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.

Monosodium Urate Crystal-Induced Pyroptotic Cell Death in Neutrophil and Macrophage Facilitates the Pathological Progress of Gout.

Monosodium urate (MSU) crystal deposition in joints can lead to the infiltration of neutrophils and macrophages, and their activation plays a critical role in the pathological progress of gout. However, the role of MSU crystal physicochemical properties in inducing cell death in neutrophil and macrophage is still unclear. In this study, MSU crystals of different sizes are synthesized to explore the role of pyroptosis in gout. It is demonstrated that MSU crystals induce size-dependent pyroptotic cell death in bone marrow-derived neutrophils (BMNs) and bone marrow-derived macrophages (BMDMs) by triggering NLRP3 inflammasome-dependent caspase-1 activation and subsequent formation of N-GSDMD. Furthermore, it is demonstrated that the size of MSU crystal also determines the formation of neutrophil extracellular traps (NETs) and aggregated neutrophil extracellular traps (aggNETs), which are promoted by the addition of interleukin-1ß (IL-1ß). Based on these mechanistic understandings, it is shown that N-GSDMD oligomerization inhibitor, dimethyl fumarate (DMF), inhibits MSU crystal-induced pyroptosis in BMNs and J774A.1 cells, and it further alleviates the acute inflammatory response in MSU crystals-induced gout mice model. This study elucidates that MSU crystal-induced pyroptosis in neutrophil and macrophage is critical for the pathological progress of gout, and provides a new therapeutic approach for the treatment of gout.

Are patients with gout with negative dual-energy computed tomography for monosodium urate crystal deposition easy-to-treat?

OBJECTIVES: To determine the clinical associations and predictive value of two thresholds of negative dual-energy CT (DECT) for MSU crystal deposition in gout patients initiating urate lowering therapy (ULT), and identify which threshold is more clinically relevant. METHODS: Patients from the CRYSTALILLE cohort with a diagnosis of gout naive to ULT with baseline DECT scans of knees and feet were selected. Two thresholds of positivity for DECT detection of MSU crystal deposition were considered (<0.01 cm3 and <0.1 cm3). Baseline characteristics and the prediction of key outcomes after ULT initiation including reaching serum urate (SU) levels <6.0 and 5.0 mg/dl and occurrence of flares at 6, 12 and 24 months, associated with both thresholds of negative DECTs were compared with those of. PATIENT: s having positive DECT scans. RESULTS: 211 patients aged 66.2 years [57; 75.8] with a symptom duration of 3 years [0; 7.8] were included. 38/211 (18%) and 90/211 (43%) had negative DECT scans for the 0.01 and 0.1 cm3 thresholds, respectively. Factors associated with negative DECT scans were younger age, shorter symptom duration, and absence of cardiovascular disease for both volume thresholds. 9/39 (23.1%), 3/26 (11.5%), and 1/18 (5.6%) of patients with <0.1 cm3 MSU crystals had flares at 6, 12 and 24 months, respectively, compared with 18/45 (40.0%), 9/36 (25.0%) and 2/18 (11.1%) patients with ≥0.1 cm3 (p> 0.05).Overall, 95 patients (68.3%) reached SU levels <6.0 mg/dl and 68 (48.9%) <5.0 mg/dl, without any difference between positive and negative DECTs, with ULT dosages which tended to be lower in patients with negative DECT. CONCLUSION: The 0.1 cm3 threshold was better correlated to clinical presentation and evolution than 0.01 cm3. Patients with gout with negative DECTs exhibit milder disease and a lower comorbidity burden. They do not exhibit particularly easy-to-treat hyperuricemia, but may have a lower risk of flares.

Major cardiovascular events in patients with cardiovascular monosodium urate deposits in atherosclerotic plaques.

OBJECTIVE: To determine the association of cardiovascular atherosclerotic plaque monosodium urate deposits with the occurrence of major cardiovascular events in gout and hyperuricemia patients. METHODS: This retrospective cohort study included patients with clinically suspicion of gout, who performed a dual energy computed tomography of the affected limb and thorax between June 1st, 2012 and December 5th, 2019. Clinical and laboratory parameters were retrieved from patients charts. Established cardiovascular risk factors were evaluated. Medical history review identified the presence of major adverse cardiac events with a median follow up time of 33 months (range 0-108 months) after the performed computed tomography scan. RESULTS: Full data sets were available for 189 patients: 131 (69.3%) gout patients, 40 (21.2%) hyperuricemia patients, and 18 (9.5%) controls. Patients with cardiovascular monosodium urate deposits (n = 85/189, 45%) revealed increased serum acute phase reactants, uric acid levels and calcium scores in computed tomography compared with patients without cardiovascular monosodium urate deposits. Major adverse cardiac events were observed in 35 patients (18.5%) with a higher prevalence in those patients revealing cardiovascular monosodium urate deposits (n = 22/85, 25.9%) compared with those without cardiovascular monosodium urate deposits (n = 13/104, 12.5%, OR 2.4, p= 0.018). CONCLUSION: This is the first study demonstrating the higher hazard of major adverse cardiac events in patients with dual energy computed tomography-verified cardiovascular monosodium urate deposits. The higher prevalence of cardiac events in patients with cardiovascular monosodium urate deposits may facilitate risk stratification of gout patients, as classical cardiovascular risk scores or laboratory markers fail in their proper identification.

The Clinical Significance of Serum Interleukin-36α Levels in Patients with Gout.

BACKGROUND: Gout is a chronic inflammatory diseases caused by monosodium urate crystal deposition. However, the role of interleukin (IL)-36 in gout has not dbeen elucidated. METHODS: We enrolled 75 subjects, including 20 healthy controls (HC), 30 patients with acute gout attack and 25 patients in remission. Baseline data were obtained through clinical interrogation and laboratory data were obtained through tests of blood samples. Serum levels of IL-36α were detected using enzyme-linked immunosorbent assay. Spearman correlation analysis was used to investigate the correlation of IL-36α with other parameters. The diagnostic value of IL-36α was demonstrated using a receiver operating characteristic curve. RESULTS: The serum IL-36α level of gout patients in acute attack and remission stage was significantly higher than that of HC. Serum IL-36α was positively correlated with alanine transaminase (ALT) and aspartate transaminase (AST). Serum amyloid A (SAA) levels positively correlated with C-reactive protein levels and erythrocyte sedimentation rates. Glutamyl transpeptidase levels positively correlated with AST and ALT levels. CONCLUSION: In conclusion, serum IL-36α levels were elevated in patients with gout and correlated with the clinical markers of inflammation. Our findings suggest that IL-36α may be a novel inflammatory indicator for gout.

Review: The Role of Dual-Energy Computed Tomography in Detecting Monosodium Urate Deposits in Vascular Tissues.

PURPOSE OF REVIEW: To highlight novel findings in the detection of monosodium urate deposits in vessels using dual energy computed tomography, and to discuss the potential clinical implications for gout and hyperuricemia patients. RECENT FINDINGS: Gout is an independent risk factor for cardiovascular disease. However, classical risk calculators do not take into account these hazards, and parameters to identify patients at risk are lacking. Monosodium urate measured by dual energy computed tomography is a well-established technology for the detection and quantification of monosodium urate deposits in peripheral joints and tendons. Recent findings also suggest its applicability to identify vascular urate deposits. Dual energy computed tomography is a promising tool for detection of cardiovascular monosodium urate deposits in gout patients, to better delineate individuals at increased risk for cardiovascular disease.

IL1A regulates the inflammation in gout through the Toll-like receptors pathway.

Objective: Gout is a dangerous metabolic condition related to monosodium urate (MSU). Our aim is to study the molecular mechanisms underlying gout and to identify potential clinical biomarkers by bioinformatics analysis and experimental validation. Methods: In this study, we retrieved the overlapping genes between GSE199950-Differential Expressed Genes (DEGs) dataset and key module in Weighted Gene Co-Expression Network Analysis (WGCNA) on GSE199950. These genes were then analyzed by protein-protein interaction (PPI) network, expression and Gene Set Enrichment Analysis to identify the hub gene related to gout. Then, the gene was investigated by peripheral blood mononuclear cells (PBMCs), immunoassay and cell experiments like western blotting to uncover its underlying mechanism in gout cells. Results: From the turquoise module and 83 DEGs, we identified 62 overlapping genes, only 11 genes had mutual interactions in PPI network and these genes were highly expressed in MSU-treated samples. Then, it was found that the IL1A (interleukin 1 alpha) was the only one gene related to Toll-like receptor signaling pathway that was associated with the occurrence of gout. Thus, IL1A was determined as the hub gene in this study. In immunoassay, IL1A was significantly positively correlated with B cells and negatively correlated with macrophages. Moreover, IL1A is highly expressed in gout patients,it has a good clinical diagnostic value. Finally, the results of in vitro experiments showed that after knocking down IL1A, the expressions of pro-inflammatory cytokines and Toll-like receptor signaling pathway-related proteins (TLR2, TLR4, MyD88) were all reduced. Conclusion: It is confirmed that IL1A is a promoting gene in gout with a good diagnostic value, and specifically it affects the inflammation in gout through Toll-like receptor pathway. Our research offers fresh perspectives on the pathophysiology of gout and valuable directions for future diagnosis and treatment.

Synovial fluid crystal analysis with compensated polarization using a gout analyser in clinical practice.

OBJECTIVE: To validate the gout analyzer as a clinical method of synovial fluid crystal analysis. METHODS: Thirty knee synovial fluid samples with suspected calcium pyrophosphate (CPP) crystals were analyzed. Within 48 hours after collection, each non-centrifuged sample was examined blindly and independently by one or more rheumatologists in the following order: 1) with an optical microscope under ordinary light, 2) with the same microscope under compensated polarization provided by a gout analyzer, and 3) with a fully equipped compensated polarized microscope with a rotating stage as the gold standard. As a reference, laboratory technicians analyzed fresh, centrifuged synovial fluid using a gout analyzer. RESULTS: Of the 30 samples analyzed, CPP and monosodium urate (MSU) crystals were detected in 11 and four, non-centrifuged samples, respectively, using a fully equipped compensated polarized microscope. The rheumatologists' detection rate of crystals in the non-centrifuged synovial fluid under ordinary light and with a gout analyzer was 73.3% and 80%, respectively. The laboratory technicians' detection rate in fresh centrifuged synovial fluid using a gout analyzer was 100%. CONCLUSION: A gout analyzer may be used to diagnose gout and calcium pyrophosphate deposition disease definitively if a fully equipped compensated polarized microscope is unavailable.

Baicalin and baicalein attenuate hyperuricemic nephropathy via inhibiting PI3K/AKT/NF-κB signalling pathway.

AIM: Inflammation and apoptosis are main pathological processes that lead to the development of hyperuricemic nephropathy (HN). This study aims to explore whether baicalin (BA) and baicalein (BAI) can relieve the damage through PI3K/AKT/NF-κB signal pathway and provide more reliable and precise evidence for the treatment of HN. METHODS: HN mice were induced by yeast extract with potassium oxonate (PO), and HK-2 cells were induced by monosodium urate (MSU). Molecular docking, western blot, q-PCR, and other methods were used to explore the changes of various indicators in HN mice and HK-2 cells. RESULTS: Molecular docking results showed that BA and BAI had good binding ability with PI3K, AKT, p65 and IκBα. BA and BAI significantly ameliorated the levels of renal function, decreased the p-PI3K, p-AKT and p-p65 expression, down-regulated the BAX/BCL2 and CASP3, and blunted the mRNA levels of tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-18 in both renal tissue of HN mice and HK-2 cells induced by MSU. BA and BAI also decreased the oxidative stress level of MSU-induced HK-2 cells. CONCLUSION: BA and BAI were confirmed to attenuate HN through alleviating renal inflammatory and apoptosis in cells and tissues by inhibiting PI3K/AKT/NF-κB pathway. BA and BAI were expected to be developed as new anti-HN drugs.

Crystal arthropathies and osteoarthritis-where is the link?

Osteoarthritis (OA) is one of the leading causes of disability worldwide. As our understanding of OA progressively has moved from a purely mechanical "wear and tear" concept toward a complex multi-tissue condition in which inflammation plays a central role, the possible role of crystal-induced inflammation in OA incidence and progression may be relevant. In addition to gout, which affects 4% of the US population, basic calcium phosphate and calcium pyrophosphate deposition both may induce joint inflammation and may play a role in pain in OA. This narrative review article discusses the possible mechanisms underlying the associations between crystal-induced arthropathies and OA, and the important implications of these for clinical practice and future research.

Assessing tophaceous spinal gout treatment response using dual-energy CT as a point-of-care imaging modality: case report.

We describe a case of chronic tophaceous gout affecting the spine, hands, elbows, feet, and knees in a 67-year-old man with serum urate levels at 549 µmol/L whose response to treatment was successfully mapped using dual-energy computed tomography (DECT). The patient presented with exacerbation of acute-on-chronic lumbar back pain. He had received a diagnosis of gout 3 years prior to this presentation yet was not on any urate-lowering therapy. The patient received febuxostat 80 mg and colchicine 0.3 mg once daily and underwent DECT to assess baseline monosodium urate (MSU) burden. At baseline, MSU deposits were seen in the hands, elbows, feet, knees, and lumbar spine including the left L5-S1 facet joint encroaching onto the neural foramen. After 2.5 years of treatment, serum urate level was within the target range (< 360 µmol/L), and the patient underwent a follow-up DECT that revealed almost full resolution of MSU deposition in the spine, including the MSU-burdened facet joint and neural foramen in the lumbar spine, in addition to all the affected peripheral joints. This case is the first report of radiological evidence of nearly complete resolution of MSU deposits in spinal gout on DECT after urate-lowering therapy treatment, which demonstrates the utility of this imaging modality as a non-invasive investigational point-of-care imaging modality for mapping treatment response and identifying the etiology of back pain in a patient with chronic tophaceous spinal gout.

The application of a novel hydrodynamic cavitation device to debride intra-articular monosodium urate crystals.

INTRODUCTION: Efficient and complete debridement of intra-articular deposits of monosodium urate crystals is rarely achieved by existing arthroscopic tools such as shavers or radiofrequency ablation, while cavitation technology represents a prospective solution for the non-invasive clearance of adhesions at intra-articular interfaces. METHODS: Simulation modeling was conducted to identify the optimal parameters for the device, including nozzle diameters and jet pressures. Gouty arthritis model was established in twelve rats that were equally and randomly allocated into a cavitation debridement group or a curette debridement group. A direct injection nozzle was designed and then applied on animal model to verify the effect of the cavitation jet device on the removal of crystal deposits. Image analysis was performed to evaluate the clearance efficiency of the cavitation device and the pathological features of surrounding tissue were collected in all groups. RESULTS: To maximize cavitation with the practical requirements of the operation, an experimental rig was applied, including a 1 mm direct injection nozzle with a jet pressure of 2.0 MPa at a distance of 20 mm and a nitrogen bottle as high-pressure gas source. With regards to feasibility of the device, the clearance rates in the cavitation group were over 97% and were significantly different from the control group. Pathological examination showed that the deposition of monosodium urate crystals was removed completely while preserving the normal structure of the collagen fibers. CONCLUSIONS: We developed a promising surgical device to efficiently remove intra-articular deposits of monosodium urate crystals. The feasibility and safety profile of the device were also verified in a rat model. Our findings provide a non-invasive method for the intraoperative treatment of refractory gouty arthritis.

Dual-energy CT assessment of rapid monosodium urate depletion and bone erosion remodelling during pegloticase plus methotrexate co-therapy.

OBJECTIVES: Pegloticase rapidly lowers serum urate in uncontrolled/refractory gout patients, with ≥1 tophus resolution in 70% of pegloticase responders and 28% of non-responders. Dual-energy computed tomography (DECT) non-invasively detects MSU deposition, including subclinical deposition, quantifies MSU volumes and depicts bone erosions. This report presents DECT findings in MIRROR open-label trial participants receiving pegloticase+MTX co-therapy. METHODS: Serial DECT scans were obtained during pegloticase (8 mg biweekly infusions)+oral MTX (15 mg/week) co-therapy. Bilateral hand/wrist, elbow, foot/ankle and knee images were analysed with default post-processing settings. MSU volumes were quantified and bone erosions were identified and evaluated for remodelling (decreased size, sclerosis, new bone formation). DECT and physical examination findings were compared. RESULTS: 2 patients underwent serial DECT. Patient 1 (44-year-old male) completed 52 weeks of pegloticase+MTX co-therapy (26 infusions). Baseline examination detected 4 tophus-affected joints while DECT identified 73 MSU-affected joints (total MSU volume: 128.76 cm3). At end-of-treatment, there were no clinically-affected joints and 4 joints with DECT-detected MSU deposition. MSU volume decreased by 99% and bone erosion remodelling was evident. Patient 2 (51-year-old male) had 10 weeks of therapy (5 infusions), discontinuing because of urate-lowering response loss. Baseline examination detected 7 tophus-affected joints while DECT identified 55 MSU-affected joints (total MSU volume: 59.20 cm3). At end-of-treatment, there were 5 clinically affected joints and 42 joints with DECT-detected MSU deposition. MSU volume decreased by 58% and bone erosion remodelling was evident. CONCLUSION: DECT detected subclinical MSU deposition and quantified changes over time. Rapid tophus resolution and bone erosion remodelling occurred during pegloticase+MTX co-therapy. TRIAL REGISTRATION: ClinicalTrials.gov, https://clinicaltrials.gov, NCT03635957.

Evolving Role of Dual-Energy CT in the Clinical Workup of Gout: A Retrospective Study.

BACKGROUND. Dual-energy CT (DECT) allows noninvasive detection of monosodium urate (MSU) crystal deposits and has become incorporated into the routine clinical evaluation for gout at many institutions over the past decade. OBJECTIVE. The purpose of this study was to compare two time periods over the past decade in terms of radiologists' interpretations of DECT examinations performed for the evaluation of gout and subsequent clinical actions. METHODS. This retrospective study included 100 consecutive adult patients who underwent DECT to evaluate for gout in each of two periods (one beginning in March 2013 and one beginning in September 2019). Examinations performed in 2013 were conducted using a second-generation DECT scanner (80 kV [tube A] and 140 kV [tube B] with a 0.4-mm tin filter), and those performed in 2019 were conducted using a third-generation DECT scanner (80 kV [tube A] and 150 kV [tube B] with a 0.6-mm tin filter) that provides improved spectral separation. Original DECT reports were classified as positive, negative, or equivocal for MSU crystals indicative of gout. Joint aspirations occurring after the DECT examinations were recorded on the basis of findings from medical record review. A single radiologist performed a post hoc retrospective blinded image review, classifying examinations as positive, negative, or equivocal. RESULTS. In 2013, 44.0% of DECT examinations were interpreted as positive, 23.0% as negative, and 33.0% as equivocal; in 2019, 37.0% were interpreted as positive, 47.0% as negative, and 16.0% as equivocal (p < .001). The frequency of joint aspiration after DECT was 14.0% in 2013 versus 2.0% in 2019 (p = .002), and that after DECT examinations with negative interpretations was 17.4% in 2013 versus 2.1% in 2019 (p = .02). In post hoc assessment by a single radiologist, the distribution of interpretations in 2013 was positive in 49.0%, negative in 22.0%, and equivocal in 29.0%, and in 2019 it was positive in 39.0%, negative in 50.0%, and equivocal in 11.0% (p < .001). CONCLUSION. When DECT examinations performed for gout in 2013 and 2019 were compared, the frequency of equivocal interpretations was significantly lower in 2019, possibly in relation to interval technologic improvements. Negative examinations were less frequently followed by joint aspirations in 2019, possibly reflecting increasing clinical acceptance of the DECT results. CLINICAL IMPACT. The findings indicate an evolving role for DECT in the evaluation of gout after an institution's routine adoption of the technology for this purpose.

Kv1.5 channel mediates monosodium urate-induced activation of NLRP3 inflammasome in macrophages and arrhythmogenic effects of urate on cardiomyocytes.

BACKGROUND: Gout is usually found in patients with atrial fibrillation (AF). K+ efflux is a common trigger of NLRP3 inflammasome activation which is involved in the pathogenesis of AF. We investigated the role of the K+ channel Kv1.5 in monosodium urate crystal (MSU)-induced activation of the NLRP3 inflammasome and electrical remodeling in mouse and human macrophages J774.1 and THP-1, and mouse atrial myocytes HL-1. METHODS AND RESULTS: Macrophages, primed with lipopolysaccharide (LPS), were stimulated by MSU. HL-1 cells were incubated with the conditioned medium (CM) from MSU-stimulated macrophages. Western blot, ELISA and patch clamp were used. MSU induced caspase-1 expression in LPS-primed J774.1 cells and IL-1ß secretion, suggesting NLRP3 inflammasome activation. A selective Kv1.5 inhibitor, diphenyl phosphine oxide-1 (DPO-1), and siRNAs against Kv1.5 suppressed the levels of caspase-1 and IL-1ß. MSU reduced intracellular K+ concentration which was prevented by DPO-1 and siRNAs against Kv1.5. MSU increased expression of Hsp70, and Kv1.5 on the plasma membrane. siRNAs against Hsp70 were suppressed but heat shock increased the expression of Hsp70, caspase-1, IL-1ß, and Kv1.5 in MSU-stimulated J774.1 cells. The CM from MSU-stimulated macrophages enhanced the expression of caspase-1, IL-1ß and Kv1.5 with increased Kv1.5-mediated currents that shortened action potential duration in HL-1 cells. These responses were abolished by DPO-1 and a siRNA against Kv1.5. CONCLUSIONS: Kv1.5 regulates MSU-induced activation of NLRP3 inflammasome in macrophages. MSUrelated activation of NLRP3 inflammasome and electrical remodeling in HL-1 cells are via macrophages. Kv1.5 may have therapeutic value for diseases related to gout-induced activation of the NLRP3 inflammsome, including AF.

Gout-associated monosodium urate crystal-induced necrosis is independent of NLRP3 activity but can be suppressed by combined inhibitors for multiple signaling pathways.

Monosodium urate (MSU) crystals, the etiological agent of gout, are formed in joints and periarticular tissues due to long-lasting hyperuricemia. Although MSU crystal-triggered NLRP3 inflammasome activation and interleukin 1ß (IL-1ß) release are known to have key roles in gouty arthritis, recent studies revealed that MSU crystal-induced necrosis also plays a critical role in this process. However, it remains unknown what forms of necrosis have been induced and whether combined cell death inhibitors can block such necrosis. Here, we showed that MSU crystal-induced necrosis in murine macrophages was not dependent on NLRP3 inflammasome activation, as neither genetic deletion nor pharmacological blockade of the NLRP3 pathway inhibited the necrosis. Although many cell death pathways (such as ferroptosis and pyroptosis) inhibitors or reactive oxygen species inhibitors did not have any suppressive effects, necroptosis pathway inhibitors GSK'872 (RIPK3 inhibitor), and GW806742X (MLKL inhibitor) dose-dependently inhibited MSU crystal-induced necrosis. Moreover, a triple combination of GSK'872, GW806742X, and IDN-6556 (pan-caspase inhibitor) displayed enhanced inhibition of the necrosis, which was further fortified by the addition of MCC950 (NLRP3 inhibitor), suggesting that multiple cell death pathways might have been triggered by MSU crystals. Baicalin, a previously identified inhibitor of NLRP3, inhibited MSU crystal-induced inflammasome activation and suppressed the necrosis in macrophages. Besides, baicalin gavage significantly ameliorated MSU crystal-induced peritonitis in mice. Altogether, our data indicate that MSU crystals induce NLRP3-independent necrosis, which can be inhibited by combined inhibitors for multiple signaling pathways, highlighting a new avenue for the treatment of gouty arthritis.

Anethole ameliorates inflammation induced by monosodium urate in an acute gouty arthritis model via inhibiting TLRs/MyD88 pathway.

OBJECTIVE: To assess the effects of anethole on monosodium urate (MSU)-induced inflammatory response, investigate its role in acute gouty arthritis (AGA), and verify its molecular mechanism. METHODS: Hematoxylin and eosin staining assay and time-dependent detection of degree of ankle swelling were performed to assess the effects of anethole on joint injury in MSU-induced AGA mice. Enzyme-linked-immunosorbent serologic assay was performed to demonstrate the production levels of inflammatory factors (interleukin 1ß [IL-1ß], interleukin 6 [IL-6], interleukin 8 [IL-8], tumor necrosis factor α [TNF-α], and monocyte chemo-attractant protein-1 [MCP-1]) in MSU-induced AGA mice. Western blot assays were used to confirm the effects of anethole on oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activity and the activation of toll-like receptors (TLRs)-myeloid differentiation factor 88 (MyD88) pathway in MSU-induced AGA mice. RESULTS: We observed that a significant joint injury occurred in MSU-induced AGA mice. Anethole could alleviate the pathological injury of the synovium in MSU-induced AGA mice and suppressed ankle swelling. In addition, we observed that anethole could inhibit MSU-induced inflammatory response and inflammasome activation in MSU-induced AGA mice. Moreover, we discovered that anethole enabled to inhibit the activation of TLRs/MyD88 pathway in MSU-induced AGA mice. Our findings further confirmed that anethole contributed to the inhibitory effects on progression in MSU-induced AGA mice. CONCLUSION: It confirmed that anethole ameliorated the MSU-induced inflammatory response in AGA mice in vivo via inhibiting TLRs-MyD88 pathway.

Calycosin represses AIM2 inflammasome-mediated inflammation and pyroptosis to attenuate monosodium urate-induced gouty arthritis through NF-κB and p62-Keap1 pathways.

Gouty arthritis is an inflammatory disease induced by monosodium urate (MSU), and is closely related to the activation of inflammasomes. Calycosin plays an anti-inflammatory role in arthritis. This study explored the mechanism of Calycosin in MSU-induced gouty arthritis. MSU-induced gouty arthritis mouse models with or without treatment of Calycosin were established, and physiological and pathological indicators were determined. Similarly, peripheral blood mononuclear cells (PBMCs) and THP-1 macrophages were used in vitro. Lactate dehydrogenase (LDH) was tested. The degree of centrifugal infiltration was detected by immunofluorescence. ELISA and quantitative reverse-transcription polymerase chain reaction were conducted to determine the levels of inflammatory factors. Immunohistochemistry, immunofluorescence, and flow cytometry were utilized to detect the content of caspase-1. Protein expressions of NF-κB-, p62-Keap1 pathway-, and pyroptosis-related factors were examined by western blot. In MSU-induced mouse models, calycosin increased mechanical hyperalgesia but decreased the swelling index of the mouse knee joint in a time-dependent manner. MSU treatment increased inflammatory cells and LysM-eGFP+ neutrophils recruitment in vivo, and promoted the LDH content in vitro, and meanwhile, calycosin reversed the aforementioned effects of MSU. In addition, calycosin repressed the release of inflammatory factors, promoted p62 level and diminished the levels of AIM2, caspase-1, ASC, IL-1ß, Keap1, Cleaved GSDMD, and Cleaved caspase-1 and phosphorylation of p65 and IκBα in MSU-induced mouse or cell models. Furthermore, AIM2 silencing also inhibited MSU-induced inflammation and pyroptosis. Collectively, calycosin may inhibit AIM2 inflammasomes-mediated inflammation and pyroptosis through NF-κB and p62-Keap1 pathways, ultimately playing a protective role in gouty arthritis.

Bactericidal/Permeability-Increasing Protein Downregulates the Inflammatory Response in In Vivo Models of Arthritis.

We investigated the effects of bactericidal/permeability-increasing protein (BPI) alone or in combination with hyaluronic acid (HA) in two animal models: collagen-induced arthritis (CIA) and crystal-induced inflammation. In CIA, mice were intraperitoneally injected with PBS, HA, or BPI plus or minus HA, twice a week for 2 months, and then euthanized to collect paw and blood. Arthritis was assessed in ankle joints by clinical and histological evaluation. Pathogenic crystals were intraperitoneally injected in mice plus or minus BPI, or with a composition of BPI and HA. After sacrifice, total and differential leukocyte counts were determined. Cytokine levels were measured in serum and peritoneal fluids. In CIA mice, BPI improved clinical and histological outcomes (histological scores ≥2-fold), and downregulated inflammatory mediators (47-93%). In crystal-induced inflammation, BPI reduced leukocyte infiltration (total count: ≥60%; polymorphonuclear cells: ≥36%) and inhibited cytokine production (35-74%). In both models, when mice were co-treated with BPI and HA, the improvement of all parameters was greater than that observed after administration of the two substances alone. Results show that BPI attenuates CIA and inflammation in mice, and this effect is enhanced by HA co-administration. Combined use of BPI and HA represents an interesting perspective for new potential treatments in arthritis.