How is the activity of IL-33 controlled by the speed of its release, and how could this affect obesity?

肥胖与炎性免疫环境和2型免疫反应的抑制相关。该项目将研究促进2型细胞因子IL-33在肥胖中的作用，使用一系列寄生虫来源的IL-33途径的蛋白调节剂。白细胞介素-33是一种警报细胞因子-它储存在预先形成的上皮细胞和基质细胞的细胞核中，与基因组DNA结合，并在细胞损伤时释放。它有一个n端dna结合区域，阻止它从细胞核中释放出来，直到这个区域被蛋白水解裂解。这种与DNA的结合似乎减缓了坏死上皮细胞核中IL-33的释放，从而在更长时间内维持IL-33信号传导，增强其作用。本项目旨在进一步研究IL-33的释放速度如何控制IL-33反应的强度。IL-33与肥胖有关，缺乏IL-33的小鼠比野生型小鼠明显更肥胖。IL-33的释放可激活免疫细胞，如嗜酸性粒细胞和2型先天淋巴样细胞，这些细胞在代谢调节中起重要作用。我们之前已经证明了脂肪组织基质细胞表达IL-33在控制局部免疫反应中的重要性。IL-33的作用可以通过HpARI来调节，HpARI是我们发现的一种寄生虫衍生蛋白，它与IL-33和DNA结合，阻断细胞因子2的作用。我们已经开发出一种突变型HpARI，它具有增强IL-33作用的惊人特性。它似乎是通过影响细胞因子释放的速度和有效半衰期来起作用的。我们还描述了产生IL-33信号所需的信号通路元件，这些元件可以使用小分子抑制或加剧3。在这个项目中，我们将使用我们独特的一组IL-33调节剂来确定IL-33释放的速度如何影响IL-33的响应性，以及这如何影响代谢变化和肥胖。我们将进行体外实验，我们将使用超分辨率活显微镜测量HpARI突变体处理后IL-33的释放速度。我们将评估在培养中释放速度如何影响IL-33应答细胞（如ILC2s、肥大细胞、巨噬细胞和Th2细胞）中的下游IL-33信号传导。响应细胞中信号元件的激活将通过流式细胞术染色、western blot和蛋白质组学分析来评估全局蛋白质表达和修饰。然后，我们将这些结果应用于体内实验，给小鼠高脂肪饮食诱导白色脂肪脂肪积累增加，通过使用HpARI（或IL-33受体缺陷小鼠）阻断IL-33途径，并使用HpARI突变体放大IL-33反应。我们将评估体重增加、脂肪堆积、免疫反应、葡萄糖抵抗和胰岛素敏感性。因此，该项目将把基本的细胞过程（细胞坏死过程中核蛋白的释放）与代谢和肥胖的控制联系起来。中国生物医学工程学报，2016,37 (7):12651

Obesity is associated with an inflammatory immune environment and contrasting suppression of type 2 immune responses. This project will investigate the role of the type 2-promoting cytokine IL-33 in obesity, using a range of parasite-derived protein modulators of the IL-33 pathway. Interleukin-33 is an alarmin cytokine - it is stored pre-formed, bound to genomic DNA in the nucleus of epithelial and stromal cells and released upon cell damage. It has an N-terminal DNA-binding domain, which prevents its release from the nucleus until this domain has been proteolytically cleaved. This binding to DNA appears to slow the release of IL-33 from the nucleus of necrotic epithelial cells, thus sustaining IL-33 signalling over longer periods, enhancing its effects. This project will aim to study further how the speed of release of IL-33 controls the intensity of IL-33 responses. IL-33 is implicated in obesity, with IL-33-deficient mice becoming significantly more obese than wild-type mice. IL-33 release results in the activation of immune cells such as eosinophils and type 2 innate lymphoid cells which are known to be important in regulation of metabolism. We have previously shown the importance of expression of IL-33 by adipose tissue stromal cells in control of local immune responses 1. The effects of IL-33 can be modulated using HpARI, a parasite-derived protein that we identified, and which binds to IL-33 and DNA, blocking the effects of the cytokine 2. We have developed a mutant version of HpARI, which has the surprising property of potently enhancing the effects of IL-33. It appears to act by affecting the speed and effective half-life of the cytokine's release. We have also characterised elements of the signalling pathway which are required for productive IL-33 signalling, and which can be inhibited or exacerbated using small molecules 3. In this project, we will use our unique set of IL-33 modulators to determine how the speed of IL-33 release affects IL-33 responsiveness, and how this affects metabolic changes and obesity. We will carry out in vitro experiments in which we will measure the speed of release of IL-33 after treatment with HpARI mutants, using super-resolution live microscopy. We will assess how speed of release in culture affects downstream IL-33 signalling in IL-33-responsive cells such as ILC2s, mast cells, macrophages and Th2 cells. Activation of signalling elements in responding cells will be assessed by flow cytometric staining, western blot, and proteomic analysis of global protein expression and modification. We will then apply these results to in vivo experiments administering mice with a high-fat diet to induce increases in white adipose fat accumulation, blocking the IL-33 pathway by using HpARI (or IL-33 receptor deficient mice), and amplifing IL-33 responses using HpARI mutants. We will assess weight gain, adipose fat accumulation, immune responsiveness, glucose resistance and insulin sensitivity. This project will therefore link a basic cellular process (release of nuclear proteins during cellular necrosis) and control of metabolism and obesity. Jackson-Jones, 2016, Nat Commun, 7:12651 Osbourn, 2017, Immunity, 47:739 McCarthy, 2019, Immunol Cell Biol, 97:54