Unexpected chronic lymphocytic leukemia B cell activation by bisphosphonates.

Chronic lymphocytic leukemia (CLL) is a disease of the elderly, often presenting comorbidities like osteoporosis and requiring, in a relevant proportion of cases, treatment with bisphosphonates (BPs). This class of drugs was shown in preclinical investigations to also possess anticancer properties. We started an in vitro study of the effects of BPs on CLL B cells activated by microenvironment-mimicking stimuli and observed that, depending on drug concentration, hormetic effects were induced on the leukemic cells. Higher doses induced cytotoxicity whereas at lower concentrations, more likely occurring in vivo, the drugs generated a protective effect from spontaneous and chemotherapy-induced apoptosis, and augmented CLL B cell activation/proliferation. This CLL-activation effect promoted by the BPs was associated with markers of poor CLL prognosis and required the presence of bystander stromal cells. Functional experiments suggested that this phenomenon involves the release of soluble factors and is increased by cellular contact between stroma and CLL B cells. Since CLL patients often present comorbidities such as osteoporosis and considering the diverse outcomes in both CLL disease progression and CLL response to treatment among patients, illustrating this phenomenon holds potential significance in driving additional investigations.

Mandatory role of endoplasmic reticulum and its pentose phosphate shunt in the myocardial defense mechanisms against the redox stress induced by anthracyclines.

Anthracyclines' cardiotoxicity involves an accelerated generation of reactive oxygen species. This oxidative damage has been found to accelerate the expression of hexose-6P-dehydrogenase (H6PD), that channels glucose-6-phosphate (G6P) through the pentose phosphate pathway (PPP) confined within the endoplasmic/sarcoplasmic reticulum (SR). To verify the role of SR-PPP in the defense mechanisms activated by doxorubicin (DXR) in cardiomyocytes, we tested the effect of this drug in H6PD knockout mice (H6PD-/-). Twenty-eight wildtype (WT) and 32 H6PD-/- mice were divided into four groups to be treated with intraperitoneal administration of saline (untreated) or DXR (8 mg/Kg once a week for 3 weeks). One week thereafter, survivors underwent imaging of 18F-deoxyglucose (FDG) uptake and were sacrificed to evaluate the levels of H6PD, glucose-6P-dehydrogenase (G6PD), G6P transporter (G6PT), and malondialdehyde. The mRNA levels of SR Ca2+-ATPase 2 (Serca2) and ryanodine receptors 2 (RyR2) were evaluated and complemented with Hematoxylin/Eosin staining and transmission electron microscopy. During the treatment period, 1/14 DXR-WT and 12/18 DXR-H6PD-/- died. At microPET, DXR-H6PD-/- survivors displayed an increase in left ventricular size (p < 0.001) coupled with a decreased urinary output, suggesting a severe hemodynamic impairment. At ex vivo analysis, H6PD-/- condition was associated with an oxidative damage independent of treatment type. DXR increased H6PD expression only in WT mice, while G6PT abundance increased in both groups, mismatching a generalized decrease of G6PD levels. Switching-off SR-PPP impaired reticular accumulation of Ca2+ decelerating Serca2 expression and upregulating RyR2 mRNA level. It thus altered mitochondrial ultrastructure eventually resulting in a cardiomyocyte loss. The recognized vulnerability of SR to the anthracycline oxidative damage is counterbalanced by an acceleration of G6P flux through a PPP confined within the reticular lumen. The interplay of SR-PPP with the intracellular Ca2+ exchanges regulators in cardiomyocytes configure the reticular PPP as a potential new target for strategies aimed to decrease anthracycline toxicity.

Methods to Study NLR in Human Blood Cells.

Autoinflammatory diseases are a group of inherited and multifactorial disorders characterized by an over-activation of innate immune response. In most cases, the clinical manifestations are due to increased activity of the NLRP3 inflammasome resulting in increased IL-1ß 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, 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.

High dose esomeprazole as an anti-inflammatory agent in sepsis: Protocol for a randomized controlled trial.

BACKGROUND: Sepsis is caused by dysregulated immune responses due to infection and still presents high mortality rate and limited efficacious therapies, apart from antibiotics. Recent evidence suggests that very high dose proton pump inhibitors might regulate major sepsis mediators' secretion by monocytes, which might attenuate excessive host reactions and improve clinical outcomes. This effect is obtained with doses which are approximately 50 times higher than prophylactic esomeprazole single daily administration and 17 times higher than the cumulative dose of a three day prophylaxis. We aim to perform a randomized trial to investigate if high dose esomeprazole reduces organ dysfunction in patients with sepsis or septic shock. METHODS: This study, called PPI-SEPSIS, is a multicenter, randomized, double blind, placebo-controlled clinical trial on critically ill septic patients admitted to the emergency department or intensive care unit. A total of 300 patients will be randomized to receive high dose esomeprazole (80 mg bolus followed by 12 mg/h for 72 h and a second 80 mg bolus 12 h after the first one) or equivolume placebo (sodium chloride 0.9%), with 1:1 allocation. The primary endpoint of the study will be mean daily Sequential Organ Failure Assessment (SOFA) score over 10 days. Secondary outcomes will include antibiotic-free days, single organ failure severity, intensive care unit-free days at day 28, and mortality. DISCUSSION: This trial aims to test the efficacy of high dose esomeprazole to reduce acute organ dysfunction in patients with septic shock. TRIAL REGISTRATION: This trial was registered on ClinicalTrials.gov with the trial identification NCT03452865 in March 2018.

Divergent Oxidative Stress in Normal Tissues and Inflammatory Cells in Hodgkin and Non-Hodgkin Lymphoma.

BACKGROUND: Previous studies reported mitochondrial and endoplasmic reticulum redox stress in peripheral blood mononucleated cells (PBMCs) of treatment-naïve Hodgkin lymphoma (HL) patients. Here, we assessed whether this response also applies to non-HL (NHL) patients, and whether the oxidative damage is a selective feature of PBMCs or, rather, also affects tissues not directly involved in the inflammatory response. METHODS: Isolated PBMCs of 28 HL, 9 diffuse large B cell lymphoma, 8 less aggressive-NHL, and 45 controls underwent flow cytometry to evaluate redox stress and uptake of the glucose analogue 2-NBDG. This analysis was complemented with the assay of malondialdehyde (MDA) levels and enzymatic activity of glucose-6P-dehydrogenase and hexose-6P-dehydrogenase (H6PD). In all lymphoma patients, 18F-fluoro-deoxyglucose uptake was estimated in the myocardium and skeletal muscles. RESULTS: Mitochondrial reactive oxygen species generation and MDA levels were increased only in HL patients as well as H6PD activity and 2-NBDG uptake. Similarly, myocardial FDG retention was higher in HL than in other groups as opposed to a similar tracer uptake in the skeletal muscle. CONCLUSIONS: Redox stress of PBMCs is more pronounced in HL with respect to both NHL groups. This phenomenon is coherent with an increased activity of H6PD that also extends to the myocardium.

Gene's expression underpinning the divergent predictive value of [18F]F-fluorodeoxyglucose and prostate-specific membrane antigen positron emission tomography in primary prostate cancer: a bioinformatic and experimental study.

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.

Mitochondrial Generated Redox Stress Differently Affects the Endoplasmic Reticulum of Circulating Lymphocytes and Monocytes in Treatment-Naïve Hodgkin's Lymphoma.

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.

Therapeutic efficacy of proton transport inhibitors alone or in combination with cisplatin in triple negative and hormone sensitive breast cancer models.

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.

Fundamental Role of Pentose Phosphate Pathway within the Endoplasmic Reticulum in Glutamine Addiction of Triple-Negative Breast Cancer Cells.

Cancer utilization of large glutamine equivalents contributes to diverging glucose-6-P flux toward the pentose phosphate shunt (PPP) to feed the building blocks and the antioxidant responses of rapidly proliferating cells. In addition to the well-acknowledged cytosolic pathway, cancer cells also run a largely independent PPP, triggered by hexose-6P-dehydrogenase within the endoplasmic reticulum (ER), whose activity is mandatory for the integrity of ER-mitochondria networking. To verify whether this reticular metabolism is dependent on glutamine levels, we complemented the metabolomic characterization of intermediates of the glucose metabolism and tricarboxylic acid cycle with the estimation of proliferating activity, energy metabolism, redox damage, and mitochondrial function in two breast cancer cell lines. ER-PPP activity and its determinants were estimated by the ER accumulation of glucose analogs. Glutamine shortage decreased the proliferation rate despite increased ATP and NADH levels. It depleted NADPH reductive power and increased malondialdehyde content despite a marked increase in glucose-6P-dehydrogenase. This paradox was explained by the deceleration of ER-PPP favored by the decrease in hexose-6P-dehydrogenase expression coupled with the opposite response of its competitor enzyme glucose-6P-phosphatase. The decreased ER-PPP activity eventually hampered mitochondrial function and calcium exchanges. These data configure the ER-PPP as a powerful, unrecognized regulator of cancer cell metabolism and proliferation.

The Role of Endoplasmic Reticulum in the Differential Endurance against Redox Stress in Cortical and Spinal Astrocytes from the Newborn SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis.

Recent studies reported that the uptake of [18F]-fluorodeoxyglucose (FDG) is increased in the spinal cord (SC) and decreased in the motor cortex (MC) of patients with ALS, suggesting that the disease might differently affect the two nervous districts with different time sequence or with different mechanisms. Here we show that MC and SC astrocytes harvested from newborn B6SJL-Tg (SOD1G93A) 1Gur mice could play different roles in the pathogenesis of the disease. Spectrophotometric and cytofluorimetric analyses showed an increase in redox stress, a decrease in antioxidant capacity and a relative mitochondria respiratory uncoupling in MC SOD1G93A astrocytes. By contrast, SC mutated cells showed a higher endurance against oxidative damage, through the increase in antioxidant defense, and a preserved respiratory function. FDG uptake reproduced the metabolic response observed in ALS patients: SOD1G93A mutation caused a selective enhancement in tracer retention only in mutated SC astrocytes, matching the activity of the reticular pentose phosphate pathway and, thus, of hexose-6P dehydrogenase. Finally, both MC and SC mutated astrocytes were characterized by an impressive ultrastructural enlargement of the endoplasmic reticulum (ER) and impairment in ER-mitochondria networking, more evident in mutated MC than in SC cells. Thus, SOD1G93A mutation differently impaired MC and SC astrocyte biology in a very early stage of life.

A novel knock-in mouse model of cryopyrin-associated periodic syndromes with development of amyloidosis: Therapeutic efficacy of proton pump inhibitors.

BACKGROUND: Cryopyrin-associated periodic syndromes (CAPS) are a group of autoinflammatory diseases linked to gain-of-function mutations in the NOD-like receptor family, pyrin domain containing 3 (NLRP3) gene, which cause uncontrolled IL-1ß secretion. Proton pump inhibitors (PPIs), which are commonly used as inhibitors of gastric acid production, also have anti-inflammatory properties, protect mice from sepsis, and prevent IL-1ß secretion by monocytes from patients with CAPS. OBJECTIVE: We sought to develop a novel Nlrp3 knock-in (KI) mouse model of CAPS to study amyloidosis, a severe CAPS complication, and test novel therapeutic approaches. METHODS: We generated KI mice by engineering the N475K mutation, which is associated with the CAPS phenotype, into the mouse Nlrp3 gene. KI and wild-type mice received PPIs or PBS intraperitoneally and were analyzed for survival, inflammation, cytokine secretion, and amyloidosis development. RESULTS: Mutant Nlrp3 KI mice displayed features that recapitulate the immunologic and clinical phenotype of CAPS. They showed systemic inflammation with high levels of serum proinflammatory cytokines, inflammatory infiltrates in various organs, and amyloid deposits in the spleen, liver, and kidneys. Toll-like receptor stimulated macrophages from KI mice secreted high levels of IL-1ß, IL-18, and IL-1α but low amounts of IL-1 receptor antagonist. Treatment of KI mice with PPIs had a clear clinical effect, showing a reduction in inflammatory manifestations, regression of amyloid deposits, and normalization of proinflammatory and anti-inflammatory cytokine production by macrophages. CONCLUSION: Nlrp3 KI mice displayed a CAPS phenotype with many characteristics of autoinflammation, including amyloidosis. The therapeutic effectiveness of PPIs associated with a lack of toxicity indicates that these drugs could represent relevant adjuvants to the anti-IL-1 drugs in patients with CAPS and other IL-1-driven diseases.

Oxidation of methionine residues in human apolipoprotein A-I generates a potent pro-inflammatory molecule.

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)-1ß 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-1ß 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.

Progressive waves of IL-1ß release by primary human monocytes via sequential activation of vesicular and gasdermin D-mediated secretory pathways.

IL-1ß is an essential cytokine, but its release needs to be strictly controlled to avoid severe inflammatory manifestations. Lacking a signal sequence, IL-1ß does not follow the endoplasmic reticulum-Golgi route. Several pathways have been proposed to mediate its release. One involves the translocation of pro-IL-1ß into intracellular vesicles of lysosomal origin that eventually fuse with the plasma membrane. Another exploits pores formed on the plasma membrane upon proteolytic cleavage of gasdermin D (GSDMD). Here we investigated how primary monocytes-the main source of IL-1ß in humans-control IL-1ß release in response to pro-inflammatory stimuli of increasing intensity and found that two different routes are induced depending on the strength of activation. Triggering of Toll-like receptor 4 (TLR4) by LPS induces slow IL-1ß release through LAMP2A+ vesicles. In contrast, the simultaneous stimulation of TLR2, TLR4 and TLR7/8 drives high levels of ROS, GSDMD cleavage and faster IL-1ß secretion. Drugs blocking ROS production prevent GSDMD cleavage supporting a role of oxidative stress in GSDMD-mediated secretion. Singly stimulated monocytes undergo apoptosis, whereas triple stimulation triggers pyroptosis, which might amplify inflammation. In both cases, however, IL-1ß secretion precedes cell death. Inhibition of caspases 4/5 prevents GSDMD cleavage and pore-mediated secretion, but not vesicular release. The two pathways also display other distinct pharmacologic sensitivities that reflect the underlying mechanisms. Remarkably, single TLR4 stimulation is sufficient to activate massive, GSDMD-mediated IL-1ß secretion in monocytes from patients affected by Cryopyrin Associated Periodic Syndrome (CAPS), an autoinflammatory disease linked to NLRP3 mutations. The exaggerated sensitivity to activation correlates with high basal ROS levels in CAPS monocytes. In conclusion, the vesicular pathway limits IL-1ß release upon low pathogen load while stronger stimulation or concomitant cell stress induce instead uncontrolled secretion via GSDMD leading to detrimental inflammatory manifestations.

OLT1177, a ß-sulfonyl nitrile compound, safe in humans, inhibits the NLRP3 inflammasome and reverses the metabolic cost of inflammation.

Activation of the NLRP3 inflammasome induces maturation of IL-1ß and IL-18, both validated targets for treating acute and chronic inflammatory diseases. Here, we demonstrate that OLT1177, an orally active ß-sulfonyl nitrile molecule, inhibits activation of the NLRP3 inflammasome. In vitro, nanomolar concentrations of OLT1177 reduced IL-1ß and IL-18 release following canonical and noncanonical NLRP3 inflammasome activation. The molecule showed no effect on the NLRC4 and AIM2 inflammasomes, suggesting specificity for NLRP3. In LPS-stimulated human blood-derived macrophages, OLT1177 decreased IL-1ß levels by 60% and IL-18 by 70% at concentrations 100-fold lower in vitro than plasma concentrations safely reached in humans. OLT1177 also reduced IL-1ß release and caspase-1 activity in freshly obtained human blood neutrophils. In monocytes isolated from patients with cryopyrin-associated periodic syndrome (CAPS), OLT1177 inhibited LPS-induced IL-1ß release by 84% and 36%. Immunoprecipitation and FRET analysis demonstrated that OLT1177 prevented NLRP3-ASC, as well as NLRP3-caspase-1 interaction, thus inhibiting NLRP3 inflammasome oligomerization. In a cell-free assay, OLT1177 reduced ATPase activity of recombinant NLRP3, suggesting direct targeting of NLRP3. Mechanistically, OLT1177 did not affect potassium efflux, gene expression, or synthesis of the IL-1ß precursor. Steady-state levels of phosphorylated NF-κB and IkB kinase were significantly lowered in spleen cells from OLT1177-treated mice. We observed reduced IL-1ß content in tissue homogenates, limited oxidative stress, and increased muscle oxidative metabolism in OLT1177-treated mice challenged with LPS. Healthy humans receiving 1,000 mg of OLT1177 daily for 8 d exhibited neither adverse effects nor biochemical or hematological changes.

Dysregulated IL-1ß Secretion in Autoinflammatory Diseases: A Matter of Stress?

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-1ß. 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-1ß 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.

NLR in Human Diseases: Role and Laboratory Findings.

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-1ß 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.

Clinical Characteristics of Patients Carrying the Q703K Variant of the NLRP3 Gene: A 10-year Multicentric National Study.

OBJECTIVE: The aim of our study was to analyze the clinical and functional effect of the p.Q703K (p. Q705K, c. 2107C>A) variant of the NLRP3 gene in a population of patients screened for suspected cryopyrin-associated periodic syndrome (CAPS). METHODS: Since 2002, 580 patients underwent molecular analysis for NLRP3. Data on clinical presentation, response to treatment, and longterm followup were collected using a uniform questionnaire. The pattern of cytokine secretion after lipopolysaccharide stimulation from isolated monocytes was analyzed in 3 patients carrying the p.Q703K variant and 1 patient with a chronic infantile neurologic, cutaneous, articular syndrome phenotype carrying both the p.M406I and p.Q703K, and compared with 7 patients with CAPS with sure pathogenic variants and 6 healthy controls. RESULTS: The p.Q703K variant was found in 57 screened patients with an overall allelic frequency of 5%. The frequency in normal controls was 5.5%. Clinical data at the moment of molecular analysis and at followup were available in 36 patients. Two patients displayed additional mutations of NLRP3. The mean followup was 2.5 years. Thirteen patients (39%) had a final diagnosis different from the original suspicion of CAPS. The remaining 21 patients displayed a mild phenotype mainly characterized by recurrent episodes of urticarial rash and arthralgia. Only 8 patients were treated with anti-interleukin (IL)-1 treatment, with a complete response in 5 patients. The pattern of secretion of IL-1ß and other cytokines (IL-6 and IL-1 receptor antagonist) in patients did not display the aberrancies observed in patients with CAPS and was similar to that observed in healthy controls. CONCLUSION: The present study confirms the weak clinical and functional effect of the p.Q703K variant.

Disease activity accounts for long-term efficacy of IL-1 blockers in pyogenic sterile arthritis pyoderma gangrenosum and severe acne syndrome.

OBJECTIVE: To provide a rationale for anti-IL-1 treatment in pyogenic sterile arthritis, pyoderma gangrenosum and acne (PAPA) by defining whether IL-1ß 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-1ß, IL-1α, IL-1Ra, IL-6, IL-18 and TNF-α was assessed in supernatants by ELISA. The NLRP3 requirement for IL-1ß 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-1ß secretion in PAPA is increased, requires NLRP3 and correlates with disease activity. Patients with a history of osteoarticular flares release more IL-1ß, IL-6 and TNF-α compared with those with predominant cutaneous recurrences. Monocytes from patients in anti-IL-1 treatment dramatically reduced IL-1ß 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-1ß signalling was not observed suggesting that factors other than mutation itself may play a role in regulating IL-1ß secretion and response to treatment in PAPA. CONCLUSION: PAPA patients with active lesions display increased NLRP3-mediated IL-1ß 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.

Redox stress unbalances the inflammatory cytokine network: role in autoinflammatory patients and healthy subjects.

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-1ß 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.