role and mechanisms of fibroblast activation by INIL-33, laying the groundwork for future development of rational IL-33-targeting antifibrotic strategies.