The inflammasome is an intracellular multi-protein complex, activated upon recognition of endogenous and exogenous danger signals. Assembly of the inflammasome triggers caspase 1 activation leading to gasdermin D (GSDMD) cleavage and pore formation in the cell membrane, and to IL-1B and IL-18 processing and secretion. These cytokines play major roles in inflammation, enhancing the immune response but promoting autoimmune and pro-inflammatory diseases when dysregulated. Thus, understanding how they are secreted is important to therapeutically target these cytokines. The role of GSDMD in the secretion of IL-1B and IL-18 have been shown using liposomes and in murine myeloid cells. However, whether GSDMD is involved in IL-1B and IL-18 released in human macrophages, one of the main sources of these cytokines, has not been addressed. This thesis establishes that in human macrophages stimulated with LPS and nigericin, GSDMD is processed to form pore-like structures in the cell membrane through which IL-1B and IL-18 could be released. This is supported by the fact that chemical inhibition of GSDMD pore formation in primary human macrophages, and depletion of this protein in the human macrophage-like THP1 cells impaired cytokine secretion upon NLRP3 inflammasome activation. During infection, danger signals that activate the inflammasome are also able to engage the complement system, a group of proteins that act in a cascade-like fashion, resulting in the formation of the complement component 5a (C5a) and the membrane attack complex (MAC). Although, the levels of C5a and the MAC are enhanced in IL-1B and IL-18-mediated pathologies, it is unknown whether these complement components can induce inflammasome activation in human myeloid cells. This thesis shows that sublytic concentrations of the MAC triggered dispersion of the trans-Golgi network upstream of NLRP3 inflammasome assembly, and IL-1B and IL-18 secretion in a NLRP3 and a GSDMD-dependent manner in human macrophages. Moreover, this thesis shows that to activate the NLRP3 inflammasome, the MAC needs to be internalized and that it colocalises with early endosomes at early time points and with the adapter protein ASC and NLRP3 at later time points. Overall, this shows how two major pathways of the innate immune system - the complement system and the inflammasome - are linked together, identifying a potentially druggable pathway for major diseases.
|Date of Award||1 Aug 2022|
- The University of Manchester
|Supervisor||Daniel Davis (Supervisor) & Gloria Lopez-Castejon (Supervisor)|
- Interleukin 1 beta
- Membrane Attack Complex