NLRP3 inflammasome-induced pyroptosis and serum ASC specks are increased in patients with cardiogenic shock.

Cardiogenic shock (CS) is characterized by impaired cardiac function, very high mortality, and limited treatment options. The proinflammatory signaling during different phases of CS is incompletely understood. We collected serum and plasma (n = 44) as well as freshly isolated peripheral blood mononuclear cells (PBMCs, n = 7) of patients with CS complicating acute myocardial infarction on admission and after revascularization (24, 48, and 72 h) and of healthy controls (serum and plasma, n = 75; PBMCs, n = 12). PBMCs of patients with CS had increased gene expression of NLRP3, CASP1, PYCARD, IL1B, and IL18 and showed increased rates of pyroptosis (control, 4.7 ± 0.3 vs. 9.9 ± 1.7% in patients with CS, P = 0.02). Serum interleukin (IL)-1ß levels were increased after revascularization. IL-18 and IL-6 were higher in patients with CS than in healthy controls but comparable before and after revascularization. Proinflammatory apoptosis-associated speck-like proteins containing CARD (ASC) specks were elevated in the serum of patients with CS on admission and increased after revascularization (admission, 11.1 ± 4.4 specks/µL; after 24 h, 19.0 ± 3.9, P = 0.02). ASC specks showed a significant association with 30-day mortality in patients with CS (P < 0.05). The estimated regression coefficients and odds ratios indicated a positive relationship between ASC specks and mortality (odds ratio: 1.029, 95% confidence interval, 1.000 to 1.072; P = 0.02). Pyroptosis and circulating ASC specks are increased in patients with CS and are particularly induced after reperfusion. This underscores their potential role as a biomarker for poor outcomes in patients with CS. ASC specks represent promising new therapeutic targets for patients with CS with high inflammatory burden.NEW & NOTEWORTHY The expression of NLR family pyrin domain containing-3 (NLRP3) inflammasome-related genes and the rate of pyroptosis are increased in PBMCs from patients with CS. Furthermore, patients with CS are characterized by higher serum concentrations of ASC specks and IL-1ß, IL-6, and IL-18. This current study adds circulating ASC specks to the portfolio of biomarkers for the identification of patients with a high inflammatory burden paving the way for precision medicine approaches to improve clinical outcomes.

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.

Diagnostic value of serum NLRP3, metalloproteinase-9 and interferon-γ for postoperative hydrocephalus and intracranial infection in patients with severe craniocerebral trauma.

Traumatic brain injury (TBI) is a major cause of morbidity and mortality globally. We unveiled the diagnostic value of serum NLRP3, metalloproteinase-9 (MMP-9) and interferon-γ (IFN-γ) levels in post-craniotomy intracranial infections and hydrocephalus in patients with severe craniocerebral trauma to investigate the high risk factors for these in patients with TBI, and the serological factors predicting prognosis, which had a certain clinical predictive value. Study subjects underwent bone flap resection surgery and were categorized into the intracranial infection/hydrocephalus/control (without postoperative hydrocephalus or intracranial infection) groups, with their clinical data documented. Serum levels of NLRP3, MMP-9 and IFN-γ were determined using ELISA kits, with their diagnostic efficacy on intracranial infections and hydrocephalus evaluated by receiver operating characteristic curve analysis. The independent risk factors affecting postoperative intracranial infections and hydrocephalus were analysed by logistic multifactorial regression. The remission after postoperative symptomatic treatment was counted. The intracranial infection/control groups had significant differences in Glasgow Coma Scale (GCS) scores, opened injury, surgical time and cerebrospinal fluid leakage, whereas the hydrocephalus and control groups had marked differences in GCS scores, cerebrospinal fluid leakage and subdural effusion. Serum NLRP3, MMP-9 and IFN-γ levels were elevated in patients with post-craniotomy intracranial infections/hydrocephalus. The area under the curve values of independent serum NLRP3, MMP-9, IFN-γ and their combination for diagnosing postoperative intracranial infection were 0.822, 0.722, 0.734 and 0.925, respectively, and for diagnosing hydrocephalus were 0.865, 0.828, 0.782 and 0.957, respectively. Serum NLRP3, MMP-9 and IFN-γ levels and serum NLRP3 and MMP-9 levels were independent risk factors influencing postoperative intracranial infection and postoperative hydrocephalus, respectively. Patients with hydrocephalus had a high remission rate after postoperative symptomatic treatment. Serum NLRP3, MMP-9 and IFN-γ levels had high diagnostic efficacy in patients with postoperative intracranial infection and hydrocephalus, among which serum NLRP3 level played a major role.

Fluctuation trend of inflammatory indexes related to gestational diabetes mellitus from second trimester to third trimester of pregnancy.

OBJECTIVE: This study aims to assess the prognostic and diagnostic value of inflammatory indexes related to gestational diabetes mellitus (GDM) from the second trimester to the third trimester of pregnancy. MATERIALS AND METHODS: In this study, we randomly selected 65 pregnant women diagnosed with GDM at our hospital from December 2022 to June 2023 to form the GDM group (n = 65). Additionally, 65 pregnant women at the same gestational weeks without GDM were selected as the Normal group (n = 65). We collected gestational information and serum samples at 24 and 36 weeks of gestation from the participants. The levels of NLRP3, IL-1Ra, and TBP-2 were determined using enzyme-linked immunosorbent assay (ELISA) to explore their changes during pregnancy. Further, this study analyzed the changes in the levels of NLRP3, IL-1Ra, and TBP-2 at 24 and 36 weeks of gestation in GDM patients and their correlation with gestational diabetes mellitus. RESULTS: The study showed that pre-pregnancy body mass index (BMI), neonatal weight, gestational hypertension, and macrosomia are significantly associated with the occurrence of GDM (P < 0.05). Statistical analysis comparing the normal and GDM groups found no significant changes in the levels of NLRP3, IL-1Ra, and TBP-2 with the progression of gestation in the normal group. In contrast, in the GDM group, the levels of IL-1Ra in serum samples at 24 and 36 weeks were significantly increased (P < 0.05) while the levels of NLRP3 and TBP-2 were significantly reduced (P < 0.05). At 36 weeks, there was a positive correlation between the levels of NLRP3, IL-1Ra, and TBP-2. Compared to the normal group, the overall levels of NLRP3, IL-1Ra, and TBP-2 in the GDM group were lower (P < 0.05) and the weight of the newborns was significantly correlated with these three indicators (P < 0.05), specifically newborn weight increased with the levels of NLRP3 and TBP-2 but decreased with the increase of IL-1Ra (P < 0.05). Multifactorial logistic regression analysis further revealed that NLRP3 is an independent factor influencing GDM (P < 0.05). ROC curve analysis of the NLRP3 level at 24 weeks of gestation found that NLRP3 has a good value in predicting GDM (AUC = 0.720, 95%CI 0.630-0.809, P < 0.001) and the combined prediction of NLRP3, IL-1Ra, and TBP-2 also showed a good predictive value for GDM. CONCLUSION: The changes in NLRP3, IL-1Ra, and TBP-2 persisted throughout the 24 to 36 weeks of gestation, playing an important role in predicting the occurrence of GDM and the weight of the newborn.

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.

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.

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.

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.

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.

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.

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.

The role of microglia mediated pyroptosis in neonatal hypoxic-ischemic brain damage.

Neonatal hypoxic-ischemic encephalopathy (HIE) often leads to neonatal death or severe, irreversible neurological deficits. Pathologically, the occurrence of massive cell death and subsequent inflammation suggested that pyroptosis, an inflammation associated programed cell death, might play a role in HIE. Here, by measuring changes of key molecules in pyroptosis pathway in HIE patients, we discovered that their elevation levels tightly correlate with the severity of HIE. Next, we demonstrated that application of MCC950, a small molecule to inhibit NLRP3 inflammasome and thus pyroptosis, substantially alleviated pyroptosis and the injury severity in rats with neonatal hypoxic-ischemic brain damage (HIBD). Mechanistically, we showed that NLRP-3/caspase-1/GSDMD axis is required for microglia pyroptosis and activation. Our data demonstrated that microglia mediated pyroptosis played a crucial role in neonatal HIE, which shed lights into the development of intervention avenues targeting pyroptosis to treat HIE and traumatic brain injuries.

Systemic activation of NLRP3 inflammasome and plasma α-synuclein levels are correlated with motor severity and progression in Parkinson's disease.

BACKGROUND: Emerging evidence indicates that inflammasome-induced inflammation plays a crucial role in the pathogenesis of Parkinson's disease (PD). Several proteins including α-synuclein trigger the activation of NLRP3 inflammasome. However, few studies examined whether inflammasomes are activated in the periphery of PD patients and their possible value in the diagnosis or tracking of the progress of PD. The aim of this study was to determine the association between inflammasome-induced inflammation and clinical features in PD. METHODS: There were a total of 67 participants, including 43 patients with PD and 24 controls, in the study. Participants received a complete evaluation of motor and non-motor symptoms, including Hoehn and Yahr (H-Y) staging scale. Blood samples were collected from all participants. The protein and mRNA expression levels of inflammasomes subtypes and components in peripheral blood mononuclear cells (PBMCs) were determined using western blotting and RT-qPCR. We applied Meso Scale Discovery (MSD) immunoassay to measure the plasma levels of IL-1ß and α-synuclein. RESULTS: We observed increased gene expression of NLRP3, ASC, and caspase-1 in PBMCs, and increased protein levels of NLRP3, caspase-1, and IL-1ß in PD patients. Plasma levels of IL-1ß were significantly higher in patients with PD compared with controls and have a positive correlation with H-Y stage and UPDRS part III scores. Furthermore, plasma α-synuclein levels were also increased in PD patients and have a positive correlation with both UPDRS part III scores and plasma IL-1ß levels. CONCLUSIONS: Our data demonstrated that the NLRP3 inflammasome is activated in the PBMCs from PD patients. The related inflammatory cytokine IL-1ß and total α-synuclein in plasma were increased in PD patients than controls, and both of them presented a positive correlation with motor severity in patients with PD. Furthermore, plasma α-synuclein levels have a positive correlation with IL-1ß levels in PD patients. All these findings suggested that the NLRP3 inflammasome activation-related cytokine IL-1ß and α-synuclein could serve as non-invasive biomarkers to monitor the severity and progression of PD in regard to motor function.

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.

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.

Clinical Significance of High-Mobility Group Box 1 Protein (HMGB1) and Nod-Like Receptor Protein 3 (NLRP3) in Patients with Ulcerative Colitis.

BACKGROUND The aim of this study was to investigate the clinical significance of NLRP3 and HMGB1 in patients with active ulcerative colitis. MATERIAL AND METHODS This was a prospective observational study which included a total of 62 cases with active ulcerative colitis during January 2017 to December 2018. The patients were divided into a mild/moderate group or a severe group according to Sutherland Disease Activity Index (DAI) score. Clinical activity index and endoscopic index were used to determine the severity of UC. Serum levels of NLRP3, HMGB1, endothelin-1, IL-1ß, and TNF-alpha were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS Sutherland DAI score, clinical activity index, and endoscopic index were all significantly higher in severe patients than in the mild/moderate group. NLRP3, HMGB1, endothelin-1, IL-1ß, and TNF-alpha were significantly higher in severe UC patients. NLRP3 level was positively correlated with HMGB1, ET-1, IL-1ß, and TNF-alpha levels. Both NLRP3 and HMGB1 were positively correlated with Sutherland DAI score, clinical activity index, and endoscopic index. CONCLUSIONS Both serum NLRP3 and HMGB1 were elevated in UC patients, and the serum levels of NLRP3 were positively correlated with serum levels of HMGB1, ET-1, IL-1ß, and TNF-alpha, as well as severity of UC patients.

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.

TLR4-dependent upregulation of the platelet NLRP3 inflammasome promotes platelet aggregation in a murine model of hindlimb ischemia.

Platelets play a critical role in the pathophysiology of peripheral arterial disease (PAD). The mechanisms by which muscle ischemia regulates aggregation of platelets are poorly understood. We have recently identified the Nod-like receptor nucleotide-binding domain leucine rich repeat containing protein 3 (NLRP3) expressed by platelets as a critical regulator of platelet activation and aggregation, which may be triggered by activation of toll-like receptor 4 (TLR4). In this study, we performed femoral artery ligation (FAL) in transgenic mice with platelet-specific ablation of TLR4 (TLR4 PF4) and in NLRP3 knockout (NLRP3-/-) mice. NLRP3 inflammasome activity of circulating platelets, as monitored by activation of caspase-1 and cleavage of interleukin-1ß (IL-1ß), was upregulated in mice subjected to FAL. Genetic ablation of TLR4 in platelets led to decreased platelet caspase 1 activation and platelet aggregation, which was reversed by the NLRP3 activator Nigericin. Two weeks after the induction of FAL, ischemic limb perfusion was increased in TLR4 PF4 and NLRP3-/- mice as compared to control mice. Hence, activation of platelet TLR4/NLRP3 signaling plays a critical role in upregulating platelet aggregation and interfering with perfusion recovery in muscle ischemia and may represent a therapeutic target to improve limb salvage.

Caspase-1 inflammasome activity in patients with Staphylococcus aureus bacteremia.

The inflammasome is a multiprotein complex that mediates caspase-1 activation with subsequent maturation of the proinflammatory cytokines IL-1ß and IL-18. The NLRP3 inflammasome is known to be activated by Staphylococcus aureus, one of the leading causes of bacteremia worldwide. Inflammasome activation and regulation in response to bacterial infection have been found to be of importance for a balanced host immune response. However, inflammasome signaling in vivo in humans initiated by S. aureus is currently sparsely studied. This study therefore aimed to investigate NLRP3 inflammasome activity in 20 patients with S. aureus bacteremia (SAB), by repeated measurement during the first week of bacteremia, compared with controls. Caspase-1 activity was measured in monocytes and neutrophils by flow cytometry detecting FLICA (fluorescent-labeled inhibitor of caspase-1), while IL-1ß and IL-18 was measured by Luminex and ELISA, respectively. As a measure of inflammasome priming, messenger RNA (mRNA) expression of NLRP3, CASP1 (procaspase-1), and IL1B (pro-IL-1ß) was analyzed by quantitative PCR. We found induced caspase-1 activity in innate immune cells with subsequent release of IL-18 in patients during the acute phase of bacteremia, indicating activation of the inflammasome. There was substantial interindividual variation in caspase-1 activity between patients with SAB. We also found an altered inflammasome priming with low mRNA levels of NLRP3 accompanied by elevated mRNA levels of IL1B. This increased knowledge of the individual host immune response in SAB could provide support in the effort to optimize management and treatment of each individual patient.

Glucocorticoid Regulates NLRP3 in Acute-On-Chronic Hepatitis B Liver Failure.

Acute-on-chronic hepatitis B liver failure (ACHBLF) refers to the acute deterioration of liver function during chronic hepatitis B virus infection, and is associated with high mortality, with rapid progression to death. Nucleotide-binding oligomerisation domain-like receptors (NLRs) Family Pyrin Domain Containing 3(NLRP3) inflammasome contributed to the pathogenesis of D-galactosamine and lipopolysaccharide-induced acute liver failure. However, the profile of NLRP3 in patients with ACHBLF has not been demonstrated. This study was therefore conducted to investigate the expression of NLRP3 in patients with ACHBLF and identify the effect of glucocorticoid on NLRP3. We recruited 70 patients with ACHBLF undergoing glucocorticoid treatment for 28 days, 30 patients with chronic hepatitis B (CHB), and 24 healthy controls (HCs) in this study. The relative messenger RNA (mRNA) level of NLRP3 and related genes were measured by reverse transcription polymerase chain reaction, the plasma levels of interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) were measured by enzyme-linked immunosorbent assay. The mRNA level of NLRP3 was significantly higher in patients with ACHBLF than in patients with CHB as well as HCs (P<0.05). The plasma levels of IL-1ß and IL-18 in patients with ACHBLF were significantly higher than in patients with CHB and HCs (P<0.05). The relative mRNA level of NLRP3 in surviving patients decreased significantly compared with that in patients who did not survive after glucocorticoid treatment (P<0.05). In conclusion, NLRP3 increased in patients with ACHBLF. Glucocorticoid could downregulate the expression of NLRP3 in surviving patients with ACHBLF.