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Triple negative breast cancers (TNBCs) are very aggressive and have a poor prognosis due to lack of efficacious therapies. The only effective treatment is chemotherapy that however is frequently hindered by the occurrence of drug resistance. We approached this problem in vitro and in vivo on a triple negative and a hormone sensitive breast cancer cell lines: 4T1 and TS/A. A main defense mechanism of tumors is the extrusion of intracellular protons derived from the metabolic shift to glycolysis, and necessary to maintain an intracellular pH compatible with life. The resulting acidic extracellular milieu bursts the malignant behavior of tumors and impairs chemotherapy. Therefore, we investigated the efficacy of combined therapies that associate cisplatin (Cis) with proton exchanger inhibitors, such as esomeprazole (ESO) and 5‐(N‐ethyl‐N‐isopropyl)amiloride (EIPA). Our results demonstrate that in the 4T1 triple negative model the combined therapy Cis plus EIPA is significantly more effective than the other treatments. Instead, in the TS/A tumor the best therapeutic result is obtained with ESO alone. Remarkably, in both 4T1 and TS/A tumors these treatments correlate with increase of CD8+ T lymphocytes and dendritic cells, and a dramatic reduction of M2 macrophages and other suppressor myeloid cells (MDSC) in the tumor infiltrates., We show that association of cisplatin and sodium/hydrogen exchanger one inhibitors decreases tumor growth, improves the tumor microenvironment state, and increases survival more efficiently than cisplatin alone in triple negative breast cancer‐bearing mice, supporting the exploitation of this combo therapy in chemoresistant TNBCs. At variance, esomeprazole alone is more efficacious than cisplatin or various combo therapies in hormone sensitive breast cancer in mice. Thus, proton transport inhibitors should be considered as valuable nontoxic anticancer drugs.

Background Positron Emission Tomography (PET) imaging with Prostate-Specific Membrane Antigen (PSMA) and Fluorodeoxyglucose (FDG) represent promising biomarkers for risk-stratification of Prostate Cancer (PCa). We verified whether the expression of genes encoding for PSMA and enzymes regulating FDG cellular uptake are independent and additive prognosticators in PCa. Methods mRNA expression of genes involved in glucose metabolism and PSMA regulation obtained from primary PCa specimens were retrieved from open-source databases and analyzed using an integrative bioinformatics approach. Machine Learning (ML) techniques were used to create predictive Progression-Free Survival (PFS) models. Cellular models of primary PCa with different aggressiveness were used to compare [18F]F-PSMA-1007 and [18F]F-FDG uptake kinetics in vitro. Confocal microscopy, immunofluorescence staining, and quantification analyses were performed to assess the intracellular and cellular membrane PSMA expression. Results ML analyses identified a predictive functional network involving four glucose metabolism-related genes: ALDOB, CTH, PARP2, and SLC2A4. By contrast, FOLH1 expression (encoding for PSMA) did not provide any additive predictive value to the model. At a cellular level, the increase in proliferation rate and migratory potential by primary PCa cells was associated with enhanced FDG uptake and decreased PSMA retention (paralleled by the preferential intracellular localization). Conclusions The overexpression of a functional network involving four glucose metabolism-related genes identifies a higher risk of disease progression since the earliest phases of PCa, in agreement with the acknowledged prognostic value of FDG PET imaging. By contrast, the prognostic value of PSMA PET imaging is independent of the expression of its encoding gene FOLH1. Instead, it is influenced by the protein docking to the cell membrane, regulating its accessibility to tracer binding.

Amyloid deposits of apolipoprotein A-I (apoA-I) and inflammation are common in atherosclerotic arteries. In this study, we investigated the interplay between oxidation of apoA-I methionine residues (Met(O)-ApoA-I), a known amyloidogenic modification of apoA-I, and the inflammatory response of immune cells. Soluble pre-fibrillar Met(O)-ApoA-I, but not apoA-I, induced intracellular accumulation of pro-interleukin (IL)-1β and secretion of the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and IL-6 in mouse bone marrow–derived macrophages (BMDMs) and human primary monocytes. Additionally, secretion of mature IL-1β was also activated in human monocytes. The pro-inflammatory activity of Met(O)-ApoA-I was Toll-like receptor 4 (TLR4)-dependent and CD36-independent and was solely determined by oxidation of apoA-I methionine residues, in particular Met-86 and Met-148. In contrast, amyloid fibrils or reconstituted high-density lipoproteins (HDLs) generated from Met(O)-ApoA-I did not induce cytokine production in BMDMs. Although lipid-free Met(O)-ApoA-I remained functional in extracting lipids from cells and generating HDL, it gained strong pro-inflammatory properties that may aggravate local inflammation in the arteries and atherosclerosis. Our study indicates that oxidation of apoA-I methionine residues produces a potent danger-associated molecular pattern capable of stimulating pro-inflammatory cytokine secretion at levels similar to those induced by known pathogen-associated molecular patterns, such as lipopolysaccharide.

Background. The redox stress caused by Hodgkin’s lymphoma (HL) also involves the peripheral blood mononucleated cells (PBMCs) even before chemotherapy. Here, we tested whether lymphocytes and monocytes show a different response to the increased mitochondrial generation of reactive oxygen species (ROS). Methods. PBMCs, isolated from the blood of treatment-naïve HL patients and control subjects, underwent assessment of malondialdehyde content and enzymatic activity of both hexose- and glucose-6P dehydrogenase (H6PD and G6PD) as well as flow cytometric analysis of mitochondrial ROS content. These data were complemented by evaluating the uptake of the fluorescent glucose analogue 2-NBDG that is selectively stored within the endoplasmic reticulum (ER). Results. Malondialdehyde content was increased in the whole population of HL PBMCs. The oxidative damage matched an increased activity of G6PD, and even more of H6PD, that trigger the cytosolic and ER pentose phosphate pathways, respectively. At flow cytometry, the number of recovered viable cells was selectively decreased in HL lymphocytes that also showed a more pronounced increase in mitochondrial ROS generation and 2-NBDG uptake, with respect to monocytes. Conclusions. PBMCs of HL patients display a selective mitochondrial and ER redox stress most evident in lymphocytes already before the exposure to chemotherapy toxicity.

Background Cryopyrin associated periodic syndromes (CAPS) are a group of autoinflammatory diseases linked to gain-of-function mutations in the NLRP3 gene that cause uncontrolled IL-1β secretion. CINCA syndrome is the most severe CAPS disease characterized by central nervous system disabilities with a long-term risk of secondary amyloidosis. Proton pump inhibitors (PPIs), commonly used as inhibitors of gastric acid production, also display anti-inflammatory properties, making them promising drugs in sepsis and in inflammatory disorders. Objectives To develop a novel NLRP3 knock-in (KI) mouse model of CAPS to evaluate amyloid deposition and to test alternative therapeutic approaches. Methods We generated KI mice by engineering N475K mutation associated with CAPS phenotype into mouse Nlrp3 gene. KI and Wild Type (WT) mice received PPIs or PBS intraperitoneally and were analyzed for survival, inflammation, cytokine secretion, and amyloidosis development. Cytokines secretion from bone marrow derived dendritic cells (BMDCs) and peritoneal macrophages (PMs) was evaluated by ELISA. Hystological analysis of all organs was evaluated by hematoxylin and eosin staining. Amyloid deposition was quantified through Congo Red staining. Results Mutant NLRP3 KI mice displayed features that recapitulates the immunological and clinical phenotype of CAPS. These mice had systemic inflammation, with high levels of serum pro-inflammatory cytokines compared to WT controls. Hystological analysis revealed the presence of acute and chronic inflammatory cell infiltrates and amyloid deposits in spleen, liver and kidneys. As in CAPS monocytes, BMDCs and PM from KI mice showed a strong increase in IL-1β, IL-18, and IL-1α secretion and decreased levels in interleukin-1 receptor antagonist (IL-1Ra), the naturally occurring IL-1b inhibitor. PPIs treatment of KI mice showed a clear clinical impact with improvement of inflammatory conditions and regression of amyloid deposits. Remarkably, BMDCs and PMs from PPI-treated mice presented reduced secretion of pro-inflammatory cytokines and re-established the levels of IL-1RA. Conclusion NLRP3 KI mice display a CAPS phenotype with many characteristics of autoinflammation, including amyloidosis. The therapeutic effectiveness associated with lack of toxicity indicates that PPIs could represent relevant adjuvants to the anti-IL-1 drugs in IL-1 driven diseases. References [1] Gattorno M, Martini A. Beyond the NLRP3 inflammasome: autoinflammatory diseases reach adolescence. Arthritis Rheum. 2013; [2] Brydges SD, Mueller JL, McGeough MD, Pena CA, Misaghi A, Gandhi C Putnam CD, et al. Inflammasome-mediated disease animal models reveal roles for innate but not adaptive immunity. Immunity. 2009; [3] Balza E, Piccioli P, Carta S, Lavieri R, Gattorno M, Semino Cet al. Proton pump inhibitors protect mice from acute sistemic inflammation and induce long-term cross-tolerance. Cell Death Dis. 2016. Disclosure of Interests Arinna Bertoni: None declared, Sonia Carta: None declared, Chiara Baldovini: None declared, Federica Penco: None declared, Enrica Balza: None declared, Silvia Borghini: None declared, Marco Di Duca: None declared, Emanuela Ognio: None declared, Paolo Nozza: None declared, Francesca Schena: None declared, Patrizia Castellani: None declared, Claudia Pastorino: None declared, Carola Perrone: None declared, Laura Obici: None declared, Alberto Martini Consultant for: I do not have any conflict of interest to declare since starting from 1 March 2016 I became the Scientific Director of the G. Gaslini Hospital; therefore, my role does not allow me to render private consultancies resulting in personal income. I perform consultancy activities on behalf of the Gaslini Institute for the companies listed below: AbbVie, Biogen, Boehringer Ingelheim, Bristol-Myers Squibb, EMD Serono, Janssen, Novartis, Pfizer, R-Pharm. The money received for these activities are directly transferred to the Gaslini Institute’s bank account. Before March 2016, I was the head of the Pediatric Rheumatology Department at the G. Gaslini Hospital, where the PRINTO Coordinating Centre is located. For the coordination activity of the PRINTO network, the Gaslini Hospital received contributions from the industries listed in this section. This money has been reinvested for the research activities of the hospital in fully independent manners besides any commitment with third parties., Isabella Ceccherini: None declared, Marco Gattorno Grant/research support from: MG has received unrestricted grants from Sobi and Novartis, Anna Rubartelli: None declared, Sabrina Chiesa: None declared

Infectious and sterile inflammation is induced by activation of innate immune cells. Triggering of toll-like receptors by pathogen-associated molecular pattern or damage-associated molecular pattern (PAMP or DAMP) molecules generates reactive oxygen species that in turn induce production and activation of pro-inflammatory cytokines such as IL-1β. Recent evidence indicates that cell stress due to common events, like starvation, enhanced metabolic demand, cold or heat, not only potentiates inflammation but may also directly trigger it in the absence of PAMPs or DAMPs. Stress-mediated inflammation is also a common feature of many hereditary disorders, due to the proteotoxic effects of mutant proteins. We propose that harmful mutant proteins can induce dysregulated IL-1β production and inflammation through different pathways depending on the cell type involved. When expressed in professional inflammatory cells, stress induced by the mutant protein activates in a cell-autonomous way the onset of inflammation and mediates its aberrant development, resulting in the explosive responses that hallmark autoinflammatory diseases. When expressed in non-immune cells, the mutant protein may cause the release of transcellular stress signals that trigger and propagate inflammation.

Autoinflammatory diseases are a group of inherited and multifactorial disorders characterized by an overactivation of innate immune response. In most cases, the clinical manifestations are due to increased activity of the NLRP3 inflammasome resulting in increased IL-1β secretion. Investigating inflammatory cells from subjects affected by autoinflammatory diseases presents a number of technical difficulties related to the rarity of the diseases, to the young age of most patients, and to the difficult modulation of gene expression in primary cells. However, since cell stress is involved in the pathophysiology of these diseases, the study of freshly drawn blood monocytes from patients affected by IL-1-mediated diseases strongly increases the chances that the observed phenomena is indeed pertinent to the pathogenesis of the disease and not influenced by the long-term cell culture conditions (e.g., the high O2 tension) or gene transfection in continuous cell lines that may lead to artifacts.

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. Cryopyrin-associated 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.

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.

Autoinflammatory diseases are a group of inherited and multifactorial disorders characterized by an overactivation of innate immune response. In most cases, the clinical manifestations are due to increased activity of the NLRP3 inflammasome resulting in increased IL-1β secretion. Investigating inflammatory cells from subjects affected by autoinflammatory diseases presents a number of technical difficulties related to the rarity of the diseases, to the young age of most patients, and to the difficult modulation of gene expression in primary cells. However, since cell stress is involved in the pathophysiology of these diseases, the study of freshly drawn blood monocytes from patients affected by IL-1-mediated diseases strongly increases the chances that the observed phenomena is indeed pertinent to the pathogenesis of the disease and not influenced by the long-term cell culture conditions (e.g., the high O2 tension) or gene transfection in continuous cell lines that may lead to artifacts.

OBJECTIVE To provide a rationale for anti-IL-1 treatment in pyogenic sterile arthritis, pyoderma gangrenosum and acne (PAPA) by defining whether IL-1β secretion is enhanced; requires NLRP3; and correlates with proline-serine-threonine phosphatase-interacting protein 1 mutations, disease activity and/or the clinical picture in PAPA. METHODS Monocytes were isolated from 13 patients and 35 healthy donors and studied at baseline and following activation. Secretion pattern of IL-1β, IL-1α, IL-1Ra, IL-6, IL-18 and TNF-α was assessed in supernatants by ELISA. The NLRP3 requirement for IL-1β secretion was investigated by silencing technique in PAPA and healthy donor monocytes. Long-term follow-up (mean 26 months, range 4-38) was performed in five patients enrolled in an anti-IL-1 regimen. RESULTS IL-1β secretion in PAPA is increased, requires NLRP3 and correlates with disease activity. Patients with a history of osteoarticular flares release more IL-1β, IL-6 and TNF-α compared with those with predominant cutaneous recurrences. Monocytes from patients in anti-IL-1 treatment dramatically reduced IL-1β secretion after ex vivo activation, and long-term follow-up demonstrated decreased frequency of flares and normalization of acute phase reactants in all the patients. A straightforward correlation between genotype and IL-1β signalling was not observed suggesting that factors other than mutation itself may play a role in regulating IL-1β secretion and response to treatment in PAPA. CONCLUSION PAPA patients with active lesions display increased NLRP3-mediated IL-1β secretion, and long-term efficacy of IL-1 blockade was demonstrated. Even if other mechanisms related to the complex proline-serine-threonine phosphatase-interacting protein 1 protein networking might play additional roles, this study further supports the potential of IL-1 blockade as an effective therapeutic strategy in PAPA syndrome.

The cell stress and redox responses are increasingly acknowledged as factors contributing to the generation and development of the inflammatory response. Several inflammation-inducing stressors have been identified, inside and outside of the cell. Furthermore, many hereditary diseases associate with inflammation and oxidative stress, suggesting a role for mutated proteins as stressors. The nucleotide-binding oligomerization domain, leucine-rich repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome is an important node at the crossroad between redox response and inflammation. Remarkably, monocytes from patients with mutations in the NLRP3 gene undergo oxidative stress after stimulation with minute amounts of TLR agonists, resulting in unbalanced production of IL-1β and regulatory cytokines. Similar alterations in cytokine production are found in healthy monocytes upon TLR overstimulation. This mini-review summarizes recent progress in this field, discusses the molecular mechanisms underlying the loss of control of the cytokine network following oxidative stress, and proposes new therapeutic opportunities.

A number of agents reducing interleukin-1beta (IL-1beta) 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-1beta 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-1beta but both strongly reduce the levels of extracellular IL-1beta by preventing the exocytosis of IL-1beta-containing secretory lysosomes. At nanomolar concentrations, ITF2357 reduces the secretion of IL-1beta 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-1beta 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-1beta secretion and suggest that disruption of tubulin is the mechanism by which inhibitors of HDACs reduce the secretion of IL-1beta.

The notion that the ATP-binding cassette transporter-A2 (ABCA2) may be involved in brain sterol homeostasis and is associated with early onset Alzheimer's disease led us to explore its neural expression. Our data support and extend the previous reports on ABCA2 expression by oligodendrocytes. They evidence that ABCA2 (i) is located in intracellular vesicles, identified in transfected cells as lysosome-related organelles only partially overlapping with classical endolysosomes; (ii) is a marker of neural progenitors as it is expressed in the subventricular zone of the lateral ventricle and the dentate gyrus of the hippocampal formation, sites of continual neurogenesis in the adult brain, and in nestin+ cells differentiated in vitro from embryonic stem cells; (iii) persists, in the adult rodent brain, in a subset of GABAergic and glutamatergic neurons. Considering that the latter are targets of Alzheimer's lesions, these data provide a new rationale to explore the neuropathological consequences of ABCA2 functional dysregulations.

Item does not contain fulltext Cell stress is implicated in triggering bouts of systemic inflammation in patients with autoinflammatory disorders. Blood monocytes from patients affected by NLRP3-mediated cryopyrin-associated periodic syndromes (CAPS) release greater amounts of IL-1beta than monocytes from unaffected subjects. Here we show that stress lowers the threshold of activation; blood monocytes from CAPS patients maintain the high levels of secreted IL-1beta (fivefold) and IL-18 (10-fold) when stimulated with 1,000-fold less LPS than that required for full IL-1beta secretion in control subjects. Unexpectedly, IL-1alpha secretion is increased 10-fold, indicating that inflammatory episodes in CAPS may not be entirely a result of IL-1beta but may also involve IL-1alpha. In CAPS monocytes, LPS induces the externalization of copious amounts of ATP (10-fold), which drive IL-1beta, IL-18, and IL-1alpha release via activation of the P2X purinoceptor 7. This enhanced ATP release appears to be the link between cell stress and increased cytokine secretion in CAPS. In the later phase after LPS stimulation, CAPS monocytes undergo oxidative stress, which impairs production of the anti-inflammatory IL-1 receptor antagonist (IL-1Ra). Remarkably, IL-1Ra secretion is fully restored by treatment with antioxidants. In two patients with the same NLRP3 mutation, but different disease severity, monocytes from the mildly affected patient exhibited more efficient redox response, lower ATP secretion, and more balanced cytokine production. Thus, the robustness of the individual antioxidant response increases the tolerance to stress and reduces the negative effect of the disease. Pharmacologic block of P2X purinoceptor 7 and improved stress tolerance may represent novel treatment strategies in stress-associated inflammatory diseases.

IL-1β acts in concert with anti-inflammatory cytokines, in particular, IL-1R antagonist (IL-1Ra), to ensure the correct development and outcome of the inflammation: imbalance in the IL-1β/IL-1Ra ratio is implicated in many human diseases and may lead to dramatic consequences. In this article, we show that single TLR engagement induces IL-1β and, with a little delay, IL-1Ra. Differently, costimulation of TLR2, TLR4, and TLR7/8 enhances IL-1β secretion but severely inhibits IL-1Ra production. The IL-1β/IL-1Ra unbalance after activation of multiple TLRs depends on the insurgence of oxidative stress, because of enhanced production of reactive oxygen species and failure of the antioxidant systems. Increased reactive oxygen species levels increase ATP externalization by monocytes, resulting in enhanced inflammasome activation and IL-1β secretion. Oxidative stress then induces cell responses to stress, including inhibition of protein synthesis, which, in turn, is responsible for the impaired production of IL-1Ra. IL-1Ra secretion is restored by exogenous antioxidants that oppose oxidative stress. Similar effects are evident also on other cytokines: TNF-α is induced, whereas IL-6 is inhibited by costimulation. Our findings provide a molecular basis to the imbalance between proinflammatory and regulatory cytokine circuits that occur in various pathologic conditions, and suggest new strategies for controlling inflammation.

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.

Results Monocytes isolated from PAPA patients tent to secrete higher levels of IL-1b but this was not consistent in all the patients (p = 0.144). Variability in IL-1b release occurred even in the presence of the same PSTPIP1 variant and it did not parallel disease activity when the whole cohort was taken in consideration. However, IL-1b levels varied according to the clinical picture (p = 0.0197), and it was significantly higher (P < 0.05) in patients displaying articular manifestations (N = 3) compared to those affected by cutaneous (N =6 ) or combined (N = 2) lesions. In the former, IL-1b secretion was increased in the presence of acute phase reactants elevation and active joint lesions, and treatment with Anakinra resulted in acute phase reactans normalization and symptom-free period. Silencing of of NLRP3 activity consistently inhibited LPS-induced IL-1b release in both PAPA and HD monocytes. Conclusion IL-1b secretion is higher in PAPA patients displaying prevalent articular manifestations. In these patients, IL-1b release correlates with disease activity and it is mediated by NLRP3.

Incidence of sepsis is increasing, representing a tremendous burden for health-care systems. Death in acute sepsis is attributed to hyperinflammatory responses, but the underlying mechanisms are still unclear. We report here that proton pump inhibitors (PPIs), which block gastric acid secretion, selectively inhibited tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) secretion by Toll-like receptor (TLR)-activated human monocytes in vitro, in the absence of toxic effects. Remarkably, the oversecretion of IL-1β that represents a hallmark of monocytes from patients affected by cryopyrin-associated periodic syndrome is also blocked. Based on these propaedeutic experiments, we tested the effects of high doses of PPIs in vivo in the mouse model of endotoxic shock. Our data show that a single administration of PPI protected mice from death (60% survival versus 5% of untreated mice) and decreased TNF-α and IL-1β systemic production. PPIs were efficacious even when administered after lipopolysaccharide (LPS) injection. PPI-treated mice that survived developed a long-term cross-tolerance, becoming resistant to LPS- and zymosan-induced sepsis. In vitro, their macrophages displayed impaired TNF-α and IL-1β to different TLR ligands. PPIs also prevented sodium thioglycollate-induced peritoneal inflammation, indicating their efficacy also in a non-infectious setting independent of TLR stimulation. Lack of toxicity and therapeutic effectiveness make PPIs promising new drugs against sepsis and other severe inflammatory conditions.

In this study, we show that IL-1β 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-1β. ROS production and antioxidant response are both required, because either inhibitors of NADPH oxidase and of thioredoxin reductase impair IL-1β secretion. These inhibitors also hinder cysteine release and consequently prevent reduction of the extracellular medium: addition of exogenous reducing agents restores IL-1β secretion. Not only silencing of thioredoxin, but also of the ROS scavenger superoxide dismutase 1 results in inhibition of IL-1β secretion. Thus, PAMP-induced ROS trigger an antioxidant response involving intracellular redox enzymes and release of cysteine, ultimately required for IL-1β processing and secretion.

Background Positional differences of Proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1) mutations may trigger opposite clinical outcomes in Pyogenic sterile Arthritis Pyoderma gangrenosum and Acne (PAPA) syndorme. The typical triad may not be fully displayed, and clinical picture often vary along the disease history in the same individual. PSTPIP1-ASC-pyrin interaction may lead to caspase-1 dependent Interleukin (IL)1β secretion. However, the involvement of NLRP3 and IL1β signaling itself in PAPA were explored mostly in in vitro or in vivo systems. Furthermore, due to disease rarity, anti-IL1 effectiveness was unveiled or refuted based on brief clinical reports with significant variability in response to treatment. Differences in experimental conditions and assessments being performed in possibly differing stage of patients9 diseases activity may give reason of the discrepancies observed. Thus, to date univocal insights concerning the actual role of IL1β signaling, its extent and the underlying regulatory molecular mechanisms in PAPA remain undefined, thus challenging an evidence-based application of anti-IL1 regimen. Objectives To define whether IL1β secretion in PAPA syndrome (1) is enhanced, (2) requires NLRP3, and (3) correlates with different PSTPIP1 mutations, disease activity and/or clinical picture. Methods A clinically well characterized cohort including 13 genetically confirmed PAPA subjects (E250Q+ N =10, E250K+ N =1, E256G+ N =2) and 1 patient wild-type (WT) for the common PSTPIP1 mutations were evaluated and compared with 35 healthy donors (HD). Peripheral blood monocytes were purified and studied at baseline and following in vitro TLR4 activation. Secretion pattern of IL1β, IL1α, IL1Ra, IL6, IL18 and Tumor Necrosis Factor (TNF)α was assessed in supernatants by ELISA and correlated to genotype, disease activity, ongoing therapies and clinical picture. Requirement of NLRP3 for IL1β secretion was investigated by silencing NLRP3 in monocytes purified from patients and HD. Results Increased IL1β release was found in the overall PAPA cohort (P=0.254), and silencing of NLRP3 prevented it. Patients with active disease displayed higher IL1β oversecretion (P Conclusions Variable IL1 β secretion was obserbed in PAPA cohort according to disease activity and clinical phenotype. The availability of a relevant number of patients and the chance to clinically and experimentally evaluate them in different phases of their disease history, allowed us to better define the actual behavior of IL1β secretion in this condition. Even if other mechanisms related to the complex PSTPISP1 protein networking might play additional roles, these results are in agreement with preliminary anecdotal reports on anti-IL1 regimen in PAPA and support the use of IL1 blockade as a potential effective therapeutic strategy in the clinical management of this condition. Disclosure of Interest None declared

Background CAPS are autoinflammatory diseases characterized by recurrent episodes of fever and systemic inflammation. Three nosological entities representing different phenotypes, from the milder to the most severe (FCAS, MWS, CINCA). Cryopyrin, renamed NLRP3, is part of the intracellular multiprotein complex inflammasome that mediates IL-1 processing and secretion through caspase-1 activation. NLRP3 mutations in CAPS are gain-of-function, as they enhance inflammasome activity. The result is hypersecretion of IL-1, responsible for the inflammatory clinical manifestations. Objectives –To increase the knowledge on the pathologic consequences of NLRP3 mutations in CAPS patients; –To understand the underlying molecular and regulatory mechanisms of CAPS disease; –To identify novel molecular targets for the treatment of cryopyrin/NLRP3 related disorders. Methods We have generated a KI mouse carrying the N475K mutation into the murine NLRP3 gene. This mutation corresponds to the N477K human mutation, associated to a severe CINCA phenotype with neurological complications; Phenotypical and immunological characterization of NLRP3 Knock In (KI) mice has been performed by flow cytometry; The IL1β secretion from bone marrow derived dendritic cells (BMDCs) and peritoneal macrophages (PMs) of NLRP3 Knock In Mice has been evaluated by ELISA. Results The NLRP3 KI mice that we have obtained show hair loss, presence of skin rash and reduced survival time when compared to wild type (WT). Autopsy of KI mice, prematurely dead, revealed splenomegaly and a relevant inflammatory status. We compared the IL-1 secretion of inflammatory cells from WT and KI mice. PMs and BMDCs from mutant mice did not secrete mature IL-1β spontaneously. When stimulated with 100 ng/ml of LPS KI cells secreted higher levels of IL-1b than WT cells. The kinetics of IL-1β secretion was much faster in KI cells, reaching the plateau at 3h from exposure to LPS, thus reproducing the results obtained from monocytes of CAPS patients. As in CAPS monocytes, brief exposure to ATP strongly induced the secretion of IL-1β by LPS-activated WT cells while failed to stimulate further IL-1β secretion by inflammatory cells of KI mice. Finally, PMs and BMDCs from KI are more responsive to agonists of TLRs when compared to WT cells: LPS at 0.01 ng/ml triggered high levels of IL-1β secretion (comparable to 100 ng/ml of LPS) in inflammatory cells from KI indicating that the presence of the mutation lowers the threshold of activation. The neurological studies by MRI are in progress Conclusions The NLRP3 KI mice recapitulates phenotype and functional characteristics of CAPS patients. Thus, this model will hopefully provide elucidations in the mechanisms underlying CAPS as well as other inflammasomepathies. References Brydges SD et al., Immunity. 2009. Meng et al., Immunity 2009. Gattorno M. et al., Arthritis Rheum. 2013. Disclosure of Interest None declared

The notion that the ATP-binding cassette transporter-A2 (ABCA2) may be involved in brain sterol homeostasis and is associated with early onset Alzheimer's disease led us to explore its neural expression. Our data support and extend the previous reports on ABCA2 expression by oligodendrocytes. They evidence that ABCA2 (i) is located in intracellular vesicles, identified in transfected cells as lysosome-related organelles only partially overlapping with classical endolysosomes; (ii) is a marker of neural progenitors as it is expressed in the subventricular zone of the lateral ventricle and the dentate gyrus of the hippocampal formation, sites of continual neurogenesis in the adult brain, and in nestin(+) cells differentiated in vitro from embryonic stem cells; (iii) persists, in the adult rodent brain, in a subset of GABAergic and glutamatergic neurons. Considering that the latter are targets of Alzheimer's lesions, these data provide a new rationale to explore the neuropathological consequences of ABCA2 functional dysregulations.

Based on our previous studies where we found that IFNAR2-1, the short IFNalpha/beta receptor variant, was expressed in pleomorphic sarcoma cells, we decided to determine the relative levels of expression of IFNAR2.1 versus the longer form, named IFNAR2.2, in different pleomorphic sarcoma cells in relation to their response to interferon alpha treatment. When examining a panel of PS cells isolated from surgical specimens, we found that IFNAR2.1 prevailed in 6 out 7 lines analysed and that these generally showed cell cycle arrest and low levels of apoptosis upon IFNalpha treatment. The reverse ratio, i.e. higher constitutive levels of IFNAR2.2 than IFNAR2.1, was associated with an irreversible inhibition of cell growth and pronounced apoptosis. Impairment of tumour growth by low- and high-dose IFNalpha treatment of nude mice inoculated with PS cells expressing predominantly IFNAR2.1 further asserted the effect of the cytokine also in vivo. A proteomic analysis of 120 signalling components in growth arrested, apoptotic PS cells harbouring higher levels of IFNAR2.2 revealed engagement of the canonical Jak/Stat/ISGF3-pathway, the activation of the mitochodrial apoptotic pathway and a potentially novel mechanism of cell cycle blockade unrelated to down-regulation of cyclin A/B and their interacting/regulating kinases. Our results confirm the dominant negative role of IFNAR2.1, but also suggest that the relative endogenous levels of the two IFNalpha/beta receptor isoforms may dictate the signalling pathways triggered by the ligand, such as to cause exclusively cell cycle arrest or induce programmed cell death. This parameter may be of importance for the clinical outcome of IFNalpha treatment of PS.

Although malignant fibrous histiocytoma (MFH) is one of the most diffuse and highly aggressive tumors among soft tissue sarcomas in adults, it is poorly characterized from the molecular point of view. The overt lack of expression of phenotypic markers in MFH cells and the hypothesis that MFH may originate from transformed multipotent stem/progenitor cells with mesenchymal features has led us to investigate this notion and search for 'MFH-specific' genes. To address this problem, we have undertaken a differential display-based three-pair comparative mRNA profiling of bone-marrow derived mesenchymal stem cells (MSC) and cells isolated by primary MFH, leiomyosarcoma and smooth muscle cells, fibrosarcoma and dermal fibroblasts. This approach highlighted pair-wise analogies in gene expression patterns between matched tumor and healthy cells and yielded direct access to 43 genes differentially expressed between MSC and MFH cells. Eleven of the identified genes were selected for comparative evaluation of their expression levels in other sarcoma types, as well as potential markers for the detection of circulating tumor cells. Several of these genes defined the stem/progenitor versus MFH cell and some of them have the potential to be exploited for disclosure of circulating sarcoma cells. The striking similarity in the gene expression patterns observed in the two cell types was further corroborated by a remarkable similarity in the cell phenotypic markers that these cells expressed ex vivo. The findings open now the possibility to examine, also functionally, genes not previously known to be implicated in MFH development and strengthen the hypothesis that MFH originates from a mesenchymal progenitor cell.

Background Familial Mediterranean fever (FMF) and Pyogenic Arthritis, Pyoderma gangrenosum and Acne (PAPA) syndrome are autoinflammatory disorders that are thought to share similar pathoetiology. While FMF is due to mutations of MEFV gene encoding for Pyrin, PAPA syndrome is caused by genetic variants of CD2 binding protein 1 (CD2BP1). Since Pyrin is able to bind CD2BP1 in a newly identified inflammasome called Pyroptosome, FMF and PAPA have been recently recognized as disorders of the same pathway, probably involving caspase-1 mediated interleukin (IL)-1β secretion, as suggested by the response to anti IL-1 treatment. However, a clear involvement of IL-1β is still controversial, especially because most of the data derive from studies applying not homogeneous experimental approaches. Objectives To investigate in our cohorts of FMF and PAPA patients whether 1) MEFV/CD2BP1 mutated monocytes displayed enhancedredox-stress and IL1β secretion, 2) IL1β pathway activationcorrelated with type of mutation and 3) pyroptosome-related IL1β secretion was mediated by NLRP3 inflammasome. Methods Thirteen genetically confirmed FMF pediatric patients (7 with 2 high penetrance and 6 with 1 high penetrance and 1 low penetrance or 2 low penetrance mutation) and 10 asymptomatic parents (healthy carriers, HC) were evaluated. Four PAPA (2 children and 2 adults) patients carrying different CD2BP1 mutations (E250Q, E250K and E256G) were also analyzed and compared to 25 healthy donors (HD). Peripheral blood primary human monocytes were freshly isolated and studied at baseline and after 3-6-18 hours (h) of TLR s in vitro activation. Intracellular reactive oxygen species (ROS) were examined by biochemical assay. Monocyte pattern of secretion of IL-1β, IL-18 and IL-1Ra was assessed by ELISA. The involvement of NLRP3 inflammasome was investigated by in vitro silencing. Results FMF patients carrying high penetrance MEFV mutations displayed higher ROS levels than HD at baseline. The same pattern was observed for PAPA patients carrying the E250Q and E256G mutations. Overall FMF and PAPA monocytes displayed enhanced but not anticipated IL1β and IL18 secretion, in the presence of IL1Ra levels comparable to HD. Increased IL-1β pathway activation was greater in the presence of more severe MEFV mutations and in pediatric E250Q(+) and E256G(+) PAPA patients. Silencing of NLRP3 in representative cases inhibited IL-1β secretion in both FMF (N=1), FMF HC (N=3), PAPA (N=2) and HD (N=6). Conclusions MEFV and CD2BP1 mutated monocytes are under sustained oxidative stress. This parallels an enhanced pattern of secretion of the pro-inflammatory IL1β, with greater effect in the presence of high-penetrance mutations in FMF and E249Q or E256G mutation in PAPA patients. Results from in vitro silencing experiments in freshly isolated monocytes suggest that NLRP3 inflammasome may be involved. Disclosure of Interest None Declared