Deficiency of histone deacetylases 3 in macrophage alleviates monosodium urate crystals-induced gouty inflammation in mice.

BACKGROUND: Gout is caused by monosodium urate (MSU) crystals deposition to trigger immune response. A recent study suggested that inhibition of Class I Histone deacetylases (HDACs) can significantly reduce MSU crystals-induced inflammation. However, which one of HDACs members in response to MSU crystals was still unknown. Here, we investigated the roles of HDAC3 in MSU crystals-induced gouty inflammation. METHODS: Macrophage specific HDAC3 knockout (KO) mice were used to investigate inflammatory profiles of gout in mouse models in vivo, including ankle arthritis, foot pad arthritis and subcutaneous air pouch model. In the in vitro experiments, bone marrow-derived macrophages (BMDMs) from mice were treated with MSU crystals to assess cytokines, potential target gene and protein. RESULTS: Deficiency of HDAC3 in macrophage not only reduced MSU-induced foot pad and ankle joint swelling but also decreased neutrophils trafficking and IL-1ß release in air pouch models. In addition, the levels of inflammatory genes related to TLR2/4/NF-κB/IL-6/STAT3 signaling pathway were significantly decreased in BMDMs from HDAC3 KO mice after MSU treatment. Moreover, RGFP966, selective inhibitor of HDAC3, inhibited IL-6 and TNF-α production in BMDMs treated with MSU crystals. Besides, HDAC3 deficiency shifted gene expression from pro-inflammatory macrophage (M1) to anti-inflammatory macrophage (M2) in BMDMs after MSU challenge. CONCLUSIONS: Deficiency of HDAC3 in macrophage alleviates MSU crystals-induced gouty inflammation through inhibition of TLR2/4 driven IL-6/STAT3 signaling pathway, suggesting that HDAC3 could contribute to a potential therapeutic target of gout.

Effect of Berberine on Activation of TLR4-NFκB Signaling Pathway and NLRP3 Inflammasome in Patients with Gout.

OBJECTIVE: To determine the effects of berberine (BBR) on the activation of toll-like receptor 4 (TLR4), nuclear factor (NF)κB (NF-κB) signaling and NLRP3 inflammasome in patients with gout. METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from 24 acute (AP) and 41 non-acute (NAP) phases of primary gout patients, respectively, as well as 30 healthy controls (HC). TLR4, NF-κB (p65), NLRP3, apoptosis-associated specklike protein containing a CARD (PYCARD), cysteinyl aspartate specific proteinase-1 (CASP1), and interleukin-1ß (IL-1ß) mRNA expression levels in PBMCs were measured by quantitative reverse transcriptase polymerase chain reaction. The protein levels of TLR4, myeloid differentiation factor 88 (MyD88), NF-κB (p50/65), inhibitor of kappa B kinase α/ß (IKKα/ß), NF-κB inhibitor α (IKBα), phospho-IKKα/ß (p-IKKα/ß), NLRP3, PYCARD, and CASP1 were monitored by Western blotting. Serum IL-1ß protein level was measured using enzyme-linked immunosorbent assay (ELISA). In addition, PBMCs from HC and macrophages derived from a spontaneously immortalized monocyte-like cell line (THP-1) were stimulated using monosodium urate (MSU, 100 µg/mL), 0.1% dimethyl sulfoxide, 25 µmol/L BBR, and 10, 25, and 50 µmol/L BBR+100 µg/mL MSU for different time periods. The protein levels of IL-1ß and IL-18 in cell culture supernatants was measured by ELISA, and the protein expressions of TLR4, MyD88, NF-κB (p50/p65), IKKα/ß, I κBß, p-IKKα/ß, NLRP3, PYCARD, and CASP1 in macrophages were analyzed by Western blotting. RESULTS: (1) TLR4, NF-κB (p65), PYCARD, CASP1, and IL-1ß mRNA levels in PBMCs were significantly higher in the AP group than in the HC group (P<0.05). The NLRP3 mRNA expression levels in PBMCs were found to be significantly lower in the AP and NAP groups than in the HC group (P<0.05, P<0.01). (2) The protein levels of TLR4, IKKß, MyD88, NF-κB, p-IKKα/ß, PYCARD, and CASP1 in PBMCs were significantly higher, and those of IκBα, IKKα, and NLRP3 were found to be significantly lower in the AP group than in the HC group (P<0.05 or P<0.01). (3) The serum IL-1ß protein levels were significantly higher in the AP and NAP groups than in the HC group (P<0.01). (4) The IL-1ß protein level was significantly lower in the culture supernatants of the PBMCs stimulated with MSU for 3 and 6 h in the 25 and 50 µmoL/L BBR groups compared with that in the MSU group (P<0.01). (5) The protein levels of IL-1ß and IL-18 were also significantly lower in the culture supernatants of macrophages stimulated with MSU for 3 and 6 h in BBR groups compared with those in the MSU group (P<0.01). (6) The protein levels of TLR4, MyD88, NF-κB (p50, p65, p105), IKKα/ß, p-IκBα, p-IKKα/ß, PYCARD, and CASP1 were significantly differed between the macrophages stimulated with MSU for 0.5 and 6 h in BBR groups compared with those in the MSU group (P<0.05 or P<0.01). CONCLUSIONS: Activation of TLR4-NFκB signaling and NLRP3 inflammasome by MSU crystals drives the progression of gout inflammation. BBR ameliorates gouty inflammation, which is mechanistically associated with its regulation of TLR4-NF-κB signaling and NLRP3 inflammasome expression.

microRNA-223 Deficiency Exacerbates Acute Inflammatory Response to Monosodium Urate Crystals by Targeting NLRP3.

OBJECTIVE: MicroRNAs were identified as master-switch molecules limiting acute inflammatory response. This study investigated the potential role of microRNA (miR)-223 in the mechanism of gout. METHODS: Wild-type (WT) and miR-223 knock-out (KO) mice were used to evaluate the phenotypes of gout models. Inflammatory cytokines were measured in air pouch and peritoneal cavity lavage fluid. In addition to miR-223 level in gout patients, miR-223 and pro-inflammatory genes were examined in bone marrow-derived macrophages (BMDMs) from mice as well as peripheral blood mononuclear cells from healthy controls (HC) treated with monosodium urate (MSU) crystals in vitro. RESULTS: MiR-223 was up-regulated in the early phase in BMDMs from WT mice after MSU challenge and decreased rapidly, and this was not observed in miR-223 KO mice in vitro. In addition, miR-223 was required for macrophages homeostasis. In comparison with WT mice in vivo, miR-223 deficiency exacerbated swelling index of MSU-induced inflammation in foot pad and ankle joint models. MiR-223 deficiency also markedly aggravated inflammatory cells infiltration and cytokines release including interleukin (IL)-1ß, IL-6 and monocyte chemotactic protein-1 (MCP-1) in the air pouch and peritonitis models. In the in vitro experiments, miR-223 deficiency promoted the inflammatory response by targeting NLR family pyrin domain containing protein 3 (NLRP3). Besides, miR-223 level was down-regulated in gout patients and in HC exposed to MSU in vitro. CONCLUSION: MiR-223 was down-regulated in gout patients and miR-223 deficiency exacerbated inflammatory response in diverse murine models, suggesting that up-regulation of miR-223 could be a potential therapeutic strategy for alleviating gouty inflammation.

Molecular detection of Enterocytozoon bieneusi in farm-raised pigs in Hainan Province, China: infection rates, genotype distributions, and zoonotic potential.

Enterocytozoon bieneusi is a zoonotic fungal pathogen with a high degree of host diversity that can parasitize many animals, including humans. Pigs may play an important role in the epidemiology of E. bieneusi as reservoir hosts. Nevertheless, the genotypes of E. bieneusi in pigs in China remain poorly understood. The aim of this study was to determine the prevalence of E. bieneusi infection amongst pigs raised on farms from four cities of Hainan Province, using nested polymerase chain reaction (PCR) of the partial small subunit of the ribosomal RNA gene, and to identify genotypes of E. bieneusi isolates based on sequence analysis of the ribosomal internal transcribed spacer (ITS) region. Among 188 stool samples, E. bieneusi was detected in 46.8% (88/188). Eight genotypes including four known (EbpA, CS-4, MJ14, and CHG19) and four novel (HNP-I - HNP-IV) genotypes were identified. Using phylogenetic analysis, genotypes EbpA, CS4, CHG19, HNP-III, and HNP-IV were clustered into zoonotic Group 1, while the remaining three genotypes (MJ14, HNP-I, and HNP-II) clustered into Group 10. The high prevalence of zoonotic genotypes of E. bieneusi among pigs suggests that pig farming is a potential source of human infection. Additionally, this is the first identification of genotypes in Group 10 in pigs indicating unique epidemic features of E. bieneusi in pigs in Hainan Province, the southernmost part of China.

TITLE: Détection moléculaire d'Enterocytozoon bieneusi chez les porcs d'élevage dans la province de Hainan en Chine : taux d'infection, répartition des génotypes et potentiel zoonotique. ABSTRACT: Enterocytozoon bieneusi est un pathogène fongique zoonotique avec une grande diversité d'hôte qui peut parasiter de nombreux animaux, y compris les humains. Les porcs peuvent jouer un rôle important dans l'épidémiologie d'E. bieneusi en tant qu'hôtes réservoirs. Néanmoins, les génotypes d'E. bieneusi chez le porc en Chine restent mal connus. Le but de cette étude était de déterminer la prévalence de l'infection par E. bieneusi chez les porcs élevés dans des fermes de quatre villes de la province de Hainan, en utilisant la réaction en chaîne par polymérase emboîtée (PCR) de la petite sous-unité partielle du gène de l'ARN ribosomal et de identifier les génotypes des isolats d'E. bieneusi sur la base d'une analyse de séquence de la région des espaceurs internes transcrits ribosomiques (ITS). Sur 188 échantillons de selles, E. bieneusi a été détecté dans 46,8 % (88/188). Huit génotypes, dont quatre génotypes connus (EbpA, CS-4, MJ14 et CHG19) et quatre génotypes nouveaux (HNP-I à IV), ont été identifiés. Dans une analyse phylogénétique, les génotypes EbpA, CS4, CHG19, HNP-III et HNP-IV étaient regroupés dans le groupe zoonotique 1, tandis que les trois génotypes restants (MJ14, HNP-I et HNP-II) étaient regroupés dans le groupe 10. La prévalence élevée des génotypes zoonotiques d'E. bieneusi chez les porcs suggère que l'élevage porcin est une source potentielle d'infection humaine. De plus, il s'agit de la première identification de génotypes du groupe 10 chez les porcs, indiquant des caractéristiques épidémiques uniques d'E. bieneusi chez les porcs dans la province de Hainan, la partie la plus méridionale de la Chine.

Downregulation of Transcription Factor T-Bet as a Protective Strategy in Monosodium Urate-Induced Gouty Inflammation.

Gout is sterile joint inflammation triggered by the damaging effects of monosodium urate (MSU) crystals accumulation. Previous studies suggest transcription factor T-bet plays an important role in inflammatory arthritis. Notably, mice lacking T-bet markedly reduced joint inflammation of rheumatoid arthritis models, however, the involvement of T-bet in gouty inflammation has yet to be clarified. Here, we took advantage of T-bet knockout (KO) mice to investigate the role of T-bet in the pathogenesis of MSU-induced gout inflammation. T-bet KO and wild type (WT) mice were used for models of acute inflammation induced with MSU crystals, including footpad, air pouch and peritonitis models. Inflammatory cytokines and phagocytosis were detected in bone-marrow-derived macrophages (BMDMs) from T-bet KO and WT mice treated with MSU crystals in vitro. In addition, T-bet expression in peripheral blood mononuclear cells (PBMCs) from gout patients was measured, as well as plasma inflammatory cytokines. We found that the levels of interleukin (IL)-17, IL-23, and interferon-γ were reduced, but tumor necrosis factor-α was not, in BMDMs from T-bet KO compared with WT mice after MSU challenge in vitro, as well as MSU phagocytosis. In comparison with WT mice in vivo, the swelling index of T-bet KO mice was significantly decreased in the footpad model. T-bet deficiency also dramatically relieved MSU-induced inflammatory cell infiltration in peritonitis and air pouch models in vivo, and as well as the IL-1ß levels of air pouch lavage fluid (APLF). In addition, plasma IL-17 and IL-23 levels were elevated in acute gout, whereas protein levels of T-bet were downregulated in PBMCs from acute gout patients and intercritical gout treated with MSU crystals in vitro as well. Transcription factor T-bet deficiency protects against MSU-induced gouty inflammation, suggesting that downregulation of T-bet could be a protective strategy and contribute to spontaneous remission of inflammation in acute gout.

Resveratrol ameliorates gouty inflammation via upregulation of sirtuin 1 to promote autophagy in gout patients.

BACKGROUND: Resveratrol exerts an anti-inflammatory effect on collagen-induced arthritis and osteoarthritis in rats via activation of sirtuin 1 (SIRT1). Autophagy can be induced by resveratrol and leads to amelioration of interleukin-1 beta (IL-1ß) release in vitro. We aimed to determine the anti-inflammatory mechanisms of resveratrol in patients with gout. METHODS: Blood samples were obtained from patients with acute gout, intercritical gout (IG) and healthy controls (HC). The mRNA and protein levels of SIRT1 and nuclear factor-kappa B (NF-kB) p65 were determined in peripheral blood mononuclear cells (PBMCs) lysate from these patients. In the in vitro experiment, SIRT1, autophagy-related genes (beclin-1 and microtubule-associated protein 1 light-chain 3) and key genes involved in the gouty inflammatory pathway, including NF-κB p65, NLR family pyrin domain containing 3 (NLRP3), caspase-1 and IL-1ß, were determined in PBMCs lysate or plasma from IG patients exposed to monosodium urate (MSU) crystals with or without resveratrol. RESULTS: The mRNA and protein levels of SIRT1 were downregulated in PBMCs from gout patients in comparison with HC. In the in vitro experiment, the protein levels of SIRT1 were downregulated in PBMCs from IG patients exposed to MSU crystals and were restored by resveratrol in a dose-dependent manner. Furthermore, high doses of resveratrol ameliorated the release of the inflammatory cytokine IL-1ß. In addition, the mRNA levels of NLRP3 and NF-κB p65 were regulated by resveratrol, but caspase-1 and IL-1ß were not. Furthermore, resveratrol promoted MSU-induced autophagy in PBMCs from patients with gout. CONCLUSION: These findings suggest that resveratrol ameliorates gouty inflammation via upregulation of SIRT1 to promote autophagy in patients with gout.

High Levels of Serum Uric Acid, Cystain C and Lipids Concentration and their Clinical Significance in Primary Gouty Arthritis Patients.

OBJECTIVE: To investigate the changes of serum Uric Acid (sUA), lipids and Cystatin C (CysC) in primary gout patients, and to explore the clinical significance in gout patients. METHODS: sUA, CysC, high-sensitivity C-reactive Protein (hsCRP) and other biochemical parameters were measured in 326 gout patient and 210 healthy control subjects, blood cell counts were also detected. Clinical data were collected from gout patients. RESULTS: sUA, CysC, hsCRP, Body Mass Index (BMI), White Blood Cell (WBC) counts, neutrophil Granulocyte (GR), Monocyte (Mo), Triglycerides (TG), plasma Total Cholesterol (TC), Very Low Density Lipoprotein (VLDL), apolipoprotein B100 (apoB100), Blood Glucose (GLU), serum Creatinine (sCr) and Urea Nitrogen (BUN) were significantly increased in gout patients compared with HC subjects (P<0.01, respectively), while lymphocyte counts and High Density Lipoprotein- Cholesterol (HDL-C) were significantly decreased in gout patients compared with HC subjects (P<0.01, respectively). Positive correlations were observed between concentration of sUA and age, TG, VLDL, sCr and CysC (P<0.05, respectively). While negative correlations were observed between the concentration of sUA and HDL-C(P<0.01). Besides, Positive correlations were observed between concentration of CysC and WBC, GR, Mo, apoA1, GLU, sCr, BUN, sUA, hsCRP (P<0.05, respectively). While negative correlations were observed between the concentration of CysC and TC, LDL-C(P<0.01, respectively). CONCLUSIONS: Blood lipid profile changes in gout patients. Gout patients who suffer from lipid metabolism disorder and vascular diseases might be associated with hyperuricemia, which leads to endothelial cell damage and vascular smooth muscle cell proliferation. CysC might be a marker for renal function damage and inflammation. Hyperuricemia is the risk factor of renal disorder in gout patients.

Systemic lupus erythematosus complicated by noncirrhotic portal hypertension: A case report and review of literature.

A 48 year-old Chinese woman suffering from polyarthritis, irregular fever and trichomadesis was admitted to the hospital. A diagnosis of systemic lupus erythematosus (SLE) was made based on polyarthritis, pancytopenia, reduced complement 3, multiple positive autoantibodies, a positive Coomb's test and protein in her urine. In addition, splenomegaly was detected during physical examination and confirmed by abdominal ultrasonography and magnetic resonance imaging, indicating that the patient had SLE and portal hypertension. Further negative investigations ruled out the possibility of cirrhosis. The patient was diagnosed with active SLE complicated by noncirrhotic portal hypertension (NCPH) without liver histopathology, due to the patient's refusal for liver biopsy. Portal vein diameter and splenomegaly decreased following treatment with methylprednisolone, hydroxychloroquine and metoprolol tartrate. To date, SLE complicated by NCPH has rarely been reported, as it is under-recognized clinically as well as pathologically. Here we describe a case of SLE complicated by NCPH and review the literature for its characteristics, which may contribute to improving the recognition of NCPH and reducing missed and delayed diagnosis of this disorder.

Mice with miR-146a deficiency develop severe gouty arthritis via dysregulation of TRAF 6, IRAK 1 and NALP3 inflammasome.

BACKGROUND: MicroRNAs (miRNAs) serve as important regulators of inflammatory and immune responses and are implicated in several immune disorders including gouty arthritis. The expression of miR-146a is upregulated in the peripheral blood mononuclear cells of patients with inter-critical gout when compared to normouricemic and hyperuricemic controls and those patients with acute gout flares. However, the role of miR-146a in the development of gout remains unknown. Here, we used miR-146a knockout (KO) mice to test miR-146a function in a monosodium urate (MSU)-induced gouty arthritis model. METHODS: The footpad or ankle joint of miR-146a KO and wild-type (WT) mice were injected with an MSU suspension to induce acute gouty arthritis. Bone marrow-derived macrophages (BMDMs) were stimulated with MSU and the gene expression of miR-146a; interleukin 1 beta (IL-1ß); tumor necrosis factor-α (TNF-α); and the NACHT, LRR and PYD domains-containing protein 3 (NALP3) inflammasome was evaluated. TNF-α and IL-1ß protein levels in BMDMs were assessed by fluorescence-activated cell sorting and western blot analyses. Gene and protein levels of TNF receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase (IRAK1), the targets of miR-146a, were also measured. RESULTS: Significantly increased paw swelling and index and ankle joint swelling were observed in miR-146a KO mice compared to WT controls after MSU treatment. MiR-146a expression in BMDMs from WT mice was dramatically upregulated at 4 h following MSU stimulation. Additionally, the expression of IL-1ß, TNF-α, and NALP3 was higher in BMDMs from miR-146a KO mice after exposure to MSU crystals compared to those from WT mice. Consistent with the observed gene expression, the IL-1ß and TNF-α proteins were upregulated in miR-146a KO mice. Additionally quantitative RT-PCR and western blot demonstrated that TRAF6 and IRAK1 were dramatically upregulated in BMDMs from miR-146 KO mice compared to those from WT mice. CONCLUSIONS: Collectively, these observations suggest that miR-146a provides negative feedback regulation of gouty arthritis development and lack of miR-146a enhances gouty arthritis via upregulation of TRAK6, IRAK-1, and the NALP3 inflammasome function.

Association of NLRP3 polymorphisms with susceptibility to primary gouty arthritis in a Chinese Han population.

The NLRP3-interleukin1ß (IL1ß) signaling pathway is involved in monosodium urate (MSU)-mediated inflammation. The aim of this present study was to determine whether single nucleotide polymorphisms (SNPs) in the NLRP3 gene are associated with susceptibility to gouty arthritis (GA) and whether these SNPs alter the expression of components of the NLRP3-IL1ß signaling pathway. The rs10754558, rs4612666, and rs1539019 SNPs were detected in 583 patients with GA and 459 healthy subjects. NLRP3 and IL1ß mRNA levels in peripheral blood mononuclear cells (PBMCs) and serum IL1ß levels were measured in different genotype carriers, and correlations between the NLRP3 SNPs and NLRP3 mRNA, IL1ß mRNA, and serum IL1ß levels were investigated. The GG genotype of NLRP3 rs10754558 was found to be significantly associated with patients with GA compared to the healthy control subjects via multivariate logistic regression analysis (adjusted OR = 2.68, P = 0.006). The CGA haplotypes were independently associated with patients with GA compared to the healthy control subjects (adjusted OR = 1.968, P = 0.02). The levels of NLRP3 mRNA, IL1ß mRNA, and serum IL1ß in the patients with GA were significantly different among the three genotypes of rs10754558 (all P < 0.01). The GG genotype of rs10754558 and the CGA haplotype of rs4612666-C, rs10754558-G, and rs1539019-A are both independent risk factors for primary GA development. The rs10754558 polymorphism might participate in regulating immune and inflammation responses in patients with GA by influencing the expression of components of the NLRP3 inflammasome. Future multicenter studies aimed at replicating these findings in an independent population as well as functional tests will aid in further defining the role of these SNPs in the development of GA.