Britannin as a novel NLRP3 inhibitor, suppresses inflammasome activation in macrophages and alleviates NLRP3-related diseases in mice.

Overactivation of the NLRP3 inflammasomes induces production of pro-inflammatory cytokines and drives pathological processes. Pharmacological inhibition of NLRP3 is an explicit strategy for the treatment of inflammatory diseases. Thus far no drug specifically targeting NLRP3 has been approved by the FDA for clinical use. This study was aimed to discover novel NLRP3 inhibitors that could suppress NLRP3-mediated pyroptosis. We screened 95 natural products from our in-house library for their inhibitory activity on IL-1ß secretion in LPS + ATP-challenged BMDMs, found that Britannin exerted the most potent inhibitory effect with an IC50 value of 3.630 µM. We showed that Britannin (1, 5, 10 µM) dose-dependently inhibited secretion of the cleaved Caspase-1 (p20) and the mature IL-1ß, and suppressed NLRP3-mediated pyroptosis in both murine and human macrophages. We demonstrated that Britannin specifically inhibited the activation step of NLRP3 inflammasome in BMDMs via interrupting the assembly step, especially the interaction between NLRP3 and NEK7. We revealed that Britannin directly bound to NLRP3 NACHT domain at Arg335 and Gly271. Moreover, Britannin suppressed NLRP3 activation in an ATPase-independent way, suggesting it as a lead compound for design and development of novel NLRP3 inhibitors. In mouse models of MSU-induced gouty arthritis and LPS-induced acute lung injury (ALI), administration of Britannin (20 mg/kg, i.p.) significantly alleviated NLRP3-mediated inflammation; the therapeutic effects of Britannin were dismissed by NLRP3 knockout. In conclusion, Britannin is an effective natural NLRP3 inhibitor and a potential lead compound for the development of drugs targeting NLRP3.

TREM-1 induces pyroptosis in cardiomyocytes by activating NLRP3 inflammasome through the SMC4/NEMO pathway.

Sepsis often causes cell death via pyroptosis and hence results in septic cardiomyopathy. Triggering receptors expressed in myeloid cells-1 (TREM-1) may initiate cellular cascade pathways and, in turn, induce cell death and vital organ dysfunction in sepsis, but the evidence is limited. We set to investigate the role of TREM-1 on nucleotide-binding oligomerization domain-like receptors with pyrin domain-3 (NLRP3) inflammasome activation and cardiomyocyte pyroptosis in sepsis models using cardiac cell line (HL-1) and mice. In this study, TREM-1 was found to be significantly increased in HL-1 cells challenged with lipopolysaccharide (LPS). Pyroptosis was also significantly increased in the HL-1 cells challenged with lipopolysaccharide and an NLRP3 inflammasome activator, nigericin. The close interaction between TREM-1 and structural maintenance of chromosome 4 (SMC4) was also identified. Furthermore, inhibition of TREM-1 or SMC4 prevented the upregulation of NLRP3 and decreased Gasdermin-D, IL-1ß and caspase-1 cleavage. In mice subjected to caecal ligation and puncture, the TREM-1 inhibitor LR12 decreased the expression of NLRP3 and attenuated cardiomyocyte pyroptosis, leading to improved cardiac function and prolonged survival of septic mice. Our work demonstrates that, under septic conditions, TREM-1 plays a critical role in cardiomyocyte pyroptosis. Targeting TREM-1 and its associated molecules may therefore lead to novel therapeutic treatments for septic cardiomyopathy.

Callintegers A and B, Unusual Tricyclo[4.4.0.09,10]tetradecane Clerodane Diterpenoids from Callicarpa integerrima with Inhibitory Effects on NLRP3 Inflammasome Activation.

Callintegers A (1) and B (2), unprecedented clerodane norditerpenoids based on a novel carbon skeleton, were isolated from Callicarpa integerrima. Compounds 1 and 2 possess a novel 6/6/6-fused tricyclic ring system. Their structures and absolute configurations were determined by quantum chemical calculations, spectroscopic analysis, and single-crystal X-ray diffraction methods. Biological evaluation showed that compound 2 inhibited IL-1ß secretion in a dose-dependent manner with an IC50 value of 5.5 ± 3.2 µM. Caspase-1 maturation and IL-1ß secretion were also reduced, indicating that compound 2 impaired NLRP3 inflammasome activation.

Amiodarone, Unlike Dronedarone, Activates Inflammasomes via Its Reactive Metabolites: Implications for Amiodarone Adverse Reactions.

Amiodarone is a benzofuran derivative used to treat arrhythmias, but its use is limited by adverse reactions. There is evidence that some of the severe adverse reactions such as liver injury and interstitial lung disease are immune-mediated; however, details of the mechanism have not been elucidated. We tested the ability of amiodarone to induce the release of danger-associated molecular patterns (DAMPs) that activate inflammasomes. Human hepatocarcinoma functional liver cell-4 (FLC-4) cells were used for drug bioactivation, and the detection of inflammasome activation was performed with the human macrophage cell line, THP-1 cells. Amiodarone is known to be oxidized to reactive quinone metabolites. The supernatant from the incubation of amiodarone with FLC-4 cells for 7 days increased caspase-1 activity and production of IL-1ß by THP-1 cells. In the supernatant of FLC-4 cells with amiodarone, the heat shock protein (HSP) 40 was significantly increased. Addition of a cytochrome P450 inhibitor to the FLC-4 cells prevented the release of HSP40 from the FLC-4 cells and activation of THP-1 inflammasomes by the FLC-4 supernatant. These results suggested that the reactive quinone metabolites of amiodarone can cause the release of DAMPs from hepatocytes which can activate inflammasomes. Dronedarone, a safer analog of amiodarone, did not activate inflammasomes. Inflammasome activation may be an important step in the activation of the immune system by amiodarone, which in some patients, can cause immune-related adverse events. In addition, our data suggest that drugs that block the effects or the formation of IL-1ß would provide better treatment of amiodarone-induced immune-related adverse reactions.

Chemical mitochondrial uncouplers share common inhibitory effect on NLRP3 inflammasome activation through inhibiting NFκB nuclear translocation.

Activation of NLRP3 inflammasome is implicated in varieties of pathologies, the aim of the present study is to characterize the effect and mechanism of mitochondrial uncouplers on NLRP3 inflammasome activation by using three types of uncouplers, niclosamide, CCCP and BAM15. Niclosamide, CCCP and BAM15 inhibited LPS plus ATP-induced increases of NLRP3 protein and IL-1ß mRNA levels in RAW264.7 macrophages and THP-1 derived macrophages. Niclosamide, CCCP and BAM15 inhibited LPS plus ATP-induced increase of NFκB (P65) phosphorylation, and inhibited NFκB (P65) nuclear translocation in RAW264.7 macrophages. Niclosamide and BAM15 inhibited LPS-induced increase of IκBα phosphorylation in RAW264.7 macrophages, and the inhibitory effect was dependent on increased intracellular [Ca2+]i; however, CCCP showed no significant effect on IκBα phosphorylation in RAW264.7 macrophages stimulated with LPS. In conclusion, chemical mitochondrial uncouplers niclosamide, CCCP and BAM15 share common inhibitory effect on NLRP3 inflammasome activation through inhibiting NFκB nuclear translocation.

The Human-Specific STING Agonist G10 Activates Type I Interferon and the NLRP3 Inflammasome in Porcine Cells.

Pigs have anatomical and physiological characteristics comparable to those in humans and, therefore, are a favorable model for immune function research. Interferons (IFNs) and inflammasomes have essential roles in the innate immune system. Here, we report that G10, a human-specific agonist of stimulator of interferon genes (STING), activates both type I IFN and the canonical NLRP3 inflammasome in a STING-dependent manner in porcine cells. Without a priming signal, G10 alone transcriptionally stimulated Sp1-dependent p65 expression, thus triggering activation of the nuclear factor-κB (NF-κB) signaling pathway and thereby priming inflammasome activation. G10 was also found to induce potassium efflux- and NLRP3/ASC/Caspase-1-dependent secretion of IL-1ß and IL-18. Pharmacological and genetic inhibition of NLRP3 inflammasomes increased G10-induced type I IFN expression, thereby preventing virus infection, suggesting negative regulation of the NLRP3 inflammasome in the IFN response in the context of STING-mediated innate immune activation. Overall, our findings reveal a new mechanism through which G10 activates the NLRP3 inflammasome in porcine cells and provide new insights into STING-mediated innate immunity in pigs compared with humans.

NLRP3 Sensing of Diverse Inflammatory Stimuli Requires Distinct Structural Features.

The NLRP3 inflammasome is central to host defense and implicated in various inflammatory diseases and conditions. While the favored paradigm of NLRP3 inflammasome activation stipulates a unifying signal intermediate that de-represses NLRP3, this view has not been tested. Further, structures within NLRP3 required for inflammasome activation are poorly defined. Here we demonstrate that while the NLRP3 LRRs are not auto-repressive and are not required for inflammasome activation by all agonists, distinct sequences within the NLRP3 LRRs positively and negatively modulate inflammasome activation by specific ligands. In addition, elements within the HD1/HD2 "hinge" of NLRP3 and the nucleotide-binding domain have contrasting functions depending upon the specific agonists. Further, while NLRP3 1-432 is minimally sufficient for inflammasome activation by all agonists tested, the pyrin, and linker domains (1-134) function cooperatively and are sufficient for inflammasome activation by certain agonists. Conserved cysteines 8 and 108 appear important for inflammasome activation by sterile, but not infectious insults. Our results define common and agonist-specific regions of NLRP3 that likely mediate ligand-specific responses, discount the hypothesis that NLRP3 inflammasome activation has a unified mechanism, and implicate NLRP3 as an integrator of agonist-specific, inflammasome activating signals.

Supraphysiological Levels of Testosterone Induce Vascular Dysfunction via Activation of the NLRP3 Inflammasome.

Background: Both supraphysiological and subphysiological testosterone levels are associated with increased cardiovascular risk. Testosterone consumption at supraphysiological doses has been linked to increased blood pressure, left ventricular hypertrophy, vascular dysfunction, and increased levels of inflammatory markers. Activation of the NLRP3 inflammasome contributes to the production of proinflammatory cytokines, leading to cardiovascular dysfunction. We hypothesized that supraphysiological levels of testosterone, via generation of mitochondrial reactive oxygen species (mROS), activates the NLRP3 inflammasome and promotes vascular dysfunction. Methods: Male, 12 week-old C57Bl/6J (WT) and NLRP3 knockout (NLRP3-/-) mice were used. Mice were treated with testosterone propionate [TP (10 mg/kg) in vivo] or vehicle for 30 days. In addition, vessels were incubated with testosterone [Testo (10-6 M, 2 h) in vitro]. Testosterone levels, blood pressure, vascular function (thoracic aortic rings), pro-caspase-1/caspase-1 and interleukin-1ß (IL-1ß) expression, and generation of reactive oxygen species were determined. Results: Testosterone increased contractile responses and reduced endothelium-dependent vasodilation, both in vivo and in vitro. These effects were not observed in arteries from NLRP3-/- mice. Aortas of TP-treated WT mice (in vivo), as well as aortas from WT mice incubated with testo (in vitro), exhibited increased mROS levels and increased caspase-1 and IL-1ß expression. These effects were not observed in arteries from NLRP3-/- mice. Flutamide [Flu, 10-5 M, androgen receptor (AR) antagonist], carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 10-6 M, mitochondrial uncoupler) and MCC950 (MCC950, 10-6 M, a NLRP3 receptor inhibitor) prevented testosterone-induced mROS generation. Conclusion: Supraphysiological levels of testosterone induce vascular dysfunction via mROS generation and NLRP3 inflammasome activation. These events may contribute to increased cardiovascular risk.

Therapeutic Potential of Pharmacological Targeting NLRP3 Inflammasome Complex in Cancer.

Introduction: Dysregulation of NLRP3 inflammasome complex formation can promote chronic inflammation by increased release of IL-1ß. However, the effect of NLRP3 complex formation on tumor progression remains controversial. Therefore, we sought to determine the effect of NLRP3 modulation on the growth of the different types of cancer cells, derived from lung, breast, and prostate cancers as well as neuroblastoma and glioblastoma in-vitro. Method: The effect of Caspase 1 inhibitor (VX765) and combination of LPS/Nigericin on NLRP3 inflammasome activity was analyzed in A549 (lung cancer), MCF-7 (breast cancer), PC3 (prostate cancer), SH-SY5Y (neuroblastoma), and U138MG (glioblastoma) cells. Human fibroblasts were used as control cells. The effect of VX765 and LPS/Nigericin on NLRP3 expression was analyzed using western blot, while IL-1ß and IL-18 secretion was detected by ELISA. Tumor cell viability and progression were determined using Annexin V, cell proliferation assay, LDH assay, sphere formation assay, transmission electron microscopy, and a multiplex cytokine assay. Also, angiogenesis was investigated by a tube formation assay. VEGF and MMPs secretion were detected by ELISA and a multiplex assay, respectively. Statistical analysis was done using one-way ANOVA with Tukey's analyses and Kruskal-Wallis one-way analysis of variance. Results: LPS/Nigericin increased NRLP3 protein expression as well as IL-1ß and IL-18 secretion in PC3 and U138MG cells compared to A549, MCF7, SH-SY5Y cells, and fibroblasts. In contrast, MIF expression was commonly found upregulated in A549, PC3, SH-SY5Y, and U138MG cells and fibroblasts after Nigericin treatment. Nigericin and a combination of LPS/Nigericin decreased the cell viability and proliferation. Also, LPS/Nigericin significantly increased tumorsphere size in PC3 and U138MG cells. In contrast, the sphere size was reduced in MCF7 and SH-SY5Y cells treated with LPS/Nigericin, while no effect was detected in A549 cells. VX765 increased secretion of CCL24 in A549, MCF7, PC3, and fibroblasts as well as CCL11 and CCL26 in SH-SY5Y cells. Also, VX765 significantly increased the production of VEGF and MMPs and stimulated angiogenesis in all tumor cell lines. Discussion: Our data suggest that NLRP3 activation using Nigericin could be a novel therapeutic approach to control the growth of tumors producing a low level of IL-1ß and IL-18.

The Nlrp3 Inflammasome Orchestrates Mobilization of Bone Marrow-Residing Stem Cells into Peripheral Blood.

Mobilization of stem cells from bone marrow (BM) into peripheral blood (PB) in response to tissue or organ injury, infections, strenuous exercise, or mobilization-inducing drugs is as we postulated result of a "sterile inflammation" in the BM microenvironment that triggers activation of the Complement Cascade (ComC). Therefore, we became interested in the role of the Nlrp3 inflammasome in this process and show for the first time that its activation in ATP-dependent manner orchestrates BM egress of hematopoietic stem/progenitor cells (HSPCs) as well as other stem cells, including mesenchymal stroma cells (MSCs), endothelial progenitor cells (EPCs), and very small embryonic-like stem cells (VSELs). To explain this extracellular ATP is a potent activator of the Nrlp3 inflammasome, which leads to the release of interleukin 1ß and interleukin 18, as well as several danger-associated molecular pattern molecules (DAMPs) that activate the mannan-binding lectin (MBL) pathway of the ComC, from cells of the innate immunity network. In support of this mechanism, we demonstrate that the Nlrp3 inflammasome become activated in innate immunity cells by granulocyte colony stimulating factor (G-CSF) and AMD3100 in an ATP-dependent manner. Moreover, administration of the Nlrp3 inflammasome activator nigericin induces mobilization in mice, and the opposite effect is obtained by administration of an Nlrp3 inhibitor (MCC950) to mice mobilized by G-CSF or AMD3100. In summary, our results further support the crucial role of innate immunity, BM sterile inflammation, and novel role of the ATP-Nlrp3-ComC axis in the egress of stem cells into PB.

Simvastatin Suppresses Interleukin Iß Release in Human Peripheral Blood Mononuclear Cells Stimulated With Cholesterol Crystals.

Statins are mainstream therapy in the treatment and prevention of cardiovascular disease through inhibitory effects on cholesterol synthesis. However, statins' beneficial effects in cardiovascular disease may also be attributable to their role as anti-inflammatory mediators. Here, we investigated the effects of simvastatin treatment on expression levels of interleukin (IL) 1ß in both patient with hyperlipidemia and healthy human peripheral blood mononuclear cells (PBMCs) using cholesterol crystals (CC), a cardiovascular pathogenic stimulus for activation of the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome. Cholesterol crystal-induced NLRP3 inflammasome activation was used to trigger maturation and release of IL-1ß in PBMCs. Specifically, isolated PBMCs from patients with hyperlipidemia at baseline and following 8 weeks of in vivo treatment with simvastatin (10-20 mg) daily were stimulated with lipopolysaccharide (LPS; 100 ng/mL) for 3 hours to induce proIL-Iß expression followed by CC (2 mg/mL) stimulation for further 18 hours to activate the NLRP3 inflammasome complex to induce maturation/activation of IL-1ß. Peripheral blood mononuclear cells were also isolated from healthy donors and stimulated in vitro with simvastatin (50, 25, 5, and 2 µmol/L) prior to stimulation with LPS and CC as described above. The effects of simvastatin treatment on levels of IL-1ß expression were determined by enzyme-linked immunosorbent assay and western blot. Both in vitro and in vivo treatments with simvastatin led to a significant reduction in the levels of expression of IL-1ß in response to stimulation with CC. Simvastatin inhibits the expression and activation of IL-1ß induced by CC in PBMCs, which may contribute to its protective role in patients with cardiovascular disease.

Saturated Fatty Acids Undergo Intracellular Crystallization and Activate the NLRP3 Inflammasome in Macrophages.

OBJECTIVE: Inflammation provoked by the imbalance of fatty acid composition, such as excess saturated fatty acids (SFAs), is implicated in the development of metabolic diseases. Recent investigations suggest the possible role of the NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3) inflammasome, which regulates IL-1ß (interleukin 1ß) release and leads to inflammation, in this process. Therefore, we investigated the underlying mechanism by which SFAs trigger NLRP3 inflammasome activation. APPROACH AND RESULTS: The treatment with SFAs, such as palmitic acid and stearic acid, promoted IL-1ß release in murine primary macrophages while treatment with oleic acid inhibited SFA-induced IL-1ß release in a dose-dependent manner. Analyses using polarized light microscopy revealed that intracellular crystallization was provoked in SFA-treated macrophages. As well as IL-1ß release, the intracellular crystallization and lysosomal dysfunction were inhibited in the presence of oleic acid. These results suggest that SFAs activate NLRP3 inflammasome through intracellular crystallization. Indeed, SFA-derived crystals activated NLRP3 inflammasome and subsequent IL-1ß release via lysosomal dysfunction. Excess SFAs also induced crystallization and IL-1ß release in vivo. Furthermore, SFA-derived crystals provoked acute inflammation, which was impaired in IL-1ß-deficient mice. CONCLUSIONS: These findings demonstrate that excess SFAs cause intracellular crystallization and subsequent lysosomal dysfunction, leading to the activation of the NLRP3 inflammasome, and provide novel insights into the pathogenesis of metabolic diseases.

Trimethylamine-N-Oxide Induces Vascular Inflammation by Activating the NLRP3 Inflammasome Through the SIRT3-SOD2-mtROS Signaling Pathway.

BACKGROUND: Trimethylamine-N-oxide (TMAO) has recently been identified as a novel and independent risk factor for promoting atherosclerosis through inducing vascular inflammation. However, the exact mechanism is currently unclear. Studies have established a central role of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome in the pathogenesis of vascular inflammation. Here, we examined the potential role of the NLRP3 inflammasome in TMAO-induced vascular inflammation in vitro and in vivo and the underlying mechanisms. METHODS AND RESULTS: Experiments using liquid chromatography-tandem mass spectrometry, Western blot, and fluorescent probes showed that TMAO-induced inflammation in human umbilical vein endothelial cells (HUVECs) and aortas from ApoE-/- mice. Moreover, TMAO promoted NLRP3 and activated caspase-1 p20 expression and caspase-1 activity in vitro and in vivo. Notably, a caspase-1 inhibitor (YVAD), an NLRP3 inhibitor (MCC950), as well as NLRP3 short interfering RNA attenuated TMAO-induced activation of the NLRP3 inflammasome, subsequently leading to suppression of inflammation in HUVECs. TMAO additionally stimulated reactive oxygen species (ROS) generation, in particular, mitochondrial ROS, while inhibiting manganese superoxide dismutase 2 (SOD2) activation and sirtuin 3 (SIRT3) expression in HUVECs and aortas from ApoE-/- mice. TMAO-induced endothelial NLRP3 inflammasome activation was ameliorated by the mitochondrial ROS scavenger Mito-TEMPO, or SIRT3 overexpression in HUVECs. Conversely, TMAO failed to further inhibit SOD2 and activate the NLRP3 inflammasome or induce inflammation in SIRT3 short interfering RNA-treated HUVECs and aortas from SIRT3-/- mice. CONCLUSIONS: TMAO promoted vascular inflammation by activating the NLRP3 inflammasome, and the NLRP3 inflammasome activation in part was mediated through inhibition of the SIRT3-SOD2-mitochondrial ROS signaling pathway.

WP1066 suppresses macrophage cell death induced by inflammasome agonists independently of its inhibitory effect on STAT3.

The compound WP1066 was originally synthesized by modifying the structure of AG490, which inhibits the activation of signal transducer and activator of transcription 3 (STAT3) by directly targeting Janus kinases (JAKs). WP1066 exhibits stronger anti-cancer activity than AG490 against malignant glioma and other cancer cells and is regarded as a promising therapeutic agent. By screening a small library of target-known compounds, we identified WP1066 as an inhibitor of macrophage cell death induced by agonists of the NLRP3 inflammasome, an intracellular protein complex required for the processing of the proinflammatory cytokine interleukin (IL)-1ß. WP1066 strongly inhibited cell death as well as extracellular release of IL-1ß induced by inflammasome agonists in mouse peritoneal exudate cells and human leukemia monocytic THP-1 cells that were differentiated into macrophagic cells by treatment with PMA. However, inflammasome agonists did not increase STAT3 phosphorylation, and another JAK inhibitor, ruxolitinib, did not inhibit cell death, although it strongly inhibited basal STAT3 phosphorylation. Thus, WP1066 appears to suppress macrophage cell death independently of its inhibitory effect on STAT3. In contrast, WP1066 itself induced the death of undifferentiated THP-1 cells, suggesting that WP1066 differentially modulates cell death in a context-dependent manner. Consistent with previous findings, WP1066 induced the death of human glioma A172 and T98G cells. However, neither ruxolitinib nor AG490, the former of which completely suppressed STAT3 phosphorylation, induced the death of these glioma cells. These results suggest that WP1066 targets cell death-modulating molecules other than those involved in JAK-STAT3 signaling.

Mechanism and Regulation of NLRP3 Inflammasome Activation.

Members of the nucleotide-binding domain and leucine-rich repeat (LRR)-containing (NLR) family and the pyrin and HIN domain (PYHIN) family can form multiprotein complexes termed 'inflammasomes'. The biochemical function of inflammasomes is to activate caspase-1, which leads to the maturation of interleukin 1 beta (IL-1ß) and IL-18 and the induction of pyroptosis, a form of cell death. Unlike other inflammasomes, the NLRP3 inflammasome can be activated by diverse stimuli. The importance of the NLRP3 inflammasome in immunity and human diseases has been well documented, but the mechanism and regulation of its activation remain unclear. In this review we summarize current understanding of the mechanism and regulation of NLRP3 inflammasome activation as well as recent advances in the noncanonical and alternative inflammasome pathways.

Agmatine Reverses Sub-chronic Stress induced Nod-like Receptor Protein 3 (NLRP3) Activation and Cytokine Response in Rats.

The activation of Nod-like receptor protein 3 (NLRP3) has lately been implicated in stress and depression as an initiator mechanism required for the production of interleukin (IL)-1ß and IL-18. Agmatine, an endogenous polyamine widely distributed in mammalian brain, is a novel neurotransmitter/neuromodulator, with antistress, anxiolytic and antidepressant-like effects. In this study, we examined the effect of exogenously administered agmatine on NLRP3 inflammasome pathway/cytokine responses in rats exposed to restraint stress for 7 days. The rats were divided into three groups: stress, stress+agmatine (40 mg/kg; i.p.) and control groups. Agmatine significantly down-regulated the gene expressions of all stress-induced NLRP3 inflammasome components (NLRP3, NF-κB, PYCARD, caspase-1, IL-1ß and IL-18) in the hippocampus and prefrontal cortex (PFC) and reduced pro-inflammatory cytokine levels not only in both brain regions, but also in serum. Stress-reduced levels of IL-4 and IL-10, two major anti-inflammatory cytokines, were restored back to normal by agmatine treatment in the PFC. The findings of the present study suggest that stress-activated NLRP3 inflammasome and cytokine responses are reversed by an acute administration of agmatine. Whether antidepressant-like effect of agmatine can somehow, at least partially, be mediated by the inhibition of NLRP3 inflammasome cascade and relevant inflammatory responses requires further studies in animal models of depression.

Sintered indium-tin oxide particles induce pro-inflammatory responses in vitro, in part through inflammasome activation.

Indium-tin oxide (ITO) is used to make transparent conductive coatings for touch-screen and liquid crystal display electronics. As the demand for consumer electronics continues to increase, so does the concern for occupational exposures to particles containing these potentially toxic metal oxides. Indium-containing particles have been shown to be cytotoxic in cultured cells and pro-inflammatory in pulmonary animal models. In humans, pulmonary alveolar proteinosis and fibrotic interstitial lung disease have been observed in ITO facility workers. However, which ITO production materials may be the most toxic to workers and how they initiate pulmonary inflammation remain poorly understood. Here we examined four different particle samples collected from an ITO production facility for their ability to induce pro-inflammatory responses in vitro. Tin oxide, sintered ITO (SITO), and ventilation dust particles activated nuclear factor kappa B (NFκB) within 3 h of treatment. However, only SITO induced robust cytokine production (IL-1ß, IL-6, TNFα, and IL-8) within 24 h in both RAW 264.7 mouse macrophages and BEAS-2B human bronchial epithelial cells. Our lab and others have previously demonstrated SITO-induced cytotoxicity as well. These findings suggest that SITO particles activate the NLRP3 inflammasome, which has been implicated in several immune-mediated diseases via its ability to induce IL-1ß release and cause subsequent cell death. Inflammasome activation by SITO was confirmed, but it required the presence of endotoxin. Further, a phagocytosis assay revealed that pre-uptake of SITO or ventilation dust impaired proper macrophage phagocytosis of E. coli. Our results suggest that adverse inflammatory responses to SITO particles by both macrophage and epithelial cells may initiate and propagate indium lung disease. These findings will provide a better understanding of the molecular mechanisms behind an emerging occupational health issue.

Involvement of endoplasmic reticulum stress in albuminuria induced inflammasome activation in renal proximal tubular cells.

Albuminuria contributes to the progression of tubulointerstitial fibrosis. Although it has been demonstrated that ongoing albuminuria leads to tubular injury manifested by the overexpression of numerous proinflammatory cytokines, the mechanism remains largely unknown. In this study, we found that the inflammasome activation which has been recognized as one of the cornerstones of intracellular surveillance system was associated with the severity of albuminuria in the renal biopsies specimens. In vitro, bovine serum albumin (BSA) could also induce the activation of NLRP3 inflammasome in the cultured kidney epithelial cells (NRK-52E). Since there was a significant overlap of NLRP3 with the ER marker calreticulin, the ER stress provoked by BSA seemed to play a crucial role in the activation of inflammasome. Here, we demonstrated that the chemical chaperone taurine-conjugated ursodeoxycholic acid (TUDCA) which was proved to be an enhancer for the adaptive capacity of ER could attenuate the inflammasome activation induced by albuminuria not only in vitro but also in diabetic nephropathy. Taken together, these data suggested that ER stress seemed to play an important role in albuminuria-induced inflammasome activation, elimination of ER stress via TUDCA might hold promise as a novel avenue for preventing inflammasome activation ameliorating kidney epithelial cells injury induced by albuminuria.

Novel role of PKR in inflammasome activation and HMGB1 release.

The inflammasome regulates the release of caspase activation-dependent cytokines, including interleukin (IL)-1ß, IL-18 and high-mobility group box 1 (HMGB1). By studying HMGB1 release mechanisms, here we identify a role for double-stranded RNA-dependent protein kinase (PKR, also known as EIF2AK2) in inflammasome activation. Exposure of macrophages to inflammasome agonists induced PKR autophosphorylation. PKR inactivation by genetic deletion or pharmacological inhibition severely impaired inflammasome activation in response to double-stranded RNA, ATP, monosodium urate, adjuvant aluminium, rotenone, live Escherichia coli, anthrax lethal toxin, DNA transfection and Salmonella typhimurium infection. PKR deficiency significantly inhibited the secretion of IL-1ß, IL-18 and HMGB1 in E. coli-induced peritonitis. PKR physically interacts with several inflammasome components, including NOD-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), NLRP1, NLR family CARD domain-containing protein 4 (NLRC4), absent in melanoma 2 (AIM2), and broadly regulates inflammasome activation. PKR autophosphorylation in a cell-free system with recombinant NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC, also known as PYCARD) and pro-caspase-1 reconstitutes inflammasome activity. These results show a crucial role for PKR in inflammasome activation, and indicate that it should be possible to pharmacologically target this molecule to treat inflammation.