Correlation analysis between the early diagnosis and severity of neuropathic pain and the activation level of NLRP3 inflammasome: A retrospective study.

To investigate the correlation between neuropathic pain's early diagnosis, severity, and nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome activation, we retrospectively evaluated 50 patients with neuropathic pain and 50 healthy individuals. Activation of the NLRP3 inflammasome was measured in blood samples, as well as pain levels and clinical markers. Neuropathic pain patients exhibited elevated NLRP3 inflammasome activation. Pain intensity positively correlated with activation. Correlation was also observed with inflammatory markers and pain-related biomarkers. NLRP3 inflammasome demonstrated high diagnostic sensitivity. In conclusion, NLRP3 inflammasome activation influences neuropathic pain initiation and progression. Measuring activation levels may serve as an early diagnostic indicator and severity gauge for neuropathic pain.

Serum NLRP3: A potential marker for identifying high-risk coronary arterial aneurysm in children with Kawasaki disease.

BACKGROUND: Kawasaki disease (KD) is a vasculitis of unknown etiology in children aged under 5 years. Coronary arterial aneurysm (CAA) is the major complication of KD. It is no longer though to be a self-limiting disease because its cardiovascular sequelae might persist into adulthood. NLRP3 is a key protein of the NLRP3 inflammasome that participates in sterile inflammatory disease. This study investigated the serum levels of NLRP3 in patients with KD at different stages to explore the relationships between serum NLRP3 and clinical parameters. METHODS: A total of 247 children enrolled in this study. There were 123 patients in the acute stage of KD, and 93 healthy children made up the healthy control (HC) group. Among the acute KD patients, 52 had coronary arterial aneurysm (KD-CAA) and 71 did not (KD-NCAA). 36 patient samples were collected after IVIG and aspirin treatment. Additionally, 29 patients were in the cardiovascular sequelae stage. Enzyme-linked immunosorbent assay was used to measure serum NLRP3 levels in all subjects. RESULTS: Serum NLRP3 was elevated in the KD group and was even higher in the KD-CAA subgroup than in the KD-NCAA subgroup of acute-stage patients. Serum NLRP3 declined when the patients were treated with IVIG and aspirin, but during the convalescent (coronary sequelae) stage, serum NLRP3 re-increased. Serum NLRP3 was higher in the ≥ 6-mm-coronary-arterial-diameter group than that the < 6-mm-diameter group. The ROC curve of serum NLRP3 indicated its utility in the prediction of both KD and KD-CAA. CONCLUSIONS: NLRP3 may be involved in the development of KD and CAA in children with KD. Targeting NLRP3 might mitigate CAA, thereby reducing the risk of cardiovascular events in adulthood.

Gene Polymorphisms of NLRP3 Associated With Plasma Levels of 4ß-Hydroxycholesterol, an Endogenous Marker of CYP3A Activity, in Patients With Asthma.

Inflammation decreases the activity of cytochrome P450 3A (CYP3A). Nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) is responsible for regulating the inflammatory response, and its genetic polymorphisms have been linked to inflammatory diseases such as asthma. However, there have been few studies on the effect of NLRP3 on CYP3A activity. We aimed to investigate the association between polymorphisms in the NLRP3 gene and plasma 4ß-hydroxycholesterol (4ßOHC), an endogenous marker of CYP3A activity, in patients with asthma. In this observational study including 152 adult asthma patients, we analyzed 10 NLRP3 gene single-nucleotide polymorphisms (SNPs). Plasma 4ßOHC levels were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results showed that five SNPs were associated with significantly lower plasma 4ßOHC concentrations. Among these SNPs, rs3806265, rs4612666, rs1539019, and rs10733112 contributed to a significant increase in plasma IL-6 concentrations. Moreover, a multivariate regression model showed that the rs3806265 TT, rs4612666 CC, rs1539019 AA, and rs10733112 TT genotypes were significant factors for decreased plasma 4ßOHC, even after including patient background factors and CYP3A5*3 (rs776746) gene polymorphisms as covariates. These results were also observed when plasma 4ßOHC concentrations were corrected for cholesterol levels. We conclude that NLRP3 gene polymorphisms are involved in increasing plasma IL-6 concentrations and decreasing plasma 4ßOHC concentrations in patients with asthma. Therefore, NLRP3 gene polymorphisms may be a predictive marker of CYP3A activity in inflammatory diseases such as asthma.

Unraveling the blood microbiome: novel insights into inflammasome responses in Crohn's disease.

OBJECTIVE: Crohn's disease (CD), an inflammatory bowel disease with unknown etiology, is influenced by genetic, environmental, and immunological factors. This study aimed to analyze the blood microbiome and inflammasome responses, emphasizing NLRP3 protein expression and IL-1ß and IL-18 plasma levels, between Crohn's patients and healthy subjects. METHODS: A total of 40 volunteers were included in this study. The 16S rRNA technique was used to sequence the V3-V4 regions of the blood sample. NLRP3 protein levels in plasma were ascertained through Western Blot, and IL-1ß and IL-18 plasma profiles were examined using ELISA. RESULTS: Analysis highlighted five unique phyla in patients' plasma, emphasizing the role of the blood microbiome in CD. Compared to controls, Crohn's patients exhibited elevated NLRP3 protein expression. Plasma IL-1ß levels were diminished in patients ( P  = 0.0041), whereas IL-18 levels were comparably higher ( P  = 0.8209). In patients with CD, the presence of Staphylococcus sciuri in blood samples highlights its potential role in the disease's onset. The study also underscored the interplay between dietary habits, specifically increased meat consumption, and the progression of CD. CONCLUSION: Our pioneering research discerns the variations in the blood microbiome and inflammasome responses between Crohn's patients and healthy individuals. Significant microbiome alterations and the detection of the Staphylococcus sciuri pathogen in Crohn's patients were notable. The pronounced NLRP3 protein in patients suggests its potential as a diagnostic biomarker. Future explorations into IL-1ß and IL-18 pathways promise to unveil innovative insights into CD.

Serum NLRP3: A biomarker for identifying high-risk septic patients.

BACKGROUND: Over-activation of the NLRP3 inflammasome can lead to sepsis. NLRP3 is an essential protein in the classical pathway of pyroptosis. This study assessed the use of serum NLRP3 level as a potential inflammatory biomarker in septic patients. METHODS: Patients were categorized into five groups: healthy controls (n = 30), ICU controls (n = 22), infection (n = 19), septic non-shock (n = 33), and septic shock (n = 83). Serum NLRP3 levels were measured by enzyme-linked immunosorbent assay for all patients upon enrollment. Clinical parameters and laboratory test data (APACHE II, SOFA, and lactate) were also assessed. Moreover, the ability of serum NLRP3 levels to predict sepsis was determined by the area under the curve (AUC) analysis. RESULTS: The NLRP3 levels in the septic shock group was significantly higher (431.89, 386.61-460.21 pg/mL) than that in the healthy control group (23.24, 9.38-49.73 pg/mL), ICU control group (74.82, 62.71-85.93 pg/mL), infection group (114.34, 99.21-122.56 pg/mL), and septic non-shock group (136.99, 128.80-146.98 pg/mL; P＜0.001 for all comparisons). Additionally, the AUC indicated that the ability of serum NLRP3 levels to predict sepsis and septic shock incidences was not lower than that of the SOFA score. Patients with higher NLRP3 serum levels (>147.72 pg/mL) had significantly increased 30-day mortality rate. CONCLUSIONS: NLRP3 is useful for the early identification of high-risk septic patients, particularly septic shock patients. Moreover, elevated NRLP3 levels could result in poor septic prediction outcomes.

Predictive Value of miR-146a rs2431697 Polymorphism to Myelofibrosis Progression in Patients with Myeloproliferative Neoplasm.

BACKGROUND: Bone marrow myelofibrosis (BMF) that develop on top of Polycythaemia vera (PV) and essential thrombocythemia leads to shortening of the patient's overall survival. This study aimed to address the impact of miR-146a rs2431697 polymorphism on inflammatory biomarkers and genes expression and the hazards of myelofibrosis progression. PATIENTS AND METHODS: The study included 88 myeloproliferative neoplasm (40 PV; 27 ET; 21 MF) and 90 healthy controls. For all investigated subjects miR-146a rs2431697 genotypes were identified by sequencing and the expression of miR-146a; IL-1ß; NF-κB; a NOD-like receptor family, pyrin domain containing 3 (NLRP3) (NLRP3) genes were estimated by real time PCR. RESULTS: miR146a genotypes revealed that there was significant association between TT and TC genotypes with MF. The degree of miR146a expression was significantly reduced in MF as compared to both PV and ET. In contrast; the levels of IL-1ß; NF-κB; NLRP3 genes expression were significantly elevated in MF patients group as compared to PV and ET patients' group. Multivariate analysis identified TT genotype as poor predictor of MF progression. CONCLUSION: miR-146a rs2431697 TT genotype is associated with high risk of MF progression in MPN patients. Targeting of IL-1ß; NF-κB; NLRP3 genes might help  in hindering of  MF progression in  MPN patients,
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Characterizing the NLRP3 Inflammasome in Mood Disorders: Overview, Technical Development, and Measures of Peripheral Activation in Adolescent Patients.

The NOD-, LRR-, and pyrin-domain-containing protein 3 (NLRP3) inflammasome is a node of intracellular stress pathways and a druggable target which integrates mitochondrial stress and inflammatory cascades. While a body of evidence suggests the involvement of the NLRP3 inflammasome in numerous diseases, a lack of reliable measurement techniques highlights the need for a robust assay using small quantities of biological samples. We present a literature overview on peripheral activation of the NLRP3 inflammasome in mood disorders, then outline a process to develop and validate a robust assay to measure baseline and activated intracellular levels of "apoptosis-associated speck-like protein containing a CARD" (ASC) as a key component of an inflammatory profile in peripheral blood mononuclear cells (PBMC). A consistent association between high NLRP3 mRNA levels and relevant cytokines was seen in the literature. Using our method to measure ASC, stimulation of PBMC with lipopolysaccharide and nigericin or adenosine triphosphate resulted in microscopic identification of intracellular ASC specks, as well as interleukin 1 (IL-1) beta and caspase-1 p10 in the periphery. This was abolished by dose-dependent pre-treatment with 100 nM MCC950. We also report the use of this technique in a small pilot sample from patients with bipolar disorder and depressive disorders. The results show that levels of intracellular ASC and IL-1 beta are sensitive to change upon activation and maintained over time, which may be used to improve the detection of NLRP3 activation and guide personalized therapeutic strategy in the treatment of patients.

Soluble LAG-3 and Toll-interacting protein: Novel upstream neuro-inflammatory markers in Parkinson's disease.

INTRODUCTION: There is some evidence regarding the role of LAG-3, TLR mediated neuroinflammation in PD. METHODS: sLAG-3, TOLLIP, NLRP3 levels were measured in PD and healthy controls. RESULTS: These markers were significantly higher in PD and were associated with progression. CONCLUSION: sLAG3 and TOLLIP are involved in the NLRP3 mediated inflammatory activation in PD.

Association between serum NLRP3 and malignant brain edema in patients with acute ischemic stroke.

BACKGROUND: We aimed to explore the association of serum level of the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) and its related inflammatory biomarkers (hypoxia inducible factor-1α, cathepsin B, caspase-1 and matrix metalloproteinase-9) with malignant brain edema (MBE) in patients with acute ischemic stroke. METHODS: We prospectively enrolled patients with acute ischemic stroke admitted < 24 h from onset of symptoms. Brain CT was performed on admission and blood samples were collected. Repeated brain CT/MRI was performed < 7 days of admission to identify the presence of MBE, defined as neurological deterioration with imaging signs of midline shift or compressed basal cisterns. Logistic regression analysis was performed to assess the association between inflammatory biomarkers and MBE, adjusted for age and National Institutes of Health Stroke Scale (NIHSS). RESULTS: 200 patients (69.3 ± 14.3 years; male 55 %) were included for analysis, of whom 26 patients developed MBE (median time from stroke onset to MBE 32.5 h). Compared with patients without MBE, those with MBE had higher level of serum concentration of NLRP3 (median time from onset to blood collection 3 h, 1.85 ng/ml vs. 1.11 ng/ml, P = 0.026). NLRP3 level was positively correlated with NIHSS on admission (Spearman ρ = 0.18, P = 0.01) and the association between NLRP3 and MBE was attenuated (OR 1.47, 95 % CI 0.88-2.46, P = 0.138) after adjusting for age and NIHSS. There was no significant difference in other biomarkers between MBE and non-MBE groups. CONCLUSIONS: There was a trend of association between a higher level of serum concentration of NLRP3 and an increased risk of MBE after ischemic stroke, possibly confounded by the severity of stroke, which is worth further validation in large cohort studies.

Possible mechanisms of cholesterol elevation aggravating COVID-19.

Importance: Despite the availability of a vaccine against the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), humans will have to live with this virus and the after-effects of the coronavirus disease 2019 (COVID-19) infection for a long time. Cholesterol plays an important role in the infection and prognosis of SARS-CoV-2, and the study of its mechanism is of great significance not only for the treatment of COVID-19 but also for research on generic antiviral drugs. Observations: Cholesterol promotes the development of atherosclerosis by activating NLR family pyrin domain containing 3 (NLRP3), and the resulting inflammatory environment indirectly contributes to COVID-19 infection and subsequent deterioration. In in vitro studies, membrane cholesterol increased the number of viral entry sites on the host cell membrane and the number of angiotensin-converting enzyme 2 (ACE2) receptors in the membrane fusion site. Previous studies have shown that the fusion protein of the virus interacts with cholesterol, and the spike protein of SARS-CoV-2 also requires cholesterol to enter the host cells. Cholesterol in blood interacts with the spike protein to promote the entry of spike cells, wherein the scavenger receptor class B type 1 (SR-B1) plays an important role. Because of the cardiovascular protective effects of lipid-lowering therapy and the additional anti-inflammatory effects of lipid-lowering drugs, it is currently recommended to continue lipid-lowering therapy for patients with COVID-19, but the safety of extremely low LDL-C is questionable. Conclusions and Relevance: Cholesterol can indirectly increase the susceptibility of patients to SARS-CoV-2 and increase the risk of death from COVID-19, which are mediated by NLRP3 and atherosclerotic plaques, respectively. Cholesterol present in the host cell membrane, virus, and blood may also directly participate in the virus cell entry process, but the specific mechanism still needs further study. Patients with COVID-19 are recommended to continue lipid-lowering therapy.

Associations between exercise, inflammation and symptom severity in those with mental health disorders.

AIM: This study aimed to investigate the effects of 6-weeks of moderate intensity aerobic exercise on markers of inflammation and symptom severity in those undergoing management of a mental health disorder. METHOD: Twenty six participants were allocated into two groups, those reporting as apparently healthy (AH, n = 13) or those undergoing the management of a mental health disorder (MI, n = 13). Following a baseline testing and familiarization session, participants commenced the 6-week aerobic training intervention, involving stationary cycling at 65% heart rate reserve for 35 min progressing to 70% for 40 min. Measures of aerobic fitness (VO2peak), anthropometric variables, symptom questionnaires and venous blood were collect pre- and post-intervention. Venous blood was assessed for nod-like receptor pyrin containing-3, interleukin (IL)-6, tumor necrosis factor-alpha (TNF-α), IL-1ß, C-reactive protein (CRP) and brain-derived neurotropic factor (BDNF). RESULTS: There were no baseline differences between groups, however following the intervention the AH demonstrated lower TNF-α (p = 0.049) than the MI group. Within change was observed for the MI group with an increase in VO2peak (p = 0.049) and declines in symptom severity (p = 0.00-0.005). Significant correlations between variables indicated a positive association between body fat, body fat percentage, CRP and symptom severity (p = 0.01-0.04). Conversely, symptom severity and CRP were inversely associated with VO2peak values (p = 0.02-0.04). CONCLUSION: Six-weeks of moderate intensity aerobic exercise increases VO2peak and reduces symptom severity in those currently undergoing management of a mental health disorder. Further, there may be a physiological link between aerobic capacity, symptom severity, inflammation and adiposity, however greater exploration is required.

The Inflammasome Signaling Pathway Is Actively Regulated and Related to Myocardial Damage in Coronary Thrombi from Patients with STEMI.

BACKGROUND: The Nod-Like-Receptor-Protein-3 (NLRP3) inflammasome and the Interleukin-6 (IL-6) pathways are central mechanisms of the inflammatory response in myocardial reperfusion injury. Expanding our knowledge about the inflammasome signaling axis is important to improve treatment options. In a cross-sectional study, we aimed to study presence, localization, and genetic expression of inflammasome- and IL-6- signaling-related proteins in coronary thrombi and circulating leukocytes from ST-elevation myocardial infarction (STEMI) patients, with relation to myocardial injury and time from symptoms to PCI. METHODS: Intracoronary thrombi were aspirated from 33 STEMI patients. Blood samples were drawn. mRNA of Toll-Like-Receptor-4 (TLR4), NLRP3, caspase 1, Interleukin-1ß (IL1-ß), Interleukin-18 (IL-18), IL-6, IL-6-receptor (IL-6R), and glycoprotein 130 (gp130) were isolated from thrombi and circulating leukocytes and relatively quantified by RT-PCR. A part of each thrombus was embedded in paraffin for histology and immunohistochemistry analyses. RESULTS: Genes encoding the 8 markers were present in 76-100% of thrombi. Expression of TLR4 in thrombi significantly correlated to troponin T (r = 0.455, p = 0.013), as did NLRP3 (r = 0.468, p = 0.024). Troponin T correlated with expression in circulating leukocytes of TLR4 (r = 0.438, p = 0.011), NLRP3 (r = 0.420, p = 0.0149), and IL-1ß (r = 0.394, p = 0.023). IL-6R expression in thrombi correlated significantly to troponin T (r = 0.434, p = 0.019), whereas gp130 was inversely correlated (r = -0.398, p = 0.050). IL-6 in circulating leukocytes correlated inversely to troponin T (r = -0.421, p = 0.015). There were no significant correlations between genes expressed in thrombi and time from symptom to PCI. CONCLUSIONS: The inflammasome signaling pathway was actively regulated in coronary thrombi and in circulating leukocytes from patients with STEMI, in association with myocardial damage measured by troponin T. This supports the strategy of medically targeting this pathway in treating myocardial infarction and contributes to sort out optimal timing and targets for anti-inflammatory treatment. The study is registered at clinicaltrials.gov with identification number NCT02746822.

Inflammatory cytokines derived from peripheral blood contribute to the modified electroconvulsive therapy-induced cognitive deficits in major depressive disorder.

Little is known about the pathophysiology of memory deficits in patients with major depressive disorder (MDD) treated with modified electroconvulsive therapy (MECT). This study examined the profiles of cytokines, the memory function, and their association in MECT-treated MDD patients. Forty first-episode, drug-free MDD patients and 40 healthy controls were recruited. MECT was started with antidepressant treatment at a stable initial dose. The Wechsler Memory Scale (WMS) and Hamilton Rating Scale for Depression 17 (HRSD-17) were used to assess the cognitive function. MDD patients were divided into the memory impairment group (WMS < 50) and the non-memory impairment group (WMS ≥ 50) based on the total WMS scores after MECT. The levels of NOD-like receptor 3 (NLRP3) inflammasome, interleukin-18 (IL-18) and nuclear factor kappa-B (NF-κB) in the serum were measured. MDD patients showed significantly higher levels of NLRP3 inflammasome, IL-18 and NF-κB than that in the controls prior to MECT, and the levels also significantly increased after MECT. In MDD patients, the serum levels of these inflammatory cytokines were negatively associated with the total WMS scores and likely contributed to the scores independently. The receiver operating characteristic curve showed that the serum levels of these inflammatory cytokines may predict the cognitive impairment risk in MDD patients receiving MECT. Abnormal levels of NLRP3 inflammasome, IL-18 and NF-κB reflecting the disturbed balance of pro-inflammatory and anti-inflammatory mechanisms likely contribute to the MECT-induced cognitive deficits in MDD patients.

Serum Copeptin, NLPR3, and suPAR Levels among Patients with Autosomal-Dominant Polycystic Kidney Disease with and without Impaired Renal Function.

BACKGROUND: The pathophysiology of renal disease progression in autosomal-dominant polycystic kidney disease (ADPKD) involves not only cystogenesis but also endothelial dysfunction, leading to the activation of inflammatory and fibrotic pathways. This study evaluated the levels of biomarkers related to osmoregulation, immune system activation, and tubular injury in ADPKD patients with impaired or preserved renal function. METHODS: This study included 26 ADPKD patients with modestly impaired renal function (estimated glomerular filtration rate [eGFR] 45-70 mL/min/1.73 m2; Group A), 26 age- and sex-matched ADPKD patients with relatively preserved renal function (eGFR >70 mL/min/1.73 m2; Group B), and 26 age- and sex-matched controls (Group C). Serum levels of copeptin, the inflammasome nucleotide-binding and oligomerization domain-like receptors pyrin domain-containing protein 3 (NLRP3), and soluble urokinase-type plasminogen activator receptor (suPAR) were measured with ELISA techniques. RESULTS: Patients in Group A had higher levels of copeptin (median [interquartile range]: 50.44 [334.85] pg/mL), NLRP3 (5.86 [3.89] ng/mL), and suPAR (390.05 [476.53] pg/mL) compared to patients in Group B (32.38 [58.33], p = 0.042; 2.42 [1.96], p < 0.001; and 313.78 [178.85], p = 0.035, respectively) and Group C (6.75 [6.43]; 1.09 [0.56]; and 198.30 [28.53], respectively; p < 0.001 for all comparisons). Levels of all studied markers were also significantly higher in Group B patients compared to controls (p < 0.001), despite having similar eGFR. In patients with ADPKD, all studied biomarkers were correlated positively with asymmetric-dimethylarginine (ADMA) and endocan levels, and negatively with eGFR. ADMA and endocan levels were the only parameters independently associated with increased copeptin levels. CONCLUSIONS: This study showed that ADPKD patients with impaired and preserved renal function had higher copeptin, NLRP3, and suPAR levels than controls. Such findings support that cystogenesis and inflammation are associated with endothelial dysfunction, even in the early stages of ADKPD.

Peripheral blood expression levels of inflammasome complex components in two different focal epilepsy syndromes.

BACKGROUND: Although the role of inflammation in epilepsy pathogenesis has been extensively investigated, the inflammasome complex, a key component of neuroinflammation, has been understudied in epilepsy patients. METHODS: To better understand the involvement of this system in epilepsy, levels of inflammasome complex components (NLRP1, NLRP3, CASP1, ASC), end-products of inflammasome complex activity [IL-1ß, IL-18, nitric oxide synthase (NOS) isoforms] and other inflammatory factors (NFκB, IL-6, TNF-α) were measured in peripheral blood of patients with focal epilepsy of unknown cause (FEoUC) (n = 47), mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) (n = 35) and healthy controls using real time qPCR and/or ELISA. RESULTS: Inflammasome complex associated factors were either downregulated or unchanged in epilepsy patients. Likewise, flow cytometry studies failed to show an increase in ratios of NLRP3-expressing CD3+ and CD14+ peripheral blood mononuclear cells (PBMC) in epileptic patients. Anti-neuronal antibody positive epilepsy patients showed increased NLRP1 and neuronal NOS mRNA expression levels, whereas patients under poly-therapy showed reduced serum inflammasome levels. FEoUC patients demonstrated increased PBMC NFκB mRNA expression levels and serum IL-1ß and IL-6 levels. Both MTLE-HS and FEoUC patients displayed higher ratios of NFκB-expressing CD14+ PBMC than healthy controls. CONCLUSIONS: Although previous clinical studies have implicated increased inflammasome complex expression levels in epilepsy, our results indicate suppressed inflammasome complex activity in the peripheral blood of focal epilepsy patients. Alternatively, the IL-6-NFκB signaling pathway, appears to be activated in focal epilepsy, suggesting that factors of this pathway might be targeted for future theranostic applications.

Systemic juvenile idiopathic arthritis and recurrent macrophage activation syndrome due to a CASP1 variant causing inflammasome hyperactivation.

OBJECTIVES: We investigated a patient with systemic juvenile idiopathic arthritis (sJIA) and recurrent macrophage activation syndrome (MAS) to discover genetic and immunological contributing factors. METHODS: Severe recurrent MAS motivated whole exome sequencing (WES) to identify genetic variants potentially involved in disease pathogenesis. In vitro peripheral blood mononuclear cell (PBMC) stimulations for cytokine expression and caspase-1 activity assays as well as NF-κB reporter luciferase assays were performed to functionally characterize variants. RESULTS: WES revealed an extremely rare heterozygous missense variant, c.482G>A, p.R161H in the CASP1 gene encoding pro-caspase-1. Lipopolysaccharide (LPS) stimulation of patient PBMCs induced high levels of IL-6 compared to controls, and activation of the NLRP3 inflammasome resulted in increased production of IL-1ß and IL-18 as well as significantly elevated caspase-1 activity. Constitutive and inducible levels of IL-18 and IFNγ in whole blood were markedly elevated. Expression of the CASP1 variant in an NF-κB reporter luciferase assay induced increased NF-κB activation in a RIP2-dependent manner. The disease course of the patient was complicated by severe recurrent MAS. However, dual IL-1 and IL-6 blockade caused disease remission. CONCLUSION: For the first time, we demonstrate the involvement of a CASP1 variant in sJIA and recurrent MAS. This variant is gain-of-function for both inflammasome and NF-κB activation leading to increased production of IL-6, IL-1ß and IL-18. Although dual IL-1 and IL-6 blockade may be beneficial in patients, in whom single treatment is not sufficient to control MAS, caution should be practiced, since interstitial lung disease may progress despite apparent clinical and biochemical remission.

NLRP3 Inflammasome Gene Polymorphisms Variably Associated with its Serum Levels in Acute Myocardial Infarction.

BACKGROUND AND OBJECTIVE: Nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) plays a pivotal role in initiation of inflammation. genetic variation in NLRP3 gene have been proposed to predispose several inflammatory diseases. This study aimed to evaluate the risk of NLRP3 (exon 3) gene polymorphisms and its relation with serum NLRP3 among myocardial infarction. MATERIALS AND METHODS: Case-control study involved 69 patients with Myocardial Infarction and 53 controls, from each subject 3 mL were collected and used for DNA extraction then the amplified exon 3 genes were sequenced by Sanger method. Serum NLRP3 was quantified using sandwich ELISA. RESULTS: According to the results Q705K found to possess a 16.21 times risk for MI incidence compared with controls. In addition, 44 novel single nucleotide polymorphisms have been identified at the position 14347, 14261, 14240 and 14229 and their allelic variants as a risk factor for MI incidence as 3.92, 8.6, 2.04 and 4.57 when compared with their relevant allele in controls respectively. Statistically high level of serum NLRP3 (1.7 ng mL-1) among MI patients compared to controls (0.71 ng mL-1). The 0.75 ng mL-1 considered as a good predictor for MI with ECG findings. Only Q705K and 14229 genetic variant alleles were significantly associated with high NLRP3 protein serum level among MI patients. CONCLUSION: Four novel SNPs in exon 3 of NLRP3 gene in addition to previously reported Q705K conferring risk for development of MI among Iraqis. Only variants allele of Q705K and gene position 14229 was associated with elevated serum NLRP3 protein among MI patients.

Interleukin-31 interaction with inflammasome: A promising diagnostic and prognostic panel for early sepsis identification in critically ill patients.

PURPOSE: This case-control study aimed to evaluate the ability to use a panel of IL-31, IL-1ß and NLRP3 to differentiate sepsis from systemic inflammatory response syndrome (SIRS) and to predict septic shock. METHODS: Serum levels of IL-31, IL-1ß and NLRP3 were measured by ELISA in 149 participants; 38 with sepsis, 51 with SIRS, 30 with septic shock and 30 healthy controls. RESULTS: Lower levels of IL-31 were found in sepsis (10.21 ± 4.34 pg/ml) compared to SIRS (16.74 ± 3.18 pg/ml) and to controls with the lowest levels detected in septic shock (6.26 ± 2.72 pg/ml). IL-1ß and NLRP3 levels were higher in sepsis (54.99 ± 14.11 pg/ml and 9.93 ± 2.38 ng/ml) compared to SIRS (27.8 ± 6.94 pg/ml and 4.86 ± 1.33 ng/ml) with the highest levels seen in septic shock (125.1 ± 32.79 pg/ml and 19.43 ± 6.48 ng/ml) respectively. IL-31 discriminated sepsis in patients showing SIRS with 80% sensitivity and 70% specificity and, identified septic shock with 78.6% sensitivity and 60.3% specificity. IL-1ß identified sepsis from SIRS with 93.3% and 83.3% specificity. NLRP3 discriminated sepsis from SIRS with 94.5% sensitivity and 93.3% specificity. And, with sensitivity 99.1% and 90.1% and specificity 98.9% and 80% IL-1ß and NLRP3 could respectively define septic shock. A panel of combined markers provided 100% sensitivity and specificity. The three biomarkers proved to be independent prognostic biomarkers. At 95% CI, IL-31 hazard ratio (HR) was 0.716, p = 0.001; IL-1ß HR was 1.023, p ≤ 0.001; and NLRP3 HR was 1.114, p ≤ 0.001. Additionally, IL-1ß proved to be an independent predictor of septic shock (ß = 0.355; p = 0.035). CONCLUSION: The cross-relation between IL-31, IL-1ß and NLRP3 in sepsis can provide a promising diagnostic and prognostic panel.

Quartz Dust Exposure Affects NLRP3 Inflammasome Activation and Plasma Levels of IL-18 and IL-1Ra in Iron Foundry Workers.

PURPOSE: To study the association between inhalation of particulate matter or quartz in Swedish iron foundries and the effects on NLRP3 inflammasome activation. METHODS: Particle exposure measurements were performed during an eight-hour work day for 85 foundry workers at three Swedish iron foundries. Personal sampling was used for measurement of respirable quartz and dust and stationary measurements to obtain exposure measurements for inhalable dust and PM10. The NLRP3 inflammasome markers, interleukin- (IL-) 1ß and IL-18, and inhibitors IL-1 receptor antagonist (IL-1Ra) and IL-18 binding protein (IL-18BP) were measured in plasma. Inflammasome activation was measured by caspase-1 enzymatic activity in monocytes in whole blood by flow cytometry, and expression of inflammasome-related genes was quantified using real-time PCR. Multiple linear regression analysis was used to investigate associations between PM exposures and inflammatory markers. Sex, age, smoking, current infection, BMI, and single nucleotide polymorphism in the inflammasome regulating genes CARD8 (C10X) and NLRP3 (Q705K) were included as covariates. RESULTS: The average exposure levels of respirable dust and quartz were 0.85 and 0.052 mg/m3, respectively. A significant exposure-response was found for respirable dust and IL-18 and for inhalable dust and IL-1Ra. Whole blood, drawn from study participants, was stimulated ex vivo with inflammasome priming stimuli LPS or Pam3CSK4, resulting in a 47% and 49% increase in caspase-1 enzymatic activity in monocytes. This increase in caspase-1 activity was significantly attenuated in the higher exposure groups for most PM exposure measures. CONCLUSIONS: The results indicate that exposure levels of PM in the iron foundry environment can affect the NLRP3 inflammasome and systemic inflammation.