Investigation of inflammasome regulation

: the influence of priming and CARD-only proteins

Abstract

Inflammasomes are immunological sensors that detect microbial- and host-derived signals to trigger the releaseof pro-inflammatory cytokines and pyroptosis. Inflammasome sensors are expressed primarily in monocytes/macrophages but our current in vitro macrophage models have considerable limitations. Induced pluripotent stem cell-derived macrophages (iMacs) have great potential to be used in inflammasome studies but have not been widely used.

Inflammasome activation must be tightly regulated to prevent excessive/damaging inflammation. For NLRP3 activation, this involves the requirement for two signals, termed priming and activation. However, whether priming through different Toll-like receptors (TLR) can influence the signalling outcomes of NLRP3 or other inflammasomes, such as NLRP1 or AIM2, is not clear. Additional inflammasome regulation in humans occurs through caspase-recruitment domain (CARD)-only proteins (COPs) which are believed to disrupt CARD-CARD interactions to prevent inflammasome activation. However, conflicting studies suggest that COPs, including CARD16, can promote inflammasome activation so the role of these proteins remains unclear.

The data presented here show successful differentiation of iMacs that express high levels of macrophage-specific markers, display macrophage-like phagocytic activity, and respond robustly to a wide range of inflammasome stimuli. In addition, iPSC-derived myeloid precursor cells were differentiated into iPSC-derived microglia that displayed significant NLRP3 activity. In addition priming through different TLR pathways was shown to influence both NLRP1 and NLRP3 responses through IFN signalling. LPS-induced IFN signalling limited NLRP3 and NLRP1 activation in a time-dependent manner. This effect was dependent on IL-10 and to a lesser extent STAT3.

However, mass spectrometry experiments revealed that LPS-induced IFN signalling did not regulate inflammasome activity through altering inflammasome-related protein expression, suggesting a role for transcription-independent mechanisms. Finally, CARD16 was shown to increase NLRP3 and NLRP1 inflammasome activation in a time-dependent manner. CARD16 also regulated cytokine secretion in an NF-κBindependent manner, suggesting a role for CARD16 in the regulation of other transcription factors.

Thesis is embargoed until 31 December 2026.

Date of Award	Dec 2023
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Northern Ireland Department for the Economy
Supervisor	Rebecca Coll (Supervisor) & Paul Moynagh (Supervisor)