Unmasking the NLRP3 inflammasome in dendritic cells as a potential therapeutic target for autoimmunity, cancer, and infectious conditions.

Proper and functional immune response requires a complex interaction between innate and adaptive immune cells, which dendritic cells (DCs) are the primary actors in this coordination as professional antigen-presenting cells. DCs are armed with numerous pattern recognition receptors (PRRs) such as nucleotide-binding and oligomerization domain-like receptors (NLRs) like NLRP3, which influence the development of their activation state upon sensation of ligands. NLRP3 is a crucial component of the immune system for protection against tumors and infectious agents, because its activation leads to the assembly of inflammasomes that cause the formation of active caspase-1 and stimulate the maturation and release of proinflammatory cytokines. But, when NLRP3 becomes overactivated, it plays a pathogenic role in the progression of several autoimmune disorders. So, NLRP3 activation is strictly regulated by diverse signaling pathways that are mentioned in detail in this review. Furthermore, the role of NLRP3 in all of the diverse immune cells' subsets is briefly mentioned in this study because NLRP3 plays a pivotal role in modulating other immune cells which are accompanied by DCs' responses and subsequently influence differentiation of T cells to diverse T helper subsets and even impact on cytotoxic CD8+ T cells' responses. This review sheds light on the functional and therapeutic role of NLRP3 in DCs and its contribution to the occurrence and progression of autoimmune disorders, prevention of diverse tumors' development, and recognition and annihilation of various infectious agents. Furthermore, we highlight NLRP3 targeting potential for improving DC-based immunotherapeutic approaches, to be used for the benefit of patients suffering from these disorders.

[Preparation and assessment of a monoclonal antibody against mouse NLRP3].

Objective To prepare a monoclonal antibody (mAb) against mouse NOD-like receptor family pyrin domain-containing 3 (NLRP3) and assess its specificity. Methods A gene fragment encoding mouse NLRP3 exon3 (Ms-N3) was inserted into the vector p36-G3-throhFc to construct a recombinant plasmid named Ms-N3-throhFc. This plasmid was then transfected into HEK293F cells for eukaryotic expression. NLRP3-/- mice were immunized with Ms-N3 protein purified using a protein A chromatography column, and splenocytes from the immunized mice were fused with SP2/0 myeloma cells to generate hybridoma cells. Specific mAbs against murine NLRP3 from hybridoma cells were screened using ELISA and immunofluorescence assay(IFA). Results The Ms-N3-throhFc recombinant plasmid was successfully constructed and exhibited stable expression in HEK293F cells. Twelve hybridoma cell lines were initially screened using ELISA. IFA revealed that the mAb secreted by the 9-B8-3-2-C5 cell line specifically recognized the native form of mouse NLRP3 protein. The heavy and light chain subtypes of this mAb were identified as IgM and κ, respectively. Conclusion A monoclonal antibody against mouse NLRP3 has been successfully prepared.

The non-pathogenic protozoon Leishmania tarentolae interferes with the activation of NLRP3 inflammasome in human cells: new perspectives in the control of inflammation.

Background: Innate immune responses against infectious agents can act as triggers of inflammatory diseases. On the other hand, various pathogens have developed mechanisms for the evasion of the immune response, based on an inhibition of innate immunity and inflammatory responses. Inflammatory diseases could thus be controlled through the administration of pathogens or pathogen-derived molecules, capable of interfering with the mechanisms at the basis of inflammation. In this framework, the NLRP3 inflammasome is an important component in innate antimicrobial responses and a major player in the inflammatory disease. Parasites of the genus Leishmania are master manipulators of innate immune mechanisms, and different species have been shown to inhibit inflammasome formation. However, the exploitation of pathogenic Leishmania species as blockers of NLRP3-based inflammatory diseases poses safety concerns. Methods: To circumvent safety issues associated with pathogenic parasites, we focused on Leishmania tarentolae, a species of Leishmania that is not infectious to humans. Because NLRP3 typically develops in macrophages, in response to the detection and engulfment microorganisms, we performed our experiments on a monocyte-macrophage cell line (THP-1), either wild type or knockout for ASC, a key component of NLRP3 formation, with determination of cytokines and other markers of inflammation. Results: L. tarentolae was shown to possess the capability of dampening the formation of NLRP3 inflammasome and the consequent expression of pro-inflammatory molecules, with minor differences compared to effects of pathogenic Leishmania species. Conclusion: The non-pathogenic L. tarentolae appears a promising pro-biotic microbe with anti-inflammatory properties or a source of immune modulating cellular fractions or molecules, capable of interfering with the formation of the NLRP3 inflammasome.

Intestinal microflora promotes Th2-mediated immunity through NLRP3 in damp and heat environments.

Background: With the worsening of the greenhouse effect, the correlation between the damp-heat environment (DH) and the incidence of various diseases has gained increasing attention. Previous studies have demonstrated that DH can lead to intestinal disorders, enteritis, and an up-regulation of NOD-like receptor protein 3 (NLRP3). However, the mechanism of NLRP3 in this process remains unclear. Methods: We established a DH animal model to observe the impact of a high temperature and humidity environment on the mice. We sequenced the 16S rRNA of mouse feces, and the RNA transcriptome of intestinal tissue, as well as the levels of cytokines including interferon (IFN)-γ and interleukin (IL)-4 in serum. Results: Our results indicate that the intestinal macrophage infiltration and the expression of inflammatory genes were increased in mice challenged with DH for 14 days, while the M2 macrophages were decreased in Nlrp3 -/- mice. The alpha diversity of intestinal bacteria in Nlrp3 -/- mice was significantly higher than that in control mice, including an up-regulation of the Firmicutes/Bacteroidetes ratio. Transcriptomic analysis revealed 307 differentially expressed genes were decreased in Nlrp3 -/- mice compared with control mice, which was related to humoral immune response, complement activation, phagocytic recognition, malaria and inflammatory bowel disease. The ratio of IFN-γ/IL-4 was decreased in control mice but increased in Nlrp3 -/- mice. Conclusions: Our study found that the inflammation induced by DH promotes Th2-mediated immunity via NLRP3, which is closely related to the disruption of intestinal flora.

Molecular regulation of NLRP3 inflammasome activation during parasitic infection.

Parasitic diseases are a serious global health concern, causing many common and severe infections, including Chagas disease, leishmaniasis, and schistosomiasis. The NLRP3 inflammasome belongs to the NLR (nucleotide-binding domain leucine-rich-repeat-containing proteins) family, which are cytosolic proteins playing key roles in the detection of pathogens. NLRP3 inflammasomes are activated in immune responses to Plasmodium, Leishmania, Toxoplasma gondii, Entamoeba histolytica, Trypanosoma cruzi, and other parasites. The role of NLRP3 is not fully understood, but it is a crucial component of the innate immune response to parasitic infections and its functions as a sensor triggering the inflammatory response to the invasive parasites. However, while this response can limit the parasites' growth, it can also result in potentially catastrophic host pathology. This makes it essential to understand how NLRP3 interacts with parasites to initiate the inflammatory response. Plasmodium hemozoin, Leishmania glycoconjugate lipophosphoglycan (LPG) and E. histolytica Gal/GalNAc lectin can stimulate NLRP3 activation, while the dense granule protein 9 (GRA9) of T. gondii has been shown to suppress it. Several other parasitic products also have diverse effects on NLRP3 activation. Understanding the mechanism of NLRP3 interaction with these products will help to develop advanced therapeutic approaches to treat parasitic diseases. This review summarizes current knowledge of the NLRP3 inflammasome's action on the immune response to parasitic infections and aims to determine the mechanisms through which parasitic molecules either activate or inhibit its action.

Activation of the NLRP3 inflammasome by human adenovirus type 7 L4 100-kilodalton protein.

Human adenovirus type 7 (HAdV-7) is a significant viral pathogen that causes respiratory infections in children. Currently, there are no specific antiviral drugs or vaccines for children targeting HAdV-7, and the mechanisms of its pathogenesis remain unclear. The NLRP3 inflammasome-driven inflammatory cascade plays a crucial role in the host's antiviral immunity. Our previous study demonstrated that HAdV-7 infection activates the NLRP3 inflammasome. Building upon this finding, our current study has identified the L4 100 kDa protein encoded by HAdV-7 as the primary viral component responsible for NLRP3 inflammasome activation. By utilizing techniques such as co-immunoprecipitation, we have confirmed that the 100 kDa protein interacts with the NLRP3 protein and facilitates the assembly of the NLRP3 inflammasome by binding specifically to the NACHT and LRR domains of NLRP3. These insights offer a deeper understanding of HAdV-7 pathogenesis and contribute to the development of novel antiviral therapies.

Inflammasome Activation and IL-1ß Release Triggered by Nanosecond Pulsed Electric Fields in Murine Innate Immune Cells and Skin.

Although electric field-induced cell membrane permeabilization (electroporation) is used in a wide range of clinical applications from cancer therapy to cardiac ablation, the cellular- and molecular-level details of the processes that determine the success or failure of these treatments are poorly understood. Nanosecond pulsed electric field (nsPEF)-based tumor therapies are known to have an immune component, but whether and how immune cells sense the electroporative damage and respond to it have not been demonstrated. Damage- and pathogen-associated stresses drive inflammation via activation of cytosolic multiprotein platforms known as inflammasomes. The assembly of inflammasome complexes triggers caspase-1-dependent secretion of IL-1ß and in many settings a form of cell death called pyroptosis. In this study we tested the hypothesis that the nsPEF damage is sensed intracellularly by the NLRP3 inflammasome. We found that 200-ns PEFs induced aggregation of the inflammasome adaptor protein ASC, activation of caspase-1, and triggered IL-1ß release in multiple innate immune cell types (J774A.1 macrophages, bone marrow-derived macrophages, and dendritic cells) and in vivo in mouse skin. Efflux of potassium from the permeabilized cell plasma membrane was partially responsible for nsPEF-induced inflammasome activation. Based on results from experiments using both the NRLP3-specific inhibitor MCC950 and NLRP3 knockout cells, we propose that the damage created by nsPEFs generates a set of stimuli for the inflammasome and that more than one sensor can drive IL-1ß release in response to electrical pulse stimulation. This study shows, to our knowledge, for the first time, that PEFs activate the inflammasome, suggesting that this pathway alarms the immune system after treatment.

Distinct SARS-CoV-2 specific NLRP3 and IL-1ß responses in T cells of aging patients during acute COVID-19 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease 2019 (COVID-19) that presents with varied clinical manifestations ranging from asymptomatic or mild infections and pneumonia to severe cases associated with cytokine storm, acute respiratory distress syndrome (ARDS), and even death. The underlying mechanisms contributing to these differences are unclear, although exacerbated inflammatory sequelae resulting from infection have been implicated. While advanced aging is a known risk factor, the precise immune parameters that determine the outcome of SARS-CoV-2 infection in elderly individuals are not understood. Here, we found aging-associated (age ≥61) intrinsic changes in T cell responses when compared to those from individuals aged ≤ 60, even among COVID-positive patients with mild symptoms. Specifically, when stimulated with SARS-CoV-2 peptides in vitro, peripheral blood mononuclear cell (PBMC) CD4+ and CD8+ T cells from individuals aged ≥61 showed a diminished capacity to produce IFN-γ and IL-1ß. Although they did not have severe disease, aged individuals also showed a higher frequency of PD-1+ cells and significantly diminished IFN-γ/PD-1 ratios among T lymphocytes upon SARS-CoV-2 peptide stimulation. Impaired T cell IL-1ß expression coincided with reduced NLRP3 levels in T lymphocytes. However, the expression of these molecules was not affected in the monocytes of individuals aged ≥61. Together, these data reveal SARS-CoV-2-specific CD4+ and CD8+ T-cell intrinsic cytokine alterations in the individuals older than 61 and may provide new insights into dysregulated COVID-directed immune responses in the elderly.

The Role of NLRP3 Inflammasomes in Trained Immunity.

Inflammasomes are cytosolic multi-protein complexes that play an important role in the innate immune system, inducing cytokine maturation and pyroptosis. Trained immunity is the induction of memory in innate immune cells by epigenetic reprogramming due to repeated inflammatory stimuli that alter the inflammatory response and increase resistance to infection or disease. Although it is speculated that nucleotide-binding oligomerization domain (NOD), leucine-rich repeat (LRR), and the NLR family pyrin domain containing 3 (NLRP3) inflammasomes respond to various inflammatory stimuli and are associated with trained immunity, the exact relationship is still unclear. This paper aims to introduce data from recent research on the role of inflammasomes in trained immunity through cellular immunometabolic and epigenetic reprogramming. It also suggests a new therapeutic strategy for inflammatory diseases through the complementary regulation of inflammasomes and trained immunity.

Seasonal coronavirus infections trigger NLRP3 inflammasome activation in macrophages but is therapeutically targetable.

Seasonal coronaviruses widely circulate in the global population, and severe complications can occur in specific vulnerable populations. Little is known on their pathogenic mechanisms and no approved treatment is available. Here, we present anecdotal evidence that the level of IL-1ß, a hallmark of inflammasome activation, appears elevated in a subset of seasonal coronavirus infected patients. We found that cultured human macrophages support the full life cycle of three cultivatable seasonal coronaviruses. Their infections effectively activate NLRP3 inflammasome activation through TLR4 ligation and NF-κB activation. This activation can be attenuated by specific pharmacological inhibitors and clinically used medications including dexamethasone and flufenamic acid. Interestingly, combination of antiviral and anti-inflammatory drugs simultaneously inhibit seasonal coronavirus-triggered inflammatory response and viral replication. Collectively, these findings show that the TLR4/NF-κB/NLRP3 axis drives seasonal coronavirus triggered-inflammatory response, which in turn represents a viable therapeutic target.

Deletion of African Swine Fever Virus (ASFV) H240R Gene Attenuates the Virulence of ASFV by Enhancing NLRP3-Mediated Inflammatory Responses.

African swine fever (ASF) is a highly contagious infectious disease of domestic pigs and wild boars caused by African swine fever virus (ASFV), with a mortality rate of up to 100%. In order to replicate efficiently in macrophages and monocytes, ASFV has evolved multiple strategies to evade host antiviral responses. However, the underlying molecular mechanisms by which ASFV-encoded proteins execute immune evasion are not fully understood. In this study, we found that ASFV pH240R strongly inhibits transcription, maturation, and secretion of interleukin-1ß (IL-1ß). Importantly, pH240R not only targeted NF-κB signaling but also impaired NLRP3 inflammasome activation. In this mechanism, pH240R interacted with NF-kappa-B essential modulator (NEMO), a component of inhibitor of kappa B kinase (IKK) complex and subsequently reduced phosphorylation of IκBα and p65. In addition, pH240R bonded to NLRP3 to inhibit NLRP3 inflammasome activation, resulting in reduced IL-1ß production. As expected, infection with H240R-deficient ASFV (ASFV-ΔH240R) induced more inflammatory cytokine expression both in vitro and in vivo than its parental ASFV HLJ/18 strain. Consistently, H240R deficiency reduced the viral pathogenicity in pigs compared with its parental strain. These findings reveal that the H240R gene is an essential virulence factor, and deletion of the H240R gene affects the pathogenicity of ASFV HLJ/18 by enhancing antiviral inflammatory responses, which provides insights for ASFV immune evasion mechanisms and development of attenuated live vaccines and drugs for prevention and control of ASF. IMPORTANCE African swine fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious and acute hemorrhagic viral disease of domestic pigs, with a high mortality approaching 100%. ASFV has spread rapidly worldwide and caused huge economic losses and ecological consequences. However, the pathogenesis and immune evasion mechanisms of ASFV are not fully understood, which limits the development of safe and effective ASF attenuated live vaccines. Therefore, investigations are urgently needed to identify virulence factors that are responsible for escaping the host antiviral innate immune responses and provide a new target for development of ASFV live-attenuated vaccine. In this study, we determined that the H240R gene is an essential virulence factor, and its depletion affects the pathogenicity of ASFV by enhancing NLRP3-mediated inflammatory responses, which provides theoretical support for the development of an ASFV attenuated live vaccine.

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.

m(6)A methyltransferase METTL3 relieves cognitive impairment of hyperuricemia mice via inactivating MyD88/NF-κB pathway mediated NLRP3-ASC-Caspase1 inflammasome.

BACKGROUND: Recent studies have uncovered that hyperuricemia (HUA) leads to cognitive deficits, which are accompanied by neuronal damage and neuroinflammation. Here, we aim to explore the role of methyltransferase-like 3 (METTL3) in HUA-mediated neuronal apoptosis and microglial inflammation. METHODS: A HUA mouse model was constructed. The spatial memory ability of the mice was assessed by the Morris water maze experiment (MWM), and neuronal apoptosis was analyzed by the TdT-mediated dUTP nick end labeling (TUNEL) assay. Besides, enzyme-linked immunosorbent assay (ELISA) was utilized to measure the contents of inflammatory factors (IL-1ß, IL-6, and TNF-α) and oxidative stress markers (MDA, SOD, and CAT) in the serum of mice. In vitro, the mouse hippocampal neuron (HT22) and microglia (BV2) were treated with uric acid (UA). Flow cytometry was applied to analyze HT22 and BV2 cell apoptosis, and ELISA was conducted to observe neuroinflammation and oxidative stress. In addition, the expression of MyD88, p-NF-κB, NF-κB, NLRP3, ASC and Caspase1 was determined by Western blot. RESULTS: METTL3 and miR-124-3p were down-regulated, while the MyD88-NF-κB pathway was activated in the HUA mouse model. UA treatment induced neuronal apoptosis in HT22 and stimulated microglial activation in BV2. Overexpressing METTL3 alleviated HT22 neuronal apoptosis and resisted the release of inflammatory cytokines and oxidative stress mediators in BV2 cells. METTL3 repressed MyD88-NF-κB and NLRP3-ASC-Caspase1 inflammasome. In addition, METTL3 overexpression enhanced miR-124-3p expression, while METTL3 knockdown aggravated HT22 cell apoptosis and BV2 cell overactivation. CONCLUSION: METTL3 improves neuronal apoptosis and microglial activation in the HUA model by choking the MyD88/NF-κB pathway and up-regulating miR-124-3p.

CD11c+ myeloid cell exosomes reduce intestinal inflammation during colitis.

Intercellular communication is critical for homeostasis in mammalian systems, including the gastrointestinal (GI) tract. Exosomes are nanoscale lipid extracellular vesicles that mediate communication between many cell types. Notably, the roles of immune cell exosomes in regulating GI homeostasis and inflammation are largely uncharacterized. By generating mouse strains deficient in cell-specific exosome production, we demonstrate deletion of the small GTPase Rab27A in CD11c+ cells exacerbated murine colitis, which was reversible through administration of DC-derived exosomes. Profiling RNAs within colon exosomes revealed a distinct subset of miRNAs carried by colon- and DC-derived exosomes. Among antiinflammatory exosomal miRNAs, miR-146a was transferred from gut immune cells to myeloid and T cells through a Rab27-dependent mechanism, targeting Traf6, IRAK-1, and NLRP3 in macrophages. Further, we have identified a potentially novel mode of exosome-mediated DC and macrophage crosstalk that is capable of skewing gut macrophages toward an antiinflammatory phenotype. Assessing clinical samples, RAB27A, select miRNAs, and RNA-binding proteins that load exosomal miRNAs were dysregulated in ulcerative colitis patient samples, consistent with our preclinical mouse model findings. Together, our work reveals an exosome-mediated regulatory mechanism underlying gut inflammation and paves the way for potential use of miRNA-containing exosomes as a novel therapeutic for inflammatory bowel disease.

miR-99a-5p: A Potential New Therapy for Atherosclerosis by Targeting mTOR and Then Inhibiting NLRP3 Inflammasome Activation and Promoting Macrophage Autophagy.

Objective: MicroRNAs have been revealed to be involved in the development of atherosclerosis. The present study is aimed at exploring the potential of miR-99a-5p as a therapy for atherosclerosis. We suspected that miR-99a-5p might inhibit NLRP3 inflammasome activation and promote macrophage autophagy via constraining mTOR, therefore, alleviating atherosclerosis. Methods: The cell viability in ox-LDL-induced THP-1 macrophages was assessed by CCK-8 assay. Bioinformatic analysis was used to predict the target genes of miR-99a-5p. The binding between miR-99a-5p and mTOR was confirmed by luciferase reporter assay. In vivo, a high-fat-diet-induced atherosclerosis model was established in apolipoprotein E knockout mice. Hematoxylin-eosin, oil red O, and Sirius red staining were performed for the determination of atherosclerotic lesions. MTOR and associated protein levels were detected by Western blot analysis. Results: miR-99a-5p inhibited NLRP3 inflammasome activation and promoted macrophage autophagy by targeting mTOR. Enforced miR-99a-5p significantly reduced the levels of inflammasome complex and inflammatory cytokines. Furthermore, miR-99a-5p overexpression inhibited the expression of mTOR, whereas mTOR overexpression reversed the trend of the above behaviors. In vivo, the specific overexpression of miR-99a-5p significantly reduced atherosclerotic lesions, accompanied by a significant downregulation of autophagy marker CD68 protein expression. Conclusion: We demonstrated for the first time that miR-99a-5p may be considered a therapy for atherosclerosis. The present study has revealed that miR-99a-5p might inhibit NLRP3 inflammasome activation and promote macrophage autophagy by targeting mTOR, therefore, alleviating atherosclerosis.

Cell-specific Deletion of NLRP3 Inflammasome Identifies Myeloid Cells as Key Drivers of Liver Inflammation and Fibrosis in Murine Steatohepatitis.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease worldwide. The NLRP3 inflammasome, a platform for caspase-1 activation and release of interleukin 1ß, is increasingly recognized in the induction of inflammation and liver fibrosis during NAFLD. However, the cell-specific contribution of NLRP3 inflammasome activation in NAFLD remains unknown. METHODS: To investigate the role of NLRP3 inflammasome activation in hepatocytes, hepatic stellate cells (HSCs) and myeloid cells, a conditional Nlrp3 knock-out mouse was generated and bred to cell-specific Cre mice. Both acute and chronic liver injury models were used: lipopolysaccharide/adenosine-triphosphate to induce in vivo NLRP3 activation, choline-deficient, L-amino acid-defined high-fat diet, and Western-type diet to induce fibrotic nonalcoholic steatohepatitis (NASH). In vitro co-culture studies were performed to dissect the crosstalk between myeloid cells and HSCs. RESULTS: Myeloid-specific deletion of Nlrp3 blunted the systemic and hepatic increase in interleukin 1ß induced by lipopolysaccharide/adenosine-triphosphate injection. In the choline-deficient, L-amino acid-defined high-fat diet model of fibrotic NASH, myeloid-specific Nlrp3 knock-out but not hepatocyte- or HSC-specific knock-out mice showed significant reduction in inflammation independent of steatosis development. Moreover, myeloid-specific Nlrp3 knock-out mice showed ameliorated liver fibrosis and decreased HSC activation. These results were validated in the Western-type diet model. In vitro co-cultured studies with human cell lines demonstrated that HSC can be activated by inflammasome stimulation in monocytes, and this effect was significantly reduced if NLRP3 was downregulated in monocytes. CONCLUSIONS: The study provides new insights in the cell-specific role of NLRP3 in liver inflammation and fibrosis. NLRP3 inflammasome activation in myeloid cells was identified as crucial for the progression of NAFLD to fibrotic NASH. These results may have implications for the development of cell-specific strategies for modulation of NLRP3 activation for treatment of fibrotic NASH.

Adipose­derived mesenchymal stem cell­derived HCAR1 regulates immune response in the attenuation of sepsis.

Sepsis serves as a leading cause of admission to and death of patients in the intensive care unit (ICU) and is described as a systemic inflammatory response syndrome caused by abnormal host response to infection. Adipose­derived mesenchymal stem cells (ADSCs) have exhibited reliable and promising clinical application potential in multiple disorders. However, the function and the mechanism of ADSCs in sepsis remain elusive. In the present study, the crucial inhibitory effect of ADSC­derived hydroxy­carboxylic acid receptor 1 (HCAR1) on sepsis was identified. Reverse transcription quantitative­PCR determined that the mRNA expression of HCAR1 was reduced while the mRNA expression of Toll­like receptor 4 (TLR4), major histocompatibility complex class II (MHC II), NOD­like receptor family pyrin domain containing 3 (NLRP3), and the levels of interleukin­1ß (IL­1ß), tumor necrosis factor­α (TNF­α), interleukin­10 (IL­10), and interleukin­18 (IL­18) were enhanced in the peripheral blood of patients with sepsis. The expression of HCAR1 was negatively correlated with TLR4 (r=­0.666), MHC II (r=­0.587), and NLRP3 (r=­0.621) expression and the expression of TLR4 was positively correlated with NLRP3 (r=0.641), IL­1ß (r=0.666), TNF­α (r=0.606), and IL­18 (r=0.624) levels in the samples. Receiver operating characteristic (ROC) curve analysis revealed that the area under the ROC curve (AUC) of HCAR1, TLR4, MHC II and NLRP3 mRNA expression was 0.830, 0.853, 0.735 and 0.945, respectively, in which NLRP3 exhibited the highest diagnostic value, and the AUC values of IL­1ß, IL­18, TNF­α, and IL­10 were 0.751, 0.841, 0.924 and 0.729, respectively, in which TNF­α exhibited the highest diagnostic value. A sepsis rat model was established by injecting lipopolysaccharide (LPS) and the rats were randomly divided into 5 groups, including a normal control group (NC group; n=6), a sepsis model group (LPS group; n=6), an ADSC transplantation group (L + M group; n=6), a combined HCAR1 receptor agonist group [L + HCAR1 inducer (Gi) + M group; n=6], and a combined HCAR1 receptor inhibitor group [L + HCAR1 blocker (Gk) + M group; n=6]. Hematoxylin and eosin staining determined that ADSCs attenuated the lung injury of septic rats and ADSC­derived HCAR1 enhanced the effect of ADSCs. The expression of HCAR1, TLR4, MHC II, NLRP3, IL­1ß, IL­18 and TNF­α levels were suppressed by ADSCs and the effect was further induced by ADSC­derived HCAR1. However, ADSC­derived HCAR1 induced the levels of anti­inflammatory factor IL­10. The negative correlation of HCAR1 expression with TLR4, MHC II, and NLRP3 expression in the peripheral blood and lung tissues of the rats was then identified. It is thus concluded that ADSC­derived HCAR1 regulates immune response in the attenuation of sepsis. ADSC­derived HCAR1 may be a promising therapeutic strategy for sepsis.

PLCL1 regulates fibroblast-like synoviocytes inflammation via NLRP3 inflammasomes in rheumatoid arthritis.

BACKGROUND: Phospholipase C-like 1 (PLCL1), a protein that lacks catalytic activity, has similar structures to the PLC family. The aim of this research was to find the function and underlying mechanisms of PLCL1 in fibroblast-like synoviocyte (FLS) of rheumatoid arthritis (RA). METHODS: In this study, we first analyzed the expression of PLCL1 in the synovial tissue of RA patients and K/BxN mice by immunohistochemical staining. Then silencing or overexpressing PLCL1 in FLS before stimulating by TNF-α. The levels of IL-6, IL-1ß and CXCL8 in FLS and supernatants were detected by Western Blot (WB), Real-Time Quantitative PCR and Enzyme Linked Immunosorbent Assay. We used INF39 to specifically inhibit the activation of NLRP3 inflammasomes, and detected the expression of NLRP3, Cleaved Caspase-1, IL-6 and IL-1ß in FLS by WB. RESULT: When PLCL1 was silenced, the level of IL-6, IL-1ß and CXCL8 were down-regulated. When PLCL1 was overexpressed, the level of IL-6, IL-1ß and CXCL8 were unregulated. The previous results demonstrated that the mechanism of PLCL1 regulating inflammation in FLS was related to NLRP3 inflammasomes. INF39 could counteract the release of inflammatory cytokines caused by overexpression of PLCL1. CONCLUSION: Result showed that the function of PLCL1 in RA FLS might be related to the NLRP3 inflammasomes. We finally confirmed our hypothesis with the NLRP3 inhibitor INF39. Our results suggested that PLCL1 might promote the inflammatory response of RA FLS by regulating the NLRP3 inflammasomes.

The Role of NLRP3 Inflammasome Activation Pathway of Hepatic Macrophages in Liver Ischemia-Reperfusion Injury.

Ischemia-reperfusion injury (IRI) is considered an inherent component involved in liver transplantation, which induce early organ dysfunction and failure. And the accumulating evidences indicate that the activation of host innate immune system, especially hepatic macrophages, play a pivotal role in the progression of LIRI. Inflammasomes is a kind of intracellular multimolecular complexes that actively participate in the innate immune responses and proinflammatory signaling pathways. Among them, NLRP3 inflammasome is the best characterized and correspond to regulate caspase-1 activation and the secretion of proinflammatory cytokines in response to various pathogen-derived as well as danger-associated signals. Additionally, NLRP3 is highly expressed in hepatic macrophages, and the assembly of NLRP3 inflammasome could lead to LIRI, which makes it a promising therapeutic target. However, detailed mechanisms about NLRP3 inflammasome involving in the hepatic macrophages-related LIRI is rarely summarized. Here, we review the potential role of the NLRP3 inflammasome pathway of hepatic macrophages in LIRI, with highlights on currently available therapeutic options.