Engagement of tumor necrosis factor receptor 1 signals two diametrically opposed pathways: survival-inflammation and cell death. An additional switch decides, depending on the cellular context, ...between caspase-dependent apoptosis and RIP kinase (RIPK)-mediated necrosis, also termed necroptosis. We explored the contribution of both cell death pathways in TNF-induced systemic inflammatory response syndrome (SIRS). Deletion of apoptotic executioner caspases (caspase-3 or -7) or inflammatory caspase-1 had no impact on lethal SIRS. However, deletion of RIPK3 conferred complete protection against lethal SIRS and reduced the amounts of circulating damage-associated molecular patterns. Pretreatment with the RIPK1 kinase inhibitor, necrostatin-1, provided a similar effect. These results suggest that RIPK1-RIPK3-mediated cellular damage by necrosis drives mortality during TNF-induced SIRS. RIPK3 deficiency also protected against cecal ligation and puncture, underscoring the clinical relevance of RIPK kinase inhibition in sepsis and identifying components of the necroptotic pathway that are potential therapeutic targets for treatment of SIRS and sepsis.
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► Necroptosis is a crucial process during lethal SIRS and sepsis ► RIPK3 ablation and RIPK1 inhibition protect mice against TNF-induced lethal SIRS ► Reduced amounts of DAMP release correlate with protection against lethality ► RIPK3 ablation protects against lethal sepsis induced by ceacal ligation and puncture
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The gut microbiota regulate susceptibility to multiple human diseases. The Nlrp6-ASC inflammasome is widely regarded as a hallmark host innate immune axis that shapes the gut microbiota composition. ...This notion stems from studies reporting dysbiosis in mice lacking these inflammasome components when compared with non-littermate wild-type animals. Here, we describe microbial analyses in inflammasome-deficient mice while minimizing non-genetic confounders using littermate-controlled Nlrp6-deficient mice and ex-germ-free littermate-controlled ASC-deficient mice that were all allowed to shape their gut microbiota naturally after birth. Careful microbial phylogenetic analyses of these cohorts failed to reveal regulation of the gut microbiota composition by the Nlrp6- and ASC-dependent inflammasomes. Our results obtained in two geographically separated animal facilities dismiss a generalizable impact of Nlrp6- and ASC-dependent inflammasomes on the composition of the commensal gut microbiota and highlight the necessity for littermate-controlled experimental design in assessing the influence of host immunity on gut microbial ecology.
•Non-littermate controls fail to define host genetic impacts on the gut microbiota•Littermate separation does not reveal ASC-Nlrp6 impacts on the gut microbiota•Lifetime littermate separation does not reveal Nlrp6 impacts on DSS colitis
Inflammasomes were proposed to shape gut ecology based on dysbiosis in mutant mice versus non-littermate wild-types. Mamantopoulos et al. show that inflammasomes do not affect gut microbiota composition when controlling for non-genetic confounders. This finding dismisses the suggested role for inflammasomes in controlling host health through regulation of intestinal ecology.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Neutrophils are the most prevalent immune cells in circulation, but the repertoire of canonical inflammasomes in neutrophils and their respective involvement in neutrophil IL‐1β secretion and ...neutrophil cell death remain unclear. Here, we show that neutrophil‐targeted expression of the disease‐associated gain‐of‐function Nlrp3A350V 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)‐1β 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‐1β 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/Gp91Phox‐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.
Synopsis
Canonical inflammasomes in neutrophils promote caspase‐1‐dependent cleavage of proIL‐1β and GSDMD. GSDMD mediates secretion of IL‐1β and canonical inflammasome‐induced neutrophil pyroptosis, whereas GSDMD is dispensable for NETosis induction by the phorbol ester PMA.
CAPS mutant NLRP3 in neutrophils drives autoinflammatory pathology in mice.
NLRP1b, NLRP3, NLRC4, AIM2 and Pyrin inflammasomes promote caspase‐1‐mediated pyroptosis and IL‐1β secretion in neutrophils.
Caspase‐1‐dependent cleavage of GSDMD mediates neutrophil pyroptosis and secretion of IL‐1β and DAMPs.
GSDMD is dispensable for PMA‐induced NETosis.
Canonical inflammasomes in neutrophils promote caspase‐1‐dependent cleavage of proIL‐1β and GSDMD. GSDMD mediates the secretion of IL‐1β and canonical inflammasome‐induced neutrophil pyroptosis, whereas GSDMD is dispensable for NETosis induction by the phorbol ester PMA.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Abstract The TNF signaling pathway is a valuable target in the therapy of autoimmune diseases, and anti-TNF drugs are successfully used to treat diseases such as rheumatoid arthritis, Crohn's disease ...and psoriasis. By their ability to interfere with inflammatory processes at multiple levels, these TNF blockers have become invaluable tools to inhibit the inflammation induced damage and allow recovery of the affected tissues. Unfortunately this therapy has some drawbacks, including increased risk of infection and malignancy, and remarkably, the onset of new auto-immune diseases. Some of these effects are caused by the unwanted abrogation of beneficial TNF signaling. More specific targeting of the pathological TNF-induced signaling might lead to broader applicability and improved safety. Specificity might be increased by inhibiting the soluble TNF/TNFR1 axis while leaving the often beneficial transmembrane TNF/TNFR2 signaling untouched. This approach looks promising because it inhibits the pathological effects of TNF and reduces the side effects, and it opens the way for the treatment of other diseases in which TNFR2 inhibition is detrimental. In this review we give an overview of in vivo mouse studies of TNF mediated pathologies demonstrating that the blockade or genetic deletion of sTNF or TNFR1 is preferable over total TNF blockade.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The nucleotide-binding-domain (NBD)-and leucine-rich repeat (LRR)-containing (NLR) family, pyrin-domain-containing 3 (NLRP3) inflammasome drives pathological inflammation in a suite of autoimmune, ...metabolic, malignant, and neurodegenerative diseases. Additionally, NLRP3 gain-of-function point mutations cause systemic periodic fever syndromes that are collectively known as cryopyrin-associated periodic syndrome (CAPS). There is significant interest in the discovery and development of diarylsulfonylurea Cytokine Release Inhibitory Drugs (CRIDs) such as MCC950/CRID3, a potent and selective inhibitor of the NLRP3 inflammasome pathway, for the treatment of CAPS and other diseases. However, drug discovery efforts have been constrained by the lack of insight into the molecular target and mechanism by which these CRIDs inhibit the NLRP3 inflammasome pathway. Here, we show that the NAIP, CIITA, HET-E, and TP1 (NACHT) domain of NLRP3 is the molecular target of diarylsulfonylurea inhibitors. Interestingly, we find photoaffinity labeling (PAL) of the NACHT domain requires an intact (d)ATP-binding pocket and is substantially reduced for most CAPS-associated NLRP3 mutants. In concordance with this finding, MCC950/CRID3 failed to inhibit NLRP3-driven inflammatory pathology in two mouse models of CAPS. Moreover, it abolished circulating levels of interleukin (IL)-1β and IL-18 in lipopolysaccharide (LPS)-challenged wild-type mice but not in Nlrp3L351P knock-in mice and ex vivo-stimulated mutant macrophages. These results identify wild-type NLRP3 as the molecular target of MCC950/CRID3 and show that CAPS-related NLRP3 mutants escape efficient MCC950/CRID3 inhibition. Collectively, this work suggests that MCC950/CRID3-based therapies may effectively treat inflammation driven by wild-type NLRP3 but not CAPS-associated mutants.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Familial Mediterranean fever (FMF) is the most common monogenic autoinflammatory disease worldwide. It is caused by mutations in the inflammasome adaptor Pyrin, but how FMF mutations alter signaling ...in FMF patients is unknown. Herein, we establish Clostridium difficile and its enterotoxin A (TcdA) as Pyrin-activating agents and show that wild-type and FMF Pyrin are differentially controlled by microtubules. Diverse microtubule assembly inhibitors prevented Pyrin-mediated caspase-1 activation and secretion of IL-1β and IL-18 from mouse macrophages and human peripheral blood mononuclear cells (PBMCs). Remarkably, Pyrin inflammasome activation persisted upon microtubule disassembly in PBMCs of FMF patients but not in cells of patients afflicted with other autoinflammatory diseases. We further demonstrate that microtubules control Pyrin activation downstream of Pyrin dephosphorylation and that FMF mutations enable microtubule-independent assembly of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) micrometer-sized perinuclear structures (specks). The discovery that Pyrin mutations remove the obligatory requirement for microtubules in inflammasome activation provides a conceptual framework for understanding FMF and enables immunological screening of FMF mutations.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
The caspase activation and recruitment domain (CARD)-based inflammasome sensors NLRP1b and NLRC4 induce caspase-1-dependent pyroptosis independent of the inflammasome adaptor ASC. Here, we show that ...NLRP1b and NLRC4 trigger caspase-8-mediated apoptosis as an alternative cell death program in caspase-1−/− macrophages and intestinal epithelial organoids (IECs). The caspase-8 adaptor FADD was recruited to ASC specks, which served as cytosolic platforms for caspase-8 activation and NLRP1b/NLRC4-induced apoptosis. We further found that caspase-1 protease activity dominated over scaffolding functions in suppressing caspase-8 activation and induction of apoptosis of macrophages and IECs. Moreover, TLR-induced c-FLIP expression inhibited caspase-8-mediated apoptosis downstream of ASC speck assembly, but did not affect pyroptosis induction by NLRP1b and NLRC4. Moreover, unlike during pyroptosis, NLRP1b- and NLRC4-elicited apoptosis retained alarmins and the inflammasome-matured cytokines interleukin 1β (IL-1β) and IL-18 intracellularly. This work identifies critical mechanisms regulating apoptosis induction by the inflammasome sensors NLRP1b and NLRC4 and suggests converting pyroptosis into apoptosis as a paradigm for suppressing inflammation.
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•NLRP1b and NLRC4 induce apoptosis in casp1−/− macrophages and epithelial organoids•NLRP1b/NLRC4-induced apoptosis requires ASC-dependent caspase-8 activation•Caspase-1 activity is dominant over scaffolding in suppressing apoptosis induction•TLR-induced c-FLIP levels inhibit NLRP1b/NLRC4-induced apoptosis in primed cells
Van Opdenbosch et al. find that CARD-based inflammasome sensors drive ASC- and caspase-8-dependent apoptosis as an alternative cell death program when caspase-1 activation is impaired. TLR-mediated upregulation of c-FLIP is identified as a second checkpoint that regulates ASC/caspase-8-mediated apoptosis. Moreover, apoptosis differs from pyroptosis in retaining inflammasome-dependent cytokines and alarmins intracellularly.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Tumor necrosis factor (TNF) has remarkable antitumor effects, but its systemic therapeutic use is prevented by its lethal inflammatory effects. TNFR1 (P55) is essential for both the antitumor and ...toxic effects because both of them are absent in P55‐deficient mice. In previous work we demonstrated that P55+/− mice are completely resistant to TNF toxicity, while the antitumor effects induced by TNF combined with interferon gamma (IFNγ) remain fully functional in these mice. Hence, a high dose of TNF/IFNγ has an excellent therapeutic potential when P55 levels are reduced, because TNF induces tumor regression without systemic toxicity. Here, we provide proof of principle for therapeutic application of this approach by using antisense oligonucleotides (ASOs). Treatment of mice with ASOs targeting P55 resulted in a strong reduction in P55 protein levels in liver, small intestine and blood mononuclear cells. This P55 downregulation was associated with significant protection of mice against acute TNF toxicity as measured by hypothermia, systemic inflammation and lethality. This treatment also protected mice against toxicity of TNF/IFNγ treatment in several cancer models: B16Bl6, Lewis lung carcinoma and a lung colony model. Our results confirm the therapeutic value of this strategy, which could lead to the development of a safer and more effective TNF/IFNγ antitumor therapy.
What's new?
Tumor necrosis factor (TNF) can produce potent antitumor effects as well as potentially life‐threatening systemic inflammation, which has limited its therapeutic use. Different strategies to overcome systemic toxicity have been explored, including the inhibition of mediators of TNF‐induced toxicity. Here, antisense oligonucleotides targeted against TNFR1 (P55), the primary mediating receptor in models of both acute TNF toxicity and TNF/IFNγ antitumor activity, were found to effectively lower TNFR1 protein levels in select tissues and thereby reduce toxicity associated with TNF/IFNγ therapy, without affecting antitumor activity. The findings provide new evidence for the applicability and safety of TNF/IFNγ therapy.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Inflammasomes are multi-protein complexes that guard against cellular stress and microbial infections. Inflammasome activation studies frequently require delivery of pathogen-derived virulence ...factors into the cytosol of macrophages and other innate immune cells. This is a challenging requirement since primary macrophages are difficult-to-transfect, especially when it comes to the intracellular delivery of proteins. Here, we report on the use of nanoparticle-sensitized photoporation as a promising upcoming intracellular delivery technology for delivering proteins of various molecular weights into the cytosol of primary macrophages. While 60-70 nm gold nanoparticles are the most commonly used sensitizing nanoparticles for photoporation, here we find that 0.5 μm iron oxide nanoparticles perform markedly better on primary macrophages. We demonstrate that LFn-FlaA or lipopolysaccharides can be delivered in primary macrophages resulting in activation of the NLRC4 or the non-canonical inflammasome, respectively. We furthermore show that photoporation can be used for targeted delivery of these toxins into selected cells, opening up the possibility to study the interaction between inflammasome activated cells and surrounding healthy cells. Taken together, these results show that nanoparticle-sensitized photoporation is very well suited to deliver pathogenic virulence factors in primary macrophages, thus constituting an effective new enabling technology for inflammasome activation studies.
Laser-induced nanoparticle-sensitized photoporation of hard-to-transfect primary macrophages for the
ex vivo
delivery of inflammasome inducing toxins.
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