Markers Of Inflammasome Activation Research Articles

Beneficial Effects of Human Schwann Cell-Derived Exosomes in Mitigating Secondary Damage After Penetrating Ballistic-Like Brain Injury.

There is a growing body of evidence that the delivery of cell-derived exosomes normally involved in intracellular communication can reduce secondary injury mechanisms after brain and spinal cord injury and improve outcomes. Exosomes are nanometer-sized vesicles that are released by Schwann cells and may have neuroprotective effects by reducing post-traumatic inflammatory processes as well as promoting tissue healing and functional recovery. The purpose of this study was to evaluate the beneficial effects of human Schwann-cell exosomes (hSC-Exos) in a severe model of penetrating ballistic-like brain injury (PBBI) in rats and investigate effects on multiple outcomes. Human Schwann cell processing protocols followed Current Good Manufacturing Practices (cGMP) with exosome extraction and purification steps approved by the Food and Drug Administration for an expanded access single ALS patient Investigational New Drug. Anesthetized male Sprague-Dawley rats (280-350g) underwent PBBI surgery or Sham procedures and, starting 30 min after injury, received either a dose of hSC-Exos or phosphate-buffered saline through the jugular vein. At 48h after PBBI, flow cytometry analysis of cortical tissue revealed that hSC-Exos administration reduced the number of activated microglia and levels of caspase-1, a marker of inflammasome activation. Neuropathological analysis at 21 days showed that hSC-Exos treatment after PBBI significantly reduced overall contusion volume and decreased the frequency of Iba-1 positive activated and amoeboid microglia by immunocytochemical analysis. This study revealed that the systemic administration of hSC-Exos is neuroprotective in a model of severe TBI and reduces secondary inflammatory injury mechanisms and histopathological damage. The administration of hSC-Exos represents a clinically relevant cell-based therapy to limit the detrimental effects of neurotrauma or other progressive neurological injuries by impacting multiple pathophysiological events and promoting neurological recovery.

Markers of inflammasome activation in Coronavirus disease 2019 (COVID-19) Egyptian patients: Impact on disease severity

There are conflicting data regarding the relationship between coronavirus disease 2019 (COVID-19) severity and Caspase-1 (Casp-1), interleukin-1ß (IL-1β), and IL-18. Our study sought to quantify the levels of IL-18, IL-1ß, and Casp-1 as indicators for inflammasome activation in COVID-19 patients at Assiut University Hospitals and to correlate their levels with parameters of disease severity in COVID-19 patients. Serum levels of Casp-1, IL-1β and IL-18 were measured in 63 COVID-19 patients and 26 normal controls by an enzyme linked immunosorbent assay (ELISA). Also, arterial blood gas analysis and laboratory parameters including hemoglobin, platelets, lymphocyte count, liver function test, kidney function test, C-reactive protein (CRP), D-dimer, ferritin and LDH were estimated. Serum levels of Casp-1, IL-1β and IL-18 were significantly higher in the COVID-19 group as compared to controls (p= 0.04, p=0.001 and p=0.03, respectively). Although the three markers were higher in the severe group, yet only IL-1ß showed a significant difference as compared to the non-severe group (p=0.04). IL-18 had significant positive correlations with CRP and ferritin (p = 0.04 and p = 0.02, respectively). IL-1β was positively correlated with alanine aminotransferase. Casp-1 had significant positive correlations with CRP and lactate dehydrogenase (p=0.045 and p=0.001, respectively). Patients showed weak positive correlations between serum level of Casp-1 and each of IL-1β and IL-18. Also, a strong positive correlation was found between IL-1β and IL-18 (p < 0.0001). In conclusion, inflammasome activation was a hallmark in COVID-19 patients. The markers of activation were positively correlated with many parameters of inflammation, may suggest their important roles in the pathophysiology of the disease and its progression. IL-1β was the only marker to be correlated with disease severity and therefore may be suggested as a potential marker for identifying severe COVID-19 patients.

Elevated Systemic Levels of Markers Reflecting Intestinal Barrier Dysfunction and Inflammasome Activation Are Correlated in Severe Mental Illness.

Gut microbiota alterations have been reported in severe mental illness (SMI) but fewer studies have probed for signs of gut barrier disruption and inflammation. We hypothesized that gut leakage of microbial products due to intestinal inflammation could contribute to systemic inflammasome activation in SMI. We measured plasma levels of the chemokine CCL25 and soluble mucosal vascular addressin cell adhesion molecule-1 (sMAdCAM-1) as markers of T cell homing, adhesion and inflammation in the gut, lipopolysaccharide binding protein (LBP) and intestinal fatty acid binding protein (I-FABP) as markers of bacterial translocation and gut barrier dysfunction, in a large SMI cohort (n = 567) including schizophrenia (SCZ, n = 389) and affective disorder (AFF, n = 178), relative to healthy controls (HC, n = 418). We assessed associations with plasma IL-18 and IL-18BPa and leukocyte mRNA expression of NLRP3 and NLRC4 as markers of inflammasome activation. Our main findings were: (1) higher levels of sMAdCAM-1 (P = .002), I-FABP (P = 7.6E-11), CCL25 (P = 9.6E-05) and LBP (P = 2.6E-04) in SMI compared to HC in age, sex, BMI, CRP and freezer storage time adjusted analysis; (2) the highest levels of sMAdCAM-1 and CCL25 (both P = 2.6E-04) were observed in SCZ and I-FABP (P = 2.5E-10) and LBP (3) in AFF; and (3), I-FABP correlated with IL-18BPa levels and LBP correlated with NLRC4. Our findings support that intestinal barrier inflammation and dysfunction in SMI could contribute to systemic inflammation through inflammasome activation.

Caspase-1-responsive fluorescence biosensors for monitoring endogenous inflammasome activation

The activation of inflammasome leads to secretion of inflammatory factors and cell pyroptosis that are critical in the pathogenesis of various chronic and acute inflammatory diseases. Recruitment and activation of caspase-1 is a marker of inflammasome activation. However, there is still lack of real-time and efficient methods to detect the activation of inflammasome, especially in vivo. Herein, we developed two activatable caspase-1-responsive fluorescence biosensors, WEHD-HCy and YVAD-HCy, to specifically monitor the activation of inflammasome in vivo. Our in vitro study demonstrated that WEHD-HCy and YVAD-HCy can sensitively and specifically respond to caspase-1 activation. Moreover, these biosensors can efficiency and specifically activated in the common inflammatory disease model, including inflammatory bowel disease, Salmonella infection, and acute arthritis. In particular, WEHD-HCy is more advantageous than YVAD-HCy to specifically image of caspase-1 activity both in vitro and in vivo. These caspase-1-responsive fluorescence biosensors provide an efficient, rapid, and in situ tool for monitoring inflammasome activation, and have the potential to be suitable for clinical diagnosis of various inflammatory diseases associated with inflammasome activation.

Baseline plasma IL-18 may predict simvastatin treatment response in patients with ARDS: a secondary analysis of the HARP-2 randomised clinical trial

BackgroundInterleukin (IL)-18 is a marker of inflammasome activation, and high baseline plasma IL-18 is associated with increased mortality in patients with sepsis-induced ARDS. The aim of this analysis was to determine if simvastatin was associated with benefit in patients with ARDS and high plasma IL-18.MethodsIn this secondary analysis of the HARP-2 study, we compared 28-day mortality and response to simvastatin according to baseline plasma IL-18 using cox proportional hazards analysis. Separately, monocyte-derived macrophages from healthy volunteers were pre-incubated with simvastatin or rosuvastatin before stimulation with ATP and LPS, and the effect on secreted IL-18 and IL-1β compared.Results511 patients from HARP-2 had available data. High baseline plasma IL-18 (≥ 800 pg/ml) was associated with increased 28-day mortality (high IL-18 30.6% vs. low IL-18 17.5%; HR 1.89 [95% CI 1.30–2.73]; p = 0.001). Allocation to simvastatin in patients with high baseline plasma IL-18 was associated with a lower probability of 28-day mortality compared with placebo (24.0% vs 36.8%; p = 0.01). Finally, simvastatin, but not rosuvastatin, reduced stimulated macrophage secretion of IL-18 and IL-1β.ConclusionIn patients with high baseline plasma IL-18, simvastatin is associated with a higher probability of survival, and this effect may be due to reduced inflammasome activation. These data suggest that baseline plasma IL-18 may allow a personalised treatment approach by identifying patients with ARDS who could benefit from simvastatin therapy.

Trikafta Rescues CFTR and Lowers Monocyte P2X7R-induced Inflammasome Activation in Cystic Fibrosis.

Rationale: Cystic fibrosis (CF) is caused by mutations in the CFTR (CF transmembrane conductance regulator) gene and is characterized by sustained inflammation. ATP triggers IL-1β secretion via P2X7R (P2X7 receptor) and activation of the NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3) inflammasome. Objectives: To explore the effect of the CFTR modulator elexacaftor/tezacaftor/ivacaftor (Trikafta) on CFTR expression and the ATP/P2X7R signaling axis in monocytes and on circulating proinflammatory markers. Methods: Inflammatory mediators were detected in blood from 42 patients with CF before and after 3 months of Trikafta therapy. Markers of inflammasome activation and IL-1β secretion were measured in monocytes before and after stimulation with ATP and LPS, in the presence or absence of the P2X7R inhibitor A438079. Measurements and Main Results: P2X7R is overexpressed in CF monocytes, and receptor inhibition decreased NLRP3 expression, caspase-1 activation, and IL-1β secretion. In vitro and invivo, P2X7R expression is regulated by CFTR function and intracellular chloride (Cl-) levels. Trikafta therapy restored CFTR expression yet decreased P2X7R in CF monocytes, resulting in normalized Cl- and potassium efflux, and reduced intracellular calcium levels. CFTR modulator therapy decreased circulating levels of ATP and LPS and reduced inflammasome activation and IL-1β secretion. Conclusions: P2X7R expression is regulated by intracellular Cl- levels and in CF monocytes promotes inflammasome activation. Trikafta therapy significantly increased CFTR protein expression and reduced ATP/P2X7R-induced inflammasome activation. P2X7R may therefore be a promising target for reducing inflammation in patients with CF who are noneligible for Trikafta or other CFTR modulator therapy.

Overactivation of the NLRP3 Inflammasome in Chronic Myelomonocytic Leukemia KRAS Mutated Patients Can be Detected By the Apoptosis-Associated Speck-like Protein (ASC) and Reverted By IL1β Inhibitors

It has been recently shown that RAS mutations, which occur in 11-38% of Chronic Myelomonocytic Leukemia (CMML), do not only act via RAS/MEK/ERK signaling, but contribute to the disease through NLRP3 inflammasome activation (Hamarsheh, Nat Comm 2020). Despite a therapeutic approach based on NLRP3/IL1β axis blockade, as bring to a stem cell transplantation (SCT) has been proposed, data on the efficacy of IL1β inhibitors in hematopoietic neoplasms is limited.A 55 year old man with previous autoinflammatory episodes (constrictive pericarditis) was diagnosed on September 2020 of CMML-1 KRAS G12D (Inter-2). Due to worsening (orchiepidedymitis, pneumonitis, cellulitis), and the impossibility of performing an SCT at that time, on December 02 2020 he started anakinra (a IL1β receptor antagonist) with good response. Due to new episodes of autoinflammation, anakinra was discontinued (12 April 2021) with severe clinical worsening (heart failure) and no response to diuretic/corticosteroid. After anakinra was restarted (04 May 2021), a progressive improvement was seen, allowing a successful pericardiectomy before an SCT. We obtained blood samples from this patient (at different times) and plasma and whole blood samples from 11 and 5 other CMML KRAS mut patients, respectively. We also included CMML patients without KRAS mutations (KRAS wt) (n=8), with sepsis (n=5) and healthy individuals (n=9).Plasma levels of 15 inflammatory cytokines associated with NLRP3 inflammasome and NFkB pathways were measured using a customized MILLIPLEX ® kit. The inflammasome marker activation assays were conducted as previously published (Martínez García JJ, Nature Comm 2019).Compared to healthy controls, KRAS wt CMML patients did not show differences in any cytokine tested, except IL6, while KRAS mut patients showed significantly higher levels of IL1α, IL1ra, IL18, IL12p40 (associated with NLRP3 inflammasome), IL6, IL8 (associated with NFkB pathway) and M-CSF (Fig. 1A B). Compared to KRAS wt CMML patients, those with KRAS mut showed higher levels of cytokines associated with both the NLRP3 and NFkB pathways, reaching statistical significance for those related with NLRP3 inflammasome. We also observed changes in inflammasome related cytokines before and after anakinra (Table 1).This cytokine profile in the plasma made us analyze the oligomerization of ASC as a marker of inflammasome activation in monocytes of KRAS mut CMML. We found that in all cases of KRAS mut CMML patients around 30 to 80% of monocytes presented oligomers of ASC measured by the time of flight assay, while in healthy donors and KRAS wt CMML patients, ASC oligomerization occurred upon NLRP3 inflammasome activation with lipopolysaccharide (LPS) + ATP or Pyrin inflammasome activation with LPS and Clostridium difficile B toxin (TcdB) (Fig. 2A). Ex vivo activation of PBMCs from KRAS mut CMML patients showed that despite the high percentage of cells with ASC oligomers, very low levels of IL1b released from these cells, even when NLRP3 was activated with LPS+ATP (Fig. 2B), suggesting that this inflammasome is activated in vivo and could not be further activated ex vivo. As control, Pyrin inflammasome activation in PBMCs from KRAS mut CMML was able to induce IL1b release similarly to healthy controls (Fig. 2B).We then found that anakinra treatment of the KRAS mut CMML patient followed in this study, resulted in a decrease of the percentage of monocytes with basal active inflammasomes (Fig. 2C). A little ex vivo activation of the NLRP3 inflammasome was obtained when cells were treated with LPS+ATP, while Pyrin inflammasome was activated at normal levels after LPS+TcdB treatment (Fig. 2D). The inflammasome basal activation increased in the monocytes of the KRAS mut CMML patient after anakinra withdraw and during clinical deterioration and restarting anakinra (second arrow) decreased the basal percentage of monocytes with ASC oligomers (Fig. 2C). Since ASC oligomers are associated to pyroptosis via caspase 1 activation and gasdermin D processing, we then analyzed pyroptotic markers in the plasma of the patient during the time. ASC was increased when monocytes presented elevated percentage of ASC oligomers (Fig. 2E), suggesting that ASC detection could be a promising biomarker.Overall, we show that, in vivo, the NLRP3 inflammasome activation of KRAS mut CMML patients may revert with IL1β blockers. ASC could identify those candidates to receive this therapy.PI18/00316 [Display omitted] DisclosuresJerez: Novartis: Consultancy; BMS: Consultancy; GILEAD: Research Funding. Bellosillo: Thermofisher Scientific: Consultancy, Speakers Bureau; Roche: Research Funding, Speakers Bureau; Qiagen: Consultancy, Speakers Bureau. Hernández-Rivas: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ferrer Marin: Cty: Research Funding; Incyte: Consultancy, Research Funding; Novartis: Speakers Bureau.

AIM2 inflammasome mediates apoptotic and pyroptotic death in the cerebellum following chronic hypoperfusion

Vascular dementia (VaD) is the second most common form of dementia and is caused by vascular pathologies resulting in chronic cerebral hypoperfusion (CCH)- induced brain injury, and ultimately cognitive impairment and memory loss. Several lines of evidence have demonstrated chronic inflammation may be involved in VaD disease progression. It is now recognized that a major contributor to cerebral and systemic chronic inflammation involves the activation of innate immune molecular complexes termed inflammasomes. Whilst previous studies on animal models of VaD have focused on the cortex, hippocampus and striatum, few studies have investigated the effect of CCH on the cerebellum. Emerging studies have found new roles of the cerebellum in cognition, based on its structural interconnectivity with other brain regions and clinical relevance in neuropsychological deficits. In the present study, we conducted our investigation on the cerebellum using a CCH mouse model of VaD following bilateral common carotid artery stenosis (BCAS). This study is the first to characterize an increased expression of inflammasome receptors, adaptor and effector proteins, markers of inflammasome activation, proinflammatory cytokines, and apoptotic and pyroptotic cell death proteins in the cerebellum following CCH. Furthermore, in AIM2 knockout mice, we observed attenuated inflammasome-mediated production of proinflammatory cytokines, apoptosis, and pyroptosis in the cerebellum following CCH. Collectively, our findings provide novel evidence that AIM2 inflammasome activation promotes apoptosis and pyroptosis in the cerebellum following chronic hypoperfusion in a mouse model of VaD.

Elevated markers of gut leakage and inflammasome activation in COVID-19 patients with cardiac involvement.

BackgroundA high proportion of COVID‐19 patients have cardiac involvement, even those without known cardiac disease. Downregulation of angiotensin converting enzyme 2 (ACE2), a receptor for SARS‐CoV‐2 and the renin‐angiotensin system, as well as inflammatory mechanisms have been suggested to play a role. ACE2 is abundant in the gut and associated with gut microbiota composition. We hypothesized that gut leakage of microbial products, and subsequent inflammasome activation could contribute to cardiac involvement in COVID‐19 patients.MethodsPlasma levels of a gut leakage marker (LPS‐binding protein, LBP), a marker of enterocyte damage (intestinal fatty acid binding protein, IFABP), a gut homing marker (CCL25, ligand for chemokine receptor CCR9) and markers of inflammasome activation (IL‐1β, IL‐18 and their regulatory proteins) were measured at three time points (day 1, 3–5 and 7–10) in 39 hospitalized COVID‐19 patients and related to cardiac involvement.ResultsCompared to controls, COVID‐19 patients had elevated plasma levels of LBP and CCL25 but not IFABP, suggesting impaired gut barrier function and accentuated gut homing of T cells without excessive enterocyte damage. Levels of LBP were twice as high at baseline in patients with elevated cardiac markers compared with those without and remained elevated during hospitalization. Also, markers of inflammasome activation were moderately elevated in patients with cardiac involvement. LBP was associated with higher NT‐pro‐BNP levels, whereas IL‐18, IL‐18BP and IL‐1Ra were associated with higher troponin levels.ConclusionPatients with cardiac involvement had elevated markers of gut leakage and inflammasome activation, suggestive of a potential gut‐heart axis in COVID‐19.

Extracellular DNA, Neutrophil Extracellular Traps, and Inflammasome Activation in Severe Asthma.

Rationale: Extracellular DNA (eDNA) and neutrophil extracellular traps (NETs) are implicated in multiple inflammatory diseases. NETs mediate inflammasome activation and IL-1β secretion from monocytes and cause airway epithelial cell injury, but the role of eDNA, NETs, and IL-1β in asthma is uncertain. Objectives: To characterize the role of activated neutrophils in severe asthma through measurement of NETs and inflammasome activation. Methods: We measured sputum eDNA in induced sputum from 399 patients with asthma in the Severe Asthma Research Program-3 and in 94 healthy control subjects. We subdivided subjects with asthma into eDNA-low and -high subgroups to compare outcomes of asthma severity and of neutrophil and inflammasome activation. We also examined if NETs cause airway epithelial cell damage that can be prevented by DNase. Measurements and Main Results: We found that 13% of the Severe Asthma Research Program-3 cohort is "eDNA-high," as defined by sputum eDNA concentrations above the upper 95th percentile value in health. Compared with eDNA-low patients with asthma, eDNA-high patients had lower Asthma Control Test scores, frequent history of chronic mucus hypersecretion, and frequent use of oral corticosteroids for maintenance of asthma control (all P values <0.05). Sputum eDNA in asthma was associated with airway neutrophilic inflammation, increases in soluble NET components, and increases in caspase 1 activity and IL-1β (all P values <0.001). In in vitro studies, NETs caused cytotoxicity in airway epithelial cells that was prevented by disruption of NETs with DNase. Conclusions: High extracellular DNA concentrations in sputum mark a subset of patients with more severe asthma who have NETs and markers of inflammasome activation in their airways.

243 - Modulation of NLRP3 inflammasome activation in human macrophages by slow release hydrogen sulfide compounds

The NLRP3 inflammasome complex undergoes priming and activation leading to pro-inflammatory IL-1β and IL-18 synthesis. This study investigated the potential role of mitochondrial redox modulation in activation of the NLRP3 inflammasome in human cells. THP-1 cells were differentiated to macrophages with PMA for 24 hours and the NLRP3 inflammasome primed with LPS (0.1µg/ml) followed by activation with bzATP. NLRP3 and pro-IL1β were detected by Western blotting and ASC speck formation by immunofluorescence as markers of NLRP3 inflammasome priming. IL-1β and IL-18 in cell media were quantified by ELISA as markers of inflammasome activation. Under the conditions employed, intracellular oxidant generation (mitoparaquat and paraquat; Pq) assessed by mitoSOX fluorescence, was not sufficient to prime macrophages and did not affect priming mediated by LPS. At a relatively high extracellular concentration (5 µM) mitoPq, but not Pq, was able to increase IL-1β production in LPS-primed macrophages, suggesting promotion of inflammatory responses; IL-18 production was not affected. In LPS-primed and bzATP activated cells, the non-targeted slow release H2S compound, GYY4137, slightly reduced IL-1β production but was able to reduce IL-18 synthesis by approximately 50%. Although the mitochondria-targeted, slow release H2S compound AP39 did not affect inflammasome priming, at 300 nM (final media concentration) it reduced IL-1β and IL-18 synthesis in primed and activated cells. These data support a role for redox active oxygen and sulfur species, particularly when targeted to mitochondria, in modulating inflammatory cytokine synthesis in human macrophages with pro- and anti-inflammatory activities respectively.

MROS-TXNIP axis activates NLRP3 inflammasome to mediate renal injury during ischemic AKI.

Ischemia/reperfusion (I/R) is a critical risk factor for acute kidney injury (AKI). Recent studies provided evidence that tubular epithelial cells (TEC)-associated inflammation aggravates kidney injury and impairs tissue repair after I/R injury. Here we demonstrated that the Nod-like receptor protein 3 (NLRP3) inflammasome is activated by mitochondrial reactive oxygen species (mROS) during I/R injury via direct interactions between the inflammasome and thioredoxin-interacting protein (TXNIP). Firstly, we found that NLRP3 inflammasome activation was induced by I/R injury, peaking at day 3 after reperfusion. Consistent with this observation, NLRP3 deletion significantly attenuated I/R-induced kidney damage and markers of inflammasome activation. Then, we observed mitochondrial dysfunction, characterized by ultrastructural changes and cytochrome C (Cyt c) redistribution. Mitochondria-targeted antioxidant MitoTEMPO prevented mROS overproduction and the decline in mitochondrial membrane potential (MMP) in vitro. MitoTEMPO treatment also inhibited NLRP3 inflammasome activation and co-localization of NLRP3 and TXNIP after simulated ischemia/reperfusion (SI/R) injury. Finally, we transfected HK-2 cells with TXNIP siRNA to explore the role of TXNIP in mROS-induced NLRP3 inflammasome activation. We found that TXNIP siRNA significantly inhibited NLRP3 inflammasome activation. These results demonstrate that NLRP3 inflammasome is activated through the mROS-TXNIP-NLRP3 pathway and provide a potential therapeutic target in ischemic AKI.

Negative regulation of NLRP3 inflammasome by SIRT1 in vascular endothelial cells

NLRP3 inflammasome not only functions as a critical effector in innate immunity, but also triggers the production of proinflammatory cytokines involved in inflammation-associated diseases. Sirtuin 1 (SIRT1) plays an important role in the regulation of cellular inflammation. However, whether the activation of NLRP3 inflammasome is regulated by SIRT1 remains unknown. In this study, we investigated the regulatory effect of SIRT1 on NLRP3 inflammasome and the underlying mechanisms. We found that lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced the activation of NLRP3 inflammasome in human umbilical vein endothelial cells (HUVECs). Activation of SIRT1 inhibited NLRP3 inflammasome activation and subsequent caspase-1 cleavage as well as interleukin (IL)-1β secretion, whereas SIRT1 knockdown obviously enhanced the activation of NLRP3 inflammasome in HUVECs. Importantly, gene silencing of SIRT1 abrogated the inhibitory effect of SIRT1 activator on NLRP3 inflammasome formation and IL-1β production in HUVECs stimulated with LPS plus ATP. Further study indicated that cluster of differentiation 40 (CD40) may be involved in the regulation of NLRP3 inflammasome by SIRT1. In vivo studies indicated that implantation of the periarterial carotid collar increased the arterial expression levels of CD40 and CD40 Ligand (CD40L), but inhibited arterial SIRT1 expression in the rabbits. Moreover, treatment with SIRT1 activator decreased CD40 and CD40L levels in collared arteries. Meanwhile, serum IL-1β level, the marker of inflammasome activation, was also inhibited by SIRT1 activation. Taken together, these findings revealed a novel regulatory mechanism of NLRP3 inflammasome by SIRT1, which may be related to suppression of CD40.

Role of dietary fatty acids in liver injury caused by vinyl chloride metabolites in mice

BackgroundVinyl chloride (VC) causes toxicant-associated steatohepatitis at high exposure levels. Recent work by this group suggests that underlying liver disease may predispose the liver to VC hepatotoxicity at lower exposure levels. The most common form of underlying liver disease in the developed world is non-alcoholic fatty liver disease (NAFLD). It is well-known that the type of dietary fat can play an important role in the pathogenesis of NAFLD. However, whether the combination of dietary fat and VC/metabolites promotes liver injury has not been studied. MethodsMice were administered chloroethanol (CE - a VC metabolite) or vehicle once, 10weeks after being fed diets rich in saturated fatty acids (HSFA), rich in poly-unsaturated fatty acids (HPUFA), or the respective low-fat control diets (LSFA; LPUFA). ResultsIn control mice, chloroethanol caused no detectable liver injury, as determined by plasma transaminases and histologic indices of damage. In HSFA-fed mice, chloroethanol increased HSFA-induced liver damage, steatosis, infiltrating inflammatory cells, hepatic expression of proinflammatory cytokines, and markers of endoplasmic reticulum (ER) stress. Moreover, markers of inflammasome activation were increased, while markers of inflammasome inhibition were downregulated. In mice fed HPUFA all of these effects were significantly attenuated. ConclusionsChloroethanol promotes inflammatory liver injury caused by dietary fatty acids. This effect is far more exacerbated with saturated fat, versus poly-unsaturated fat; and strongly correlates with a robust activation of the NLRP3 inflammasome in the saturated fed animals only. Taken together these data support the hypothesis that environmental toxicant exposure can exacerbate the severity of NAFLD/NASH.

Aberrant Inflammasome Activation Characterizes Tuberculosis-Associated Immune Reconstitution Inflammatory Syndrome.

Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) complicates combination antiretroviral therapy (cART) in up to 25% of patients with HIV/TB coinfection. Monocytes and IL-18, a signature cytokine of inflammasome activation, are implicated in TB-IRIS pathogenesis. In this study, we investigated inflammasome activation both pre- and post-cART in TB-IRIS patients. HIV/TB patients exhibited higher proportions of monocytes expressing activated caspase-1 (casp1) pre-cART, compared with HIV patients without TB, and patients who developed TB-IRIS exhibited the greatest increase in casp1 expression. CD64(+) monocytes were a marker of increased casp1 expression. Furthermore, IL-1β, another marker of inflammasome activation, was also elevated during TB-IRIS. TB-IRIS patients also exhibited greater upregulation of NLRP3 and AIM2 inflammasome mRNA, compared with controls. Analysis of plasma mitochondrial DNA levels showed that TB-IRIS patients experienced greater cell death, especially pre-cART. Plasma NO levels were lower both pre- and post-cART in TB-IRIS patients, providing evidence of inadequate inflammasome regulation. Plasma IL-18 levels pre-cART correlated inversely with NO levels but positively with monocyte casp1 expression and mitochondrial DNA levels, and expression of IL-18Rα on CD4(+) T cells and NK cells was higher in TB-IRIS patients, providing evidence that IL-18 is a marker of inflammasome activation. We propose that inflammasome activation in monocytes/macrophages of HIV/TB patients increases with ineffective T cell-dependent activation of monocytes/macrophages, priming them for an excessive inflammatory response after cART is commenced, which is greatest in patients with TB-IRIS.

Anti-Inflammatory Role of MicroRNA-146a in the Pathogenesis of Diabetic Nephropathy.

Inflammation has a critical role in the pathogenesis of diabetic complications, including diabetic nephropathy (DN). MicroRNAs have recently emerged as important regulators of DN. However, the role of microRNAs in the regulation of inflammation during DN is poorly understood. Here, we examined the in vivo role of microRNA-146a (miR-146a), a known anti-inflammatory microRNA, in the pathogenesis of DN. In a model of streptozotocin-induced diabetes, miR-146a(-/-) mice showed significantly exacerbated proteinuria, renal macrophage infiltration, glomerular hypertrophy, and fibrosis relative to the respective levels in control wild-type mice. Diabetes-induced upregulation of proinflammatory and profibrotic genes was significantly greater in the kidneys of miR-146a(-/-) than in the kidneys of wild-type mice. Notably, miR-146a expression increased in both peritoneal and intrarenal macrophages in diabetic wild-type mice. Mechanistically, miR-146a deficiency during diabetes led to increased expression of M1 activation markers and suppression of M2 markers in macrophages. Concomitant with increased expression of proinflammatory cytokines, such as IL-1β and IL-18, markers of inflammasome activation also increased in the macrophages of diabetic miR-146a(-/-) mice. These studies suggest that in early DN, miR-146a upregulation exerts a protective effect by downregulating target inflammation-related genes, resulting in suppression of proinflammatory and inflammasome gene activation. Loss of this protective mechanism in miR-146a(-/-) mice leads to accelerated DN. Taken together, these results identify miR-146a as a novel anti-inflammatory noncoding RNA modulator of DN.

Resveratrol alleviates vascular inflammatory injury by inhibiting inflammasome activation in rats with hypercholesterolemia and vitamin D2 treatment.

Atherosclerosis (AS) is an inflammatory disease involved in vascular inflammatory injury. The inflammasome is an important part of inflammatory diseases and participates in the vascular inflammatory injury. Resveratrol (RSV) possesses anti-inflammatory activities, but its effects on inflammasomes during vascular injury remain unclear. This study focused on the effects and mechanisms of RSV on inflammasomes during vascular injury. Male Sprague-Dawley rats were treated with a purified diet or cholesterol-enriched diet combined with vitamin D2 (VD; 1.8 million units/kg/days, Po) and saline or RSV (50mg/kg/days, Po) daily for 5weeks. The concentrations and enzyme activities of related indicators were measured by a spectrophotometer or ELISA kit. Their gene and protein expression levels were analyzed by reverse transcription-polymerase chain reaction and Western blot, respectively. Upon administration with RSV, rats with combined hyper cholesterol and VD demonstrated the following changes: the vascular histopathological changes were relieved, and the level of the von Willebrand factor decreased. The level of serum IL-1β, a marker of inflammasome activation, significantly decreased. The mRNA and protein expression levels of the three components of inflammasomes, namely, NOD-like receptor pyrin domain containing 3, apoptosis-associated speck-like protein containing a caspase-recruitment domain, and caspase-1, were downregulated. The effects of RSV were closely related to hypolipidemia (decrease in the levels of total cholesterol, triglycerides, and low-density lipoprotein cholesterol combined with the expression of the lectin-like ox-LDL receptor and increase in high-density lipoprotein cholesterol), antioxidation (decrease in MDA levels and increase in SOD and GPx activities), and anti-inflammation (downregulation of the expression of IL-1β, intracellular adhesion molecule-1, and monocyte chemotactic protein-1). The mechanisms for the downregulation of NF-κB p65 and p38 MAPK expression, as well as the upregulation of SIRT1 expression, were analyzed. This study proved that RSV inhibited inflammasome activation to protect vascular injury in vivo. RSV exhibited therapeutic potential in the treatment of vascular injury.

The role of SRC kinase in the caspase-1 pathway after hypoxia in the brain of newborn piglets.

Hypoxia induces a cerebral inflammatory response, which contributes to brain injury. Inflammasomes are complex intracellular molecular structures that initiate the inflammatory cascade. Caspase-1 and interleukin 1-β (IL-1β), have been established as markers of inflammasome activation. Src kinase, a cytosolic non-receptor protein tyrosine kinase, is linked to cell proliferation and differentiation and is up regulated during hypoxia. The role of Src kinase in the above pathway is not fully understood. The present study tests the hypothesis that inhibition of Src kinase, by a selective inhibitor, PP2, will prevent the activation of caspase-1 and production of IL-1β acutely, as well as at 1 and 15 days after hypoxia in the cerebral cortex of the newborn piglet. Piglets were divided into: Normoxia (Nx), Hypoxia acute (Hx), Hypoxia-day 1 (Hx-day 1), and Hypoxia day 15 (Hx-day 15). Piglets pretreated with Src kinase inhibitor, PP2, 1 mg/kg IV, 30 min prior to hypoxia were divided into: Hypoxia acute (Hx + PP2), 1 day (Hx + PP2-day 1), and day 15 (Hx + PP2-day 15). Hypoxia was induced by exposing the piglets to an FiO2 of 0.07 for 1 hour. Caspase-1 activity and expression were determined with spectrophotometry and Western blot respectively, while IL-1β levels were measured by solid phase ELISA. Caspase-1 activation was achieved immediately (within 1 h) after hypoxia and persisted for 15 days. IL-1β level was also increased after hypoxia reaching a maximum level at 24 h following hypoxia and returned to baseline by 15 days. Administration of PP2 attenuated the activity acutely, but not the expression of the caspase-1. IL-1β level at 24 h after hypoxia returned to baseline in piglets that were pretreated with PP2. We provide evidence that inhibition of Src kinase in the acute phase after hypoxia involves changes in the production or processing of caspase-1 subunits. Our data suggest that Src kinase mediates hypoxia-induced caspase-1 activation in the cerebral cortex of newborn piglets. Inhibition of Src kinase may attenuate the neuroinflammatory response and could represent a potential target for neuroprotection after hypoxic injury.

Artefactual nanoparticle activation of the inflammasome platform: in vitro evidence with a nano-formed calcium phosphate.

To determine whether in vitro experimental conditions dictate cellular activation of the inflammasome by apatitic calcium phosphate nanoparticles. The responses of blood-derived primary human cells to in situ-formed apatite were investigated under different experimental conditions to assess the effect of aseptic culture, cell rest and duration of particle exposure. Cell death and particle uptake were assessed, while IL-1β and caspase 1 responses, with and without lipopolysaccharide prestimulation, were evaluated as markers of inflammasome activation. Under carefully addressed experimental conditions, apatitic nanoparticles did not induce cell death or engage the inflammasome platform, although both could be triggered through artefacts of experimentation. In vitro studies often predict that engineered nanoparticles, such as synthetic apatite, are candidates for inflammasome activation and, hence, are toxic. However, the experimental setting must be very carefully considered as it may promote false-positive outcomes.

Normal inflammasome activation and low production of IL-23 by monocyte-derived macrophages from subjects with a history of reactive arthritis

Objectives: The pathogenesis of reactive arthritis (ReA) is incompletely understood but may involve aberration(s) in the host’s innate immune response towards infecting microbes. We therefore studied the production of interleukin (IL)-1β, a marker of inflammasome activation, and of IL-6, IL-12, IL-23, and tumour necrosis factor (TNF)-α, promoters of T-cell differentiation, by peripheral blood mononuclear cells (PBMNs) and monocyte-derived macrophages from healthy subjects with a history of ReA.Method: The study included 10 human leucocyte antigen (HLA)-B27-positive healthy subjects with previous ReA triggered by Yersinia enterocolitica O:3 infection and 20 healthy reference subjects, of whom 10 were HLA-B27 positive. PBMNs and macrophages were cultured for 18 h with bacterial lipopolysaccharide (LPS), muramyl dipeptide (MDP), Yersinia, or their appropriate combinations. PBMNs were also stimulated with monosodium urate (MSU) crystals. Cytokine levels were measured using an enzyme-linked immunosorbent assay (ELISA) and the Luminex system.Results: IL-1β secretion was similar from cells of the ReA group and from the HLA-B27-positive and -negative reference groups. TNF-α production from macrophages upon co-stimulation of LPS and MDP increased in the order ReA group < HLA-B27-positive reference group < HLA-B27-negative reference group (p for a trend = 0.027). Similarly, Yersinia-induced TNF-α and IL-23 production increased in the same order (p for trend for TNF-α = 0.036; p for trend for IL-23 = 0.026).Conclusions: PBMNs and macrophages from healthy subjects with previous ReA show normal inflammasome activation and low TNF-α and IL-23 production. This low cytokine production may impair bacterial elimination and thereby contribute to the triggering of ReA.