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IL1β is a central mediator of inflammation. Secretion of IL1β typically requires proteolytic maturation by the inflammasome and formation of membrane pores by gasdermin D (GSDMD). Emerging evidence suggests an important role for IL1β in promoting cancer progression in patients, but the underlying mechanisms are ill-defined. Here, we have shown a key role for IL1β in driving tumor progression in two distinct mouse tumor models. Notably, activation of the inflammasome, caspase-8, as well as the pore-forming proteins GSDMD and mixed lineage kinase domain–like protein in the host were dispensable for the release of intratumoral bioactive IL1β. Inflammasome-independent IL1β release promoted systemic neutrophil expansion and fostered accumulation of T-cell–suppressive neutrophils in the tumor. Moreover, IL1β was essential for neutrophil infiltration triggered by antiangiogenic therapy, thereby contributing to treatment-induced immunosuppression. Deletion of IL1β allowed intratumoral accumulation of CD8+ effector T cells that subsequently activated tumor-associated macrophages. Depletion of either CD8+ T cells or macrophages abolished tumor growth inhibition in IL1β-deficient mice, demonstrating a crucial role for CD8+ T-cell–macrophage cross-talk in the antitumor immune response. Overall, these results support a tumor-promoting role for IL1β through establishing an immunosuppressive microenvironment and show that inflammasome activation is not essential for release of this cytokine in tumors.

Neutrophils are the most prevalent immune cells in circulation, but the repertoire of canonical inflammasomes in neutrophils and their respective involvement in neutrophil IL-1 beta secretion and neutrophil cell death remain unclear. Here, we show that neutrophil-targeted expression of the disease-associated gain-of-function Nlrp3(A350V) mutant suffices for systemic autoinflammatory disease and tissue pathology in vivo. We confirm the activity of the canonical NLRP3 and NLRC4 inflammasomes in neutrophils, and further show that the NLRP1b, Pyrin and AIM2 inflammasomes also promote maturation and secretion of interleukin (IL)-1 beta in cultured bone marrow neutrophils. Notably, all tested canonical inflammasomes promote GSDMD cleavage in neutrophils, and canonical inflammasome-induced pyroptosis and secretion of mature IL-1 beta are blunted in GSDMD-knockout neutrophils. In contrast, GSDMD is dispensable for PMA-induced NETosis. We also show that Salmonella Typhimurium-induced pyroptosis is markedly increased in Nox2/Gp91(Phox)-deficient neutrophils that lack NADPH oxidase activity and are defective in PMA-induced NETosis. In conclusion, we establish the canonical inflammasome repertoire in neutrophils and identify differential roles for GSDMD and the NADPH complex in canonical inflammasome-induced neutrophil pyroptosis and mitogen-induced NETosis, respectively.

IL1β is a central mediator of inflammation. Secretion of IL1β typically requires proteolytic maturation by the inflammasome and formation of membrane pores by gasdermin D (GSDMD). Emerging evidence suggests an important role for IL1β in promoting cancer progression in patients, but the underlying mechanisms are ill-defined. Here, we have shown a key role for IL1β in driving tumor progression in two distinct mouse tumor models. Notably, activation of the inflammasome, caspase-8, as well as the pore-forming proteins GSDMD and mixed lineage kinase domain-like protein in the host were dispensable for the release of intratumoral bioactive IL1β. Inflammasome-independent IL1β release promoted systemic neutrophil expansion and fostered accumulation of T-cell-suppressive neutrophils in the tumor. Moreover, IL1β was essential for neutrophil infiltration triggered by antiangiogenic therapy, thereby contributing to treatment-induced immunosuppression. Deletion of IL1β allowed intratumoral accumulation of CD8+ effector T cells that subsequently activated tumor-associated macrophages. Depletion of either CD8+ T cells or macrophages abolished tumor growth inhibition in IL1β-deficient mice, demonstrating a crucial role for CD8+ T-cell-macrophage cross-talk in the antitumor immune response. Overall, these results support a tumor-promoting role for IL1β through establishing an immunosuppressive microenvironment and show that inflammasome activation is not essential for release of this cytokine in tumors. ispartof: CANCER IMMUNOLOGY RESEARCH vol:9 issue:3 pages:309-323 ispartof: location:United States status: published

Interleukin-1β (IL-1β) is a central mediator of inflammation whose secretion typically requires proteolytic maturation by the inflammasome and formation of membrane pores by gasdermin D (GSDMD). Emerging evidence suggests an important role for IL-1β in promoting cancer progression in patients, but the underlying mechanisms are little understood. Here, we show a key role for IL-1β in driving tumor progression in two distinct mouse tumor models. Notably, inflammasome activation and GSDMD were dispensable for the production of intratumoral bioactive IL-1β, which promoted systemic mobilization and infiltration of neutrophils into tumors. Neutrophils recruited via IL-1β suppressed the acquisition of an effector T-cell phenotype and subsequent antitumor immune response. Moreover, IL-1β was essential for neutrophil accumulation upon antiangiogenic therapy, thereby contributing to therapy-induced immunosuppression. Antitumor immunity in the absence of IL-1β-dependent neutrophil recruitment relied on immunostimulatory macrophages which promoted the infiltration and activation of cytotoxic T-cells. Overall, these results support a tumor-promoting role for IL-1β through establishing an immunosuppressive microenvironment and show that inflammasome activation is not essential for its release in tumors.

Rheumatoid arthritis is a chronic autoinflammatory disease that affects 1-2% of the world's population and is characterized by widespread joint inflammation. Interleukin-1 is an important mediator of cartilage destruction in rheumatic diseases, but our understanding of the upstream mechanisms leading to production of interleukin-1β in rheumatoid arthritis is limited by the absence of suitable mouse models of the disease in which inflammasomes contribute to pathology. Myeloid-cell-specific deletion of the rheumatoid arthritis susceptibility gene A20/Tnfaip3 in mice (A20(myel-KO) mice) triggers a spontaneous erosive polyarthritis that resembles rheumatoid arthritis in patients. Rheumatoid arthritis in A20(myel-KO) mice is not rescued by deletion of tumour necrosis factor receptor 1 (ref. 2). Here we show, however, that it crucially relies on the Nlrp3 inflammasome and interleukin-1 receptor signalling. Macrophages lacking A20 have increased basal and lipopolysaccharide-induced expression levels of the inflammasome adaptor Nlrp3 and proIL-1β. As a result, A20-deficiency in macrophages significantly enhances Nlrp3 inflammasome-mediated caspase-1 activation, pyroptosis and interleukin-1β secretion by soluble and crystalline Nlrp3 stimuli. In contrast, activation of the Nlrc4 and AIM2 inflammasomes is not altered. Importantly, increased Nlrp3 inflammasome activation contributes to the pathology of rheumatoid arthritis in vivo, because deletion of Nlrp3, caspase-1 and the interleukin-1 receptor markedly protects against rheumatoid-arthritis-associated inflammation and cartilage destruction in A20(myel-KO) mice. These results reveal A20 as a novel negative regulator of Nlrp3 inflammasome activation, and describe A20(myel-KO) mice as the first experimental model to study the role of inflammasomes in the pathology of rheumatoid arthritis.

Background: Chronic inflammation in the tumor microenvironment (TME) sustained by immune cells has a crucial role both in tumor initiation and progression. One of the central cytokines of inflammation, IL-1β, is produced as a biologically inactive precursor that requires proteolytic processing by caspase-1. Activation of caspase-1 is triggered by the formation of inflammasomes, multiprotein complexes that detect microbial and endogenous danger signals primarily via NOD-like receptors, such as NLRP3 and NLRC4. Biologically active IL-1β is believed to be released through membrane pores formed by gasdermin D during a lytic form of cell death called pyroptosis. Although IL-1β-mediated inflammation has been shown to have a detrimental role in tumor progression, the signaling pathway controlling IL-1β release in the TME and the exact effect of the cytokine on antitumor T-cell responses have not been fully elucidated. A better understanding of how IL-1β release is controlled in tumors will also pave the way towards the therapeutic utilization of small-molecule inhibitors available to target NOD-like receptors and caspase-1. Methods: First, we characterized the impact of IL-1β in the TME by assessing the immune cell composition and vasculature of Lewis lung carcinomas (LLC) and E0771 breast carcinomas in IL-1β-deficient mice using flow cytometry and histologic analysis. Next, we used mice deficient in different inflammasome components, including NLRP3, NLRC4 and caspase-1, to investigate the involvement of these proteins in controlling IL-1β release in LLC and E0771 tumors. Using immunoblots and small-molecule inhibitors, we further characterized the activation of alternative enzymatic pathways and their involvement in IL-1β release by tumor-associated myeloid cells. Finally, we examined the role of pyroptosis and necroptosis in IL-1β release using gasdermin D- and MLKL-deficient mice, respectively. Release of IL-1β was assessed using ELISA and immunoblots. Results: We found that IL-1β secretion was restricted to myeloid cells and promoted tumor progression in mouse models of lung and breast carcinoma. IL-1β deletion abrogated the tumor-induced mobilization of immunosuppressive neutrophils and normalized the tumor vasculature, thereby alleviating hypoxia. Consequently, proliferation of effector T-cells in the TME was enhanced, leading to higher CD4+ and CD8+ T-cell abundance in the absence of IL-1β. We observed that, although the NLRP3 inflammasome was active in tumor-infiltrating myeloid cells, NLRP3 and caspase-1 were not essential for the proteolytic maturation of pro-IL-1β and secretion of biologically active IL-1β in the TME. Inhibition or genetic deletion of caspase-8 reduced inflammasome-independent IL-1β release, indicating that caspase-8 provides an alternative pathway for proteolytic activation and secretion of IL-1β in tumor-infiltrating myeloid cells. Moreover, IL-1β release by tumor-infiltrating myeloid cells was independent of lytic cell death modalities including gasdermin D-mediated pyroptosis and MLKL-mediated necroptosis, suggesting an alternative release mechanism for the cytokine in the TME. Conclusions: Overall, our results demonstrate that tumor-infiltrating myeloid cells are able to release IL-1β independently of inflammasomes. We show that proteolytic maturation of IL-1β via caspase-8 in myeloid cells acts as an important driver of immune suppression in the TME through vascular destabilization, recruitment of immunosuppressive neutrophils and consequential inhibition of antitumor T-cell responses. We also show, that, unlike in autoinflammation, gasdermin D-mediated pyroptosis is not essential for the release of IL-1β in tumors. These results suggest that therapeutic inhibition of inflammasomes or pyroptosis will likely not be beneficial in certain tumor types due to the presence of an alternative caspase-8-mediated IL-1β release pathway in tumor-associated myeloid cells. Citation Format: Máté Kiss, Lieselotte Vande Walle, Helena Van Damme, Aleksandar Murgaski, Evangelia Bolli, Jiri Keirsse, Maria Solange Martins, Yvon Elkrim, Amelie Fossoul, Jens Serneels, Massimiliano Mazzone, Mohamed Lamkanfi, Jo A. Van Ginderachter, Damya Laoui. Inflammasome-independent IL-1β release by myeloid cells promotes vessel destabilization and immune suppression in the tumor microenvironment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A083.

Despite its clinical importance in infection and autoimmunity, the activation mechanisms of the NLRP1b inflammasome remain enigmatic. Here we show that deletion of the inflammasome adaptor ASC in BALB/c mice and in C57BL/6 macrophages expressing a functional NLRP1b prevents anthrax lethal toxin (LeTx)-induced caspase-1 autoproteolysis and speck formation. However, ASC−/− macrophages undergo normal LeTx-induced pyroptosis and secrete significant amounts of interleukin (IL)-1β. In contrast, ASC is critical for caspase-1 autoproteolysis and IL-1β secretion by the NLRC4, NLRP3 and AIM2 inflammasomes. Notably, LeTx-induced inflammasome activation is associated with caspase-1 ubiquitination, which is unaffected in ASC-deficient cells. In vivo, ASC-deficient mice challenged with LeTx produce significant levels of IL-1β, IL-18 and HMGB1 in circulation, although caspase-1 autoproteolysis is abolished. As a result, ASC−/− mice are sensitive to rapid LeTx-induced lethality. Together, these results demonstrate that ASC-driven caspase-1 autoprocessing and speck formation are dispensable for the activation of caspase-1 and the NLRP1b inflammasome., The NLRP1b inflammasome activation may lead to pyroptosis and secretion of the inflammatory cytokines IL-1ß and IL-18 but the mechanisms behind these processes are not fully understood. Here, the authors show that they can occur independently of the inflammasome adaptor ASC and without caspase-1 autoprocessing.