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.

Inflammasome and Caspase-1 Activity Characterization and Evaluation: An Imaging Flow Cytometer-Based Detection and Assessment of Inflammasome Specks and Caspase-1 Activation.

Inflammasome dysregulation is a hallmark of various inflammatory diseases. Evaluating inflammasome-associated structures (ASC specks) and caspase-1 activity by microscopy is time consuming and limited by small sample size. The current flow cytometric method, time of flight inflammasome evaluation (TOFIE), cannot visualize ASC specks or caspase-1 activity, making colocalization studies of inflammasome components and enzymatic activity impossible. We describe a rapid, high-throughput, single-cell, fluorescence-based image analysis method utilizing the Amnis ImageStreamX instrument that quantitatively and qualitatively characterizes the frequency, area, and cellular distribution of ASC specks and caspase-1 activity in mouse and human cells. Unlike TOFIE, this method differentiates between singular perinuclear specks and false positives. With our technique we also show that the presence of NLRP3 reduces the size of ASC specks, which is further reduced by the presence of active caspase-1. The capacity of our approach to simultaneously detect and quantify ASC specks and caspase-1 activity, both at the population and single-cell level, renders it the most powerful tool available for visualizing and quantifying the impact of mutations on inflammasome assembly and activity.

NLRP3 inflammasome signal pathway involves in Vibrio harveyi-induced inflammatory response in murine peritoneal macrophages in vitro.

Vibrio harveyi, an important zoonotic pathogen, can infect wounds and cause inflammatory response. Understanding the inflammatory response pathways could facilitate the exploration of molecular mechanisms for treating V. harveyi infection. NLR family pyrin domain-containing 3 (NLRP3) inflammasome is involved in the interaction between hosts and pathogenic microorganisms and could be sensed by various pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). Nonetheless, the function of NLRP3 inflammasome in V. harveyi infection remains unclear. In the present study, we established a V. harveyi infection model using murine peritoneal macrophages (PMs). Various techniques, including western blot analysis, enzyme-linked immunosorbent assay (ELISA), RT-qPCR, immunofluorescence, and inhibition assays, were used to explore the molecular mechanism of V. harveyi-induced inflammation. The results showed that many inflammatory cytokines participated in V. harveyi infection, with interleukin (IL)-1ß being the most abundant. Pan-caspase inhibitor pretreatment significantly decreased the secretion of IL-1ß in murine PMs. Moreover, the identification of V. harveyi involved a large number of NLR molecules, especially the NLRP3 receptor, and further studies revealed that NLPR3 inflammasome was activated by V. harveyi infection, as evidenced by puncta-like NLRP3 surrounding cell nuclear, ASC specks in the nucleus and cytoplasm, and ASC oligomerization. Inhibition of NLRP3 inflammasome impaired the release of mature IL-1ß in V. harveyi-infected murine PMs. Furthermore, blocking the secretion of mature IL-1ß could markedly decrease the release of other proinflammatory cytokines, including IL-6, IL-12, and tumor necrosis factor-α. Overall, these data indicated that NLRP3 inflammasome was activated in response to V. harveyi infection and enhanced inflammatory response by promoting IL-1ß secretion in murine PMs.

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.

Activation of the NLRP3 Inflammasome by Particles from the Echinococcus granulosus Laminated Layer.

The interaction of dendritic cells and macrophages with a variety of rigid noncellular particles triggers activation of the NLRP3 inflammasome and consequent secretion of interleukin 1ß (IL-1ß). Noncellular particles can also be generated in the context of helminth infection, since these large pathogens often shed their outermost structures during growth and/or molting. One such structure is the massive, mucin-based, soft, flexible laminated layer (LL), which protects the larval stages of cestodes of the genus Echinococcus We show that particles from the Echinococcus granulosus LL (pLL) trigger NLRP3- and caspase-1-dependent IL-1ß in lipopolysaccharide (LPS)-primed mouse bone marrow-derived dendritic cells (BMDC). This response can be elicited by pLL too large for phagocytosis and nonetheless requires actin dynamics, Syk, and phosphatidylinositol 3-kinase (PI3K). These three requirements had already been observed in our previous study on the alteration by pLL of CD86, CD40, IL-10, and IL-12 responses to LPS in BMDC; however, we now show that these alterations are independent of NLRP3 and caspase-1. In other words, an initial interaction with particles requiring actin dynamics, Syk, and PI3K, but not phagocytosis, elicits both NLRP3-dependent and NLRP3-independent responses. Intraperitoneal injection of pLL induced IL-1ß, suggesting that contact with LL materials induces IL-1ß in the E. granulosus infection setting. Our results extend our understanding of NLRP3 inflammasome activation by noncellular particulate materials both to helminth-derived materials and to flexible/soft materials.

Angiotensin (1-7) inhibits arecoline-induced migration and collagen synthesis in human oral myofibroblasts via inhibiting NLRP3 inflammasome activation.

Arecoline induces oral submucous fibrosis (OSF) via promoting the reactive oxygen species (ROS). Angiotensin (1-7) (Ang-(1-7)) protects against fibrosis by counteracting angiotensin II (Ang-II) via the Mas receptor. However, the effects of Ang-(1-7) on OSF remain unknown. NOD-like receptors (NLRs) family pyrin domain containing 3 (NLRP3) inflammasome is identified as the novel mechanism of fibrosis. Whereas the effects of arecoline on NLRP3 inflammasome remain unclear. We aimed to explore the effect of Ang-(1-7) on NLRP3 inflammasome in human oral myofibroblasts. In vivo, activation of NLRP3 inflammasomes with an increase of Ang-II type 1 receptor (AT1R) protein level and ROS production in human oral fibrosis tissues. Ang-(1-7) improved arecoline-induced rats OSF, reduced protein levels of NADPH oxidase 4 (NOX4) and the NLRP3 inflammasome. In vitro, arecoline increased ROS along with upregulation of the angiotensin-converting enzyme (ACE)/Ang-II/AT1R axis and NLRP3 inflammasome/interleukin-1ß axis in human oral myofibroblasts, which were reduced by NOX4 inhibitor VAS2870, ROS scavenger N-acetylcysteine, and NOX4 small interfering RNA (siRNA). Furthermore, arecoline induced collagen synthesis or migration via the Smad or RhoA-ROCK pathway respectively, which could be inhibited by NLRP3 siRNA or caspase-1 blocker VX-765. Ang-(1-7) shifted the balance of RAS toward the ACE2/Ang-(1-7)/Mas axis, inhibited arecoline-induced ROS and NLRP3 inflammasome activation, leading to attenuation of migration or collagen synthesis. In summary, Ang-(1-7) attenuates arecoline-induced migration and collagen synthesis via inhibiting NLRP3 inflammasome in human oral myofibroblasts.

Gypenosides improve diabetic cardiomyopathy by inhibiting ROS-mediated NLRP3 inflammasome activation.

NLRP3 inflammasome activation plays an important role in diabetic cardiomyopathy (DCM), which may relate to excessive production of reactive oxygen species (ROS). Gypenosides (Gps), the major ingredients of Gynostemma pentaphylla (Thunb.) Makino, have exerted the properties of anti-hyperglycaemia and anti-inflammation, but whether Gps improve myocardial damage and the mechanism remains unclear. Here, we found that high glucose (HG) induced myocardial damage by activating the NLRP3 inflammasome and then promoting IL-1ß and IL-18 secretion in H9C2 cells and NRVMs. Meanwhile, HG elevated the production of ROS, which was vital to NLRP3 inflammasome activation. Moreover, the ROS activated the NLRP3 inflammasome mainly by cytochrome c influx into the cytoplasm and binding to NLRP3. Inhibition of ROS and cytochrome c dramatically down-regulated NLRP3 inflammasome activation and improved the cardiomyocyte damage induced by HG, which was also detected in cells treated by Gps. Furthermore, Gps also reduced the levels of the C-reactive proteins (CRPs), IL-1ß and IL-18, inhibited NLRP3 inflammasome activation and consequently improved myocardial damage in vivo. These findings provide a mechanism that ROS induced by HG activates the NLRP3 inflammasome by cytochrome c binding to NLRP3 and that Gps may be potential and effective drugs for DCM via the inhibition of ROS-mediated NLRP3 inflammasome activation.

Prolonged fasting suppresses mitochondrial NLRP3 inflammasome assembly and activation via SIRT3-mediated activation of superoxide dismutase 2.

Twenty-four hours of fasting is known to blunt activation of the human NLRP3 inflammasome. This effect might be mediated by SIRT3 activation, controlling mitochondrial reactive oxygen species. To characterize the molecular underpinnings of this fasting effect, we comparatively analyzed the NLRP3 inflammasome response to nutrient deprivation in wild-type and SIRT3 knock-out mice. Consistent with previous findings for human NLRP3, prolonged fasting blunted the inflammasome in wild-type mice but not in SIRT3 knock-out mice. In SIRT3 knock-out bone marrow-derived macrophages, NLRP3 activation promoted excess cytosolic extrusion of mitochondrial DNA along with increased reactive oxygen species and reduced superoxide dismutase 2 (SOD2) activity. Interestingly, the negative regulatory effect of SIRT3 on NLRP3 was not due to transcriptional control or priming of canonical inflammasome components but, rather, occurred via SIRT3-mediated deacetylation of mitochondrial SOD2, leading to SOD2 activation. We also found that siRNA knockdown of SIRT3 or SOD2 increased NLRP3 supercomplex formation and activation. Moreover, overexpression of wild-type and constitutively active SOD2 similarly blunted inflammasome assembly and activation, effects that were abrogated by acetylation mimic-modified SOD2. Finally, in vivo administration of lipopolysaccharide increased liver injury and the levels of peritoneal macrophage cytokines, including IL-1ß, in SIRT3 KO mice. These results support the emerging concept that enhancing mitochondrial resilience against damage-associated molecular patterns may play a pivotal role in preventing inflammation and that the anti-inflammatory effect of fasting-mimetic diets may be mediated, in part, through SIRT3-directed blunting of NLRP3 inflammasome assembly and activation.

NLRP3-dependent synaptic plasticity deficit in an Alzheimer's disease amyloidosis model in vivo.

Pro-inflammatory mechanisms have recently emerged as an important component of early Alzheimer's disease (AD) pathogenesis. A particularly attractive therapeutic strategy is to selectively prevent the disruptive effects of activation of the innate immune system in the brain at an early transitional stage by reducing the production or directly neutralizing pro-inflammatory cytokines, in particular IL-1ß and TNF-α. Here we tested their in vivo effects on synaptic plasticity deficits, which provide sensitive and robust measures of synaptic failure, in a rat model of AD amyloidosis. Using electrophysiological techniques we longitudinally studied the effects of the NLRP3 inflammasome inhibitor Mcc950, the IL-1 receptor antagonist (anakinra) and an anti-TNF-α agent (etanercept) in awake freely moving transgenic rats overexpressing AD associated ß-amyloid precursor protein at a pre-plaque stage of amyloidosis. Repeated treatment with Mcc950 reversibly abrogated the inhibition of long-term potentiation. The IL-1 receptor antagonist and etanercept also had a similar beneficial effect on the deficit in synaptic plasticity. Our findings support the clinical development of Mcc950 and clinically available IL-1- and TNF-α-neutralizing agents in early AD.

Atypical Gasdermin D and Mixed Lineage Kinase Domain-like Protein Leakage Aggravates Tetrachlorobenzoquinone-Induced Nod-like Receptor Protein 3 Inflammasome Activation.

Our previous study showed that tetrachlorobenzoquinone (TCBQ) mediated the activation of Nod-like receptor protein 3 (NLRP3) inflammasome, which involves K+ efflux, reactive oxygen species (ROS) production, and mitochondrial DNA damage. In addition, TCBQ down-regulates NLRP3 ubiquitination and promotes the activation of NLRP3 inflammasome. However, the induction of NLRP3 inflammasome by atypical pathways has not yet been characterized. Using human umbilical vein endothelial cells (HUVEC), we discovered that TCBQ activates caspase 1/4/5 and cleaves gasdermin D (GSDMD) into N-terminal and C-terminal cleavage products. In parallel, TCBQ also activates receptor interacting protein kinase 3 (RIPK3)/mixed lineage kinase domain-like protein (MLKL) signaling pathways. The N-terminal fragments of GSDMD and MLKL translocate from cytoplasm to cell membrane and form oligomers and membrane pores on the cell membrane. The formation of membrane pores not only promotes the extracellular secretion of interleukin 1 beta (IL-1ß) but also affects cellular ion homeostasis, in particular promotes K+ outflow, which further activates NLRP3 inflammasome and aggravates cellular inflammation. These results indicated that GSDMD and MLKL play important roles in TCBQ-induced endothelial pro-inflammatory responses, which may point to potential therapeutic approaches for TCBQ-mediated toxicity.

Stimulator of IFN genes-mediated DNA-sensing pathway is suppressed by NLRP3 agonists and regulated by mitofusin 1 and TBC1D15, mitochondrial dynamics mediators.

The stimulator of IFN genes (STING)-mediated DNA-sensing pathway plays an important role in the innate immune response to pathogen infection, autoimmunity, and cancer; however, its regulatory mechanism has not been fully elucidated, and we do not yet know whether the STING pathway is counter-regulated by other innate immune pathways. Here, we show that the NLRP3-activating agonists, ATP and nigericin, prevent STING pathway activation in association with mitochondrial fragmentation; however, the suppression of the STING pathway and mitochondria fission were not dependent on NLRP3 or potassium efflux. Although nigericin-induced mitochondria fission was rescued by knockdown of either dynamin-related protein 1 or TBC1 domain family member 15 (TBC1D15), which are two distinct mitochondria fission regulators, only TBC1D15 restored the activity of the STING pathway, which indicates that inflammasome-activating signals curtail STING pathway activation via TBC1D15. Finally, we found that deficiency of mitofusin (MFN) 1, a mediator of mitochondrial fusion, inhibited STING pathway activation, which leads to a decrease in the induction of IFN-ß and its inducible gene, ISG56, in conjunction with diminished activation of the signaling molecules, TANK-binding kinase 1 and IFN regulatory factor 3, that are downstream of STING. These results highlight the crucial role of MFN1 in maintaining the competency of the STING pathway. Collectively, our findings reveal that mitochondrial dynamics regulators modulate the activation of the STING signaling pathway.-Kwon, D., Park, E., Kang, S.-J. Stimulator of IFN genes-mediated DNA-sensing pathway is suppressed by NLRP3 agonists and regulated by mitofusin 1 and TBC1D15, mitochondrial dynamics mediators.

Amyloid ß induces NLRP3 inflammasome activation in retinal pigment epithelial cells via NADPH oxidase- and mitochondria-dependent ROS production.

Amyloid ß (Aß)-induced chronic inflammation is believed to be a key pathogenic process in early-stage age-related macular degeneration (AMD). Nucleotide oligomerization domain (NOD)-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation triggered by Aß is responsible for retinal pigment epithelium (RPE) dysfunction in the onset of AMD; however, the detailed molecular mechanism remains unclear. In this study, we investigated the involvement of NADPH oxidase- and mitochondria-derived reactive oxygen species (ROS) in the process of Aß1-40 -induced NLRP3 inflammasome activation in LPS-primed ARPE-19 cells. The results showed that Aß1-40 could induce excessive ROS generation, MAPK/NF-κB signaling activation and subsequently NLRP3 inflammasome activation in LPS-primed ARPE-19 cells. Furthermore, the inductive effect of Aß1-40 on NLRP3 inflammasome activation was mediated in a manner dependent on NADPH oxidase- and mitochondria-derived ROS. Our findings may provide a novel insight into the molecular mechanism by which Aß contributes to the early-stage AMD.

The BCL-2 inhibitor APG-2575 resets tumor-associated macrophages toward the M1 phenotype, promoting a favorable response to anti-PD-1 therapy via NLRP3 activation.

The main challenges in the use of immune checkpoint inhibitors (ICIs) are ascribed to the immunosuppressive tumor microenvironment and the lack of sufficient infiltration of activated CD8+ T cells. Transforming the tumor microenvironment (TME) from "cold" to "hot" and thus more likely to potentiate the effects of ICIs is a promising strategy for cancer treatment. We found that the selective BCL-2 inhibitor APG-2575 can enhance the antitumor efficacy of anti-PD-1 therapy in syngeneic and humanized CD34+ mouse models. Using single-cell RNA sequencing, we found that APG-2575 polarized M2-like immunosuppressive macrophages toward the M1-like immunostimulatory phenotype with increased CCL5 and CXCL10 secretion, restoring T-cell function and promoting a favorable immunotherapy response. Mechanistically, we demonstrated that APG-2575 directly binds to NF-κB p65 to activate NLRP3 signaling, thereby mediating macrophage repolarization and the activation of proinflammatory caspases and subsequently increasing CCL5 and CXCL10 chemokine production. As a result, APG-2575-induced macrophage repolarization could remodel the tumor immune microenvironment, thus improving tumor immunosuppression and further enhancing antitumor T-cell immunity. Multiplex immunohistochemistry confirmed that patients with better immunotherapeutic efficacy had higher CD86, p-NF-κB p65 and NLRP3 levels, accompanied by lower CD206 expression on macrophages. Collectively, these data provide evidence that further study on APG-2575 in combination with immunotherapy for tumor treatment is required.

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.

The APPL1-Rab5 axis restricts NLRP3 inflammasome activation through early endosomal-dependent mitophagy in macrophages.

Although mitophagy is known to restrict NLRP3 inflammasome activation, the underlying regulatory mechanism remains poorly characterized. Here we describe a type of early endosome-dependent mitophagy that limits NLRP3 inflammasome activation. Deletion of the endosomal adaptor protein APPL1 impairs mitophagy, leading to accumulation of damaged mitochondria producing reactive oxygen species (ROS) and oxidized cytosolic mitochondrial DNA, which in turn trigger NLRP3 inflammasome overactivation in macrophages. NLRP3 agonist causes APPL1 to translocate from early endosomes to mitochondria, where it interacts with Rab5 to facilitate endosomal-mediated mitophagy. Mice deficient for APPL1 specifically in hematopoietic cell are more sensitive to endotoxin-induced sepsis, obesity-induced inflammation and glucose dysregulation. These are associated with increased expression of systemic interleukin-1ß, a major product of NLRP3 inflammasome activation. Our findings indicate that the early endosomal machinery is essential to repress NLRP3 inflammasome hyperactivation by promoting mitophagy in macrophages.

Bafilomycin A1 enhances NLRP3 inflammasome activation in human monocytes independent of lysosomal acidification.

The release of interleukin (IL)-1ß from primary human monocytes in response to extracellular LPS occurs through the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome. In primary monocytes, in response to LPS, NLRP3 inflammasome activation is characterized by an independence of K+ efflux and ASC speck formation and has been termed the 'alternative' pathway. Here, we report that pharmacological inhibition of V-ATPase with bafilomycin A1 exacerbated LPS-induced NLRP3 inflammasome activation in primary human monocytes. Inhibition of V-ATPase in the presence of extracellular LPS led to NLRP3-dependent, K+ efflux-independent, ASC oligomerization and caspase-1 activation. Although V-ATPases are required for lysosomal acidification, we found that acidic lysosomal pH and protease activity were dispensable for this altered response, suggesting that V-ATPase inhibition triggered alternative signalling events. Therefore, V-ATPases may serve additional roles during NLRP3 inflammasome activation in primary human monocytes.

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.

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.

Kv1.3 channel blockade alleviates cerebral ischemia/reperfusion injury by reshaping M1/M2 phenotypes and compromising the activation of NLRP3 inflammasome in microglia.

After cerebral ischemia/reperfusion injury, pro-inflammatory M1-like and anti-inflammatory M2-like phenotypes of microglia are involved in neuroinflammation, in which NLRP3 inflammasome plays an essential role. Kv1.3 channel has been recognized as neuro-immunomodulatory target, but it is not clear as to its role in the neuroinflammation after cerebral ischemic injury. The current study aimed to investigate the issue. Middle cerebral artery occlusion/reperfusion (MCAO/R) model in rats and oxygen-glucose deprivation/ reoxygenation (OGD/R) in primary microglia were utilized to mimic disease state of ischemic stroke. Treatment with PAP-1, a Kv1.3 channel blocker, produced a significant improvement in neurological deficit scores and a decrease in infarct volume in MCAO/R model. An increased number of M2-like phenotypic microglia and a reduced number of M1-like phenotypic microglia were observed by immunofluorescent staining in the in vivo model, which was further validated by flow cytometry in vitro. Western blot showed that PAP-1 treatment profoundly reduced cleavage of caspase-1 and IL-1ß in vivo and in vitro. Furthermore, PAP-1 administration reduced the number of NLRP3+/Iba1+ cells and NLRP3 protein levels in vivo, while reduced mRNA and protein expression levels of NLRP3 in vitro. Reduced mRNA expression levels of IL-1ß in vitro and protein level of IL-1ß in vivo were also observed. Taken together, our findings suggested that Kv1.3 channel blockade effectively alleviated cerebral ischemic injury, possibly by reshaping microglial phenotypic response from M1 towards M2, compromising the activation of NLRP3 inflammasome in microglia, and inhibiting release of IL-1ß.

NLRP3 augmented resistance to gemcitabine in triple-negative breast cancer cells via EMT/IL-1ß/Wnt/ß-catenin signaling pathway.

BACKGROUND: Gemcitabine is widely used in the treatment of breast cancer (BC). However, the resistance to drugs remains a tough concern. The study explored the potential mechanism concerning gemcitabine resistance in triple-negative BC (TNBC) in vitro. METHODS: TNBC cells (TNBCC) and gemcitabine-resistance cell lines (GRC) were used. We investigated the sensitivity to gemcitabine responsive to regulation of Nod-like receptor protein 3 (NLRP3) expression in TNBCC in different gemcitabine concentrations. RT-PCR checked NLRP3 mRNA expression and MTT assessed the cell cytotoxicity. Gemcitabine resistance was studied in GRC exposed to 0, 1, 3, 5 nm gemcitabine after GRC were treated with NLRP3 agonist Nigericin sodium salt (NSS) or antagonist CY-09. Epithelial-to-mesenchymal transition (EMT) biomarkers were evaluated via RT-PCR and inflammasome IL-1ß, ß-catenin content and GSK-3ß activity were measured by ELISA methods. Last, we inactivated the signaling and examined the NLRP3, EMT mRNA expression by RT-PCR, IL-1ß, ß-catenin content and GSK-3ß activity by ELISA and cell cytotoxicity through MTT. RESULTS: NLRP3 up-regulation improved cell survival and reduced sensitivity to gemcitabine (P<0.05). NLRP3 had higher expression in GRC than TNBCC. GRC cell viability dropped as the gemcitabine concentration increased. NLRP3 up-regulation added to resistance to gemcitabine in GRC (P<0.05). NLRP3 agonist might induce EMT process, activate wnt/ß-catenin signaling and IL-1ß, while inactivation of wnt/ß-catenin signaling could result in the inhibition of NLRP3, IL-1ß and EMT as well as cell viability in GRC (P<0.05). CONCLUSION: NLRP3 could enhance resistance to gemcitabine via IL-1ß/EMT/Wnt/ß-catenin signaling, which suggested that NLRP3 antagonist CY-09 might be incorporated into gemcitabine treatment for TNBC.