Rivers, Elizabeth; (2024) Autophagy-Inflammasome Interplay in Wiskott-Aldrich Syndrome. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Background: Wiskott-Aldrich syndrome (WAS) is an X-linked severe immunodeficiency disorder resulting from impaired regulation of the actin cytoskeleton in haematopoietic cells. Clinical features include microthrombocytopaenia, recurrent infections and susceptibility to haematological malignancy, necessitating stem cell treatment in the form of haematopoietic stem cell transplantation or gene therapy. An inflammatory phenotype is seen in 70-80% of children, including severe eczema, arthritis, inflammatory bowel disease and vasculitis and up to 30% have ongoing autoinflammation post stem cell treatment, which can be challenging to treat. Emerging evidence highlights the importance of regulated autophagy and inflammasome activity in the development of autoinflammatory disease and identifies the actin cytoskeleton as a crucial player. Previous work from our laboratory suggested a key role for murine Wiskott-Aldrich syndrome protein (WASp) in autophagic processes, which are necessary for inflammasome regulation. This thesis explores the inflammatory signature in WAS and role of human WASp in the autophagy-inflammasome axis, with a view to identifying potential biomarkers for autoinflammatory disease and opportunities for targeted therapeutic interventions. / Methods: Serum inflammatory cytokine and chemokine concentrations were quantified from healthy donors and WAS patients using ELISA and correlated to clinical and laboratory markers of inflammatory disease and level of WASp correction post definitive stem cell therapy. Exploration of the autophagy-inflammasome axis was subsequently carried out in CRISPR WAS KO THP-1 and primary macrophages through infection and sterile models. Autophagic flux was studied using autophagy protein expression (LC3, p62 and LAMP-1) by western blotting and localisation through confocal microscopy, in response to rapamycin/bafilomycin. CCCP-induced mitochondrial damage was used to further explore selective mitophagy, with functional consequences investigated through evaluation of mitochondrial respiration. The role of WASp was interrogated through evaluation of primary macrophages from healthy donors and WAS patients pre- and post- in vitro and in vivo WASp reconstitution. The mechanism of WASp involvement was investigated through use of actin and actin-related protein (ARP) inhibitors, in addition to primary macrophages from ARP-deficient patients. / Results: IL-18 is significantly elevated in WAS patients pre- as well as post- definitive stem cell therapy where myeloid WASp correction is poor. This correlates reliably with the presence/ absence of inflammatory symptoms and builds on previous evidence from our laboratory for the role of WASp in inflammasome pathways. Rapamycin-induced autophagosome formation and trafficking to lysosomes in primary human macrophages is demonstrated to be dependent on WASp in an ARP2/3-actin-dependent manner. Furthermore, human WASp is identified to be crucial for maintenance of mitochondrial network integrity and mitophagy, with impaired actin cage formation around damaged mitochondria found in WASp-deficiency and significant intrinsic metabolic dysfunction in WAS. / Conclusion: This work demonstrates new and important roles of human WASp in autophagic processes and immunometabolic regulation, which may contribute to the inflammatory clinical features and complex immunophenotype seen in WAS.

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