Inflammasome function in neutrophils Kaiwen Chen Bachelor of Science (Honours Class I) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2015 Institute for Molecular Bioscience i ii Abstract The innate immune system protects against infection but also drives inflammatory disorders. Key molecular drivers of both processes are ‘inflammasomes’, multi-protein complexes that assemble in the cytosol to activate the protease, caspase-1. Active caspase-1 cleaves specific proinflammatory cytokines [e.g. interleukin (IL)-1β] into their mature, secreted forms, and initiates a form of inflammatory cell lysis called pyroptosis. Inflammasomes are assembled by select pattern recognition receptors such as NLRC4, NLRP3, AIM2, or via a non-canonical pathway involving caspase-11. Whilst inflammasomes functions have been intensely researched, the cell types mediating inflammasome signalling in distinct in vivo settings were unclear. Neutrophils are one of the first cells to arrive to a site of infection or injury, and thus have the opportunity to detect inflammasome-activating molecules in vivo, but their ability to signal by inflammasome pathways had not been closely examined. This thesis offers a detailed investigation of NLRC4, NLRP3 and caspase-11 inflammasome signalling in neutrophils during in vitro or in vivo challenge with whole microbe, purified microbial components, or adjuvant. This thesis demonstrates that acute Salmonella infection triggered NLRC4-dependent caspase-1 activation and IL-1β processing in neutrophils, and neutrophils were a major cellular compartment for IL-1β production during acute Salmonella challenge in vivo. Importantly, neutrophils did not undergo pyroptotic cell death upon NLRC4 activation, allowing these cells to sustain IL-1β production at a site of infection without compromising their crucial inflammasome-independent antimicrobial effector functions. Since IL-1β drives neutrophil recruitment and activation, the finding that neutrophils themselves produce IL- 1β suggests neutrophils perform novel and important auto-regulatory functions during infection. Neutrophil NLRP3 pathways were next examined. In macrophages, diverse molecules can trigger NLRP3 activation. Some of these agonists are insoluble molecules (particles or crystals), which activate NLRP3 through lysosomal rupture. Interestingly, the neutrophil NLRP3 inflammasome selectively responded to soluble, but not insoluble, NLRP3 agonists, and lysosomal rupture was a weak stimulus for neutrophil NLRP3 activation. This finding was replicated in vivo, where neutrophils did not significantly contribute to IL-1β production during alum-induced peritonitis. iii Prior studies in macrophages demonstrated that NLRP3 activation triggers the oligomerisation of the inflammasome adaptor ASC, which then recruits two caspases, pro- caspase-1 and -8. Inflammasome-activated caspase-8 initiates macrophage apoptosis in parallel to caspase-1-dependent pyroptosis. This thesis shows that like macrophages, neutrophil NLRP3 activation triggered ASC oligomerisation, caspase-1 cleavage and IL-1β secretion, but unlike macrophages, caspase-1 activation did not induce cell lysis, suggesting that resistance to pyroptosis is a universal feature of neutrophil inflammasome pathways. Neutrophils also appeared unable to undergo NLRP3/ASC/caspase-8- dependent apoptosis, likely due to inefficient caspase-8 self-processing. This finding prompted examination of other caspase-8/NLRP3 pathways in neutrophils. Chemical inhibition of the inhibitor of apoptosis proteins (IAPs) in LPS-primed macrophages leads to RIPK3 activation and formation of a caspase-8 activating platform, the ripoptosome, which promotes apoptosis and can cleave pro-IL-1β into its mature form. If caspase-8 activity is suppressed, RIPK3 forms a different molecular complex, the necrosome, to induce necroptosis. In macrophages, ripoptosome and necrosome signalling induces NLRP3 inflammasome activation and downstream caspase-1 activity. Surprisingly, blockade of IAP function was a weak stimulus for cell death in LPS-stimulated neutrophils, and did not trigger caspase-8-dependent IL-1β maturation or NLRP3/caspase-1 activation, suggesting that NLRP3 regulation by RIPK3/IAPs is differentially regulated in macrophages versus neutrophils. Caspase-11 functions are important for host defence against cytoplasmic Gram-negative bacteria, but also contribute to pathological immune responses in murine models of septic shock. Like caspase-1, caspase-11 induces pyroptosis. But caspase-11 appears unable to directly cleave cytokines, and instead triggers pro-IL-1β/18 processing through activation of the NLRP3 inflammasome. Because neutrophilia is a hallmark of sepsis, and neutrophils produce NLRP3-dependent IL-1β without concomitant cell death, the possibility that neutrophils contribute to septic shock was next investigated. Neutrophil exposure to intracellular LPS in vitro indeed triggered caspase-11-dependent IL-1β production but not cell death. Nlrp3-/- mice, like Casp11-/- mice, were protected from lethal endotoxin shock, suggesting that caspase-11/NLRP3-dependent cytokine production may contribute to LPS lethality. Importantly, prior neutrophil depletion protected wild type but not Casp11-/- mice from endotoxic shock. Although further investigations are required, these data suggest that iv neutrophils may be a sustained cellular source of caspase-11/NLRP3-dependent, pathogenic IL-1β during murine endotoxic shock. In summary, this thesis demonstrates that neutrophils signal via specialised inflammasome pathways to maximise host responses during pathogen challenge in vivo. The finding that neutrophils themselves secrete IL-1β to drive their own recruitment, activation and survival is in line with an emerging theme that neutrophils perform important immunoregulatory functions during infection. Although neutrophils are often regarded as pro-apoptotic cells, this thesis reports that, surprisingly, neutrophils resist all known forms of inflammasome- induced cell death. Presumably this allows neutrophils time to exert their important functions in host defence, but may also contribute to sustained cytokine production and thus pathology in inflammatory diseases such as endotoxic shock. The interplay between inflammation and neutrophil cell death warrants further examination to understand the underlying mechanisms of neutrophil-driven inflammatory disorders. v Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. vi Publications during candidature Original research articles 1. Sester, D.P., Thygesen, S.J., Sagulenko, V., Vajjhala, P.R., Cridland, J.A., Vitak, N., Chen, K.W., Osborne, G.W., Schroder, K., and Stacey, K.J. (2015). A novel flow cytometric method to assess inflammasome formation. Journal of immunology 194, 455-462. 2. Chen, K.W., Gross, C.J., Sotomayor, F.V., Stacey, K.J., Tschopp, J., Sweet, M.J., and Schroder, K. (2014). The neutrophil NLRC4 inflammasome selectively promotes IL-1β maturation without pyroptosis during acute Salmonella challenge. Cell reports 8, 570-582. 3. Luo, L., Wall, A.A., Yeo, J.C., Condon, N.D., Norwood, S.J., Schoenwaelder, S., Chen, K.W., Jackson, S., Jenkins, B.J., Hartland, E.L., et al. (2014). Rab8a interacts directly with PI3Kgamma to modulate TLR4-driven PI3K and mTOR signalling. Nature communications 5, 4407. 4. Achard, M.E., Chen, K.W., Sweet, M.J., Watts, R.E., Schroder, K., Schembri, M.A., and McEwan, A.G. (2013). An antioxidant role for catecholate siderophores in Salmonella. The Biochemical journal 454, 543-549. Reviews and book chapters 1. Boucher, D., Chen, K.W., Schroder, K. (2015). Burn the house, save the day: pyroptosis in pathogen restriction. Inflammasomes 2, 1-6. 2. Chen, K.W., and Schroder, K. (2013). Antimicrobial functions of inflammasomes. Current opinion in microbiology 16, 311-318. 3. Chen, K.W., Richards, A.A., Zamoshnikova, A, Schroder, K. (2013). Inflammasomes and Inflammation. Cancer and Inflammation Mechanisms: Chemical, Biological and Clinical