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

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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