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对比性的2型免疫反应抑制有关。该项目将利用一系列寄生虫衍生的IL-33途径蛋白调节剂，研究促进2型细胞因子IL-33在肥胖中的作用。白介素33是一种危险的细胞因子，它预先储存起来，与上皮细胞和基质细胞的细胞核中的基因组DNA结合，在细胞损伤时释放。它有一个N-末端的DNA结合域，这阻止了它从细胞核中释放，直到这个结构域被蛋白水解性切割。这种与DNA的结合似乎减缓了IL-33从坏死的上皮细胞核中释放的速度，从而在更长的时间内维持IL-33的信号，增强了其效果。该项目旨在进一步研究IL-33的释放速度如何控制IL-33的反应强度。IL-33与肥胖有关，IL-33缺陷的小鼠明显比野生型小鼠更肥胖。IL-33的释放导致免疫细胞的激活，如嗜酸性粒细胞和2型先天淋巴样细胞，它们在新陈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1。IL-33的作用可以通过HpARI调节，HpARI是我们鉴定的一种寄生虫来源的蛋白，它与IL-33和DNA结合，阻断细胞因子2的作用。我们已经开发了HpARI的突变版本，它具有惊人的特性，可以有效地增强IL-33的作用。它似乎通过影响细胞因子的释放速度和有效半衰期来发挥作用。我们还确定了产生IL-33信号所需的信号通路的元件，这些元件可以被小分子3抑制或加剧。在这个项目中，我们将使用我们独特的一组IL-33调节器来确定IL-33释放的速度如何影响IL-33的反应性，以及这如何影响代谢变化和肥胖。我们将进行体外实验，使用超分辨活显微镜测量HpARI突变体处理后IL-33的释放速度。我们将评估培养中释放的速度如何影响IL-33反应细胞(如ILC2、肥大细胞、巨噬细胞和Th2细胞)的下游IL-33信号。反应细胞中信号元件的激活将通过流式细胞仪染色、蛋白质印迹和全球蛋白质表达和修饰的蛋白质组学分析来评估。然后，我们将把这些结果应用于体内实验，用高脂饮食的小鼠诱导白色脂肪堆积增加，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，这个项目将把一个基本的细胞过程(在细胞坏死期间释放核蛋白)与新陈代谢和肥胖的控制联系起来。杰克逊-琼斯，2016年，Nat Commun，7：12651奥斯本，2017年，免疫力，47：739麦卡锡，2019年，免疫细胞生物学，97：54

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