Significance: The NLRP3 inflammasome is an intracellular oligomer regulating the activation of caspase-1 for the processing and secretion of IL-1β and IL-18. Although there is growing evidence to substantiate inflammasome inhibition as a therapeutic option for the treatment of inflammatory diseases, to date, there are no approved humans agents. OLT1177, a β-sulfonyl nitrile molecule, shown to be safe in humans, is a selective inhibitor of the NLRP3 inflammasome, with unique properties to reverse the metabolic costs of inflammation and to treat IL-1β– and IL-18–mediated diseases.

IL-1β and IL-18 are crucial mediators of inflammation, and a defective control of their release may cause serious diseases. Yet, the mechanisms regulating IL-1β and IL-18 secretion are partially undefined. Both cytokines are produced as inactive cytoplasmic precursors. Processing to the active form is mediated by caspase-1, which is in turn activated by the multiprotein complex inflammasome. Here, we show that in primary human monocytes microbial components acting on different pathogen-sensing receptors and the danger-associated molecule uric acid are all competent to induce maturation and secretion of IL-1β and IL-18 through a process that involves as a first event the extracellular release of endogenous ATP. ATP release is followed by autocrine stimulation of the purinergic receptors P2X ₇ . Indeed, antagonists of the P2X ₇ receptor (P2X ₇ R), or treatment with apyrase, prevent IL-1β and IL-18 maturation and secretion triggered by the different stimuli. At variance, blocking P2X ₇ R activity has no effects on IL-1β secretion by monocytes carrying a mutated inflammasome that does not require exogenous ATP for activation. P2X ₇ R engagement is followed by K ⁺ efflux and activation of phospholipase A ₂ . Both events are required for processing and secretion induced by all of the stimuli. Thus, stimuli acting on different pathogen-sensing receptors converge on a common pathway where ATP externalization is the first step in the cascade of events leading to inflammasome activation and IL-1β and IL-18 secretion.

Objective: To assess the clinical response to interleukin‐1 (IL‐1) blockade and in vitro IL‐1β and IL‐18 secretion in patients with systemic‐onset juvenile idiopathic arthritis (JIA).Methods: Twenty‐two patients with systemic‐onset JIA were treated with the IL‐1 receptor antagonist (IL‐1Ra) anakinra. Monocytes from 18 patients and 20 healthy donors were activated by different Toll‐like receptor ligands. Intracellular and secreted IL‐1β and IL‐18 were analyzed by Western blotting and enzyme‐linked immunosorbent assay.Results: Ten patients with systemic‐onset JIA exhibited a dramatic response to anakinra and were classified as complete responders. Eleven patients had an incomplete response or no response, and 1 patient could not be classified in terms of response. Compared with patients who had an incomplete response or no response, complete responders had a lower number of active joints (P = 0.02) and an increased absolute neutrophil count (P = 0.02). In vitro IL‐1β and IL‐18 secretion in response to various stimuli was not increased and was independent of treatment efficacy. Likewise, secretion of IL‐1Ra by monocytes from patients with systemic‐onset JIA was not impaired. An overall low level of IL‐1β secretion upon exposure to exogenous ATP was observed, unrelated to treatment responsiveness or disease activity.Conclusion: Two subsets of systemic‐onset JIA can be identified according to patient response to IL‐1 blockade. The 2 subsets appear to be characterized by some distinct clinical features. In vitro secretion of IL‐1β and IL‐18 by monocytes from patients with systemic‐onset JIA is not increased and is independent of both treatment outcome and disease activity.

Objective: To examine the synthesis, processing, and secretion of interleukin‐1β (IL‐1β), as well as the clinical and biologic effects of IL‐1 blockade, in patients with chronic infantile neurologic, cutaneous, articular (CINCA) syndrome and Muckle‐Wells syndrome (MWS), in an effort to understand the molecular mechanisms linking mutations of the CIAS1 gene and IL‐1β hypersecretion, and the underlying response to IL‐1 receptor antagonist (IL‐1Ra).Methods: Six patients with CINCA syndrome or MWS were treated with IL‐1Ra and followed up longitudinally. Monocytes obtained from the patients and from 24 healthy donors were activated with lipopolysaccharide (LPS) for 3 hours, and intracellular and secreted IL‐1β levels were determined by Western blotting and enzyme‐linked immunosorbent assay before and after exposure to exogenous ATP.Results: LPS‐induced IL‐1β secretion was markedly increased in monocytes from patients with CIAS1 mutations. However, unlike in healthy subjects, secretion of IL‐1β was not induced by exogenous ATP. Treatment with IL‐1Ra resulted in a dramatic clinical improvement, which was paralleled by an early and strong down‐regulation of LPS‐induced IL‐1β secretion by the patients' cells in vitro.Conclusion: Our results showed that the requirements of ATP stimulation for IL‐1β release observed in healthy individuals are bypassed in patients bearing CIAS1 mutations. This indicates that cryopyrin is the direct target of ATP and that the mutations release the protein from the requirement of ATP for activation. In addition, the dramatic amelioration induced by IL‐1Ra treatment is at least partly due to the strong decrease in IL‐1β secretion that follows the first injections of the antagonist. These findings may have implications for other chronic inflammatory conditions characterized by increased IL‐1β.

NLRP12 mutations have been described in patients affected with peculiar autoinflammatory symptoms. This study was undertaken to characterize NLRP12 mutations in patients with autoinflammatory syndromes, particularly a novel missense mutation, p.D294E, affecting a protein sequence crucial for ATP binding, which was identified in a Caucasian family with familial cold-induced autoinflammatory syndrome in some family members. Fifty patients were tested for NLRP12 mutations. A Caucasian family with the p.D294E missense mutation of NLRP12 in some family members was clinically characterized. In vitro analysis of the effects of the mutation on NF-κB activity was performed in HEK 293 cells after cotransfection of the cells with a luciferase NF-κB–responsive element and mutant or wild-type (WT) NLRP12 expression plasmids. NF-κB activity was also evaluated 24 hours after stimulation with tumor necrosis factor α in monocytes from individual family members carrying the mutation. Furthermore, secretion of interleukin-1β (IL-1β), production of reactive oxygen species (ROS), and activation of antioxidant systems in patient and healthy donor monocytes, under resting conditions and after stimulation with pathogen-associated molecular patterns (PAMPs), were also assessed. In the family assessed, the p.D294E mutation segregated in association with a particular sensitivity to cold exposure (especially arthralgias and myalgia), but not always with an inflammatory phenotype (e.g., urticarial rash or fever). In vitro, the mutant protein maintained the same inhibitory activity as that shown by WT NLRP12. Consistently, NLRP12-mutated monocytes showed neither increased levels of p65-induced NF-κB activity nor higher secretion of IL-1β. However, the kinetics of PAMP-induced IL-1β secretion were significantly accelerated, and high production of ROS and up-regulation of antioxidant systems were demonstrated. Even with a variable range of associated manifestations, the extreme sensitivity to cold represents the main clinical hallmark in an individual carrying the p.D294E mutation of the NLRP12 gene. Although regulation of NF-κB activity is not affected in patients, redox alterations and accelerated secretion of IL-1β are associated with this mild autoinflammatory phenotype.

A number of agents reducing interleukin-1β (IL-1β) activity are being developed as novel immunomodulatory and anti-inflammatory therapies. However, the elucidation of their molecular mechanism of action is required in the context of medical management of inflammatory diseases. Inhibitors of histone deacetylases (HDACs) are promising anticancer agents with pleiotropic activities. Of these, suberoylanilide hydroxamic acid has been reported to inhibit the production of several proinflammatory cytokines. In the present study, we investigated the effects of 2 HDAC inhibitors on IL-1β secretion: suberoylanilide hydroxamic acid and a newly developed hydroxamic acid-derived compound ITF2357. These HDAC inhibitors do not affect the synthesis or intracellular localization of IL-1β but both strongly reduce the levels of extracellular IL-1β by preventing the exocytosis of IL-1β-containing secretory lysosomes. At nanomolar concentrations, ITF2357 reduces the secretion of IL-1β following ATP activation of the P2X7 receptor. Whereas the inhibition of HDACs results in hyperacetylation of tubulin, acetylation of HSP90 was unaffected. The reduction in IL-1β secretion appears to be due to disruption of microtubules impairing lysosome exocytosis. Together, these observations indicate that a functional microtubule network is required for IL-1β secretion and suggest that disruption of tubulin is the mechanism by which inhibitors of HDACs reduce the secretion of IL-1β.