Research Andy Wullaert

Abstract

Linear ubiquitin chains are tags that can be attached but also again removed from a protein. The latter is performed by a protein termed OTULIN. Cells lacking OTULIN therefore accumulate linear ubiquitinated proteins and this provokes inflammation in mice and in humans. We identified inhibition of inflammasomes – protein complexes that control secretion of inflammatory cytokines – as a novel mechanism by which OTULIN could control inflammation. This project aims to understand how OTULIN prevents inflammasome responses on the molecular level as well as to investigate its impact on host defence against infections. First, since we found that Nlrp3 inflammasome activation in OTULIN-deficient macrophages was caused by increased necroptotic cell death, we will investigate which linear ubiquitinated proteins modulate necroptosis in these cells. Second, as also Pyrin inflammasome activation is enhanced in OTULIN-deficient macrophages, we will aim to identify the cell death mode and the OTULIN target proteins responsible for Pyrin activation in these cells. And third, extending our ex vivo macrophage findings, we will investigate how the inflammasome promoting effects of myeloid OTULIN deficiency affect the in vivo host response of mice against Nlrp3- or Pyrin-activating pathogens. Overall, this fundamental research project will investigate how the molecular interplay between OTULIN and inflammasome signalling in myeloid cells impacts on inflammatory and infectious responses.

Researcher(s)

• Promoter: Wullaert Andy

Research team(s)

Project type(s)

• Research Project

Abstract

Gain-of-function mutations in the Nlrp3 inflammasome cause auto-inflammatory diseases termed cryopyrin-associated periodic syndromes (CAPS). Expressing a CAPS-associated Nlrp3 mutant selectively in either macrophages or neutrophils results in severe inflammation in mice. While it is known that Nlrp3 activation results in lytic cell death due to facilitating Gasdermin D (GSDMD) to form pores in the cell membrane, we will investigate the cell type specific mechanisms by which GSDMD-mediated cell death in either macrophages of neutrophils contributes to Nlrp3-induced CAPS in mice. Using CAPS mice expressing Nlrp3 selectively in either macrophages or neutrophils, we will investigate how different cell death modes control Nlrp3-induced release of pro-inflammatory factors in these mice. In addition, we will analyze serum inflammatory profiles resulting from GSDMD-dependent cell death in either macrophages or neutrophils, and we will aim to identify novel inflammatory mediators produced upon Nlrp3 mutant expression in these cell types. Finally, using a genetic proof-of-principle approach in CAPS mice, we will evaluate the efficacy and safety of GSDMD targeting as a potential future treatment for CAPS patients. A better understanding of the cell type specific mechanisms by which GSDMD-mediated cell death drives Nlrp3-induced CAPS will increase our understanding of how Nlrp3-induced cell death regulates several other inflammatory diseases.

Researcher(s)

• Promoter: Wullaert Andy
• Fellow: Christiaenssen Bregje

Research team(s)

• Proteinscience, proteomics and epigenetic signaling (PPES)

Project type(s)

• Research Project

Abstract

The Nlrp3 inflammasome is a protein complex that responds to microbial structures leading to the activation of a protease called caspase-1 that induces a form of cell death termed pyroptosis. This cell death mode facilitates secretion of the pro-inflammatory cytokines IL-1β and IL-18 that contribute to mounting efficient inflammatory responses against infections. Leishmaniasis is a major neglected parasitic disease caused by Leishmania species that can lead to a broad range of clinical manifestations ranging from cutaneous and mucocutaneous inflammation to a lethal visceral leishmaniasis (VL). Mouse model observations demonstrated an involvement of Nlrp3 inflammasome signalling in cutaneous leishmaniasis, and patient observations showed that the inflammasome-generated cytokines IL-1β and IL-18 correlate with VL severity. However, the effect of downstream Nlrp3-induced pyroptosis on the more severe VL disease has not been investigated. Moreover, recent observations showed that the ability of VL parasites display different infection kinetics in different cell types and at different stages of the infection, suggesting that pyroptosis could represent one of the intracellular host defence mechanisms responsible for determining parasite survival and spreading. Therefore, in this project we aim to reveal how Nlrp3-induced pyroptosis affects visceral leishmaniasis in mice. More specifically, we will aim to reveal in which cell types VL parasites induce pyroptosis as well as to reveal the in vivo function of pyroptosis during a model of treatment relapse VL in mice. In addition, we will aim to reveal how pyroptosis correlates with the in vivo infection kinetics of VL parasites during pathogenesis, and how this form of cell death in turn impacts on the parasite. On the long term, this project might facilitate designing better cell type and disease stage specific VL treatments. In addition, mapping the capacities of different myeloid cell types for undergoing pyroptosis during VL will generate general knowledge with implications beyond parasitic infections such as in other types of infections and in inflammatory diseases associated with inflammasome activation.

Researcher(s)

• Promoter: Wullaert Andy
• Co-promoter: Caljon Guy
• Fellow: Mortelmans Silke

Research team(s)

• Proteinscience, proteomics and epigenetic signaling (PPES)

Project type(s)

• Research Project

Abstract

The current application envisages extending the infectious disease and inflammation research and drug discovery platforms at the University of Antwerp to accommodate highly flexible and versatile live cell imaging and biochemical read-outs with a possibility to upgrade from median to high throughput. The apparatus will be embedded in a high-end immunoprofiling platform and BSL-2 environment at the Laboratory of Microbiology, Parasitology and Hygiene (LMPH) with biosafety approvals for basic research on infection with microbial pathogens. The TECAN SPARK Cyto 600 is a highly versatile multimodal plate reader that enables cellular and in situ molecular assays in controlled O2/CO2, humidity cassette and temperature regulation environments with real-time absorbance, fluorescence and luminescence measurements. Three different optical measurement options exist by using filters, monochromators or fusion optics which eliminates the compromise between sensitivity and flexibility. The unique lid-lifting function enables substrate addition or immune cell priming through the included 2-channel injector. SPARK Cyto 600 is equipped with 2×, 4× and 10× objective lenses and a CMOS camera to enable live cell imaging. In addition to bright field imaging, fluorescence imaging is possible in 4 optical channels with capability of Time-Resolved Fluorescence (TRF) and Fluorescence Resonance Energy Transfer (FRET). An additional asset is the compatibility with bead-based proximity assays using Alpha Technology with optimized integrated filters and laser-based excitation. While all known competitors limit live cell imaging systems to bright field and fluorescence measurements, the SPARK Cyto 600 also allows for real-time detection of glow and flash luminescence signals and Bioluminescence Resonance Energy Transfer (BRET) applications to enable sensitive real-time follow-up of protein-protein interactions in cells. Given the high versatility and pressing need for such equipment for infection and inflammatory disease research, this unique apparatus will allow real-time imaging of cellular as well as molecular events in controlled conditions. This new infrastructure will therefore boost research of many research groups at the University of Antwerp and will contribute to fundamental insights at the cellular and molecular level as well as to the development of novel therapeutics and diagnostics.

Researcher(s)

• Promoter: Caljon Guy
• Co-promoter: Augustyns Koen
• Co-promoter: Sterckx Yann
• Co-promoter: Vanden Berghe Wim
• Co-promoter: Wullaert Andy

Research team(s)

• Laboratory for Microbiology, Parasitology and Hygiene (LMPH)

Project type(s)

• Research Project

Abstract

Familial Mediterranean Fever (FMF) is an auto-inflammatory disease caused by mutations in the gene encoding Pyrin. FMF-associated Pyrin mutants trigger inappropriate inflammasome activation allowing excessive levels of Interleukin (IL)-1β to provoke fever episodes, joint pain, abdominal pain and skin inflammation in FMF patients. We aim to uncover the cellular interactions by which inflammasome- and IL- 1β-induced signaling pathways orchestrate auto-inflammation in FMF. We will employ a genetic approach allowing to abolish either IL-1β production or IL-1β responses in a cell type specific manner in an FMF mouse model, which will experimentally identify these cellular drivers of FMF-related auto-inflammation in mice. In parallel, we will profile single cell transcriptomes as well as cell surface proteins in these FMF mice. This will specifically reveal the gene expression programs of cells displaying the IL-1β receptor on their surface. Guided by this knowledge, inflammasome activation and IL- 1β stimulation experiments will identify IL-1β producing and IL-1β responding cells, respectively, among different types of immune cells obtained from FMF patients. Understanding the cellular level of how inflammasomes and IL-1β cooperate to propel auto-inflammation may facilitate improved therapeutic approaches targeting the driver cell types in FMF, and may uncover common themes that will help to better understand other inflammatory diseases driven by inflammasome and IL-1β signalling.

Researcher(s)

• Promoter: Wullaert Andy

Research team(s)

• Proteinscience, proteomics and epigenetic signaling (PPES)

Project type(s)

• Research Project