Activation Of Inflammasome Pathway Research Articles

Abstract 219: Apolipoprotein C3 Induces Inflammasome Activation In Human And Mouse Monocytes Only In De-lipidated Form

Individuals with diabetes suffer from an elevated risk of coronary artery disease (CAD) events. Increased serum levels of apolipoprotein C3 (APOC3) predict incident CAD in individuals with type 1 diabetes (T1D). APOC3 is a small lipid-binding protein carried by lipoproteins, including VLDL, in circulation. Silencing of APOC3 in a mouse model of T1D-accelerated atherosclerosis has been demonstrated to prevent diabetes-accelerated atherosclerosis, strongly suggesting that APOC3 is a causal mediator. However, the exact mechanism whereby APOC3 promotes atherosclerosis in the setting of T1D is unclear. Mice with T1D demonstrated elevated levels of glucose, triglycerides, APOC3 (non-diabetic [ND] mice=442.6 μg/ml, diabetic [D] mice=755.9 μg/ml, p<0.01, N=21 per group), and IL-18 (ND=124.1 pg/ml, D=256.8 pg/ml, p<0.01, N=18-22 per group) in plasma. The elevated levels of plasma APOC3 and IL-18 in diabetic mice and a recent study demonstrating that de-lipidated APOC3 can stimulate inflammasome activation in human monocytes prompted us to investigate whether lipidated APOC3 has similar inflammasome-activating properties. Our results show that de-lipidated APOC3, but not VLDL (which contains APOC3) significantly stimulates IL-1β release from isolated human monocytes (p<0.01 for vehicle vs APOC3, N=3 per group) and mouse monocytes (p<0.01 for vehicle vs APOC3, N=4-7 per group). Mouse monocytes stimulated with de-lipidated APOC3 also had elevated IL-18 release (vehicle=21.2 pg/mg, APOC3=170.2 pg/mg, p<0.001), Il1b mRNA (APOC3=86.7-fold over control), and Nlrp3 mRNA (APOC3=13.8-fold over control), indicating activation of the NLRP3 inflammasome pathway. However, analysis of serum samples from subjects with T1D showed that virtually all APOC3 is bound to lipoproteins (primarily VLDL and HDL). To further confirm the effect of lipidated APOC3, we bound APOC3 to lipid-based small unilamellar vesicles to mimic the physiological state of APOC3 in plasma. The stimulatory effect of APOC3, measured by IL-1β release from mouse monocytes, was lost. Together, our findings suggest that although de-lipidated APOC3 has the ability to induce inflammasome activation in monocytes, virtually no APOC3 circulates in a lipid-free form in subjects with T1D.

Reticulon-1A mediates diabetic kidney disease progression through endoplasmic reticulum-mitochondrial contacts in tubular epithelial cells

Recent epidemiological studies suggest that some patients with diabetes progress to kidney failure without significant albuminuria and glomerular injury, suggesting a critical role of kidney tubular epithelial cell (TEC) injury in diabetic kidney disease (DKD) progression. However, the major risk factors contributing to TEC injury and progression in DKD remain unclear. We previously showed that expression of endoplasmic reticulum-resident protein Reticulon-1A (RTN1A) increased in human DKD, and the increased RTN1A expression promoted TEC injury through endoplasmic reticulum (ER) stress response. Here, we show that TEC-specific RTN1A overexpression worsened DKD in mice, evidenced by enhanced tubular injury, tubulointerstitial fibrosis, and kidney function decline. But RTN1A overexpression did not exacerbate diabetes-induced glomerular injury or albuminuria. Notably, RTN1A overexpression worsened both ER stress and mitochondrial dysfunction in TECs under diabetic conditions by regulation of ER-mitochondria contacts. Mechanistically, ER-bound RTN1A interacted with mitochondrial hexokinase-1 and the voltage-dependent anion channel-1 (VDAC1), interfering with their association. This disengagement of VDAC1 from hexokinase-1 resulted in activation of apoptotic and inflammasome pathways, leading to TEC injury and loss. Thus, our observations highlight the importance of ER-mitochondrial crosstalk in TEC injury and the salient role of RTN1A-mediated ER-mitochondrial contact regulation in DKD progression.

Ghrelin ameliorates diabetes-associated behavioral deficits and NLRP3 inflammasome activation via autophagic flux enhancement

Ghrelin has recently been associated with the development of diabetes comorbid with depression, but its underlying molecular mechanisms remains poorly understood. Here, molecular and histological methods were applied both in vivo and in vitro studies to investigate the mechanisms of ghrelin in diabetes comorbid with depression. Our results demonstrated the anti-depressive, anxiolytic, and neuroprotective effects of ghrelin, as evidenced by the amelioration of anxiety- and depression-like behaviors, reduction in apoptosis, and preservation of neuron integrity in streptozotocin (STZ)-treated rats. STZ treatment induced M1-phenotypic microglial polarization, accompanied by neuroinflammation, which was reversed by ghrelin treatment. Further exploration showed that autophagy was inhibited, the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome and nuclear factor (NF)-κB signaling pathway were activated in STZ rats. In line with the in vivo results, ghrelin could suppress the NLRP3 inflammasome and NF-κB signaling pathway activation via the amelioration of impaired autophagic flux in microglial BV2 cells. Importantly, clinical evidence further verified the anti-inflammatory and antidepressant effects of ghrelin. Collectively, these results suggested that ghrelin ameliorates diabetes-associated behavioral deficits and NLRP3 inflammasome activation via autophagic flux enhancement, highlighting the importance of ghrelin as a potential target of immune regulation in diabetes comorbid with depression.

(-)-Epicatechin Ameliorates Monosodium Urate-Induced Acute Gouty Arthritis Through Inhibiting NLRP3 Inflammasome and the NF-κB Signaling Pathway.

Background: Gouty arthritis is a common and complex inflammatory disease that will reduce the life quality of human beings (−)-Epicatechin (EC) is famous for antioxidant and anti-inflammatory activities. Thus, the aim of this study was to investigate the therapeutic effect of EC on gouty arthritis and its mechanisms. Methods and results: EC was added into a monosodium urate (MSU)-stimulated THP-1 cell that was induced by phorbol 12-myristate 13-acetate and lipopolysaccharide (LPS) in advance to establish a gout model in vitro. The efficiency of EC on acute gouty arthritis mice induced by MSU was further investigated. The results showed that EC concentration-dependently improved the cell viability of LPS and MSU stimulated THP-1 cells, and significantly alleviated MSU-induced ankle edema in mice in a dose-dependent manner. In addition, EC inhibited the infiltration of inflammatory cells and local cascular congestion in ankle joint tissue. Furthermore, the secretion of inflammatory cytokines (IL-1β, IL-18, IL-6, and TNF-α) activation of NLRP3 inflammasome and NF-κB signaling pathway were markedly suppressed by EC in vitro and in vivo. Conclusion: These results indicated that EC could effectively improve MSU-induced acute gouty arthritis via inhibiting NLRP3 inflammasome and the NF-κB signaling pathway in vitro and in vivo, which suggested that EC might be a promising active ingredient for the prevention and treatment of gouty arthritis.

MiR-223-3p contributes to suppressing NLRP3 inflammasome activation in Streptococcus equi ssp. zooepidemicus infection

Streptococcus equi subsp. zooepidemicus (SEZ) is an essential pathogen in a range of species, causing a worldwide variety of diseases, such as meningitis, endocarditis, and septicaemia. Studies have shown that microRNAs (miRNAs), which regulate target genes at the post-transcriptional level, play an important regulatory role in the organism. In this study, the infection of J774A.1 murine macrophages with SEZ up-regulated NLRP3 inflammasome and downstream pathways accompanied by miR-223–3p down-regulation. Through computational prediction and experimental confirmation, we have shown that miR-223–3p directly targets the NLRP3 mRNA. Consequently, overexpression of miR-223–3p suppressed NLRP3 inflammasome activation and downstream pathways in response to SEZ infection. The miR-223–3p inhibitor exhibited the opposite effect, causing hyperactivation of NLRP3 inflammation activation and downstream pathways. Additionally, we further demonstrated that miRNA-223–3p inhibited the secretion of IL-1β and IL-18 by regulating the NLRP3/caspase-1 pathway. Furthermore, intravenous administration of miR-223–3p significantly decreased inflammation in mice in response to SEZ. In conclusion, our results demonstrated that miR-223–3p contributes to suppressing the NLRP3 inflammasome activation in SEZ infection, contributing novel evidence to identify a therapeutic target for treating SEZ.

Targeting NLRP3 Inflammasome With Nrf2 Inducers in Central Nervous System Disorders.

The NLRP3 inflammasome is an intracellular multiprotein complex that plays an essential role in the innate immune system by identifying and eliminating a plethora of endogenous and exogenous threats to the host. Upon activation of the NLRP3 complex, pro-inflammatory cytokines are processed and released. Furthermore, activation of the NLRP3 inflammasome complex can induce pyroptotic cell death, thereby propagating the inflammatory response. The aberrant activity and detrimental effects of NLRP3 inflammasome activation have been associated with cardiovascular, neurodegenerative, metabolic, and inflammatory diseases. Therefore, clinical strategies targeting the inhibition of the self-propelled NLRP3 inflammasome activation are required. The transcription factor Nrf2 regulates cellular stress response, controlling the redox equilibrium, metabolic programming, and inflammation. The Nrf2 pathway participates in anti-oxidative, cytoprotective, and anti-inflammatory activities. This prominent regulator, through pharmacologic activation, could provide a therapeutic strategy for the diseases to the etiology and pathogenesis of which NLRP3 inflammasome contributes. In this review, current knowledge on NLRP3 inflammasome activation and Nrf2 pathways is presented; the relationship between NLRP3 inflammasome signaling and Nrf2 pathway, as well as the pre/clinical use of Nrf2 activators against NLRP3 inflammasome activation in disorders of the central nervous system, are thoroughly described. Cumulative evidence points out therapeutic use of Nrf2 activators against NLRP3 inflammasome activation or diseases that NLRP3 inflammasome contributes to would be advantageous to prevent inflammatory conditions; however, the side effects of these molecules should be kept in mind before applying them to clinical practice.

Dysfunctional telomeres through mitostress-induced cGAS/STING activation to aggravate immune senescence and viral pneumonia.

Disproportionately high incidence and mortality of respiratory infection such as influenza A virus (IAV) and SARS‐CoV‐2 have been evidenced in the elderly, but the role and the mechanism of age‐associated immune deregulation in disease exacerbation are not well defined. Using a late generation of mice deficient in telomerase RNA (Terc−/−), we herein demonstrated that aged mice were exquisitely susceptible to respiratory viral infection, with excessive inflammation and increased mortality. Furthermore, we identified the cGAS/STING pathway, which was essentially induced by the leaked mitochondrial DNA, as a biologically relevant mechanism contributing to exaggerated inflammation in Terc−/− mice following viral infection. Innate immune cells, mainly, macrophages with shortened telomeres, exhibited hallmarks of cellular senescence, mitochondrial distress, and aberrant activation of STING and NLRP3 inflammasome pathways, which predisposed mice to severe viral pneumonia during commonly mild infections. Application of STING inhibitor and, more importantly, senolytic agent, reduced the burden of stressed macrophages, improved mitochondrial integrity, and suppressed STING activation, thereby conferring the protection for Terc−/− mice against respiratory infection. Together, the findings expand our understanding of innate immune senescence and reveal the potential of the senolytics as a promising treatment to alleviate the symptom of viral pneumonia, particularly for the older population.

BAFF Blockade Attenuates DSS-Induced Chronic Colitis via Inhibiting NLRP3 Inflammasome and NF-κB Activation.

BackgroundBAFF production is increased in IBD patients. However, the specific role of BAFF in IBD is still uncovered. This study aimed to investigate the expression and function of BAFF in experimental colitis and the potential mechanisms.MethodsBAFF levels in the serum and colon tissues were measured by ELISA in DSS-induced colitis mice. Mouse-derived BAFF antibody was administered in DSS mice. The changes of body weight, disease activity index (DAI) scores, colon length, spleen weight, histopathological damage, inflammatory indicators, NF-κB signaling, and NLRP3 inflammasome were assayed in DSS mice and control. LPS-primed RAW264.7 cells and bone marrow derived macrophages (BMDMs) were treated with BAFF blockage and recombinant mouse BAFF. Inflammatory associated cytokines, NLRP3 inflammasomes and NF-κB signaling were detected among groups.ResultsBAFF production was elevated systemically and locally in colitis mice. BAFF blockade improved the body weight loss, DAI scores, colon length, spleen weight, and histopathological damage in colitis mice. Immunoflurescence analysis revealed that elevated macrophages in mucosal lamina propria were the primary source of BAFF in the colon. NLRP3 inflammasome and NF-κB signaling pathway activation were dramatically inhibited in DSS mice treated with BAFF blockage. In LPS-primed RAW264.7 cells/BMDMs, BAFF blockade decreased the activation of NLRP3 inflammasome (NLPR3, ASC, cleaved IL-1β, cleaved caspase 1) via inhibiting NF-κB signaling pathway. Moreover, LPS synergizes with BAFF to promote inflammatory factor secretion and expression of NF-κB signaling pathway in RAW264.7 cells.ConclusionsThese results suggested that BAFF blockade protected against colitis partially by relieving inflammation, inhibiting intestinal NLRP3 inflammasome and NF-κB signaling pathway from macrophages. BAFF plays an important role in inflammation regulation in IBD, thus providing a novel idea for further research on colitis and experimental evidences for novel potential therapeutic target in IBD.

Milk Fat Globule Membrane Alleviates Short Bowel Syndrome-Associated Liver Injury in Rats Through Inhibiting Autophagy and NLRP3 Inflammasome Activation.

BackgroundThe milk fat globule membrane (MFGM), a tri-layer membrane structure surrounding the milk fat globule, has been shown to have immune-modulating properties. This study aimed to investigate the effects of MFGM supplementation in a rat model of short bowel syndrome (SBS) associated liver disease and its possible mechanisms.Materials and MethodsTwenty one male Sprague-Dawley rats were randomly divided into three groups: Sham, SBS (underwent massive small bowel resection), and SBS+MFGM (SBS rats supplemented with 1.5 g/kg/d MFGM). Liver pathology, myeloperoxidase (MPO) staining, serum levels of aspartate aminotransferase (AST)/alanine aminotransferase (ALT), endotoxin concentration, protein expression of autophagy and nucleotide binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3) pathway in the liver tissue were measured.ResultsBoth SBS and SBS + MFGM groups had higher serum levels of ALT and liver endotoxin levels than the Sham group (P < 0.05), with no difference detected between each other. Compared with the SBS group, the SBS+MFGM group showed lower liver pathology scores of steatosis and inflammation, less MPO positive cells and reduced expressions of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), Caspase-1, interleukin (IL)-1β(P < 0.05) in the liver. Additionally, the expression of Beclin-1 and microtubule-associated protein1 light chain 3(LC3) B, the fluorescence intensity of NLRP3 and LC3B in the SBS + MFGM group were lower than the SBS group (P < 0.05). The LC3B expression was positively correlated with the NLRP3 level.ConclusionEnteral supplementation of MFGM help to alleviate liver injury in SBS rats, which might be related to inhibition of aberrant activation of autophagy and NLRP3 inflammasome pathways.

Pulmonary vascular inflammation with fatal coronavirus disease 2019 (COVID-19): possible role for the NLRP3 inflammasome

BackgroundPulmonary hyperinflammation is a key event with SARS-CoV-2 infection. Acute respiratory distress syndrome (ARDS) that often accompanies COVID-19 appears to have worse outcomes than ARDS from other causes. To date, numerous lung histological studies in cases of COVID-19 have shown extensive inflammation and injury, but the extent to which these are a COVID-19 specific, or are an ARDS and/or mechanical ventilation (MV) related phenomenon is not clear. Furthermore, while lung hyperinflammation with ARDS (COVID-19 or from other causes) has been well studied, there is scarce documentation of vascular inflammation in COVID-19 lungs.MethodsLung sections from 8 COVID-19 affected and 11 non-COVID-19 subjects, of which 8 were acute respiratory disease syndrome (ARDS) affected (non-COVID-19 ARDS) and 3 were from subjects with non-respiratory diseases (non-COVID-19 non-ARDS) were H&E stained to ascertain histopathological features. Inflammation along the vessel wall was also monitored by expression of NLRP3 and caspase 1.ResultsIn lungs from COVID-19 affected subjects, vascular changes in the form of microthrombi in small vessels, arterial thrombosis, and organization were extensive as compared to lungs from non-COVID-19 (i.e., non-COVID-19 ARDS and non-COVID-19 non-ARDS) affected subjects. The expression of NLRP3 pathway components was higher in lungs from COVID-19 ARDS subjects as compared to non-COVID-19 non-ARDS cases. No differences were observed between COVID-19 ARDS and non-COVID-19 ARDS lungs.ConclusionVascular changes as well as NLRP3 inflammasome pathway activation were not different between COVID-19 and non-COVID-19 ARDS suggesting that these responses are not a COVID-19 specific phenomenon and are possibly more related to respiratory distress and associated strategies (such as MV) for treatment.

GDF11 alleviates neointimal hyperplasia in a rat model of artery injury by regulating endothelial NLRP3 inflammasome activation and rapid re-endothelialization

BackgroundNeointimal hyperplasia induced by interventional surgery can lead to progressive obliteration of the vascular lumen, which has become a major factor affecting prognosis. The rate of re-endothelialization is known to be inversely related to neointima formation. Growth differentiation factor 11 (GDF11) is a secreted protein with anti-inflammatory, antioxidant, and antiaging properties. Recent reports have indicated that GDF11 can improve vascular remodeling by maintaining the differentiated phenotypes of vascular smooth muscle cells. However, it is not known whether and how GDF11 promotes re-endothelialization in vascular injury. The present study was performed to clarify the influence of GDF11 on re-endothelialization after vascular injury.MethodsAn adult Sprague–Dawley rat model of common carotid artery balloon dilatation injury was surgically established. A recombinant adenovirus carrying GDF11 was delivered into the common carotid artery to overexpress GDF11. Vascular re-endothelialization and neointima formation were assessed in harvested carotid arteries through histomolecular analysis. CCK-8 analysis, LDH release and Western blotting were performed to investigate the effects of GDF11 on endothelial NLRP3 inflammasome activation and relevant signaling pathways in vitro.ResultsGDF11 significantly enhanced re-endothelialization and reduced neointima formation in rats with balloon-dilatation injury by suppressing the activation of the NLRP3 inflammasome. Administration of an endoplasmic reticulum stress (ER stress) inhibitor, 4PBA, attenuated endothelial NLRP3 inflammasome activation induced by lysophosphatidylcholine. In addition, upregulation of LOX-1 expression involved elevated ER stress and could result in endothelial NLRP3 inflammasome activation. Moreover, GDF11 significantly inhibited NLRP3 inflammasome-mediated endothelial cell pyroptosis by negatively regulating LOX-1-dependent ER stress.ConclusionsWe conclude that GDF11 improves re-endothelialization and can attenuate vascular remodeling by reducing endothelial NLRP3 inflammasome activation. These findings shed light on new treatment strategies to promote re-endothelialization based on GDF11 as a future target.

Down-regulation of TRPM2 attenuates hepatic ischemia/reperfusion injury through activation of autophagy and inhibition of NLRP3 inflammasome pathway

AimHepatic ischemia/reperfusion (I/R) injury is a significant pathological process that contributes to high morbidity and mortality rates, although the underlying mechanism is unknown. Recent studies have shown that transient receptor potential melastatin 2 (TRPM2) plays a critical role in organ I/R injury, but the exact mechanism is elusive. This study investigates the role and mechanism of TPRM2 in hepatic I/R injury and oxygen-glucosedeprivation/reoxygenation (OGD/R) induced hepatocyte injury. MethodsWe evaluated the effects of TRPM2 on hepatic I/R injury using a knockout mouse model of hepatic I/R. In a model of OGD/R in hepatocytes, we investigated the mechanism of TPRM2 in it using the autophagy agonist and inhibitor and an NLRP3 inhibitor. ResultsWe discovered that knockout of TRPM2 protected against hepatic I/R accompanied by autophagy activation and NLRP3 inflammasome pathway inhibition. Furthermore, increasing autophagy attenuated OGD/R-induced cell injury and knockdown of TRPM2 alleviated the injury by activating autophagy. Additionally, we detected the expression of NLRP3 inflammasome pathway in the OGD/R-induced hepatocytes which had been treated with the autophagy agonist and inhibitor, and found that autophagy negatively regulated the NLRP3 inflammasome pathway. Moreover, we discovered that the administration of NLRP3-inhibitor INF39 increased cell viability and caused a decline in cell death in the OGD/R-treated hepatocytes. ConclusionsDownregulation of TRPM2 protected the liver against I/R injury and OGD/R induced injury, mediated by autophagy activation and inhibition of the NLRP3 inflammasome pathway, whereas autophagy negatively regulated the NLRP3 inflammasome pathway in this process.

The involvement of inflammasomes on the learning and memory impairment in a mouse model of embolic stroke

Post-stroke cognitive impairment (PSCI) is one of the major complications after a stroke and affects quality of life. Recently, several studies demonstrated that elevated brain proinflammatory cytokines such as TNF-α, IL-6 and IL-1β can induce cognitive impairment. However, the mechanisms underlying PSCI remain unclear. In the present study, we investigated whether post-stroke inflammasomes are involved in the development of PSCI using acetic acid-induced embolic cerebral infarct mice. Long-term learning and memory assessed by the passive avoidance test was impaired on days 7 and 14 after stroke, whereas short-term learning and memory assessed by Y-maze test showed no changes. Also, the expression of the phosphorylated AMPA receptor subunits GluR1 at serine 831 and serine 845 in the dorsal hippocampus were significantly decreased. Under these conditions, inflammasome-related proteins, including caspase-1, ASC/TMS1, IL-1β, TNF-α and IL-18, were significantly increased in the dorsal hippocampus 14 days after stroke. The present findings suggest that a decrease in phosphorylated GluR1 at ser831 and ser845 via the inflammasome activation pathway in the dorsal hippocampus may be involved in the development of learning and memory impairment after embolic stroke.

Review: Protective Immunity and Immunopathology of Ehrlichiosis.

Human monocytic ehrlichiosis, a tick transmitted infection, ranges in severity from apparently subclinical to a fatal toxic shock-like fatal disease. Models in immunocompetent mice range from an abortive infection to uniformly lethal depending on the infecting Ehrlichia species, dose of inoculum, and route of inoculation. Effective immunity is mediated by CD4+ T lymphocytes and gamma interferon. Lethal infection occurs with early overproduction of proinflammatory cytokines and overproduction of TNF alpha and IL-10 by CD8+ T lymphocytes. Furthermore, fatal ehrlichiosis is associated with signaling via TLR 9/MyD88 with upregulation of several inflammasome complexes and secretion of IL-1 beta, IL-1 alpha, and IL-18 by hepatic mononuclear cells, suggesting activation of canonical and noncanonical inflammasome pathways, a deleterious role for IL-18, and the protective role for caspase 1. Autophagy promotes ehrlichial infection, and MyD88 signaling hinders ehrlichial infection by inhibiting autophagy induction and flux. Activation of caspase 11 during infection of hepatocytes by the lethal ehrlichial species after interferon alpha receptor signaling results in the production of inflammasome-dependent IL-1 beta, extracellular secretion of HMGB1, and pyroptosis. The high level of HMGB1 in lethal ehrlichiosis suggests a role in toxic shock. Studies of primary bone marrow-derived macrophages infected by highly avirulent or mildly avirulent ehrlichiae reveal divergent M1 and M2 macrophage polarization that links with generation of pathogenic CD8 T cells, neutrophils, and excessive inflammation or with strong expansion of protective Th1 and NKT cells, resolution of inflammation and clearance of infection, respectively.

VEXAS Syndrome Is Characterized by Blood and Tissues Inflammasome Pathway Activation and Monocyte Dysregulation

VEXAS Syndrome Is Characterized by Blood and Tissues Inflammasome Pathway Activation and Monocyte Dysregulation

Metadherin silencing results in the inhibition of pyroptosis in lipopolysaccharide/adenosine triphosphate − stimulated renal tubular epithelial cells

Pyroptosis is induced following inflammation via activation of the NLRP3 inflammasome. Lipopolysaccharide (LPS)-induced acute inflammation causes pyroptosis in renal tubular epithelial cells, which aggravates kidney damage and is involved in physiopathological processes in multiple renal diseases. Metadherin (Mtdh) induces inflammation by NLRP3 inflammasome activation. Specifically, it induces inflammatory injury in the kidney by activating the nuclear factor kappa B (NF-κB) signaling pathway, which is involved in NLRP3 inflammasome activation. However, the role of Mtdh in pyroptosis in renal tubular epithelial cells is unclear. Therefore, we investigated whether Mtdh participates in pyroptosis in LPS/adenosine triphosphate (ATP)-treated NRK-52E cells by activating the NLRP3 inflammasome and NF-κB signaling pathway. We induced pyroptosis in NRK-52E cells with LPS/ATP, after which Mtdh was silenced via transfection with small interfering RNA. LPS/ATP upregulated Mtdh expression and induced pyroptosis and NLRP3 inflammasome activation in NRK-52E cells. However, downregulation of Mtdh expression resulted in the alleviation of pyroptosis in LPS/ATP-treated NRK-52E cells. Additionally, activation of the NLRP3 inflammasome and NF-κB signaling pathway was inhibited. This demonstrates that downregulation of Mtdh expression results in the inhibition of pyroptosis in LPS/ATP-treated NRK-52E cells through the suppression of NLRP3 inflammasome activation, which occurs via inhibition of the NF-κB signaling pathway.

P38-TFEB pathways promote microglia activation through inhibiting CMA-mediated NLRP3 degradation in Parkinson's disease

BackgroundParkinson’s disease (PD) is characterized by degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc), accompanied by accumulation of α-synuclein, chronic neuroinflammation and autophagy dysfunction. Previous studies suggested that misfolded α-synuclein induces the inflammatory response and autophagy dysfunction in microglial cells. The NLRP3 inflammasome signaling pathway plays a crucial role in the neuroinflammatory process in the central nervous system. However, the relationship between autophagy deficiency and NLRP3 activation induced by α-synuclein accumulation is not well understood.MethodsThrough immunoblotting, immunocytochemistry, immunofluorescence, flow cytometry, ELISA and behavioral tests, we investigated the role of p38-TFEB-NLRP3 signaling pathways on neuroinflammation in the α-synuclein A53T PD models.ResultsOur results showed that increased protein levels of NLRP3, ASC, and caspase-1 in the α-synuclein A53T PD models. P38 is activated by overexpression of α-synuclein A53T mutant, which inhibited the master transcriptional activator of autophagy TFEB. And we found that NLRP3 was degraded by chaperone-mediated autophagy (CMA) in microglial cells. Furthermore, p38-TFEB pathways inhibited CMA-mediated NLRP3 degradation in Parkinson's disease. Inhibition of p38 had a protective effect on Parkinson's disease model via suppressing the activation of NLRP3 inflammasome pathway. Moreover, both p38 inhibitor SB203580 and NLRP3 inhibitor MCC950 not only prevented neurodegeneration in vivo, but also alleviated movement impairment in α-synuclein A53T-tg mice model of Parkinson’s disease.ConclusionOur research reveals p38-TFEB pathways promote microglia activation through inhibiting CMA-mediated NLRP3 degradation in Parkinson's disease, which could be a potential therapeutic strategy for PD.Graphical abstractp38-TFEB pathways promote microglia activation through inhibiting CMA-mediated NLRP3 degradation in Parkinson's disease. In this model, p38 activates NLRP3 inflammasome via inhibiting TFEB in microglia. TFEB signaling negatively regulates NLRP3 inflammasome through increasing LAMP2A expression, which binds to NLRP3 and promotes its degradation via chaperone-mediated autophagy (CMA). NLRP3-mediated microglial activation promotes the death of dopaminergic neurons.

Investigating P2X7R-mediated inflammatory signalling in Alzheimer's disease.

Neuroinflammation is thought to contribute to the onset and/or progression of Alzheimer´s disease (AD). Astrocytes and microglia sense danger-associated molecules (e.g. Aβ species, ATP) through membrane receptors including the purinergic receptor P2X7 (P2X7R). These interactions activate pro-inflammatory pathways and lead to the release of cytokines that participate in damaging inter-cellular cascades. Inhibition of P2X7R in AD models ameliorates cognitive deficits, pointing towards a neuroprotective effect of P2X7R blockade in vivo potentially associated with the modulation of inflammatory responses. However, the action of P2X7R and downstream signals on tau-mediated neurodegeneration in AD remains elusive. We used biochemical and immunohistochemical methods in combination with in-situ hybridization technology (RNAscope) in post-mortem AD and control (Braak 0-VI) BA9 prefrontal cortex to examine early changes in P2X7R expression, determine the spatial pattern of P2X7R activation relative to Aβ and pathological tau accumulation, and evaluate the association between P2X7R and local inflammatory responses. Primary glial and organotypic slice cultures are being used to model glial responses to physiological Aβ species and assess downstream effects on neurons, including alterations in tau and synapse health. We found elevated levels of P2X7R from Braak stage II relative to controls accompanied by an increased amyloid burden, synaptic mislocalization of tau and an altered astrocyte reactivity. P2X7R was markedly expressed in astrocytes surrounding AD hallmarks and also in disease-associated microglia neighbouring Aβ plaques. Investigation of inflammatory mediators linked with P2X7R-positive glia revealed higher activation of the NLRP3 inflammasome and NFkB pathways with disease progression. Preliminary data from in vitro and ex vivo models show inflammatory changes and tau redistribution to the synapses following stimulation with oligomeric Aß. These data suggest that P2X7R expression and activation are altered in the early stages of AD, prior to marked synapse loss. Ongoing studies aim to determine if the blockade of P2X7R-mediated inflammatory signals has benefits for tau-associated neurodegeneration in AD models.

Abstract 14023: PRG4 in Calcified Atherosclerotic Plaque Induces Immune Tolerance in Macrophages, and is Implicated in Inflammation-Driven Vascular Calcification

Atherosclerotic calcification is an independent predictor for a cardiovascular event, and its development is driven by macrophage-led inflammation. PRG4 was previously shown to be the most upregulated gene in microarray data of carotid endarterectomy-derived, high vs low-calcified atherosclerotic plaques. It was shown to be a pro-calcifying factor in the disease, modulating the pro-calcifying switch of vascular smooth muscle cells (VSMC). PRG4’s role in calcified plaque is largely unknown, however, we are able to show a profound effect on macrophage immune response. Transcriptomic data of a highly calcified plaque cohort (N=18 patients) shows PRG4 expression is correlated with inflammatory gene pathways, including toll-like receptor signalling; and immunohistochemistry of this cohort displays co-localization with CD68 + cells. Despite this, PRG4 is not expressed in macrophages in plaque single-cell datasets or transcriptomics datasets, and plaque origin is likely fibroblast and VSMC. Human peripheral blood mononuclear cell (PBMC)-derived macrophage incubation with 100μg/ml recombinant human (rh)PRG4 shows strong NF-κB activation, and secretion of TNF, IL-6, IL-12p70 and IL-10. rhPRG4 stimulation also significantly enhances inflammasome activation, increasing ASC activation (p&lt;0.003) and IL-1β secretion (p&lt;0.001). Moreover, it suppressed macrophage phagocytosis of both zymosan particles (p&gt;0.001), and hydroxyapatite particles (p=0.004). Subsequently, cells stimulated with rhPRG4 develop immune tolerance, a suppressed inflammatory response, to secondary stimulation with either rhPRG4 or lipopolysaccharide - with almost complete ablation of TNF secretion (p&lt;0.001). This phenomenon is accompanied by a 5-fold A20 upregulation (p&lt;0.001), a well-established key regulator of endotoxin tolerance. This immunosuppressive effect was coupled with increased resistance to mitochondrial stress (p&lt;0.001). In conclusion, we see a PRG4-specific phenotype in macrophages; suppressed phagocytosis capability, and acute pro-inflammatory activation of NF-κB and inflammasome pathways, that over longer exposure develops into immunosuppression. This may have profound implications for the overall disease mechanism.

Paeonol reduces IL-β production by inhibiting the activation of nucleotide oligomerization domain-like receptor protein-3 inflammasome and nuclear factor-κB in macrophages.

Interleukin-1β, a key cytokine in gouty inflammation, is precisely regulated by the NLRP3 inflammasome and NF-κB. Our previous study demonstrated that paeonol suppressed IL-1β production in rats with monosodium urate (MSU)-induced arthritis. Whether NLRP3 inflammasome or NF-κB is responsible for the anti-inflammatory effect of paeonol remains unclear. In this study, J774A.1 cells induced by lipopolysaccharide (LPS) plus MSU, was used to investigate the effect of paeonol on NLRP3 inflammasome activation, and J774A.1 cells induced by LPS alone were used to investigate the effect of paeonol on NF-κB activation. In J774A.1 cells induced by LPS plus MSU, paeonol decreased the levels of IL-1β and caspase-1 and reduced the MSU-induced interaction of pro-caspase-1 and apoptosis-associated speck-like protein containing caspase recruitment domain (ASC), but did not affect the levels of pro-IL-1β and pro-caspase-1. In J774A.1 cells induced by LPS alone, paeonol reduced the levels of IL-1β, NLRP3, p-IKK, p-IκBα, and p-p65, but did not affect ASC levels. Paeonol also promoted the content of IκBα and retained more p65 in the cytoplasm. Furthermore, paeonol reduced the DNA-binding activity of p65 and lowered the levels of p-JNK, p-ERK, and p-p38. These results suggest that paeonol inhibits IL-1β production by inhibiting the activation of NLRP3 inflammasome, NF-κB, and MAPK signaling pathways.