Pros and cons of NRF2 activation as adjunctive therapy in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease with an important inflammatory component accompanied by deregulated redox-dependent signaling pathways that are feeding back into inflammation. In this context, we bring into focus the transcription factor NRF2, a master redox regulator that exerts exquisite antioxidant and anti-inflammatory effects. The review does not intend to be exhaustive, but to point out arguments sustaining the rationale for applying an NRF2-directed co-treatment in RA as well as its potential limitations. The involvement of NRF2 in RA is emphasized through an analysis of publicly available transcriptomic data on NRF2 target genes and the findings from NRF2-knockout mice. The impact of NRF2 on concurrent pathologic mechanisms in RA is explained by its crosstalk with major redox-sensitive inflammatory and cell death-related pathways, in the context of the increased survival of pathologic cells in RA. The proposed adjunctive therapy targeted to NRF2 is further sustained by the existence of promising NRF2 activators that are in various stages of drug development. The interference of NRF2 with conventional anti-rheumatic therapies is discussed, including the cytoprotective effects of NRF2 for alleviating drug toxicity. From another perspective, the review presents how NRF2 activation would be decreasing the efficacy of synthetic anti-rheumatic drugs by increasing drug efflux. Future perspectives regarding pharmacologic NRF2 activation in RA are finally proposed.

Melatonin Alters the miRNA Transcriptome of Inflammasome Activation in Murine Microglial Cells.

Systemic inflammation can have devastating effects on the central nervous system via its resident immune cells, the microglia. One of the primary mediators of this inflammation is inflammasomes, multiprotein complexes that trigger a release of inflammatory proteins when activated. Melatonin, a hormone with anti-inflammatory effects, is an attractive candidate for suppressing such inflammation. In this study, we have investigated how melatonin alters the microRNA (miRNA) transcriptome of microglial cells. For that purpose, we have performed RNA sequencing on a lipopolysaccharide and adenosine triphosphate (LPS + ATP) induced NLR family pyrin domain containing 3 (NLRP3) inflammasome activation model in the N9 mouse microglial cell line, with and without melatonin pre-treatment. We have identified 136 differentially expressed miRNAs in cells exposed to LPS + ATP compared to controls and 10 differentially expressed miRNAs in melatonin pre-treated cells compared to the inflammasome group. We have identified miR-155-3p as a miRNA that is upregulated with inflammasome activation and downregulated with melatonin treatment. We further confirmed this pattern of miR-155-3p expression in the brains of mice injected intraperitoneally with LPS. Moreover, an overexpression study with miRNA-155-3p mimic supported the idea that the protective effects of melatonin in NLRP3 inflammasome activation are partly associated with miRNA-155-3p inhibition.

Alteration of miRNAs in Small Neuron-Derived Extracellular Vesicles of Alzheimer's Disease Patients and the Effect of Extracellular Vesicles on Microglial Immune Responses.

Alzheimer's disease (AD) is one of the most severe neurodegenerative diseases observed in the elderly population. Although the hallmarks of AD have been identified, the methods for its definitive diagnosis and treatment are still lacking. Extracellular vesicles (EVs) have become a promising source for biomarkers since the identification of their content. EVs are released from multiple cell types and, when released from neurons, they pass from the brain to the blood with their cargo molecules. Hence, neuron-specific EV-resident microRNAs (miRNAs) are promising biomarkers for diagnosis of AD. This study aimed to identify altered miRNA content in small neuron-derived extracellular vesicles (sNDEVs) isolated from AD patients and healthy individuals. Furthermore, we examined the role of sNDEV-resident miRNAs in neuron-glia cellular interaction to understand their role in AD propagation. We identified 10 differentially expressed miRNAs in the sNDEVs of patients via next-generation sequencing and validated the most dysregulated miRNA, let-7e, with qRT-PCR. Let-7e was significantly increased in the sNDEVs of AD patients compared with those of healthy controls in a larger cohort. First, we evaluated the diagnostic utility of let-7e via ROC curve analysis, which revealed an AUC value of 0.9214. We found that IL-6 gene expression was increased in human microglia after treatment with sNDEVs of AD patients with a high amount of let-7e. Our study suggests that sNDEV-resident let-7e is a potential biomarker for AD diagnosis, and that AD patient-derived sNDEVs induce a neuroinflammatory response in microglia.

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

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

Role of Exosomal MicroRNAs in Cell-to-Cell Communication.

Exosomes, a type of extracellular vesicle, are small vesicles (30-100 nm) secreted into extracellular space from almost all types of cells. Exosomes mediate cell-to-cell communication carrying various biologically active molecules including microRNAs. Studies have shown that exosomal microRNAs play fundamental roles in healthy and pathological conditions such as immunity, cancer, and inflammation. In this chapter, we introduce the current knowledge on exosome biogenesis, techniques used in exosome research, and exosomal miRNA and their functions in biological and pathological processes.

Dimethyl Fumarate Alleviates NLRP3 Inflammasome Activation in Microglia and Sickness Behavior in LPS-Challenged Mice.

NLRP3 inflammasome activation contributes to several pathogenic conditions, including lipopolysaccharide (LPS)-induced sickness behavior characterized by reduced mobility and depressive behaviors. Dimethyl fumarate (DMF) is an immunomodulatory and anti-oxidative molecule commonly used for the symptomatic treatment of multiple sclerosis and psoriasis. In this study, we investigated the potential use of DMF against microglial NLRP3 inflammasome activation both in vitro and in vivo. For in vitro studies, LPS- and ATP-stimulated N9 microglial cells were used to induce NLRP3 inflammasome activation. DMF's effects on inflammasome markers, pyroptotic cell death, ROS formation, and Nrf2/NF-κB pathways were assessed. For in vivo studies, 12-14 weeks-old male BALB/c mice were treated with LPS, DMF + LPS and ML385 + DMF + LPS. Behavioral tests including open field, forced swim test, and tail suspension test were carried out to see changes in lipopolysaccharide-induced sickness behavior. Furthermore, NLRP3 and Caspase-1 expression in isolated microglia were determined by immunostaining. Here we demonstrated that DMF ameliorated LPS and ATP-induced NLRP3 inflammasome activation by reducing IL-1ß, IL-18, caspase-1, and NLRP3 levels, reactive oxygen species formation and damage, and inhibiting pyroptotic cell death in N9 murine microglia via Nrf2/NF-κB pathways. DMF also improved LPS-induced sickness behavior in male mice and decreased caspase-1/NLRP3 levels via Nrf2 activation. Additionally, we showed that DMF pretreatment decreased miR-146a and miR-155 both in vivo and in vitro. Our results proved the effectiveness of DMF on the amelioration of microglial NLRP3 inflammasome activation. We anticipate that this study will provide the foundation consideration for further studies aiming to suppress NLRP3 inflammasome activation associated with in many diseases and a better understanding of its underlying mechanisms.

Ethyl Pyruvate Attenuates Microglial NLRP3 Inflammasome Activation via Inhibition of HMGB1/NF-κB/miR-223 Signaling.

Ethyl pyruvate is a molecule with anti-inflammatory and pro-metabolic effects. Ethyl pyruvate has been shown to ameliorate the clinical and pathological findings of neurodegenerative diseases such as Alzheimer's and Parkinson's Diseases in rodents. Its anti-inflammatory and neuroprotective effects are widely investigated in animal and cellular models. Our study aimed to investigate the mechanism of the impact of Ethyl pyruvate on NLRP3 inflammasome activation in the N9 microglial cell line. Our results indicated that ethyl pyruvate significantly suppressed LPS and ATP-induced NLRP3 inflammasome activation, decreased active caspase-1 level, secretion of IL-1ß and IL-18 cytokines, and reduced the level of pyroptotic cell death resulting from inflammasome activation. Furthermore, ethyl pyruvate reduced the formation of total and mitochondrial ROS and suppressed inflammasome-induced HMGB1 upregulation and nuclear NF-κB translocation and reversed the inflammasome activation-induced miRNA expression profile for miR-223 in N9 cells. Our study suggests that ethyl pyruvate effectively suppresses the NLRP3 inflammasome activation in microglial cells regulation by miR-223 and NF-κB/HMGB1 axis.

Sulforaphane inhibits NLRP3 inflammasome activation in microglia through Nrf2-mediated miRNA alteration.

The NLRP3 inflammasome is a multiprotein complex that activates caspase-1 and triggers the release of the proinflammatory cytokines IL-1ß and IL-18 in response to diverse signals. Although inflammasome activation plays critical roles against various pathogens in host defense, overactivation of inflammasome contributes to the pathogenesis of inflammatory diseases, including acute CNS injuries and chronic neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In the current study, we demonstrated that Sulforaphane (SFN), a dietary natural product, inhibits NLRP3 inflammasome mediated IL-1ß and IL-18 secretion and pyroptosis in murine microglial cells. SFN decreased the secretion of IL-1ß and IL-18, and their mRNA levels in LPS primed microglia triggered by ATP. SFN suppressed the overexpression of cleaved caspase-1 and NLRP3 protein expressions as measured by caspase activity assay and western blot, respectively. SFN also prevented caspase-1 dependent pyroptotic cell death in microglia. Our data indicate that SFN suppresses NLRP3 inflammasome via the inhibition of NF-κB nuclear translocation and Nrf2 mediated miRNAs expression modulation in murine microglia.

Inhibitory effects of phytochemicals on NLRP3 inflammasome activation: A review.

BACKGROUND: The NLRP3 inflammasome formation and following cytokine secretion is a crucial step in innate immune responses. Internal and external factors may trigger inflammasome activation and result in inflammatory cytokine secretion. Inflammasome formation and activity play critical roles in several disease pathologies such as cardiovascular, metabolic, renal, digestive, and CNS diseases. Underlying pathways are not yet clear, but phytochemicals as alternative therapies have been extensively used for suppression of inflammatory responses. PURPOSE: In this review, we aimed to summarize in vivo and in vitro effects on NLRP3 inflammasome activation of selected phytochemicals. METHOD: Three phytochemicals; Sulforaphane, Curcumin, and Resveratrol were selected, and studies were reviewed to clarify their intracellular signaling mechanism in NLRP3 inflammasome activity. PubMed and Scopus databases are used for the search. For sulforaphane, 8 articles, for curcumin, 25 articles, and for resveratrol, 41 articles were included in the review. CONCLUSION: In vitro and in vivo studies pointed out that the selected phytochemicals have inhibitory properties on NLRP3 inflammasome activity. However, neither the mechanism is clear, nor the study designs and doses are standardized.

Microglial NLRP3 inflammasome activation in multiple sclerosis.

Multiple sclerosis (MS) is a chronic, autoimmune and neuroinflammatory disease of the central nervous system (CNS) mediated by autoreactive T cells directed against myelin antigens. Although the crucial role of adaptive immunity is well established in MS, the contribution of innate immunity has only recently been appreciated. Microglia are the main innate immune cells of the CNS. Similar to other myeloid cells, microglia recognize both exogenous and host-derived endogenous danger signals through pattern recognition receptors (PRRs) localized on their cell surface such as Toll Like receptor 4, or in the cytosol such as NLRP3. The second one is the sensor protein of the multi-molecular NLRP3 inflammasome complex in activated microglia that promotes the maturation and secretion of proinflammatory cytokines, interleukin-1ß and interleukin-18. Overactivation of microglia and aberrant activation of the NLRP3 inflammasome have been implicated in the pathogenesis of MS. Indeed, experimental data, together with post-mortem and clinical studies have revealed an increased expression of NLRP3 inflammasome complex elements in microglia and other immune cells. In this review, we focus on microglial NLRP3 inflammasome activation in MS. First, we overview the basic knowledge about MS, microglia and the NLRP3 inflammasome. Then, we summarize studies about microglial NLRP3 inflammasome activation in MS and its animal models. We also highlight experimental therapeutic approaches that target different steps of NLRP inflammasome activation. Finally, we discuss future research avenues and new methods in this rapidly evolving area.

Melatonin Attenuates LPS-Induced Acute Depressive-Like Behaviors and Microglial NLRP3 Inflammasome Activation Through the SIRT1/Nrf2 Pathway.

Inflammation is a crucial component of various stress-induced responses that contributes to the pathogenesis of major depressive disorder (MDD). Depressive-like behavior (DLB) is characterized by decreased mobility and depressive behavior that occurs in systemic infection induced by Lipopolysaccharide (LPS) in experimental animals and is considered as a model of exacerbation of MDD. We assessed the effects of melatonin on behavioral changes and inflammatory cytokine expression in hippocampus of mice in LPS-induced DLB, as well as its effects on NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome activation, oxidative stress and pyroptotic cell death in murine microglia in vitro. Intraperitoneal 5 mg/kg dose of LPS was used to mimic depressive-like behaviors and melatonin was given at a dose of 500 mg/kg for 4 times with 6 h intervals, starting at 2 h before LPS administration. Behavioral assessment was carried out at 24 h post-LPS injection by tail suspension and forced swimming tests. Additionally, hippocampal cytokine and NLRP3 protein levels were estimated. Melatonin increased mobility time of LPS-induced DLB mice and suppressed NLRP3 expression and interleukin-1ß (IL-1ß) cleavage in the hippocampus. Immunofluorescence staining of hippocampal tissue showed that NLRP3 is mainly expressed in ionized calcium-binding adapter molecule 1 (Iba1) -positive microglia. Our results show that melatonin prevents LPS and Adenosine triphosphate (ATP) induced NLRP3 inflammasome activation in murine microglia in vitro, evidenced by inhibition of NLRP3 expression, Apoptosis-associated speck-like protein containing a CARD (ASC) speck formation, caspase-1 cleavage and interleukin-1ß (IL-1ß) maturation and secretion. Additionally, melatonin inhibits pyroptosis, production of mitochondrial and cytosolic reactive oxygen species (ROS) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling. The beneficial effects of melatonin on NLRP3 inflammasome activation were associated with nuclear factor erythroid 2-related factor 2 (Nrf2) and Silent information regulator 2 homolog 1 (SIRT1) activation, which were reversed by Nrf2 siRNA and SIRT1 inhibitor treatment.

Sulforaphane Inhibits Lipopolysaccharide-Induced Inflammation, Cytotoxicity, Oxidative Stress, and miR-155 Expression and Switches to Mox Phenotype through Activating Extracellular Signal-Regulated Kinase 1/2-Nuclear Factor Erythroid 2-Related Factor 2/Antioxidant Response Element Pathway in Murine Microglial Cells.

Sulforaphane (SFN) is a natural product with cytoprotective, anti-inflammatory, and antioxidant effects. In this study, we evaluated the mechanisms of its effects on lipopolysaccharide (LPS)-induced cell death, inflammation, oxidative stress, and polarization in murine microglia. We found that SFN protects N9 microglial cells upon LPS-induced cell death and suppresses LPS-induced levels of secreted pro-inflammatory cytokines, tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6. SFN is also a potent inducer of redox sensitive transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), which is responsible for the transcription of antioxidant, cytoprotective, and anti-inflammatory genes. SFN induced translocation of Nrf2 to the nucleus via extracellular signal-regulated kinase 1/2 (ERK1/2) pathway activation. siRNA-mediated knockdown study showed that the effects of SFN on LPS-induced reactive oxygen species, reactive nitrogen species, and pro-inflammatory cytokine production and cell death are partly Nrf2 dependent. Mox phenotype is a novel microglial phenotype that has roles in oxidative stress responses. Our results suggested that SFN induced the Mox phenotype in murine microglia through Nrf2 pathway. SFN also alleviated LPS-induced expression of inflammatory microRNA, miR-155. Finally, SFN inhibits microglia-mediated neurotoxicity as demonstrated by conditioned medium and co-culture experiments. In conclusion, SFN exerts protective effects on microglia and modulates the microglial activation state.