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-1␤ 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-1␤ 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-1␤ and IL-18 by monocytes from patients with systemic-onset JIA is not increased and is independent of both treatment outcome and disease activity.

Microbe associated molecular pattern (MAMP) receptors in plants recognize MAMPs and activate basal defences; however a complete understanding of the molecular and physiological mechanisms conferring immunity remains elusive. Pathogens suppress active defence in plants through the combined action of effector proteins. Here we show that the chloroplast is a key component of early immune responses. MAMP perception triggers the rapid, large-scale suppression of nuclear encoded chloroplast-targeted genes (NECGs). Virulent Pseudomonas syringae effectors reprogramme NECG expression in Arabidopsis, target the chloroplast and inhibit photosynthetic CO 2 assimilation through disruption of photosystem II. This activity prevents a chloroplastic reactive oxygen burst. These physiological changes precede bacterial multiplication and coincide with pathogen-induced abscisic acid (ABA) accumulation. MAMP pretreatment protects chloroplasts from effector manipulation, whereas application of ABA or the herbicide DCMU inhibits the MAMP-induced chloroplastic reactive oxygen burst, and enhances growth of a P. syringae hrpA mutant that fails to secrete effectors.

Objective. To examine the synthesis, processing, and secretion of interleukin-1␤ (IL-1␤), 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-1␤ 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-1␤ levels were determined by Western blotting and enzyme-linked immunosorbent assay before and after exposure to exogenous ATP.Results. LPS-induced IL-1␤ secretion was markedly increased in monocytes from patients with CIAS1 mutations. However, unlike in healthy subjects, secretion of IL-1␤ 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-1␤ secretion by the patients' cells in vitro.Conclusion. Our results showed that the requirements of ATP stimulation for IL-1␤ 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-1␤ secretion that follows the first injections of the antagonist. These findings may have implications for other chronic inflammatory conditions characterized by increased IL-1␤.

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In healthy monocytes, Toll-like receptor (TLR) engagement induces production of reactive oxygen species (ROS), followed by an antioxidant response involved in IL-1β processing and secretion. Markers of the antioxidant response include intracellular thioredoxin and extracellular release of reduced cysteine. Cryopyrinassociated periodic syndromes (CAPS) are autoinflammatory diseases in which Nod-like receptor family pyrin domain-containing 3 (NLRP3) gene mutations lead to increased IL-1β secretion. We show in a large cohort of patients that IL-1β secretion by CAPS monocytes is much faster than that by healthy monocytes. This accelerated kinetics is caused by alterations in the basal redox state, as well as in the redox response to TLR triggering displayed by CAPS monocytes. Indeed, unstimulated CAPS monocytes are under a mild oxidative stress, with elevated levels of both ROS and antioxidants. The redox response to LPS is quickened, with early generation of the reducing conditions favoring IL-1β processing and secretion, and then rapidly exhausted. Therefore, secretion of IL-1β is accelerated, but reaches a plateau much earlier than in healthy controls. Pharmacologic inhibition of the redox response hinders IL-1β release, confirming the functional link between redox impairment and altered kinetics of secretion. Monocytes from patients with juvenile idiopathic arthritis display normal kinetics of redox response and IL-1β secretion, excluding a role of chronic inflammation in the alterations observed in CAPS. We conclude that preexisting redox alterations distinct from CAPS monocytes anticipate the pathogen-associated molecular pattern molecule-induced generation of the reducing environment favorable to inflammasome activation and IL-1β secretion.antioxidant response | inflammasome | reactive oxygen species | thioredoxin | autoinflammatory diseases

In this study, we show that IL-1beta processing and secretion induced by pathogen-associated molecular pattern (PAMP) molecules in human monocytes is regulated by a biphasic redox event including a prompt oxidative stress and a delayed antioxidant response. Namely, PAMPs induce an early generation of reactive oxygen species (ROS) followed by increase of intracellular thioredoxin and release of reduced cysteine: this antioxidant phase is paralleled by secretion of mature IL-1beta. ROS production and antioxidant response are both required, because either inhibitors of NADPH oxidase and of thioredoxin reductase impair IL-1beta secretion. These inhibitors also hinder cysteine release and consequently prevent reduction of the extracellular medium: addition of exogenous reducing agents restores IL-1beta secretion. Not only silencing of thioredoxin, but also of the ROS scavenger superoxide dismutase 1 results in inhibition of IL-1beta secretion. Thus, PAMP-induced ROS trigger an antioxidant response involving intracellular redox enzymes and release of cysteine, ultimately required for IL-1beta processing and secretion.

Human myeloid cells activate the NLRP3 inflammasome and secrete interleukin (IL)-1β in response to various Toll-like receptor (TLR) ligands, but the rate of secretion is much higher in primary human monocytes than in cultured macrophages or THP-1 cells. The different myeloid cells also display different redox status under resting conditions and redox response to TLR activation. Resting monocytes display a balanced redox state, with low production of reactive oxygen species (ROS) and antioxidants. TLR engagement induces an effective redox response with increased ROS generation followed by a sustained antioxidant response, parallelled by efficient IL-1β secretion. Drugs blocking ROS production or the antioxidant response prevent the secretion of mature IL-1β but not the biosynthesis of pro-IL-1β, indicating that redox remodeling is responsible for IL-1β processing and release. Unlike monocytes, THP-1 cells and cultured macrophages have up-regulated antioxidant systems that buffer the oxidative hit provided by TLR triggering and suppress the consequent redox response. This aborted redox remodeling is paralleled by low efficiency IL-1β processing and secretion. High doses (5 mm) of H2O2 overcome the high antioxidant capacity of THP-1 cells, restore an efficient redox response, and increase the rate of IL-1β secretion. Together these data indicate that a tightly controlled redox homeostasis in resting cells is a prerequisite for a robust redox response to TLR ligands, in turn necessary for the efficient inflammasome activation. Inflammasome activation by bacterial DNA is not modulated by redox responses, suggesting that redox-dependent regulation of IL-1β secretion is restricted to some inflammasomes including NLRP3 but excluding AIM-2.

ObjectiveNLRP12 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.MethodsFifty 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.ResultsIn 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.ConclusionEven 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.