Expression Of Inflammasome Components Research Articles

Streptococcus mutans outer membrane vesicles affect inflammasome activation and the glycolysis of macrophages

Recent studies indicate that bacterial outer membrane vesicles (OMVs) play a significant role in bacterial virulence and pathogenicity. Streptococcus mutans (S. mutans), a principal pathogen in dental caries, secretes a substantial number of OMVs. However, the impact of S. mutans OMVs on oral health and their underlying pathogenic mechanisms remain poorly understood. Macrophages were the initial innate immune cells to respond to bacterial invaders and their products. Therefore, we purified S. mutans OMVs, which stimulated macrophages. Compared to controls, RT-PCR and ELISA analyses revealed that S. mutans OMVs significantly increased the production of IL-1β, IL-6, TNF-α and IL-8, with IL-1β being notably elevated. IL-1β production and secretion are tightly regulated by the inflammasome. Western blot analyses demonstrated that S. mutans OMVs upregulated the expression of inflammasome components, including NLRP3, NLRC4, ASC and AIM2, with a marked increase in NLRP3 expression. Silencing different inflammasome components with siRNA revealed a reduction in IL-1β secretion induced by S. mutans OMVs, particularly through NLRP3. Additionally, ATP production and K+ efflux were found to be crucial for NLRP3 activation. Prolonged stimulation with S. mutans OMVs resulted in increased lactate production and elevated expression of glycolysis-related genes Glut-1, PFKFB3, and HK I, indicating that S. mutans OMVs significantly induce macrophage glycolysis. Furthermore, S. mutans OMVs were shown to enhance biofilm formation, increase S. mutans colonisation on epithelial cells, and inhibit macrophage phagocytosis, thereby improving the survival of S. mutans in the oral cavity. In summary, S. mutans OMVs promote the survival of S. mutans in the mouth through multiple mechanisms, potentially influencing the development of dental caries.

Assessment of the role of high-density lipoproteins and their immunomodulatory activity in systemic lupus erythematosus immunopathology

ObjectiveTo explore the potential associations between high-density lipoprotein (HDL) levels and inflammasome components in the context of systemic lupus erythematosus (SLE).MethodsA cross-sectional study was conducted. A group of 50 patients...

Coarse particulate matter (PM10) induce an inflammatory response through the NLRP3 activation

IntroductionPM exposure can induce inflammatory and oxidative responses; however, differences in these adverse effects have been reported depending on the chemical composition and size. Moreover, inflammatory mechanisms such as NLRP3 activation by PM10 have yet to be explored.ObjectiveTo assess the impact of PM10 on cell cytotoxicity and the inflammatory response through in vitro and in vivo models.MethodologyPeripheral blood mononuclear cells (PBMCs) from healthy donors were exposed to PM10. Cytotoxicity was determined using the LDH assay; the expression of inflammasome components and the production of pro-inflammatory cytokines were quantified through qPCR and ELISA, respectively; and the formation of ASC complexes was examined using confocal microscopy. For in vivo analysis, male C57BL6 mice were intranasally challenged with PM10 and bronchoalveolar lavage fluid was collected to determine cell counts and quantification of pro-inflammatory cytokines by ELISA. RNA was extracted from lung tissue, and the gene expression of inflammatory mediators was quantified.ResultsPM10 exposure induced significant cytotoxicity at concentrations over 100 µg/mL. Moreover, PM10 enhances the gene expression and release of pro-inflammatory cytokines in PBMCs, particularly IL-1β; and induces the formation of ASC complexes in a dose-dependent manner. In vivo, PM10 exposure led to cell recruitment to the lungs, which was characterized by a significant increase in polymorphonuclear cells compared to control animals. Furthermore, PM10 induces the expression of several inflammatory response-related genes, such as NLRP3, IL-1β and IL-18, within lung tissue.ConclusionBriefly, PM10 exposure reduced the viability of primary cells and triggered an inflammatory response, involving NLRP3 inflammasome activation and the subsequent production of IL-1β. Moreover, PM10 induces the recruitment of cells to the lung and the expression of multiple cytokines; this phenomenon could contribute to epithelial damage and, thus to the development and exacerbation of respiratory diseases such as viral infections.

Amino Sugar-Enriched Fraction of Korean Red Ginseng Extract Induces the Priming Step of NLRP3 Inflammasome.

Intracellular protein complexes, known as inflammasomes, activate caspase-1 and induce the secretion of pro-inflammatory cytokines, namely interleukin (IL)-1β and -18. Korean Red Ginseng extract (RGE) is a known immunomodulator and a potential candidate for the regulation of inflammasomes. The saponins, such as ginsenosides, of RGE inhibit inflammasome signaling, while non-saponin substances containing amino sugars promote the priming step, up-regulating inflammasome components (pro-IL-1β, NLRP3, caspase-1, and Asc). In this study, the amino sugar-enriched fraction (ASEF), which increases only non-saponin components, including amino sugars, without changing the concentration of saponin substances, was used to investigate whether saponin or non-saponin components of RGE would have a greater impact on the priming step. When murine macrophages were treated with ASEF, the gene expression of inflammatory cytokines (IL-1α, TNFα, IL-6, and IL-10) increased. Additionally, ASEF induced the priming step but did not affect the inflammasome activation step, such as the secretion of IL-1β, cleavage of caspase-1, and formation of Asc pyroptosome. Furthermore, the upregulation of gene expression of inflammasome components by ASEF was blocked by inhibitors of Toll-like receptor 4 signaling. Maltol, the main constituent of ASEF, promoted the priming step but inhibited the activation step of the inflammasome, while arginine, sugars, arginine-fructose-glucose, and fructose-arginine, the other main constituents of ASEF, had no effect on either step. Thus, certain amino sugars in RGE, excluding maltol, are believed to be the components that induce the priming step. The priming step that prepares the NLRP3 inflammasome for activation appears to be induced by amino sugars in RGE, thereby contributing to the immune-boosting effects of RGE.

HSP90β controls NLRP3 autoactivation.

Gain-of-function mutations in NLRP3 are linked to cryopyrin-associated periodic syndromes (CAPS). Although NLRP3 autoinflammasome assembly triggers inflammatory cytokine release, its activation mechanisms are not fully understood. Our study used a functional genetic approach to identify regulators of NLRP3 inflammasome formation. We identified the HSP90β-SGT1 chaperone complex as crucial for autoinflammasome activation in CAPS. A deficiency in HSP90β, but not in HSP90α, impaired the formation of ASC specks without affecting the priming and expression of inflammasome components. Conversely, activating NLRP3 with stimuli such as nigericin or alum bypassed the need for SGT1 and HSP90β, suggesting the existence of alternative inflammasome assembly pathways. The role of HSP90β was further demonstrated in PBMCs derived from CAPS patients. In these samples, the pathological constitutive secretion of IL-1β could be suppressed using a pharmacological inhibitor of HSP90β. This finding underscores the potential of SGT1-HSP90β modulation as a therapeutic strategy in CAPS while preserving NLRP3's physiological functions.

Age-related exacerbation of lung damage after trauma is associated with increased expression of inflammasome components.

Trauma, a significant global cause of mortality and disability, often leads to fractures and hemorrhagic shock, initiating an exaggerated inflammatory response, which harms distant organs, particularly the lungs. Elderly individuals are more vulnerable to immune dysregulation post-trauma, leading to heightened organ damage, infections, and poor health outcomes. This study investigates the role of NF-κB and inflammasomes in lung damage among aged mice post-trauma. Twelve male C57BL/6J mice underwent hemorrhagic shock and a femoral fracture (osteotomy) with external fixation (Fx) (trauma/hemorrhage, THFx), while another 12 underwent sham procedures. Mice from young (17-26 weeks) and aged (64-72 weeks) groups (n=6) were included. After 24h, lung injury was assessed by hematoxylin-eosin staining, prosurfactant protein C (SPC) levels, HMGB1, and Muc5ac qRT-PCR. Gene expression of Nlrp3 and Il-1β, and protein levels of IL-6 and IL-1β in lung tissue and bronchoalveolar lavage fluid were determined. Levels of lung-infiltrating polymorphonuclear leukocytes (PMNL) and activated caspase-3 expression to assess apoptosis, as well as NLRP3, ASC, and Gasdermin D (GSDMD) to assess the expression of inflammasome components were analyzed via immunostaining. To investigate the role of NF-κB signaling, protein expression of phosphorylated and non-phosphorylated p50 were determined by western blot. Muc5ac, and SPC as lung protective proteins, significantly declined in THFx versus sham. THFx-aged exhibited significantly lower SPC and higher HMGB1 levels versus THFx-young. THFx significantly increased activated caspase-3 versus both sham groups, and THFx-aged had significantly more caspase-3 positive cells versus THFx-young. IL-6 significantly increased in both sham and THFx-aged groups versus corresponding young groups. THFx significantly enhanced PMNL in both groups versus corresponding sham groups. This increase was further heightened in THFx-aged versus THFx-young. Expression of p50 and phosphorylated p50 increased in all aged groups, and THFx-induced p50 phosphorylation significantly increased in THFx-aged versus THFx-young. THFx increased the expression of inflammasome markers IL-1β, NLRP3, ASC and GSDMD versus sham, and aging further amplified these changes significantly. This study's findings suggest that the aging process exacerbates the excessive inflammatory response and damage to the lung following trauma. The underlying mechanisms are associated with enhanced activation of NF-κB and increased expression of inflammasome components.

Regulated cell death and inflammasome activation in gut injury following traumatic surgery in vitro and in vivo: implication for postoperative death due to multiorgan dysfunction

Postoperative multi-organ dysfunction (MOD) is associated with significant mortality and morbidity. Necroptosis has been implicated in different types of solid organ injury; however, the mechanisms linking necroptosis to inflammation require further elucidation. The present study examines the involvement of necroptosis and NLR family pyrin domain containing 3 (NLRP3) inflammasome in small intestine injury following traumatic surgery. Kidney transplantation in rats and renal ischaemia-reperfusion (I/R) in mice were used as traumatic and laparotomic surgery models to study necroptosis and inflammasome activation in the small intestinal post-surgery; additional groups also received receptor-interacting protein kinase 1 (RIPK1) inhibitor necrostatin-1s (Nec-1s). To investigate whether necroptosis regulates inflammasome activity in vitro, necroptosis was induced in human colonic epithelial cancer cells (Caco-2) by a combination of tumour necrosis factor-alpha (TNFα), SMAC mimetic LCL-161 and pan-caspase inhibitor Q-VD-Oph (together, TLQ), and necroptosis was blocked by Nec-1s or mixed lineage kinase-domain like (MLKL) inhibitor necrosulfonamide (NSA). Renal transplantation and renal ischaemia-reperfusion (I/R) upregulated the expression of necroptosis mediators (RIPK1; RIPK3; phosphorylated-MLKL) and inflammasome components (P2X purinoceptor subfamily 7, P2X7R; NLRP3; caspase-1) in the small intestines at 24 h, and Nec-1s suppressed the expression of inflammasome components. TLQ treatment induced NLRP3 inflammasome, promoted cleavage of caspase-1 and interleukin-1 beta (IL-1β), and stimulated extracellular ATP release from Caco-2 cells, and MLKL inhibitor NSA prevented TLQ-induced inflammasome activity and ATP release from Caco-2 cells. Our work suggested that necroptosis and inflammasome interactively promote remote postoperative small intestinal injury, at least in part, through ATP purinergic signalling. Necroptosis-inflammasome axis may be considered as novel therapeutic target for tackling postoperative MOD in the critical care settings.

The Comparative Effects of Schwann Cells and Wharton's Jelly Mesenchymal Stem Cells on the AIM2 Inflammasome Activity in an Experimental Model of Spinal Cord Injury

Inflammasome activation and the consequent release of pro-inflammatory cytokines play a crucial role in the development of sensory/motor deficits following spinal cord injury (SCI). Immunomodulatory activities are exhibited by Schwann cells (SCs) and Wharton's jelly mesenchymal stem cells (WJ-MSCs). In this study, we aimed to compare the effectiveness of these two cell sources in modulating the absent in melanoma 2 (AIM2) inflammasome complex in rats with SCI. The Basso, Beattie, Bresnahan (BBB) test, Nissl staining, and Luxol fast blue (LFB) staining were performed to evaluate locomotor function, neuronal survival, and myelination, respectively. Real-time polymerase chain reaction (RT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA) were employed to analyze the gene and protein expressions of inflammasome components, including AIM2, ASC, caspase-1, interleukin-1β (IL-1β), and IL-18. Both gene and protein expressions of all evaluated factors were decreased after SC or WJ-MSC treatment, with a more pronounced effect observed in the SCs group (P < 0.05). Additionally, SCs promoted neuronal survival and myelination. Moreover, the administration of 3 × 105 cells resulted in motor recovery improvement in both treatment groups (P < 0.05). Although not statistically significant, these effects were more prominent in the SC-treated animals. In conclusion, SC therapy demonstrated greater efficacy in targeting AIM2 inflammasome activation and the associated inflammatory pathway in SCI experiments compared to WJ-MSCs.

Photobiomodulation Promotes Hippocampal Neurogenesis and Improves Cognitive Function and Anti-Inflammatory Injury in Rats With Chronic Cerebral Hypoperfusion

To investigate the effect of photobiomodulation (PBM) on hippocampal neurogenesis, cognitive function, and inflammatory injury in rats with chronic cerebral hypoperfusion. Bilateral ovariectomy (OVX) was performed on female Sprague-Dawley (SD) rats. One week later, the rats were randomly assigned to three groups, Sham surgery (or Sham) group, bilateral common carotid artery occlusion (BCCAO) group, and PBM intervention (or BCCAO+PBM) group. There were 8 rats in each group. In the BCCAO group, chronic cerebral hyporeperfusion was induced by permanent ligation of bilateral common carotid arteries and no PBM was given. Rats in the Sham group underwent the same surgical procedure except for the occlusion of the two carotids arteries and no PBM was given. In addition to the BCCAO surgery, rats in the BCCAO+PBM group received 808 nm laser therapy (5 min each time at a laser dose of 20 mW/cm 2) of the frontal cortex every other day for 1 month. Between 86 and 90 days after BCCAO, Morris water maze (MWM) was used to observe the spatial learning and memory function of the rats. The rats were sacrificed on day 90 and immunofluorescence staining and Western blot were performed thereafter. Immunofluorescence staining was used to determine the expression of 5-bromodeoxyuracil nucleoside (BrdU), a cell proliferation marker, glial fibrillary acidic protein (GFAP), an astrocyte marker, doublecortin (DCX), a specific marker of newborn neuron precursor cells, NeuN, a marker of mature neurons, and Iba1, a microglia marker, in the hippocampal dentate gyrus (DG) region. Western blot was performed to analyze the protein expressions of inflammasome components, NLRP3, ASC, cleaved caspase-1, and Iba1 in the hippocampus. In the latency trial of MWM test, BCCAO+PBM rats spent shorter periods of time finding the underwater platform than the BCCAO rats did. In the probe trial, after the platform that was original placed in a quadrant was removed, the BCCAO+PBM rats spent longer periods of time exploring the quadrant than the BCCAO animals did ( P<0.05). Compared with BCCAO rats, BCCAO+PBM rats showed significant decrease in the immunofluorescence intensities of GFAP and Iba1 ( P<0.01). PBM intervention significantly increased the number of BrdU-positive cells in the hippocampal DG region compared with those of Sham and BCCAO groups ( P<0.05). Furthermore, the number of NeuN positive cells showed no significant difference among the three groups, while in BCCAO+PBM group, the number of DCX-positive cells was significantly increased ( P<0.001) and the number of DCX +/NeuN + co-located cells was significantly increased compared to that of the BCCAO group ( P<0.001). Compared with those of the BCCAO group, Western blot results showed that the protein expression levels of Iba1, NLRP3, and cleaved caspase-1 in the BCCAO+PBM group were significantly decreased ( P<0.05), while the ASC protein expression level showed no significant difference. PBM can effectively improve the spatial learning and memory function in rats with chronic cerebral hypoperfusion, inhibit the activation of glial cells, reduce inflammatory damage mediated by NLRP3 inflammasome, and promote the regeneration of endogenous neural stem cells in the hippocampal DG region of rats.

No NLRP3 inflammasome activity in kidney epithelial cells, not even when the NLRP3-A350V Muckle-Wells variant is expressed in podocytes of diabetic mice.

The NLRP3 inflammasome integrates several danger signals into the activation of innate immunity and inflammation by secreting IL-1β and IL-18. Most published data relate to the NLRP3 inflammasome in immune cells, but some reports claim similar roles in parenchymal, namely epithelial, cells. For example, podocytes, epithelial cells critical for the maintenance of kidney filtration, have been reported to express NLRP3 and to release IL-β in diabetic kidney disease, contributing to filtration barrier dysfunction and kidney injury. We questioned this and hence performed independent verification experiments. We studied the expression of inflammasome components in human and mouse kidneys and human podocytes using single-cell transcriptome analysis. Human podocytes were exposed to NLRP3 inflammasome agonists in vitro and we induced diabetes in mice with a podocyte-specific expression of the Muckle-Wells variant of NLRP3, leading to overactivation of the Nlrp3 inflammasome (Nphs2Cre;Nlrp3A350V) versus wildtype controls. Phenotype analysis included deep learning-based glomerular and podocyte morphometry, tissue clearing, and STED microscopy of the glomerular filtration barrier. The Nlrp3 inflammasome was blocked by feeding ß-hydroxy-butyrate. Single-cell transcriptome analysis did not support relevant NLRP3 expression in parenchymal cells of the kidney. The same applied to primary human podocytes in which NLRP3 agonists did not induce IL-1β or IL-18 secretion. Diabetes induced identical glomerulomegaly in wildtype and Nphs2Cre;Nlrp3A350V mice but hyperfiltration-induced podocyte loss was attenuated and podocytes were larger in Nphs2Cre;Nlrp3A350V mice, an effect reversible with feeding the NLRP3 inflammasome antagonist ß-hydroxy-butyrate. Ultrastructural analysis of the slit diaphragm was genotype-independent hence albuminuria was identical. Podocytes express low amounts of the NLRP3 inflammasome, if at all, and do not produce IL-1β and IL-18, not even upon introduction of the A350V Muckle-Wells NLRP3 variant and upon induction of podocyte stress. NLRP3-mediated glomerular inflammation is limited to immune cells.

Abstract 438: FDA-approved Drug For Alcoholism Reduces Atherosclerosis By Inhibiting Pyroptotic Executor Gasdermin D (GsdmD)

Introduction: Atherosclerosis is driven by chronic inflammation of the artery wall in response to cholesterol accumulation and defects in reverse cholesterol transport (RCT). Activation of the NLRP3 inflammasome in foam cells leads to cleavage of Gasdermin D (GsdmD), facilitating release of the mature interleukin-1beta (IL-1β). Work from our lab and others have shown that GsdmD promotes atherosclerosis. Recently, the FDA-approved drug, Disulfiram, was shown to inhibit GsdmD. Though the GsdmD/IL-1β pathway has been implicated in CVD, the efficacy of Disulfiram as an anti-atherosclerotic therapy has not been explored. Hypothesis: We aim to establish a mechanism-based role of Disulfiram as an atherosclerotic therapeutic. We hypothesize that Disulfiram inhibition of GsdmD-pore formation and subsequent IL-1β release will reduce atherosclerosis. Methods and Results: Efficacy of Disulfiram (DSF) as an GsdmD inhibitor was confirmed in inflammasome-induced macrophages and in mice, with both showing significantly reduced IL-1b release with no effect on expression of inflammasome components. To test if DSF can reduce atherosclerosis, hyperlipidemic apoE -/- mice were weaned onto a western-type diet (WTD) or WTD + 25mg/kg DSF for 15 weeks. Excitingly, DSF vs. WTD-fed mice showed smaller lesion burden (p&lt;0.01) by 26.7% for males and 29.4% for females. Total cholesterol levels were higher in DSF vs WTD males (p&lt;0.05) and females (p&lt;0.01). DSF-diet did not impact body or liver weight, white blood cells, platelets, lymphocytes, monocytes or eosinophils. Red blood cells (RBCs) and neutrophils were decreased in females (p&lt;0.01 and p&lt;0.05, respectively), but not in males. DSF-fed mice have reduced macrophage infiltration into atherosclerotic lesion sites. Conclusions: Our pre-clinical data suggests Disulfiram has the potential to be used as a stand-alone therapy or in combination with current atherosclerosis therapies.

Inhibition of LPS‑induced NLRP3 inflammasome activation by stem cell‑conditioned culture media in human gingival epithelial cells.

Interleukin (IL)‑1β is a pathogenic factor associated with the destruction of periodontal tissue in periodontitis. IL‑1β processing is regulated by cytosolic machinery known as the inflammasome. Porphyromonas gingivalis infection and lipopolysaccharide (LPS) have an important role in the destruction of periodontal tissue in periodontitis. P. gingivalis infection and LPS have been reported to activate the NOD‑like receptor family pyrin domain‑containing protein 3 (NLRP3) inflammasome in human oral cells. Stem cell therapy exhibits anti‑inflammatory effects and stem cell‑conditioned culture media (SCM) shows similar beneficial effects. The present study tested the hypothesis that SCM inhibits activation of the inflammasome and protects human gingival epithelial cells (GECs) against LPS‑induced inflammatory damage. Human GECs were treated with or without LPS plus SCM or control cell media. NLPR3 inflammasome components and inflammatory factors were measured by western blotting and immunofluorescence. The present study revealed that LPS induced an increase in the expression of inflammasome components, NLRP3, apoptosis‑associated speck‑like protein containing a caspase recruitment domain (ASC) and caspase‑1. Co‑immunoprecipitation revealed increased binding of NLRP3 and ASC, and immunofluorescence showed an increased co‑localization of ASC and caspase‑1, suggesting that LPS stimulated assembly of the NLRP3 inflammasome. SCM inhibited the overexpression and assembly of NLRP3 inflammasome components induced by LPS. Furthermore, SCM blocked the increase in IL‑1β production induced by LPS and inhibited the translocation of the inflammatory factor, NF‑κB, into the nuclei. Consequently, SCM protected cells against LPS‑induced damage, as suggested by the recovery of disturbed E‑cadherin staining pattern, which indicates a disruption in epithelial integrity. In conclusion, treatment with SCM may attenuate LPS‑induced inflammatory damage in human GECs via inhibition of NLRP3 inflammasome activation, suggesting a potential therapeutic use for SCM.

Mechanisms of systemic low-grade inflammation in HIV patients on long-term suppressive antiretroviral therapy: the inflammasome hypothesis.

We aimed to determine the contribution of inflammasome activation in chronic low-grade systemic inflammation observed in patients with HIV (PWH) on long-term suppressive antiretroviral therapy (ART) and to explore mechanisms of such activation. Forty-two PWH on long-term suppressive ART (HIV-RNA < 40 copies/ml) were compared with 10 HIV-negative healthy controls (HC). Inflammasome activation was measured by dosing mature interleukin (IL)-1β and IL-18 cytokines in patient serum. We explored inflammasome pathways through ex vivo stimulation of PWH primary monocytes with inflammasome activators; expression of inflammasome components by transcriptomic analysis; and metabolomics analysis of patient sera. Median (Q1; Q3) age, ART and viral suppression duration in PWH were 54 (48; 60), 15 (9; 20) and 7.5 (5; 12) years, respectively. Higher serum IL-18 was measured in PWH than in HC (61 (42; 77) vs. 36 (27-48 pg/ml), P = 0.009); IL-1β was detected in 10/42 PWH (0.5 (0.34; 0.80) pg/ml) but not in HC. Monocytes from PWH did not produce more inflammatory cytokines in vitro, but secretion of IL-1β in response to NLRP3 inflammasome stimulation was higher than in HC. This was not explained at the transcriptional level. We found an oxidative stress molecular profile in PWH sera. HIV infection with long-term effective ART is associated with a serum inflammatory signature, including markers of inflammasome activation, and an increased activation of monocytes upon inflammasome stimulation. Other cells should be investigated as sources of inflammatory cytokines in PWH. Oxidative stress might contribute to this chronic low-grade inflammation.

Chronic Exposure to High Fat Diet Affects the Synaptic Transmission That Regulates the Dopamine Release in the Nucleus Accumbens of Adolescent Male Rats

Obesity is a pandemic caused by many factors, including a chronic excess in hypercaloric and high-palatable food intake. In addition, the global prevalence of obesity has increased in all age categories, such as children, adolescents, and adults. However, at the neurobiological level, how neural circuits regulate the hedonic consumption of food intake and how the reward circuit is modified under hypercaloric diet consumption are still being unraveled. We aimed to determine the molecular and functional changes of dopaminergic and glutamatergic modulation of nucleus accumbens (NAcc) in male rats exposed to chronic consumption of a high-fat diet (HFD). Male Sprague-Dawley rats were fed a chow diet or HFD from postnatal day (PND) 21 to 62, increasing obesity markers. In addition, in HFD rats, the frequency but not amplitude of the spontaneous excitatory postsynaptic current is increased in NAcc medium spiny neurons (MSNs). Moreover, only MSNs expressing dopamine (DA) receptor type 2 (D2) increase the amplitude and glutamate release in response to amphetamine, downregulating the indirect pathway. Furthermore, NAcc gene expression of inflammasome components is increased by chronic exposure to HFD. At the neurochemical level, DOPAC content and tonic dopamine (DA) release are reduced in NAcc, while phasic DA release is increased in HFD-fed rats. In conclusion, our model of childhood and adolescent obesity functionally affects the NAcc, a brain nucleus involved in the hedonic control of feeding, which might trigger addictive-like behaviors for obesogenic foods and, through positive feedback, maintain the obese phenotype.

Transcriptomics and experimental validation-based approach to understand the effect and mechanism of Huangqin tang interfeience with colitis associated colorectal cancer

ContextChronic inflammation is usually caused by persistent irritation or uncontrolled infection and is characterized by ongoing tissue damage, injury-induced cellular proliferation and tissue repair. Colitis-associated colorectal cancer (CAC) isone of the classic examples of tumors that are tightly related to chronic inflammation. BackgroundTo investigated the key pharmacodynamic genes of HQT interventions in CAC by using transcriptome predictions and experiments.Materials & Methods: We used the azoxymethane/dextran sodium sulfate method to induce the mice CAC model. After preventive administration of HQT to the mice model, colonic tissues were taken for transcriptome sequencing and the transcriptome results were then experimentally validated using quantitative Real-Time PCR technique. ResultsTranscriptome sequencing revealed that the effect of the mechanism of HQT on the CAC mice model maybe related to its inhibition of accelerated epithelial mesenchymal transition and induction of pyroptosis. The levels of Matrix-metalloproteinases such as MMP-2, MMP-9 were significantly reduced in CAC mice treated with HQT; The mRNA expression for Krt17, App, CD44 and WNT pathway related sites such as Lrrc15, Cldn-1, Mpc1, Agr2 which are related factors affecting the epithelial mesenchymal transition were significantly reduced in CAC mice treated with HQT; the aberrant mRNA expression of inflammasome components that drive pyroptosis, including Nlrp3, Caspase-1, ASC, GSDMD and its mediated product IL-18 have been improved. ConclusionsOur findings provide preliminary clarification that inhibiting the progression of CAC by using HQT is effective, the mechanism of action may be relatedto the inhibition of epithelial mesenchymal transition and induction of pyroptosis during tumorigenesis.

CASP4/11 Contributes to NLRP3 Activation and COVID-19 Exacerbation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection triggers activation of the NLRP3 inflammasome, which promotes inflammation and aggravates severe COVID-19. Here, we report that SARS-CoV-2 induces upregulation and activation of human caspase-4/CASP4 (mouse caspase-11/CASP11), and this process contributes to NLRP3 activation. In vivo infections performed in transgenic hACE2 humanized mice, deficient or sufficient for Casp11, indicate that hACE2 Casp11-/- mice were protected from disease development, with the increased pulmonary parenchymal area, reduced clinical score of the disease, and reduced mortality. Assessing human samples from fatal cases of COVID-19, we found that CASP4 was expressed in patient lungs and correlated with the expression of inflammasome components and inflammatory mediators, including CASP1, IL1B, IL18, and IL6. Collectively, our data establish that CASP4/11 promotes NLRP3 activation and disease pathology, revealing a possible target for therapeutic interventions for COVID-19.

PS-B06-7: ENDOTHELIAL DYSFUNCTION EXACERBATES KIDNEY DYSFUNCTION IN AGEING KIDNEY VIA ACTIVATION OF INFLAMMASOME

Background: Ageing affects the function of the immune system and leads to immunosenescence, which is characterized by defective immune responses and increased systemic inflammation (also termed inflammageing). Inflammageing is maladaptive and results from multiple mechanisms, including aberrant inflammasome activation. The ASC is an essential component of inflammasomes that activates caspase 1. Endothelial dysfunction is also a common pathophysiologic mechanism of age-related organ damage. Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is an important mediator in the maintenance of vascular homeostasis. We have reported that the eNOS-NO pathway attenuates the progression of kidney injury via suppression of inflammasomes. However, the relationship between the eNOS-NO pathway and inflammageing in the kidney remains unclear. To determine if the eNOS-NO pathway attenuates kidney damage via the regulation of inflammasome activation in ageing kidneys, we used eNOS-deficient mice (eNOSKO) and eNOS-ASC double-knockout mice (eNOS-ASC-DKO). Methods: Wild-type mice (WT) and eNOSKO were used to determine the role of the eNOS-NO pathway in ageing kidneys. WT and eNOSKO were sacrificed at 18 months of age to harvest kidney tissue. The localization of inflammasome activation in the kidney was evaluated with immunohistochemical analyses. To determine the role of inflammasomes in ageing kidneys, we generated eNOS-ASC-DKO. The mRNA expression of inflammasome components were determined in isolated glomeruli. Results: The glomerular injury was more exacerbated in eNOSKO-18 M than in WT-18 M. In the immunohistochemical analyses, the expression of ASC coexisted with the macrophages detected by F4/80 staining in eNOSKO-18 M. These data suggested that the inflammasome activation was located in the macrophages. In the isolated glomeruli, the mRNA of the inflammasome components was higher in eNOSKO-18 M than in WT-18 M. The glomerular damage and tubulointerstitial damage were ameliorated in eNOS-ASC-DKO-18 M compared to eNOSKO-18 M. In summary, in eNOS-deficient mice, inflammasomes were activated in macrophages, and interstitial fibrosis was exacerbated. However, in eNOS-ASC-DKO-18 M mice, the tubulointerstitial damage was attenuated. Conclusion: Endothelial NOS/NO signaling ameliorates kidney damage in the aging process via the modulation of inflammageing associated with inflammasome-activation.

Increased expression of NLRP3 inflammasome components in granulosa cells and follicular fluid interleukin(IL)-1beta and IL-18 levels in fresh IVF/ICSI cycles in women with endometriosis.

The inflammasomes are a family of recently described multi-protein cytoplasmic sensors that orchestrate the inflammatory response and participate in a variety of inflammatory conditions. We hypothesized that the activation of pyrin domain‑containing protein 3 (NLRP3) inflammasome by granulosa cells (hGCs) may be activated in women with endometriosis and influence oocyte maturation and IVF outcomes. We performed a cross-sectional study to investigate the NLRP3 inflammasome status in follicular fluid (FF) and in hGCs from 44 women undergoing controlled ovarian stimulation for IVF/ICSI. Study subjects were divided into two groups according to the infertility etiology: group with tubal or male factor (control, n = 22) vs. group with endometriosis (n = 22). The FF IL-1beta and IL-18 levels in the endometriosis group were significantly higher than those in the non-endometriosis group, i.e., 5010pg/mL and 2738pg/mL, respectively (p < 0.05). No correlation was found between clinical pregnancy and live birth rate and analyzed inflammasome component levels (p > 0.05). In addition, the hGCs from endometriosis women demonstrated high expression of NLRP3 inflammasome at both protein and mRNA levels. Higher expression of inflammasome components within the ovary compartment may result from the exaggerated inflammatory state associated with endometriosis and thus impact the fertility of these women.

Trimethylamine oxide induces pyroptosis of vascular endothelial cells through ALDH2/ROS/NLRP3/GSDMD pathway.

Trimethylamine oxide (TMAO) is a metabolite of intestinal flora and is known to promote the progression of atherosclerotic plaques. However, how TMAO works, including its effect on vascular endothelial cells, is not fully understood. This study aims to explore the biological role of TMAO in human umbilical vein endothelial cells (HUVECs) and the underlying mechanism. Cell pyroptosis and the loss of plasma membrane integrity were induced under TMAO stimulation in HUVECs. The plasma membrane integrity of the cells was measured by Hoechst 33342/propidium iodide (PI) staining and lactate dehydrogenase leakage assay, and the changes in cell morphology were observed by atomic force microscope. The expression of proteins related to pyroptosis was determined by Western blotting or immunofluorescence. Mitochondrial acetaldehyde dehydrogenase 2 (ALDH2) activity in HUVECs was measured by the ALDH2 activity assay kit, and the level of reactive oxygen species (ROS) was detected by fluorescent probe DCFH-DA. TMAO induced pyroptotic cell death, manifesting by the presence of propidium iodide-positive cells, the leakage of lactate dehydrogenase, the production of N-terminal gasdermin D (GSDMD-N), and the formation of plasma membrane pores. Moreover, TMAO induced elevated expression of inflammasome components, nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1 in cells. TMAO significantly inhibited ALDH2 activity and increased intracellular ROS production. However, the activation of ALDH2 by pharmacological manipulation attenuated TMAO-induced inflammasome activation and GSDMD-N production. TMAO induces pyroptosis of vascular endothelial cells through the ALDH2/ROS/NLRP3/GSDMD signaling pathway, which may be a potential therapeutic target for improving the treatment of atherosclerosis.

Activation of NLRP3 Is Required for a Functional and Beneficial Microglia Response after Brain Trauma

Despite the numerous research studies on traumatic brain injury (TBI), many physiopathologic mechanisms remain unknown. TBI is a complex process, in which neuroinflammation and glial cells play an important role in exerting a functional immune and damage-repair response. The activation of the NLRP3 inflammasome is one of the first steps to initiate neuroinflammation and so its regulation is essential. Using a closed-head injury model and a pharmacological (MCC950; 3 mg/kg, pre- and post-injury) and genetical approach (NLRP3 knockout (KO) mice), we defined the transcriptional and behavioral profiles 24 h after TBI. Wild-type (WT) mice showed a strong pro-inflammatory response, with increased expression of inflammasome components, microglia and astrocytes markers, and cytokines. There was no difference in the IL1β production between WT and KO, nor compensatory mechanisms of other inflammasomes. However, some microglia and astrocyte markers were overexpressed in KO mice, resulting in an exacerbated cytokine expression. Pretreatment with MCC950 replicated the behavioral and blood–brain barrier results observed in KO mice and its administration 1 h after the lesion improved the damage. These findings highlight the importance of NLRP3 time-dependent activation and its role in the fine regulation of glial response.