IL-1␤ and IL-18 are crucial mediators of inflammation, and a defective control of their release may cause serious diseases. Yet, the mechanisms regulating IL-1␤ 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-1␤ 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 P2X7 receptor (P2X7R), or treatment with apyrase, prevent IL-1␤ and IL-18 maturation and secretion triggered by the different stimuli. At variance, blocking P2X 7R activity has no effects on IL-1␤ secretion by monocytes carrying a mutated inflammasome that does not require exogenous ATP for activation. P2X 7R engagement is followed by K ؉ efflux and activation of phospholipase A2. 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-1␤ and IL-18 secretion.inflammation ͉ nonclassical secretion ͉ pathogen-associated molecular patterns ͉ processing

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.

Background:Cytokine secretion has unwanted consequences in malignant and in inflammatory disorders. The deacetylase SIRT6 has pro-inflammatory activity, but the underlying mechanisms and its biological significance remain unclear. The relationship between inflammation and carcinogenesis has been known for many years (1). Chronic inflammation is a risk factor for cancer development. In addition, even in those cancers that do not develop in inflamed tissues, an inflammatory component is usually observed, and it is now known to be an essential part of the malignant microenvironment (2, 3). Inflammation contributes to tumorigenesis and cancer progression by supplying growth factors that sustain cancer cell proliferation and/or survival, proangiogenic factors, extracellular matrix-modifying enzymes that promote invasion and metastasis, and signals that lead to epithelial-mesenchymal transition (2, 4, 5). Moreover, increased circulating levels of pro-inflammatory cytokines are responsible for systemic manifestations of disease, such as cachexia, fever, and sweats (6 -9). Among other forms of cancer, pancreatic ductal adenocarcinoma (PDAC) 2 is well known for its propensity to secrete high levels of pro-inflammatory factors that contribute to its clinical aggressiveness and to its metastatic potential (10). The mechanisms controlling cyto-/chemokine production by inflammatory and cancer cells are only partially understood. A more detailed understanding of the molecular pathways leading to cancer-associated inflammation may lead to new therapeutic strategies with a strong impact on patient quality of life.Previous studies showed that intracellular nicotinamide adenine dinucleotide (NAD ϩ ) levels influence the capacity of inflammatory cells to secrete cytokines, such as tumor necrosis factor ␣ (TNF), interleukin 6 (IL6), IL1␤, interferon ␥ (IFN-␥), * This work was supported in part by the Associazione Italiana per la Ricerca sul Cancro (AIRC, Code 6108) (to A. N.), by the European Seventh Framework Program (Project 256986, PANACREAS) (to A. N.), by Ministero della Salute Grant GR-2008-1135635 (to A. N.)

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.

BackgroundT helper 17 cells (TH-17) represent a lineage of effector T cells critical in host defence and autoimmunity. In both mouse and human IL-1β has been indicated as a key cytokine for the commitment to TH-17 cells. Cryopyrin-associated periodic syndromes (CAPS) are a group of inflammatory diseases associated with mutations of the NLRP3 gene encoding the inflammasome component cryopyrin. In this work we asked whether the deregulated secretion of IL-1β secondary to mutations characterizing these patients could affect the IL-23/IL-17 axis.Methodology/Principal FindingsA total of 11 CAPS, 26 systemic onset juvenile idiopathic arthritis (SoJIA) patients and 20 healthy controls were analyzed. Serum levels of IL-17 and IL-6 serum were assessed by ELISA assay. Frequency of TH17 cells was quantified upon staphylococcus enterotoxin B (SEB) stimulation. Secretion of IL-1β, IL-23 and IL-6 by monocyte derived dendritic cells (MoDCs), were quantified by ELISA assay. A total of 8 CAPS and 11 SoJIA patients were also analysed before and after treatment with IL-1β blockade. Untreated CAPS patients showed significantly increased IL-17 serum levels as well as a higher frequency of TH17 compared to control subjects. On the contrary, SoJIA patients displayed a frequency of TH17 similar to normal donors, but were found to have significantly increased serum level of IL-6 when compared to CAPS patients or healthy donors. Remarkably, decreased IL-17 serum levels and TH17 frequency were observed in CAPS patients following in vivo IL-1β blockade. On the same line, MoDCs from CAPS patients exhibited enhanced secretion of IL-1β and IL-23 upon TLRs stimulation, with a reduction after anti-IL-1 treatment.Conclusion/SignificanceThese findings further support the central role of IL-1β in the differentiation of TH17 in human inflammatory conditions.

Background: Nicotinamide phosphoribosyltransferase (NAMPT) acts both as an enzyme in the production of the coenzyme NAD ϩ and as a secreted cytokine.

Nicotinamide adenine dinucleotide (NAD+) biosynthesis from nicotinamide is used by mammalian cells to replenish their NAD+ stores and to avoid unwanted nicotinamide accumulation. Pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the key enzyme in this biosynthetic pathway, almost invariably leads to intracellular NAD+ depletion and, when protracted, to ATP shortage and cell demise. Cancer cells and activated immune cells express high levels of NAMPT and are highly susceptible to NAMPT inhibitors, as shown by the activity of these agents in models of malignant and inflammatory disorders. As the spectrum of conditions which could benefit from pharmacological NAMPT inhibition becomes broader, the mechanisms accounting for their activity are also eventually becoming apparent, including the induction of autophagy and the impairment of Ca2+--and NF-κB-dependent signaling. Here, we discuss the rationales for exploiting NAMPT inhibitors in cancer and inflammatory diseases and provide an overview of the preclinical and clinical studies in which these agents have been evaluated.

Dendritic cells (DCs) are antigen-presenting cells that critically influence decisions about immune activation or tolerance. Impaired DC function is at the core of common chronic disorders and contributes to reduce immunocompetence during aging. Knowledge on the mechanisms regulating DC generation and function is necessary to understand the immune system and to prevent disease and immunosenescence. Here we show that the sirtuin Sirt6, which was previously linked to healthspan promotion, stimulates the development of myeloid, conventional DCs (cDCs). Sirt6-knockout (Sirt6KO) mice exhibit low frequencies of bone marrow cDC precursors and low yields of bone marrow-derived cDCs compared to wild-type (WT) animals. Sirt6KO cDCs express lower levels of class II MHC, of costimulatory molecules, and of the chemokine receptor CCR7, and are less immunostimulatory compared to WT cDCs. Similar effects in terms of differentiation and immunostimulatory capacity were observed in human monocyte-derived DCs in response to SIRT6 inhibition. Finally, while Sirt6KO cDCs show an overall reduction in their ability to produce IL-12, TNF-α and IL-6 secretion varies dependent on the stimulus, being reduced in response to CpG, but increased in response to other Toll-like receptor ligands. In conclusion, Sirt6 plays a crucial role in cDC differentiation and function and reduced Sirt6 activity may contribute to immunosenescence.