Induced Inflammasome Activation Research Articles

Psoralidin, a major component of Psoraleae Fructus, induces inflammasome activation and idiosyncratic liver injury

Idiosyncratic drug-induced liver injury (IDILI) is a rare but potentially fatal disease that is unpredictable and independent of the dose of the drug. Increasing evidence suggests that the majority of IDILI cases are immune-mediated, and the aberrant activation of inflammasome plays a vital role in progression. Psoraleae Fructus (PF), a tonic Chinese medicine, has been able to cause IDILI, but the precise mechanism of hepatotoxicity remains unclear. In this study, eight bioactive compounds involved in PF-induced inflammasome activation were investigated. The results demonstrated that psoralidin activated the inflammasomes followed by secreting caspase-1 and interleukin 1β (IL-1β) in a dose-dependent manner. Interestingly, MCC950, a potent inhibitor of the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, could not entirely suppress the psoralidin-induced inflammasome activation. Moreover, psoralidin significantly induced IL-1β maturation and caspase-1 activation in NLRP3-knockout bone marrow-derived macrophages (BMDMs), suggesting that psoralidin not only activates the NLRP3 inflammasome but also activates other types of inflammasomes. The results also demonstrated that psoralidin activated the inflammasomes by promoting the C-terminal caspase recruitment domain (ASC) oligomerization, and the production of mitochondrial reactive oxygen species (mtROS) is a decisive factor in psoralidin-induced inflammasome activation. Importantly, in vivo data revealed that psoralidin induced hepatic inflammation, increased aminotransferase activity and increased the production of IL-1β and tumor necrosis factor(TNF-α) in a susceptible mouse model of lipopolysaccharide (LPS)-mediated IDILI. In summary, these results confirmed that psoralidin causes IDILI by inducing inflammasome activation. The study suggests that psoralidin is a possible risk factor and is responsible for PF-induced IDILI.

BRLF1 suppresses RNA Pol III-mediated RIG-I inflammasome activation in the early EBV lytic lifecycle.

Latent infection with herpesviruses constitutively activates inflammasomes, while lytic replication suppresses their activation through distinct mechanisms. However, how Epstein-Barr virus (EBV) lytic replication inhibits the activation of inflammasomes remains unknown. Here, we reveal that the EBV immediate-early protein BRLF1 inhibits inflammasome activation, and BRLF1 deficiency significantly increases the activation of inflammasomes and pyroptosis during early lytic lifecycle. BRLF1 interacts with RNA polymerase III subunits to suppress immunostimulatory small RNA transcription, RIG-I inflammasome activation, and antiviral responses. Consequently, BRLF1-deficient EBV primary infection induces robust T-cell and NK cell activation and killing through IL-1β and IL-18. A BRLF1-derived peptide that inhibits inflammasome activation is sufficient to suppress T-cell and NK cell responses during BRLF1-deficient EBV primary infection in lymphocytes. These results reveal a novel mechanism involved in the evasion of inflammasome activation and antiviral responses during EBV early lytic infection and provide a promising approach for the manipulation of inflammasomes against infection of oncogenic herpesviruses.

Abstract 16855: NOX4 NADPH Oxidase Mediates Diastolic Dysfunction in Stress Cardiomyopathy via Regulation of Inflammation and Fibrosis

Acute sympathetic stress in adults results in catecholamine overload and often leads to development of stress cardiomyopathy complicated by long-lasting diastolic dysfunction. We previously reported that activation of β-adrenergic signaling in the heart increases reactive oxygen species-mediated inflammasome activation and IL-18 release, inducing myocardial inflammation, fibrosis, and dysfunction. Here we tested the hypothesis that NOX4 NADPH oxidase is a primary mediator of myocardial inflammation and diastolic dysfunction in the setting of acute stress. Acute stress in wild-type and mice with cardiomyocyte (CM)-specific Nox4 deletion ( Nox4 CM-/- ) was induced with a single dose of β-adrenergic receptor agonist isoproterenol (ISO; 5 mg/kg) injection. After 3 days, ISO-treated wild-type mice had higher myocardial H 2 O 2 , inflammasome activation and IL-1β, IL-18, IL-6, CCL2, and TNF-α levels and enhanced myocardial interstitial macrophage infiltration compared with vehicle-treated or Nox4 CM-/- mice (p<0.001). Wild-type mice left ventricles had increased presence of activated fibroblasts (periostin + ) and myofibroblasts (α-actin-2 + ) and significantly higher interstitial collagen deposition compared with Nox4 CM-/- mice 7 days post-ISO. Assessment of left ventricular function showed significantly increased mitral valve E, decreased deceleration time, decreased tissue Doppler E’ and increased E/E’ without changes in ejection fraction and end systolic or diastolic volumes, suggesting restrictive pattern of diastolic dysfunction with increased filling pressure in wild-type compared with control or Nox4 CM-/- mice. ISO treatment of ventricular CMs isolated from wild-type mice significantly increased levels of H 2 O 2 and expression of NLRP3 and cleaved caspase-1 and IL-18 as compared with Nox4 -deficient or wild-type CMs pretreated with NOX4/NOX1 inhibitor GKT137831. These data suggest that NOX4 NADPH oxidase is a critical mediator of CM sympathetic overstimulation inducing inflammasome activation and cytokine release, myocardial inflammation, and fibrosis, resulting in impaired diastolic function. Inhibiting myocardial NOX4 during acute sympathetic stress may be beneficial to preserve cardiac function.

MiR-223-3p inhibits rTp17-induced inflammasome activation and pyroptosis by targeting NLRP3.

The incidence of syphilis caused by Treponema pallidum subsp pallidum (T pallidum) infection is accompanied by inflammatory injuries of vascular endothelial cells. Studies have revealed that T pallidum infection could induce inflammasome activation and pyroptosis in macrophages. MicroRNA‐223‐3p (miR‐223‐3p) was reported to be a negative regulator in inflammatory diseases. The present study aimed to explore whether miR‐223‐3p regulates T pallidum‐induced inflammasome activation and pyroptosis in vascular endothelial cells, and determine the mechanisms which underlie this process. MiR‐223‐3p levels in syphilis and control samples were determined. The biological function of miR‐223‐3p in the NLRP3 inflammasome and pyroptosis was evaluated in T pallidum‐infected human umbilical vein endothelial cells (HUVECs). We observed a dramatic decrease in miR‐223‐3p levels in syphilis patients (n = 20) when compared to healthy controls (n = 20). Moreover, miR‐223‐3p showed a notable inhibitory effect on recombinant Tp17 (rTP17)‐induced caspase‐1 activation, resulting in decrease in IL‐1β production and pyroptosis, which was accompanied by the release of lactate dehydrogenase (LDH) in HUVECs. Additionally, the dual‐luciferase assay confirmed that NLRP3 is a direct target of miR‐223‐3p. Moreover, NLRP3 overexpression or knockdown largely blocked the effects of miR‐223‐3p on T pallidum‐induced inflammasome activation and pyroptosis in HUVECs. Most importantly, a notable negative correlation was observed between miR‐223‐3p and NLRP3, caspase‐1, and IL‐1β, respectively, in the serum of syphilis patients and healthy controls. Taken together, our results reveal that miR‐223‐3p targets NLRP3 to suppress inflammasome activation and pyroptosis in T pallidum‐infected endothelial cells, implying that miR‐223‐3p could be a potential target for syphilis patients.

The Mitochondrial Redistribution of eNOS is involved in Lipopolysaccharide Induced Inflammasome Activation During Acute Lung Injury

Acute lung injury (ALI) is a devastating clinical syndrome with no effective therapies. Inflammasome activation has been reported to play a critical role in the initiation and progression of ALI. The molecular mechanisms involved in regulating the activation of inflammasome in ALI remains unresolved, although increases in mitochondrial derived reactive oxygen species (mito-ROS) are involved. Our previous work has shown that the mitochondrial redistribution of uncoupled eNOS impairs mitochondrial bioenergetics and increases mito-ROS generation. Thus, the focus of our study was to determine if lipopolysaccharide (LPS)-mediated inflammasome activation involves the mitochondrial redistribution of uncoupled eNOS. Our data show that the increase in mito-ROS involved in LPS-mediated inflammasome activation is associated with the disruption of mitochondrial bioenergetics in human lung microvascular endothelial cells (HLMVEC) and the mitochondrial redistribution of eNOS. These effects are dependent on RhoA-ROCK signaling and are mediated via increased phosphorylation of eNOS at Threonine (T)-495. A derivative of the mitochondrial targeted Szeto-Schiller peptide (SSP) attached to the antioxidant Tiron (T-SSP), significantly attenuated LPS-mediated mito-ROS generation and inflammasome activation in HLMVEC. Further, T-SSP attenuated mitochondrial superoxide production in a mouse model of sepsis induced ALI. This in turn significantly reduced the inflammatory response and attenuated lung injury. Thus, our findings show that the mitochondrial redistribution of uncoupled eNOS is intimately involved in the activation of the inflammatory response in ALI and implicate attenuating mito-ROS as a therapeutic strategy in humans.

Membrane nanotubes facilitate the propagation of inflammatory injury in the heart upon overactivation of the \u03b2-adrenergic receptor

Acute sympathetic stress quickly induces cardiac inflammation and injury, suggesting that pathogenic signals rapidly spread among cardiac cells and that cell-to-cell communication may play an important role in the subsequent cardiac injury. However, the underlying mechanism of this response is unknown. Our previous study demonstrated that acute β-adrenergic receptor (β-AR) signaling activates inflammasomes in the heart, which triggers the inflammatory cascade. In the present study, β-AR overactivation induced inflammasome activation in both the cardiomyocytes and cardiac fibroblasts (CFs) of mice hearts following a subcutaneous injection of isoproterenol (ISO, 5 mg/kg body weight), a selective agonist of β-AR. In isolated cardiac cells, ISO treatment only activated the inflammasomes in the cardiomyocytes but not the CFs. These results demonstrated that inflammasome activation was propagated from cardiomyocytes to CFs in the mice hearts. Further investigation revealed that the inflammasomes were activated in the cocultured CFs that connected with cardiomyocytes via membrane nanotubes (MNTs), a novel membrane structure that mediates distant intercellular connections and communication. Disruption of the MNTs with the microfilament polymerization inhibitor cytochalasin D (Cyto D) attenuated the inflammasome activation in the cocultured CFs. In addition, the MNT-mediated inflammasome activation in the CFs was blocked by deficiency of the inflammasome component NOD-like receptor protein 3 (NLRP3) in the cardiomyocytes, but not NLRP3 deficiency in the CFs. Moreover, ISO induced pyroptosis in the CFs cocultured with cardiomyocytes, and this process was inhibited by disruption of the MNTs with Cyto D or by the NLRP3 inhibitor MCC950 and the caspase-1 inhibitor Z-YVAD-FMK (FMK). Our study revealed that MNTs facilitate the rapid propagation of inflammasome activation among cardiac cells to promote pyroptosis in the early phase of β-adrenergic insult. Therefore, preventing inflammasome transfer is a potential therapeutic strategy to alleviate acute β-AR overactivation-induced cardiac injury.

LncRNA MALAT1 facilitates inflammasome activation via epigenetic suppression of Nrf2 in Parkinson\u2019s disease

The goal of the present study was to elucidate the mechanism by which long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (lncRNA MALAT1) promotes inflammation in Parkinson’s disease (PD). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was used to induce PD development in C57BL/6 mice, and tyrosine hydroxylase (TH) expression was analysed by immunohistochemical analysis. Western blot and qPCR analyses were conducted to assess the expression of protein and mRNA levels, respectively. Lipopolysaccharide/adenosine triphosphate (LPS/ATP) was used to activate microglia in vitro. Chromatin immunoprecipitation (ChIP), RNA pull-down and RNA immunoprecipitation chip (RIP) assays were performed to investigate the interaction among specific molecules. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate cell viability and proliferation. Flow cytometry was performed to analyse cell apoptosis after staining. The dichlorofluorescein diacetate (DCFH-DA) assay was used to measure the generation of reactive oxygen species (ROS) in cells. The results showed that MALAT1 was highly expressed in the brains of MPTP-induced PD model mice and in LPS/ATP-induced microglia cells. Knockdown of MALAT1 inhibited elevated nuclear factor (erythroid-derived 2)-like-2 factor (NRF2) expression, thereby inhibiting inflammasome activation and ROS production. MALAT1 was shown to promote neuroinflammation by recruiting enhancer of zeste homologue 2 (EZH2) to the promoter of NRF2, suppressing Nrf2 expression. In summary, MALAT1 epigenetically inhibits NRF2, thereby inducing inflammasome activation and reactive oxygen species (ROS) production in PD mouse and microglial cell models.

Mice Lacking the Purinergic Receptor P2X5 Exhibit Defective Inflammasome Activation and Early Susceptibility to Listeria monocytogenes.

P2X5 is a member of the P2X purinergic receptor family of ligand-gated cation channels and has recently been shown to regulate inflammatory bone loss. In this study, we report that P2X5 is a protective immune regulator during Listeria monocytogenes infection, as P2X5-deficient mice exhibit increased bacterial loads in the spleen and liver, increased tissue damage, and early (within 3-6 d) susceptibility to systemic L. monocytogenes infection. Whereas P2X5-deficient mice experience normal monocyte recruitment in response to L. monocytogenes, P2X5-deficient bone marrow-derived macrophages (BMMs) exhibit defective cytosolic killing of L. monocytogenes We further showed that P2X5 is required for L. monocytogenes-induced inflammasome activation and IL-1β production and that defective L. monocytogenes killing in P2X5-deficient BMMs is substantially rescued by exogenous IL-1β or IL-18. Finally, in vitro BMM killing and in vivo L. monocytogenes infection experiments employing either P2X7 deficiency or extracellular ATP depletion suggest that P2X5-dependent anti-L. monocytogenes immunity is independent of the ATP-P2X7 inflammasome activation pathway. Together, our findings elucidate a novel and specific role for P2X5 as a critical mediator of protective immunity.

Membrane Cholesterol Is Crucial for Clostridium difficile Surface Layer Protein Binding and Triggering Inflammasome Activation

Clostridium difficile, an obligate anaerobic gram-positive bacillus, generates spores and is commonly found colonizing the human gut. Patients with C. difficile infection (CDI) often exhibit clinical manifestations of pseudomembranous colitis or antibiotic-associated diarrhea. Surface layer proteins (SLPs) are the most abundant proteins in the C. difficile cell wall, suggesting that they might involve in immune recognition. Our previous results demonstrated that C. difficile triggers inflammasome activation. Here, we found SLPs as well as C. difficile induced inflammasome activation, and in a dose-dependent manner. In addition, the cholesterol-rich microdomains on the cell membrane (also referred to as lipid rafts) are thought to be crucial for bacterial adhesion and signal transduction. We demonstrated that lipid rafts participated in C. difficile SLPs binding to the cell membrane. Fluorescence microscopy showed that membrane cholesterol depletion by methyl-β-cyclodextrin (MβCD) reduced the association of SLPs with the cell surface. The coalescence of SLPs in the cholesterol-rich microdomains was confirmed in C. difficile-infected cells. Furthermore, the inflammasome activations induced by SLPs or C. difficile were abrogated by MβCD. Our results demonstrate that SLPs recruit the lipid rafts, which may be a key step for C. difficile colonization and inducing inflammasome activation.

MO046MICROBIOTA DERIVED SHORT CHAIN FATTY ACIDS, PROPIONATE AND BUTYRATE, CONTRIBUTE TO MODULATE THE INFLAMMATORY RESPONSE IN CHRONIC KIDNEY DISEASE

Abstract Background and Aims Dysbiosis, or changes in the gut microbiota composition, had been related to the developed of several pathologies, such as chronic kidney disease. Until now, multiple studies have focused on the influence of diet on outcomes of patients with CKD. These patients with advanced disease are recommended a restricted intake of vegetable fiber due to the phosphorus and potassium levels, and low proteins to avoid the generation of uremic toxins. It is known that dietary changes lead to alterations in gut microbiota, but also in microbial metabolites production, some of which could be beneficial for the host. A recent and exciting area of research has begun to explore the role of microbiota-derived metabolites in the renal physiology. Short-chain fatty acids (SCFA, acetate, propionate and butyrate) are a type metabolite produced from dietary fiber by gut microbiota that enter in the bloodstream leading to distal effects, such as modulation of the immune cells. SCFAs are essential to maintain the permeability of the intestinal epithelial barrier, the metabolic functions and have potent anti-inflammatory effects. The aim of this study was to identify the SCFAs levels during the progression of CKD and determinate the functional role of these metabolites in the renal inflammation. Method SFCAs (acetate, propionate and butyrate) levels were determined using gas chromatography-mass spectrometry in fecal samples collected from patients with different stages of CKD (n=60) and age-matched healthy control (n=20). Moreover, common bacterial families were determined by quantitative PCR. Additionally, the in-vitro effect of the three SCFAs was evaluated in the human tubular epithelial cell line HK2 using RNA-seq, specific silencing with siRNAs and histone deacetylases (HDAC) inhibitors. To evaluate the effect in immune cells, monocyte and macrophages were treated with LPS and ATP /Nigericin to induce inflammasome activation. Results The SCFAs levels were significantly lower in patients with CKD than in healthy controls, mainly propionate and butyrate. Moreover, these levels progressively decreased with the developed of the disease, showing the patients with stage 5 (CKD5) have the lowest levels that correlates with a lesser abundance of Clostridium IV family. According to the renal function, butyrate levels were positively correlated with the glomerular filtration rate and negatively with the blood urea nitrogen and creatinine levels. Surprisingly, high propionate levels correlate with the most elevated serum calcidiol concentrations. Functionally, propionate and butyrate show a similar pattern in the modulation of inflammatory genes in HK2 cells. Most regulated pathways are associated with Inflammatory response (GO:0006954: IL6, TNF, CCL2, RELB, IRAK2, NFKB1,CCL20) and immune response (GO:0006955: CSF2, CXCL3, CD40, IL7R, LIF). Additionally, both SCFAs regulates the expression of multiple epigenetic enzymes involves in the chromatin remodeling, mainly in histone acetylation. In monocytes/macrophages, propionate and butyrate inhibits the IL1B, CASP, and ASC gene transcription damaging the IL-1β secretion. We determined that the effect of SCFAs in these in-vitro models is mediated by inhibition of HDAC although also change other histone modifications (H3K9me3, H3K27me3) and through the GPR109A receptor. Conclusion Our initial results showed that patients with advanced CKD have low levels of SCFAs, and those were correlated with the renal function. Treatment of human renal and immune cells with propionate and butyrate induces profound changes in the chromatin structure, changing the whole-genome gene expression and modulating key pathways in the renal pathology. Increasing the SCFAs levels in those patients could be a potential therapeutic strategy to slow down the disease progression.

NLRC4, ASC and Caspase-1 Are Inflammasome Components that Are Mediated by P2Y2R Activation in Breast Cancer Cells.

The inflammasomes are reported to be associated with tumor progression. In our previous study, we determined that extracellular ATP enhances invasion and tumor growth by inducing inflammasome activation in a P2Y purinergic receptor 2 (P2Y2R)-dependent manner. However, it is not clear which inflammasome among the diverse complexes is associated with P2Y2R activation in breast cancer. Thus, in this study, we determined which inflammasome components are regulated by P2Y2R activation and are involved in tumor progression in breast cancer cells and radiotherapy-resistant (RT-R)-breast cancer cells. First, we found that NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3); NLR family caspase activation and recruitment domain (CARD) containing 4 (NLRC4); apoptosis-associated speck-like protein containing a CARD complex (ASC); and caspase-1 mRNA levels were upregulated in RT-R-MDA-MB-231 cells compared to MDA-MB-231 cells, whereas tumor necrosis factor-α (TNF-α) or ATP treatment induced NLRC4, ASC, and caspase-1 but not NLRP3 protein levels. Moreover, TNF-α or ATP increased protein levels of NLRC4, ASC, and caspase-1 in a P2Y2R-dependent manner in MDA-MB-231 and RT-R-MDA-MB-231 cells. In addition, P2Y2R activation by ATP induced the secretion of IL-1β and VEGF-A, as well as invasion, in MDA-MB-231 and RT-R-MDA-MB-231 cells, which was inhibited by NLRC4, ASC, and caspase-1 small interfering RNA (siRNA). Taken together, this report suggests that P2Y2R activation by ATP induces tumor invasion and angiogenesis through inflammasome activation, specifically by regulating the inflammasome components NLRC4, ASC, and caspase-1.

Damage-induced pyroptotic cell death facilitates regeneration of the thymus

Abstract Endogenous thymus regeneration is a critical process that enables the restoration of immune competence after damage caused by infection, age and common cancer therapies; however, the molecular mechanisms regulating this process are poorly understood. Previously, we identified that depletion of CD4+CD8+ thymocytes (DPs) after injury precedes the induction of regenerative factors such as BMP4 from endothelial cells (ECs), and IL23 from dendritic cells (DCs), and that the presence of homeostatic apoptotic DPs can suppress BMP4 and IL23. Together with our findings that the metabolic needs of key thymus populations alter drastically following injury due to damage-induced metabolic remodeling, we hypothesize that further to the loss of DP-specific suppression after damage, metabolic dysfunction in DPs triggers mitochondrial-induced pyroptotic cell death, which can directly promote the production of regenerative factors. Here we demonstrate a disruption in glycolytic flux, increased mitochondrial biogenesis, an induction of ROS, and increased cleavage of caspase 1 in murine DPs after damage; key processes that can induce inflammasome activation, pyroptosis and the release of cellular DAMPs such as ATP and HMGB1. Interestingly, these metabolic perturbations were not observed in the regeneration-inducing ECs and DCs, suggesting metabolic stability may facilitate their radio-resistance. Secreted HMGB1 levels are increased in the thymus after damage, and extracellular stimulation of ECs in vitro with ATP increased Bmp4 expression. These studies describe a novel regulatory mechanism of metabolic-mediated tissue regeneration, and have the potential to lead to a superior therapeutic strategy to boost thymic function.

Inflammasome activation by members of the Bacillus genus

Abstract Inflammasome activation induced by microbial components leads to an effective innate immune response. Bacillus anthracis is able to induce activation of the NLRP1b inflammasome, whereas B. cereus can induce activation of the NLRP3 inflammasome. However, whether different strains of B. cereus have differential ability to induce activation of the inflammasome has not been investigated. Further, whether other members of the Bacillus genus can induce activation of the inflammasome has remained unknown. Here, we screened 46 B. cereus strains from environmental and clinical sources to determine whether novel inflammasomes are activated. We infected primary mouse bone marrow-derived macrophages with bacteria or bacteria-free supernatant and observed for pyroptosis and other classical features of inflammasome activation. Strikingly, over 90% (42/46) of the B. cereus strains induced inflammasome activation as measured by caspase-1 cleavage and robust secretion of IL-1b, IL-18 and LDH. We identified that the inflammasome sensor NLRP3 was necessary to drive this response. Further, we confirmed that HBL and/or NHE toxins were expressed in all 42 B. cereus strains, suggesting a conserved inflammasome activation pattern amongst B. cereus strains. We also identified that four pathogenic Bacillus species induced inflammasome activation in an NLRP3-dependent manner, including B. idriensis, B. mycoides, B. pseudomycoides and B. thurigiensis. These findings highlight that the NLRP3 inflammasome sensor is important for immune recognition of several Bacillus species and suggest that therapeutic modulation of NLRP3 may enhance host protection against these Bacillus infections.

Arginase-1 production by myeloid cells in tumor microenvironment induce inflammasome activation and activate PI3K pathway in tumor-associated macrophages in head and neck cancer

Abstract Tumor-associated macrophages (TAMs) express cytokines and mediators that can suppress T cell recruitment and activation in the tumor microenvironment, support tumor growth, and promote resistance to therapy. We found that Arginase-1 is produced in high quantities by TAMs and tumor cells in head and neck cancer tumors with different background mutations. High levels of Arginase-1 were found in tumor samples and the circulation of patients and animal models of head and neck cancer. Arginase-1 is released into the cellular microenvironment and packed into the exosomes in a metabolically active form. Mechanistically, Arginase-1 further propagated the anti-inflammatory macrophage phenotype in the tumor microenvironment and induced the epithelial-mesenchymal transition (EMT) by activation of STAT3 and beta-catenin. Arginase-1 released from the tumor further induced activation of the autophagy cascade and the PI3K pathway in TAMs. Mechanistic results showed that STAT3 activation by Arginase-1 containing exosomes has a positive feedback effect on the expression of Arginase-1, Il-1β, and lead to inflammasome activation. The silencing of STAT3 negated the effect of Arginase-1 containing exosomes and led to modulation of the inflammasome activation in the head and neck cancer tumor microenvironment. These results indicate a mechanistic role of TAMs and Arginase-1 containing exosomes in the tumor microenvironment by activation of autophagy and inflammasome, and induction of the EMT. The mechanistic links presented in this study regarding the role of arginase in head and neck cancer pathogenesis could be targeted as therapeutic strategies.

ATP-P2X7–Induced Inflammasome Activation Contributes to Melanocyte Death and CD8+ T-Cell Trafficking to the Skin in Vitiligo

Extracellular adenosine 5'-triphosphate (ATP) is a well-known inflammasome-activating signal. Emerging evidence demonstrates a critical role for inflammasome activation in vitiligo pathogenesis. However, the specific molecular mechanism of inflammasome-dependent melanocyte degeneration in vitiligo is still not clear. This study presents how extracellular ATP, released from keratinocytes by oxidative stress, affects melanocyte survival in vitiligo skin. H2O2-induced oxidative injury increased ATP release from keratinocytes and skin tissues. The high concentration of extracellular ATP induced both ROS production and cell death in melanocytes. Treatment with ATP caused the activation of caspase-1 as well as the production of active forms of IL-1β and IL-18 via P2X7 receptor in keratinocytes and melanocytes. Lesional and perilesional skin of vitiligo showed higher levels of ATP as well as upregulation of active caspase-1 compared with nonlesional skin, suggesting its possible role in inflammasome activation in vitiligo. Moreover, the elevated expression of CXCL9 in keratinocytes, mediated through ATP/P2X7 receptor-dependent inflammasome activation, was responsible for CLA+CD8+ T-cell chemotaxis into the skin. These results demonstrate that extracellular ATP as a danger signal activates the inflammasome pathway and increases cutaneous chemotaxis of CD8+ T cells via CXCL9 in vitiligo. Therefore, targeting ATP-P2X7 signaling may be a potential strategy for vitiligo treatment.

Orchestration of human macrophage NLRP3 inflammasome activation by Staphylococcus aureus extracellular vesicles

Release of extracellular vesicles (EVs) is a common feature among eukaryotes, archaea, and bacteria. However, the biogenesis and downstream biological effects of EVs released from gram-positive bacteria remain poorly characterized. Here, we report that EVs purified from a community-associated methicillin-resistant Staphylococcus aureus strain were internalized into human macrophages in vitro and that this process was blocked by inhibition of the dynamin-dependent endocytic pathway. Human macrophages responded to S. aureus EVs by TLR2 signaling and activation of NLRP3 inflammasomes through K+ efflux, leading to the recruitment of ASC and activation of caspase-1. Cleavage of pro-interleukin (IL)-1β, pro-IL-18, and gasdermin-D by activated caspase-1 resulted in the cellular release of the mature cytokines IL-1β and IL-18 and induction of pyroptosis. Consistent with this result, a dose-dependent cytokine response was detected in the extracellular fluids of mice challenged intraperitoneally with S. aureus EVs. Pore-forming toxins associated with S. aureus EVs were critical for NLRP3-dependent caspase-1 activation of human macrophages, but not for TLR2 signaling. In contrast, EV-associated lipoproteins not only mediated TLR2 signaling to initiate the priming step of NLRP3 activation but also modulated EV biogenesis and the toxin content of EVs, resulting in alterations in IL-1β, IL-18, and caspase-1 activity. Collectively, our study describes mechanisms by which S. aureus EVs induce inflammasome activation and reveals an unexpected role of staphylococcal lipoproteins in EV biogenesis. EVs may serve as a novel secretory pathway for S. aureus to transport protected cargo in a concentrated form to host cells during infections to modulate cellular functions.

DOP53 PTPN2 and TiO2 in the pathogenesis of inflammatory bowel disease

Abstract Background Inflammatory bowel disease (IBD) is caused by a complex interaction among genetic, immunological, bacterial and environmental factors. In this scenario, protein tyrosine phosphatase non-receptor type-2 (PTPN2) has been recognised as a risk factor for the development of IBD and functional studies revealed a major role for this protein in the development of experimental colitis through the regulation of the inflammasome, among other processes. In the same way, a potential relationship between diet components and IBD was suggested. In fact, it was reported that the food additive titanium dioxide (TiO2) was able to induce inflammasome activation in vitro and increase colitis severity in vivo. These observations led us to hypothesise a putative relationship between PTPN2 and TiO2 that could explain the effects of this microparticle in the pathogenesis of bowel inflammation. Methods DSS colitis model was performed in mice lacking PTPN2 in myeloid cells and their wild-type littermates, treated or not with titanium dioxide. After that, histology studies, flow cytometry, expression analysis, ELISA and barrier function experiments were performed. Also, bone marrow-derived macrophages (BMDMs) were used for in vitro studies. Results Titanium dioxide was able to exacerbate DSS-induced colitis, especially in mice lacking PTPN2 in myeloid cells. Flow cytometry analysis of the lamina propria revealed significant changes in different immune cell populations such as macrophages. In vitro experiments using BMDMs revealed that TiO2 induced the activation of the inflammasome. Also, this microparticle down-regulated the expression of the anti-inflammatory cytokine IL-10 and these effects were mainly mediated by JNK and ERK kinases. Conclusions The food additive titanium dioxide seems to play a negative role in colitis development by affecting the production of pro- and anti-inflammatory mediators in macrophages. This study reveals a new mechanism by which a certain component of the diet modulates intestinal inflammation.

Inflammasome Activation Induced by a Snake Venom Lys49-Phospholipase A2 Homologue.

Background: Snake venom phospholipases A2 (PLA2s) have hemolytic, anticoagulant, myotoxic, oedematogenic, bactericidal, and inflammatory actions. BthTX-I, a Lys49-PLA2 isolated from Bothrops jararacussu venom, is an example of Lys49-PLA2 that presents such actions. NLRP3 is a cytosolic receptor from the NLR family responsible for inflammasome activation via caspase-1 activation and IL-1β liberation. The study of NLRs that recognize tissue damage and activate the inflammasome is relevant in envenomation. Methods: Male mice (18–20 g) received an intramuscular injection of BthTX-I or sterile saline. The serum was collected for creatine-kinase (CK), lactate dehydrogenase (LDH), and interleukin-1β (IL-1β) assays, and muscle was removed for inflammasome activation immunoblotting and qRT-PCR expression for nucleotide and oligomerization domain, leucine-rich repeat-containing protein family, pyrin-containing domain 3 receptor (NLRP3) inflammasome components. Results: BthTX-I-induced inflammation and myonecrosis, shown by intravital microscope, and LDH and CK release, respectively. Mouse treatment with A438079, a P2X7 receptor antagonist, did not modify these effects. BthTX-I induced inflammasome activation in muscle, but P2X7R participation in this effect was not observed. Conclusion: Together, the results showed for the first time that BthTX-I in gastrocnemius muscle induces inflammation and consequently, inflammasome activation via NLRP3 with caspase-1 activation and IL-1β liberation.

Alcohol induced NLRP3 inflammasome activation in the brain of rats is attenuated by ATRA supplementation

Alcohol abuse affects several neurological pathways and causes significant alterations in the brain. Abstention from alcohol is an effective intervention against alcohol related diseases. But the recovery of the damaged cells to normal presents a major problem in those who have stopped alcohol consumption. Hence therapeutic interventions are needed. Our previous studies have shown that all trans retinoic acid (ATRA) is effective in reducing alcohol induced neuro toxicity. Chronic alcohol administration up-regulates and activates the NLRP3 inflammasome leading to caspase-1 activation and IL-1β production causing neuroinflammation. Hence, we investigated whether ATRA has any impact on NLRP3 inflammasomes activation. Rats were divided into two groups and were maintained for 90 days as control and ethanol group (4 ​g/kg body weight). After 90 days, ethanol administration was stopped and animals in the control group were divided into control and control ​+ ​ATRA (100 ​μg/kg body weight per day) groups; those in the ethanol group as ethanol abstention and ATRA (100 ​μg/kg body weight per day) and maintained for 30 days. Administration of ATRA reduced reactive oxygen species and endotoxins which were elevated in alcoholic rats. There was also reduction in the expression of NLRP3 inflammasome and caspase 1. Our results suggested ATRA down regulated NLRP3 activation with concomitant decrease in the release of caspase −1 and production of IL1β. However, all these parameters were higher in abstention in comparison with ATRA supplemented group. In short therapeutic intervention with ATRA regressed alcohol induced inflammasome activation better than abstention.

Resvega alleviates oxidative stress and inflammasome activation in retinal pigment epithelial cells

AbstractPurposeOxidative stress and inflammation have been shown to associate with the pathogenesis of age‐related macular degeneration. Oxidative stress is known to induce inflammasome activation. The aim of the study was to investigate the effects of the resveratrol‐containing Resvega on the inflammasome activation in retinal pigment epithelial (RPE) cells exposed to oxidative stress.MethodsFor the investigation of intracellular reactive oxygen species (ROS) production and inflammasome activation, ARPE‐19 cells were exposed to hydroquinone or MG‐132 and bafilomycin A1 primed with IL‐1α. Experiments were performed with and without pre‐treatment with Resvega (Laboratoires Théa, France). ROS production was measured using the 2′,7′‐dichlorofluorescin diacetate (DCFDA) probe. Caspase‐1 activity was determined using a specific fluorochrome inhibitor of caspases (FLICA). The amount of secreted IL‐1β and NLRP3 were measured using the enzyme‐linked immunosorbent assay (ELISA) method. Cell viability and cytotoxicity were determined using the thiazolyl blue tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays.ResultsHydroquinone induced intracellular ROS production, which was reduced by pre‐treatment with Resvega. However, reduction did not result from increased cytotoxicity or declined cell viability. Inflammasome activation by MG‐132+bafilomycin A1 in IL‐1α ‐primed cells was detected by the activation of caspase‐1 and increased secretion of IL‐1β and NLRP3. All those were reduced by the pre‐treatment with Resvega, which had no impact on the cell viability.ConclusionsOur results suggest that Resvega has potential to alleviate oxidative stress and inflammation in human retinal pigment epithelial cells.