Mechanistic insights into the C-type lectin receptor CLEC12A-mediated immune recognition of monosodium urate crystal.

CLEC12A, a member of the C-type lectin receptor family involved in immune homeostasis, recognizes MSU crystals released from dying cells. However, the molecular mechanism underlying the CLEC12A-mediated recognition of MSU crystals remains unclear. Herein, we reported the crystal structure of the human CLEC12A-C-type lectin-like domain (CTLD) and identified a unique "basic patch" site on CLEC12A-CTLD that is necessary for the binding of MSU crystals. Meanwhile, we determined the interaction strength between CLEC12A-CTLD and MSU crystals using single-molecule force spectroscopy. Furthermore, we found that CLEC12A clusters at the cell membrane and seems to serve as an internalizing receptor of MSU crystals. Altogether, these findings provide mechanistic insights for understanding the molecular mechanisms underlying the interplay between CLEC12A and MSU crystals.

Crystal structure of the complex of CLEC12A and an antibody that interferes with binding of diverse ligands.

C-type lectin receptors (CLRs) are a family of pattern recognition receptors, which detect a broad spectrum of ligands via small carbohydrate-recognition domains (CRDs). CLEC12A is an inhibitory CLR that recognizes crystalline structures such as monosodium urate crystals. CLEC12A also recognizes mycolic acid, a major component of mycobacterial cell walls, and suppresses host immune responses. Although CLEC12A could be a therapeutic target for mycobacterial infection, structural information on CLEC12A was not available. We report here the crystal structures of human CLEC12A (hCLEC12A) in ligand-free form and in complex with 50C1, its inhibitory antibody. 50C1 recognizes human-specific residues on the top face of hCLEC12A CRD. A comprehensive alanine scan demonstrated that the ligand-binding sites of mycolic acid and monosodium urate crystals may overlap with each other, suggesting that CLEC12A utilizes a common interface to recognize different types of ligands. Our results provide atomic insights into the blocking and ligand-recognition mechanisms of CLEC12A and leads to the design of CLR-specific inhibitors.

Associations between serum urate and telomere length and inflammation markers: Evidence from UK Biobank cohort.

Objective: Hyperuricemia and gout have become gradually more common. The effect of serum urate on organism aging and systematic inflammation is not determined. This study aims to evaluate whether serum urate is causally associated with cellular aging markers and serum inflammation markers. Methods: A Mendelian randomization study was performed on summary-level data from the largest published genome-wide association studies. Single nucleotide polymorphisms with a genome-wide significance level were selected as instrumental variables for leukocyte telomere length (LTL), and serum soluble makers of inflammation (CRP, IL-6, TNF-α, and IGF-1). Standard inverse variance weighted (IVW) method was used as the primary statistical method. The weighted median, MR-Egger regression, and MR-PRESSO methods were used for sensitivity analysis. Results: An inverse causal association of genetically predicted serum urate levels and LTL was found using IVW method (OR: 0.96, 95%CI 0.95, 0.97; ß=-0.040; SE=0.0072; P=4.37×10-8). The association was also supported by MR results using MR-Egger method and weighted median method. The MR-PRESSO analysis and leave-one-out sensitivity analysis supported the robustness of the combined results. In terms of other aging-related serum biomarkers, there was no evidence supporting a causal effect of serum urate on CRP, IL-6, TNF-α, or IGF-1 levels. Conclusions: Serum urate levels are negatively associated with telomere length but are not associated with serum soluble indicators of inflammation. Telomere length may be a critical marker that reflects urate-related organismal aging and may be a mechanism in the age-related pathologies and mortality caused by hyperuricemia.

The E3 Ubiquitin Ligase TRIM65 Negatively Regulates Inflammasome Activation Through Promoting Ubiquitination of NLRP3.

The dysregulation of NLRP3 inflammasome plays a critical role in pathogenesis of various human inflammatory diseases, thus NLRP3 inflammasome activation must be tightly controlled at multiple levels. However, the underlying mechanism regulating NLRP3 inflammasome activation remains unclear. Herein, the effects of Tripartite motif-containing protein 65 (TRIM65) on NLRP3 inflammasome activation and the underlying molecular mechanism were investigated in vitro and in vivo. Inhibition or deletion of Trim65 could significantly strengthen agonist induced NLRP3 inflammasome activation in THP-1 cells and BMDMs, indicated by increased caspase-1 activation and interleukin-1ß secretion. However, TRIM65 had no effect on poly (dA: dT)-induced AIM2 inflammasome activation or flagellin-induced IPAF inflammasome activation. Mechanistically, immunoprecipitation assays demonstrated that TRIM65 binds to NACHT domain of NLRP3, promotes lys48- and lys63- linked ubiquitination of NLRP3 and restrains the NEK7-NLRP3 interaction, thereby inhibiting NLRP3 inflammasome assembly, caspase-1 activation, and IL-1ß secretion. In vivo, three models of inflammatory diseases were used to confirm the suppression role of TRIM65 in NLRP3 inflammasome activation. TRIM65-deficient mice had a higher production of IL-1ß induced by lipopolysaccharide in sera, and more IL-1ß secretion and neutrophil migration in the ascites, and more severity of joint swelling and associated IL-1ß production induced by monosodium urate, suggesting that TRIM65 deficiency was susceptible to inflammation. Therefore, the data elucidate a TRIM65-dependent negative regulation mechanism of NLRP3 inflammasome activation and provide potential therapeutic strategies for the treatment of NLRP3 inflammasome-related diseases.

CD300a contributes to the resolution of articular inflammation triggered by MSU crystals by controlling neutrophil apoptosis.

Gout is an inflammatory disease triggered by deposition of monosodium urate (MSU) crystals in the joints, resulting in high neutrophil influx and pain. Here, we studied the role of the inhibitory receptor CD300a in the resolution process in a murine model of gout. We found increased CD300a expression on neutrophils emigrated to the joint. When compared to WT mice, CD300a-/- mice had persistent neutrophil influx till 24 hr after MSU injection. This was associated with increased concentration of IL-1ß and greater tissue damage in the joints of CD300a-/- mice. There was an increase in the percentage of apoptotic neutrophils in the synovial lavage of WT mice, as compared to CD300a-/- mice. This difference was reflected in the decline of efferocytic events in the synovial cavity of CD300a-/- mice 24 hr after MSU injection. A CD300a agonistic antibody was shown, for the first time, to increase apoptosis of human neutrophils, and this was associated with cleavage of caspase-8. In conclusion, our results reveal an important role of CD300a in the control of leucocyte infiltration, IL-1ß production and caspase-8 cleavage in neutrophils, contributing to the resolution of inflammation triggered by MSU injection.

The Anti-Inflammatory and Uric Acid Lowering Effects of Si-Miao-San on Gout.

Background: Si-Miao-San (SMS) is a well-known traditional Chinese medicine. This study aims to evaluate the anti-inflammatory effects of SMS on gouty arthritis and its potential mechanism of action. Methods: The effects and mechanism of SMS were evaluated in monosodium urate (MSU)-treated mice or macrophages. The expression of cytokines and PI3K/Akt was analyzed using real-time PCR and Western blotting analyses. Macrophage polarization was assessed with immunofluorescence assays, real-time PCR, and Western blotting. Mass spectrometry was used to screen the active ingredients of SMS. Results: Pretreatment with SMS ameliorated MSU-induced acute gouty arthritis in mice with increased PI3K/Akt activation and M2 macrophage polarization in the joint tissues. In vitro, SMS treatment significantly inhibited MSU-triggered inflammatory response, increased p-Akt and Arg-1 expression in macrophages, and promoted M2 macrophage polarization. These effects of SMS were inhibited when PI3K/Akt activation was blocked by LY294002 in the macrophages. Moreover, SMS significantly reduced serum uric acid levels in the hyperuricemia mice. Using mass spectrometry, the plant hormones ecdysone and estrone were detected as the potentially effective ingredients of SMS. Conclusion: SMS ameliorated MSU-induced gouty arthritis and inhibited hyperuricemia. The anti-inflammatory mechanism of SMS may exert anti-inflammatory effects by promoting M2 polarization via PI3K/Akt signaling. Ecdysone and estrone might be the potentially effective ingredients of SMS. This research may provide evidence for the application of SMS in the treatment of gout.

Screening of Fv Antibodies with Specific Binding Activities to Monosodium Urate and Calcium Pyrophosphate Dihydrate Crystals for the Diagnosis of Gout and Pseudogout.

To date, medical diagnosis of gout and pseudogout has been performed by observing the crystals in the joint fluid of patients under a polarized microscope. Conventional diagnostic methods using a polarized microscope have disadvantages, such as time-consuming analysis, a high false negative rate, and difficulty in distinguishing gout with monosodium urate (MSU) crystals and pseudogout with calcium pyrophosphate dihydrate (CPPD) crystals in synovial fluids. In this study, a chromogenic assay for the diagnosis of gout and pseudogout, without the requirement of a polarized microscope and trained experts, was proposed using Fv antibodies with specific binding activities to MSU and CPPD crystals. The IgG VH chain Fv library with randomized complementarity-determining region 3 (CDR3) region was expressed on the outer membrane of Escherichia coli using autodisplay technology. The target Fv antibodies with binding activity to MSU and CPPD crystals were screened from the autodisplayed Fv library on the E. coli outer membrane, and five clones were selected. On the basis of the binding properties of the screened Fv antibodies, peptides with the selected clone of amino acid sequences of the CDR3 region (15 residues) were chemically synthesized. The binding properties of the synthetic peptides with amino acid sequences of CDR3 regions from the selected clones were analyzed using fluorescence imaging and flow cytometry, and the affinity constants (Kd) of each peptide for binding to MSU and CPPD crystals were calculated by fitting based on the isotherm model. A chromogenic assay configuration for gout and pseudogout was developed using synthetic peptides. In this chromogenic assay, synthetic peptides labeled with biotin and streptavidin-horseradish peroxidase (HRP) complex were used, and crystal detection was possible using a chromogenic reaction between HRP and a chromogenic substrate (TMB). Finally, gout and pseudogout were diagnosed by detecting MSU and CPPD crystals in the synovial fluid in the concentration range of 0-300 µg/mL.

Uric acid drives intestinal barrier dysfunction through TSPO-mediated NLRP3 inflammasome activation.

BACKGROUND AND AIM: Intestinal epithelial dysfunction is the foundation of various intestinal and extra-intestinal diseases, while the effects and mechanism of uric acid on the intestinal barrier are little known. TSPO has been shown to be related to the generation of ROS and is involved in regulating inflammation, whether uric acid drives intestinal epithelial dysfunction through TSPO-mediated NLRP3 inflammasome activation is unknown. METHODS: UOX gene knockout mouse (UOX-/-) were used for models of hyperuricemia. Fluorescein isothiocyanate (FITC)-labeled dextran was used to assess in vivo intestinal permeability. Serum lipopolysaccharide (LPS) and culture supernatants IL-1ß were measured using ELISA Kit. IEC-6 exposed to different concentrations of uric acid was used for in vitro experiment. Protein content and mRNA were assessed using Western blotting and Q-PCR, respectively. Intracellular ROS was determined using flow cytometry and fluorescence microscope. Mitochondrial membrane potential was detected on an immunofluorescence. Small interfering RNA transfection was used to assess the interaction between translocator protein (TSPO) and NLRP3 inflammasome. N-acetyl-L-cysteine (NAC) was used as ROS scavenger. RESULTS: Our results showed that hyperuricemia mice were characteristic by increased intestinal permeability. Hyperuricemia upregulated TSPO, increased production of ROS and activated NLRP3 inflammasome, which resulted in lower expression of occludin and claudin-1. In vitro, we showed that soluble uric acid alone increased the expression of TSPO, depolarized mitochondrial membrane potential, increased ROS release and activated NLRP3 inflammasome, which further reduced the expression of occludin and claudin-1. Silencing TSPO suppressed NLRP3 inflammasome activation and increased expression of claudin-1 and occludin, which was accompanied by lower levels of ROS. Scavenging ROS also significantly inhibited NLRP3 inflammasome activation without change of TSPO, indicating that TSPO-mediated NLRP3 inflammasome activation was dependent on ROS. CONCLUSIONS: In conclusion, uric acid drives intestinal barrier dysfunction through TSPO-mediated NLRP3 inflammasome.

Gout and pseudo-gout-related crystals promote GLUT1-mediated glycolysis that governs NLRP3 and interleukin-1ß activation on macrophages.

OBJECTIVE: Macrophage activation by monosodium urate (MSU) and calcium pyrophosphate (CPP) crystals mediates an interleukin (IL)-1ß-dependent inflammation during gout and pseudo-gout flare, respectively. Since metabolic reprogramming of macrophages goes along with inflammatory responses dependently on stimuli and tissue environment, we aimed to decipher the role of glycolysis and oxidative phosphorylation in the IL-1ß-induced microcrystal response. METHODS: Briefly, an in vitro study (metabolomics and real-time extracellular flux analysis) on MSU and CPP crystal-stimulated macrophages was performed to demonstrate the metabolic phenotype of macrophages. Then, the role of aerobic glycolysis in IL-1ß production was evaluated, as well in vitro as in vivo using 18F-fluorodeoxyglucose positron emission tomography imaging and glucose uptake assay, and molecular approach of glucose transporter 1 (GLUT1) inhibition. RESULTS: We observed that MSU and CPP crystals led to a metabolic rewiring toward the aerobic glycolysis pathway explained by an increase in GLUT1 plasma membrane expression and glucose uptake on macrophages. Also, neutrophils isolated from human synovial fluid during gout flare expressed GLUT1 at their plasma membrane more frequently than neutrophils isolated from bloodstream. Both glucose deprivation and treatment with either 2-deoxyglucose or GLUT1 inhibitor suppressed crystal-induced NLRP3 activation and IL-1ß production, and microcrystal inflammation in vivo. CONCLUSION: In conclusion, we demonstrated that GLUT1-mediated glucose uptake is instrumental during the inflammatory IL-1ß response induced by MSU and CPP crystals. These findings open new therapeutic paths to modulate crystal-related inflammation.

CD44 Receptor Mediates Urate Crystal Phagocytosis by Macrophages and Regulates Inflammation in A Murine Peritoneal Model of Acute Gout.

Gout is a chronic arthritis caused by the deposition of poorly soluble monosodium urate monohydrate (MSU) crystals in peripheral joints. Resident macrophages initiate inflammation in response to MSU mediated by NF-κB nuclear translocation and NLRP3 inflammasome activation. We investigated the role of CD44, a transmembrane receptor, in mediating MSU phagocytosis by macrophages. We used an antibody that sheds the extracellular domain (ECD) of CD44 to study the role of the receptor and its associated protein phosphatase 2A (PP2A) in macrophage activation. We also studied the significance of CD44 in mediating MSU inflammation in-vivo. Cd44-/- BMDMs showed reduced MSU phagocytosis, LDH release, IL-1ß expression and production compared to Cd44+/+ BMDMs. Elevated CD44 staining was detected intracellularly and CD44 colocalized with α-tubulin as a result of MSU exposure and ECD-shedding reduced MSU phagocytosis in murine and human macrophages. Anti-CD44 antibody treatment reduced NF-κB p65 subunit nuclear levels, IL-1ß expression, pro-IL-1ß and IL-8 production in MSU stimulated THP-1 macrophages (p < 0.01). The effect of the antibody was mediated by an enhancement in PP2A activity. CD44 ECD-shedding reduced the conversion of procaspase-1 to active caspase-1, caspase-1 activity and resultant generation of mature IL-1ß in macrophages. Neutrophil and monocyte influx and upregulated production of IL-1ß was evident in wildtype mice. MSU failed to trigger neutrophil and monocyte recruitment in Cd44-/- mice and lower IL-1ß levels were detected in peritoneal lavages from Cd44-/- mice (p < 0.01). Anti-CD44 antibody treatment reduced neutrophil and monocyte recruitment and resulted in reduced lavage IL-1ß levels in the same model. CD44 plays a biologically significant role in mediating phagocytosis of MSU and downstream inflammation and is a novel target in gout treatment.

Rare genetic variants in interleukin-37 link this anti-inflammatory cytokine to the pathogenesis and treatment of gout.

OBJECTIVE: Gout is characterised by severe interleukin (IL)-1-mediated joint inflammation induced by monosodium urate crystals. Since IL-37 is a pivotal anti-inflammatory cytokine suppressing the activity of IL-1, we conducted genetic and functional studies aimed at elucidating the role of IL-37 in the pathogenesis and treatment of gout. METHODS: Variant identification was performed by DNA sequencing of all coding bases of IL37 using molecular inversion probe-based resequencing (discovery cohort: gout n=675, controls n=520) and TaqMan genotyping (validation cohort: gout n=2202, controls n=2295). Predictive modelling of the effects of rare variants on protein structure was followed by in vitro experiments evaluating the impact on protein function. Treatment with recombinant IL-37 was evaluated in vitro and in vivo in a mouse model of gout. RESULTS: We identified four rare variants in IL37 in six of the discovery gout patients; p.(A144P), p.(G174Dfs*16), p.(C181*) and p.(N182S), whereas none emerged in healthy controls (Fisher's exact p-value=0.043). All variants clustered in the functional domain of IL-37 in exon 5 (p-value=5.71×10-5). Predictive modelling and functional studies confirmed loss of anti-inflammatory functions and we substantiated the therapeutic potential of recombinant IL-37 in the treatment of gouty inflammation. Furthermore, the carrier status of p.(N182S)(rs752113534) was associated with increased risk (OR=1.81, p-value=0.031) of developing gout in hyperuricaemic individuals of Polynesian ancestry. CONCLUSION: Here, we provide genetic as well as mechanistic evidence for the role of IL-37 in the pathogenesis of gout, and highlight the therapeutic potential of recombinant IL-37 for the treatment of gouty arthritis.

The uric acid crystal receptor Clec12A potentiates type I interferon responses.

The detection of microbes and damaged host cells by the innate immune system is essential for host defense against infection and tissue homeostasis. However, how distinct positive and negative regulatory signals from immune receptors are integrated to tailor specific responses in complex scenarios remains largely undefined. Clec12A is a myeloid cell-expressed inhibitory C-type lectin receptor that can sense cell death under sterile conditions. Clec12A detects uric acid crystals and limits proinflammatory pathways by counteracting the cell-activating spleen tyrosine kinase (Syk). Here, we surprisingly find that Clec12A additionally amplifies type I IFN (IFN-I) responses in vivo and in vitro. Using retinoic acid-inducible gene I (RIG-I) signaling as a model, we demonstrate that monosodium urate (MSU) crystal sensing by Clec12A enhances cytosolic RNA-induced IFN-I production and the subsequent induction of IFN-I-stimulated genes. Mechanistically, Clec12A engages Src kinase to positively regulate the TBK1-IRF3 signaling module. Consistently, Clec12A-deficient mice exhibit reduced IFN-I responses upon lymphocytic choriomeningitis virus (LCMV) infection, which affects the outcomes of these animals in acute and chronic virus infection models. Thus, our results uncover a previously unrecognized connection between an MSU crystal-sensing receptor and the IFN-I response, and they illustrate how the sensing of extracellular damage-associated molecular patterns (DAMPs) can shape the immune response.

Anti-inflammatory effect of artemisinin on uric acid-induced NLRP3 inflammasome activation through blocking interaction between NLRP3 and NEK7.

OBJECTIVE: Artemisinin is a potent anti-malarial agent that plays a potent role in regulating inflammatory disorders. NEK7 is a major interacting partner with NLRP3 in NLRP3 inflammasome. The aim of this study was to clarify the anti-inflammatory effect of artemisinin on activation of uric acid-induced NLRP3 inflammasome through regulation of NEK7. METHODS: Human macrophage U937 cells treated with lipopolysaccharide (LPS), monosodium urate (MSU) crystals, or artemisinin were used in in vitro study. Intracellular potassium (K+) level was measured in U937 cells treated with and without artemisinin. Expression of target genes or proteins NEK7, NLRP3, ASC, caspase-1, interleukin-1ß (IL-1ß), and NF-κB signaling molecules was measured. MSU crystal-induced arthritis model was used for in vivo study. RESULTS: Gout patients showed higher NLRP3 and NEK7 mRNA expression, compared to controls. Enhanced expression of NLRP3, caspase-1, and IL-1ß was noted in macrophages treated with LPS (10 ng/ml) and MSU crystals (0.1 mg/ml), which was markedly suppressed by treatment with artemisinin (1, 10, and 100 µM). Artemisinin significantly inhibited interaction between NLRP3 and NEK7 in NLRP3 inflammasome activation. Artemisinin (10 and 100 µM) attenuated intracellular K+ efflux in macrophages stimulated with LPS and MSU crystals. Artemisinin suppressed foot and ankle swelling in MSU crystal-induced arthritis mice. CONCLUSION: This study revealed that artemisinin inhibited activation of NLRP3 inflammasome by suppressing interaction between NEK7 and NLRP3 in uric acid-induced inflammation.

TXNIP-mediated nuclear factor-κB signaling pathway and intracellular shifting of TXNIP in uric acid-induced NLRP3 inflammasome.

OBJECTIVE: The aim of this study was to assess the role of thioredoxin-interacting protein (TXNIP) in nuclear factor-κB (NF-κB) signaling and the interaction between TXNIP and NOD-like receptor protein 3 (NLRP3) in activation of the NLRP3 inflammasome in monosodium urate (MSU)-induced inflammation. METHODS: Interleukin-1ß (IL-1ß), IL-18, caspase-1, phospho-IκBα (pIκBα), phospho-NF-κB, (pNF-κB), and TXNIP in U937 macrophage-like cells treated with MSU crystals were analyzed using western blotting and real-time polymerase chain reaction (RT-PCR). Expression of these molecules was also assessed in U937 macrophages transfected with TXNIP siRNA and treated with antioxidants. RESULTS: U937 macrophages treated with MSU crystals showed increased expression of IL-1ß, IL-18, caspase-1, and TXNIP and activation of NF-κB signaling, which were strongly inhibited by addition of antioxidants or transfection with TXNIP siRNA. Intracellular translocation of TXNIP from the nucleus to mitochondria was observed in cells treated with MSU crystals. And quercetin and ascorbic acid suppressed translocation of TXNIP. Binding between TXNIP and NLRP3 under oxidative stress caused by MSU crystals was observed and was blocked by quercetin or ascorbic acid. CONCLUSION: This study showed that activation of MSU-induced NLRP3 inflammasome requires TXNIP-mediated NF-κB signaling pathway and intracellular TXNIP shifting.

The Long Pentraxin 3 Contributes to Joint Inflammation in Gout by Facilitating the Phagocytosis of Monosodium Urate Crystals.

The purpose of this study was to investigate the role of pentraxin 3 (PTX3), a pivotal component of the innate immune system, in gout. Levels of PTX3 and IL-1ß in human samples were evaluated by ELISA. Development of murine gout was evaluated through the levels of cytokines (PTX3, CXCL1, and IL-1ß) and neutrophil recruitment into the joint cavity. Phagocytosis of monosodium urate (MSU) crystals and caspase-1 activation were determined by flow cytometer. Acute gout patients showed elevated concentration of PTX3 in plasma and synovial fluid as compared with healthy and osteoarthritic subjects. Moreover, there was a positive correlation between intra-articular PTX3 and IL-1ß levels. PTX3 was induced in the periarticular tissue of mice postinjection of MSU crystals. Importantly, Ptx3-deficient mice showed reduced inflammation in response to MSU crystal injection compared with wild-type mice, including reduction of neutrophil recruitment into the joint cavity and IL-1ß and CXCL1 production. Interestingly, addition of PTX3 in vitro enhanced MSU crystal phagocytosis by monocytes and resulted in higher production of IL-1ß by macrophages. This contribution of PTX3 to the phagocytosis of MSU crystals and consequent production of IL-1ß occurred through a mechanism mainly dependent on FcγRIII. Thus, our results suggest that PTX3 acts as a humoral pattern recognition molecule in gout facilitating MSU crystal phagocytosis and contributing to the pathogenesis of gouty arthritis.

Exogenous nanoparticles and endogenous crystalline molecules as danger signals for the NLRP3 inflammasomes.

Inflammasome mechanisms are involved as some of the pathways of sterile inflammation. Inflammasomes are large multiprotein complexes in the cytosol and are a key system for the production of the pivotal inflammatory cytokines, interleukin (IL)-1ß and IL-18, and inflammatory cell death called pyroptosis. Although a number of inflammasomes have been described, the nucleotide-binding oligomerization domain-, leucine-rich repeat-, and pyrin domain-containing 3 (NLRP3) is the most extensively investigated inflammasome. Exogenous pathogen-associated molecular patterns released during infection and endogenous crystalline danger/damage-associated molecular patterns (DAMPs) are well-known activators of NLRP3 inflammasomes. In addition, nanoparticle-associated molecular patterns (NAMPs), which are mediated by synthetic materials, including nanomaterials and nanoparticles, are proposed to be new danger signals of NLRP3 inflammasomes. Importantly, NAMP- and DAMP-triggered inflammation, a defining characteristic in inflammatory diseases, is termed as sterile inflammation because it occurs in the absence of foreign pathogens. This review focuses on the role of inflammasomes in exogenous NAMP- and endogenous crystalline DAMP-mediated sterile inflammation. Moreover, many regulatory mechanisms have been identified to attenuate NLRP3 inflammasomes. Therefore, we also summarize endogenous negative regulators of NLRP3 inflammasome activation, particularly induced by NAMPs or crystalline DAMPs.

High-mobility group box 1 is responsible for monosodium urate crystal-induced inflammation in human U937 macrophages.

High-mobility group box 1 (HMGB1) was originally identified as a highly conserved non-histone DNA-binding factor and demonstrated to be a potent mediator in inflammatory diseases. We performed this study to investigate the role of HMGB1 in the pathogenesis of uric acid-induced inflammation in human U937 macrophages. To simulate uric acid-induced inflammation, human U937 macrophages were treated with monosodium urate (MSU) crystals. In addition to determining the effects of MSU crystal treatment on expression of various genes and proteins, cells were transfected with interfering RNA (siRNA) for HMGB1, or caspase-1 and then treated with MSU. Expression of interleukin-1ß (IL-1ß), IL-18, HMGB1, and caspase-1 was detected in human U937 cells and peripheral blood mononuclear cells (PBMCs) in gout patients and healthy controls by western blot analysis or quantitative real-time polymerase chain reaction. Transcript expression of IL-1ß, IL-18, caspase-1, HMGB1 in PBMCs was significantly higher in active gout patients than inactive gout patients and healthy controls. The protein levels of these molecules were significantly increased by stimulation of U937 cells with 0.2 mg/ml MSU crystals. Stimulation of U937 cells with MSU crystals induced translocation of HMGB1 from the nucleus to the cytoplasm and its extracellular release. U937 cells transfected with caspase-1 siRNA had significantly lower HMGB1 expression in the cytoplasm and supernatant than non-transfected cells. Antioxidants, such as N-acetyl-l-cysteine and quercetin, markedly inhibited the nuclear-to-cytoplasmic translocation of HMGB1 and its release into the extracellular milieu. In conclusion, HMGB1, regulated by the enzymatic activity of caspase-1, is a crucial mediator in uric acid-induced inflammation.

Urate crystals induce macrophage PAF­AH secretion which is differentially regulated by TGFß1 and hydrocortisone.

The aim of the present study was to establish the role of platelet­activating factor acetyl hydrolase (PAF­AH) in the resolution phase of gout using an established in vitro mononuclear cell model. The effects of signalling pathway inhibitors on PAF­AH secretion, as well as the effects of the common treatments hydrocortisone and colchicine, an antibody against the anti­inflammatory cytokine transforming growth factor ß1 (TGFß1), and the transcriptional inhibitor actinomycin D, were also investigated. The effect of recombinant PAF­AH on cytokine secretion by these cells was also determined. Human peripheral blood­derived monocytes were isolated and differentiated into macrophages. Monocytes and macrophages were stimulated with monosodium monohydrate urate (MSU) crystals or lipopolysaccharide in the presence or absence of AEG3482 [a c­Jun N­terminal kinase (JNK) inhibitor], MG132 (a proteasome inhibitor), hydrocortisone or colchicine. Cultures were then analysed for PAF­AH secretion using ELISA. A 6­fold upregulation of PAF­AH secretion was observed following macrophage exposure to MSU crystals for 24 h (29.3±6 vs. 5.4±0.3 ng/ml unstimulated; P<0.05). Following 72 h, PAF­AH levels decreased significantly (11.1±1.8; P<0.01). Secretion was further enhanced following pre­treatment with the JNK protein kinase inhibitor AEG3482 prior to MSU crystal stimulation (P<0.05) and was abrogated when cells were preincubated with actinomycin D or the proteasome inhibitor MG132 (50, 100 and 200 µM). The addition of recombinant PAF­AH (2.5­10 ng/ml) to MSU crystal­stimulated immature monocyte cultures significantly decreased pro­inflammatory interleukin (IL)­1ß (unstimulated 687±124 vs. stimulated 113±30 pg/ml) and IL­6 secretion (unstimulated 590±50 vs. stimulated 182±19 pg/ml). Treatment of MSU crystal­stimulated macrophages with hydrocortisone (2 µM) also significantly decreased PAF­AH release (P<0.05). Neutralising anti­TGFß1 addition decreased PAF­AH dose­dependently with the highest inhibition observed at 1 µg/ml (P<0.05). The results implicated that PAF­AH may have an anti­inflammatory role in the resolution phase of gout.

Inflammatory biomarkers and radiologic measurements in never-smokers with COPD: A cross-sectional study from the CODA cohort.

Various biomarkers have emerged as potential surrogates to represent various subgroups of chronic obstructive pulmonary disease (COPD), which manifest with different phenotypes. However, the biomarkers representing never-smokers with COPD have not yet been well elucidated. The aim of this study was to evaluate the associations of certain serum and radiological biomarkers with the presence of COPD in never-smokers. To explore the associations of serum and radiological biomarkers with the presence of COPD in never-smokers, we conducted a cross-sectional patient cohort study composed of never-smokers from the COPD in Dusty Areas (CODA) cohort, consisting of subjects living in dusty areas near cement plants in South Korea. Of the 131 never-smokers in the cohort, 77 (58.8%) had COPD. There were no significant differences in the number of subjects with high levels of inflammatory biomarkers (>90th percentile of never-smokers without COPD), including white blood cell count, total bilirubin, interleukin (IL)-6, IL-8, and C-reactive protein, or radiologic measurements (including emphysema index and mean wall area percentage) between never-smokers with COPD and those without COPD. However, the number of subjects with high uric acid was significantly higher in never-smokers with COPD than never-smokers without COPD (31.2% (24/77) vs. 11.1% (6/54); p = 0.013). In addition, multivariate analysis revealed that high uric acid was significantly associated with the presence of COPD in never-smokers (adjusted relative risk: 1.63; 95% confidence interval: 1.21, 2.18; p = 0.001). Our study suggests that high serum levels of uric acid might be a potential biomarker for assessing the presence of COPD in never-smokers.

Host-microbiota interplay in mediating immune disorders.

To maintain health, the immune system must maintain a delicate balance between eliminating invading pathogens and avoiding immune disorders such as autoimmunity and allergies. The gut microbiota provide essential health benefits to the host, particularly by regulating immune homeostasis. Dysbiosis, an alteration and imbalance of the gut microbiota, is associated with the development of several autoimmune diseases in both mice and humans. In this review, we discuss recent advances in understanding how certain factors, such as age and gender, affect the gut microbiota, which in turn can influence the development of autoimmune diseases. The age factor in microbiota-dependent immune disorders indicates a window of opportunity for future diagnostic and therapeutic approaches. We also discuss unique commensal bacteria with strong immunomodulatory activity. Finally, we provide an overview of the potential molecular mechanisms whereby gut microbiota induce autoimmunity, as well as the evidence that gut microbiota trigger extraintestinal diseases by inducing the migration of gut-derived immune cells. Elucidating the interaction of gut microbiota and the host immune system will help us understand the pathogenesis of immune disorders, and provide us with new foundations to develop novel immuno- or microbe-targeted therapies.