Uric acid-mediated inflammasome activation in IL-6 primed innate immune cells is regulated by baricitinib.

OBJECTIVES: Gout is an inflammatory arthropathy caused by the deposition of monosodium urate (MSU). The synthesis and release of IL-1ß is crucial for MSU-induced synovial inflammation. The aim of the present study was to investigate the mechanism of MSU crystal-induced autoinflammatory processes. METHODS: In vitro studies were used to evaluate the role of IL-6 in inflammasome activation in human neutrophils cultured with MSU crystals. Human neutrophils were stimulated with MSU in the presence or absence of IL-6 priming to determine NLRP3 inflammasome activation and subsequent cleaved caspase-1 induction or IL-1ß production. RESULTS: IL-6 or MSU stimulation alone did not result in the efficient IL-1ß production from human neutrophils. However, MSU stimulation induced marked IL-1ß production from IL-6-primed neutrophils. Pretreatment with baricitinib, which blocks IL-6 receptor signaling, prevented MSU-induced cleaved caspase-1 or IL-1ß induction in IL-6-primed neutrophils. Tocilizumab pretreatment also inhibited MSU-mediated IL-1ß production from IL-6-primed neutrophils. CONCLUSION: Priming of human neutrophils with IL-6 promotes uric acid-mediated IL-1ß secretion in the absence of microbial stimulation. These results suggest that an endogenous cytokine, IL-6, is involved in MSU-mediated NLRP3 inflammasome activation and subsequent IL-1ß production from innate immune cells and has a crucial role in MSU crystal-induced synovial inflammation. These findings provide insights into uric acid-mediated autoinflammation in the innate immune system.

Elevated serum levels of checkpoint molecules in patients with adult Still's disease.

BACKGROUND: The interaction between galectin-9 (Gal-9) and its ligand, T cell immunoglobulin, and mucin-containing-molecule-3 (TIM-3), one of the coinhibitory receptors, transduce the inhibitory signaling to regulate immune responses. The dysregulated expression of checkpoint molecules has been reported under various inflammatory or autoimmune conditions. The aim of this study is to investigate the levels of these checkpoint molecules and their associations between proinflammatory markers in patients with adult Still's disease (ASD). METHODS: Serum samples were collected from 47 patients with active ASD, 116 patients with rheumatoid arthritis (RA), and 37 healthy controls (HCs). Serum levels of Gal-9, soluble TIM-3 (sTIM-3), and IL-18 were determined using enzyme-linked immunosorbent assay (ELISA). Results were compared with the clinical features of ASD. RESULTS: Serum Gal-9 levels in patients with ASD (median: 21.57 ng/ml, interquartile range IQR [11.41-39.72]) were significantly higher compared to those in patients with RA (7.58 ng/ml, IQR [5.57-10.20] p < 0.001) as well as those in HCs (4.51 ng/ml, [IQR; 3.58-5.45], p < 0.001). Similarly, serum sTIM-3 levels in patients with ASD were significantly higher than those in patients with RA and HCs. Serum levels of Gal-9 or sTIM-3 showed positive correlations with IL-18 levels (Gal-9; r = 0.90, p < 0.001, sTIM-3; r = 0.78, p < 0.001) in patients with ASD. Serum levels of Gal-9 or sTIM-3 correlated with serum ferritin (Gal-9; r = 0.77, p < 0.001, sTIM-3; r = 0.71, p < 0.001) and ASD disease activity score (Pouchot's score, Gal-9; r = 0.66, p < 0.001, sTIM-3; r = 0.59, p < 0.001), whereas there was no significant correlation between serum Gal-9 or sTIM-3 and CRP. ASD patients with chronic arthritis phenotype had a significantly higher Gal-9/ferritin and sTIM-3/ferritin ratio than those without this phenotype. After immunosuppressive treatment, Gal-9 and sTIM-3 levels showed a significant decline in parallel to the disease activity scores. CONCLUSIONS: Serum levels of the coinhibitory checkpoint molecules were elevated and correlated with disease activity in patients with ASD. These coinhibitory checkpoint molecules may be implicated in the autoinflammatory process seen in ASD.

An Autopsy Case of Mixed Connective Tissue Disease Complicated by Thrombotic Thrombocytopenic Purpura.

We herein report a patient with mixed connective tissue disease (MCTD) who had been stable for years but suddenly developed thrombotic thrombocytopenic purpura (TTP). The patient showed a clinical pentad of signs of TTP, low activity of ADAMTS13, and positivity of anti-ADAMTS13 antibodies. She did not respond to plasma exchange or steroid therapy and died five days after admission. An autopsy revealed microthrombi in the brain, heart, kidney, adrenal glands, esophageal submucosa, and bone marrow as well as diffuse alveolar hemorrhaging. Physicians should bear in mind that TTP can occur in MCTD patients regardless of disease activity.

Differential regulation and correlation between galectin-9 and anti-CCP antibody (ACPA) in rheumatoid arthritis patients.

BACKGROUND: Galectin-9 (Gal-9) is involved in the regulatory process of immune responses or inflammation. The aim of the present study is to characterize circulating Gal-9 in patients with rheumatoid arthritis (RA) and its relationship with RA disease activity and phenotype. METHODS: A total of 116 RA patients and 31 age-matched healthy controls were included in this study. Disease activity of RA patients was determined by Disease Activity Score of 28 joint scoring system (DAS28-ESR). Levels of Gal-9 in serum were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Serum levels of Gal-9 were significantly higher in patients with RA compared to those in controls (median 7577 pg/ml [interquartile range (IQR) 5570-10,201] versus 4738 pg/ml [IQR 4267-5630], p = 0.001). There were significant differences in serum Gal-9 between RA patients with and without RA-ILD (9606 pg/ml [IQR 8522-12,167] versus 7078 pg/ml [IQR 5225-9447], p < 0.001) or those with and without advanced joint damage (stage II-IV, 9606 pg/ml [IQR 8522-12,167] versus 7078 pg/ml [IQR 5225-9447], p < 0.001). Although serum levels of Gal-9 correlated with the titers of ACPA (r = 0.275, p = 0.002), levels of ACPA titers conferred the different relationship, between serum Gal-9 and inflammatory mediators or RA disease activity. Although Gal-9 was correlated with ACPA titers (r = 0.508, p = 0.002), there was no correlation between Gal-9 levels and erythrocyte sedimentation rate (ESR), matrix metalloproteinase-3 (MMP-3), or DAS28-ESR in RA patients with high titers of ACPA (> 200 U/ml). Conversely, Gal-9 was correlated with MMP-3 (r = 0.300, p = 0.007) or DAS28-ESR (r = 0.331, p = 0.004) but not with ACPA titer in RA patients with low titers of ACPA titers (< 200 U/ml). CONCLUSIONS: Serum levels of Gal-9 were increased in RA patients and associated with RA disease activity in RA patients without high titers of ACPA. The levels of ACPA titers may influence the values of circulating Gal-9 in RA patients with various clinical phenotypes. These data suggest that Gal-9 possessed the properties of pro-inflammatory or arthropathic biomarker under the status of ACPA titers.

Galectin-9 as a biomarker for disease activity in systemic lupus erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by elevated interferon (IFN) signature genes. Galectin-9 (Gal-9) is a ß-galactoside-binding lectin that is reportedly useful as a biomarker for IFN gene signatures. In a cross-sectional study of Japanese patients with recent-onset SLE, we aimed to determine whether raised serum Gal-9 levels were associated with the disease activity or organ damage seen in SLE patients. METHODS: The current study included 58 Japanese patients with SLE and 31 age-matched healthy individuals. Disease activity and organ damage were assessed using SLE Disease Activity 2000 (SLEDAI-2K) and Systemic Lupus International Collaborating Clinics (SLICC) damage index. Serum and cerebrospinal fluid (CSF) Gal-9 concentrations were quantified using ELISA. Correlation analyses between Gal-9 and clinical parameters including disease activity were performed. RESULTS: Serum levels of Gal-9 were significantly increased in patients with SLE compared with the control group (16.6 ng/ml, [interquartile range (IQR); 3.6-59.7] versus 4.74 ng/ml, [IQR; 3.0-9.5], p<0.0001). Gal-9 was significantly correlated with disease activity measures in the SLEDAI-2K. Serum Gal-9 levels were significantly greater in patients with SLE-related organ involvement (23.1 ng/ml, [IQR; 5.1-59.7] versus 12.5ng/ml, [IQR; 3.6-39.0], p = 0.013). Whereas there was no difference in serum levels of CXCL10 or M2BPGi between patients with and without SLE-related organ involvement. Serum levels of Gal-9 were significantly higher in SLE patients with active renal involvement determined by BILAG renal score (A-B) compared to those without active renal involvement (C-E). Whereas there was no significant difference in serum levels of Gal-9 between SLE patients with or without active other organ involvements (neurological or hematological) determined by BILAG score. SLE patients with detectable circulating IFN-α had raised serum Gal-9 levels. Levels of Gal-9 were significantly higher in the CSF from patients with recent-onset neuropsychiatric SLE (NPSLE) than in those from non-SLE controls (3.5 ng/ml, [IQR; 1.0-27.2] versus 1.2 ng/ml, [IQR; 0.9-2.1], p = 0.009). CONCLUSIONS: Gal-9 could be a serologic marker of disease activity and organ involvement in SLE patients. Future studies evaluating the role of Gal-9 in the SLE phenotype may provide insights into SLE pathogenesis.

Effectiveness of Tocilizumab in juvenile patients with refractory Takayasu arteritis: Two case reports.

RATIONALE: Takayasu arteritis (TA) is a systemic large-vessel vasculitis which can be accompanied by the symptoms associated with vascular stenosis. PATIENT CONCERNS: We describe 2 female juveniles with TA who presented with progressive intermittent claudication. DIAGNOSIS: Contrast-enhanced computed tomography (CT) revealed the stenosis of femoral arteries and increased levels of C-reactive protein (CRP), and serum amyloid A (SAA) were noted in both patients. According to European league against rheumatism consensus criteria for the diagnosis of TA was confirmed in both patients. INTERVENTIONS: Both patients had shown resistance to glucocorticoids and treated with tocilizumab (TCZ) (subcutaneous injections, 162 mg/week). OUTCOMES: These treatments improved claudication symptoms. Follow-up imaging by enhanced CT revealed restoration of advanced stenosis of the femoral arteries in both patients. They achieved normalization of levels of the acute-phase reactants CRP and SAA. Serum levels of interleukin-6 were increased transiently after TCZ injection, but declined to within normal ranges at 12 weeks. LESSONS: Juvenile patients with TA presenting with advanced stenosis of the femoral arteries are not rare. The clinical courses of our patients suggested the beneficial effects of TCZ against the progressive vascular stenosis observed in refractory TA.

Hydroxychloroquine inhibits IL-1ß production from amyloid-stimulated human neutrophils.

BACKGROUND: Hydroxychloroquine (HCQ) is used for the treatment of patients with rheumatic diseases. We tested the hypothesis that HCQ affects the NLRP3 inflammasome, which is involved in autoinflammation. METHODS: Human neutrophils were stimulated with serum amyloid A (SAA) in vitro and measured for IL-1ß and caspase-1 (p20) secretion by ELISA. Pro-IL-1ß mRNA expression in human neutrophils was quantified by real-time RT-PCR. RESULTS: SAA stimulation induced significant production of IL-1ß in human neutrophils. SAA stimulation also induced NF-κB activation, pro-IL-1ß mRNA expression, and NLRP3 protein expression in human neutrophils. HCQ pretreatment significantly inhibited the SAA-induced IL-1ß production in human neutrophils, but did not affect the SAA-induced NF-κB activation, pro-IL-1ß mRNA expression, and NLRP3 protein expression. Furthermore, SAA stimulation induced cleaved caspase-1 (p20) secretion from human neutrophils, and this release was suppressed by HCQ pretreatment. CONCLUSIONS: Treatment with HCQ was associated with impaired production of IL-1ß in SAA-stimulated human neutrophils without affecting the priming process of the NLRP3 inflammasome such as pro-IL-1ß or NLRP3 induction. These findings suggest that HCQ affects the NLRP3 activation process, resulting in the impaired IL-1ß production in human neutrophils, as representative innate immune cells.

Intestinal Behçet disease associated with myelodysplastic syndrome accompanying trisomy 8 successfully treated with abdominal surgery followed by hematopoietic stem cell transplantation: A case report.

RATIONALE: Intestinal Behçet disease (BD) with myelodysplastic syndrome (MDS) is a rare condition that is resistant to various immunosuppressive therapies. Several cases in which hematopoietic stem cell transplantation (HSCT) was effective for intestinal BD with MDS accompanying trisomy 8 have been reported. PATIENT CONCERNS: We report an 18-year-old female with a 7-year history of BD. Colonoscopy demonstrated a huge ulcer in the cecum. Chromosomal examination revealed a karyotype of trisomy 8 in 87% of cells. Bone marrow examination revealed dysplastic cells in multilineages. DIAGNOSES: A diagnosis of intestinal BD associated with MDS accompanying trisomy 8 was made. INTERVENTIONS: The patient underwent ileocecal resection due to microperforations of ileocecal ulcers; she then underwent allogeneic peripheral blood stem cell transplantation (PBSCT) with her mother as a donor. OUTCOMES: After the PBSCT, the patient's symptoms due to BD (fever, oral aphthae, abdominal pain, and genital ulcers) completely disappeared, with no severe adverse events. LESSONS: The present case demonstrates that HSCT including PBSCT might be an effective new therapeutic option for refractory intestinal BD with MDS when immunosuppressive therapy has achieved insufficient efficacy.

Vasculo-Behçet disease complicated by conversion disorder diagnosed with 18F-fluoro-deoxy-glucose positron emission tomography combined with computed tomography (PET/CT).

Vasculitis in Behçet's disease, termed "vasculo-Behçet's disease," is a major cause of mortality and morbidity. We report a case of vasculo-Behçet's disease complicated by conversion disorder, in which 18F-fluoro-deoxy-glucose positron emission tomography combined with computed tomography (FDG PET/CT) was useful for the diagnosis. A twenty-two-year-old woman recently diagnosed with tonsillitis presented with fever, right foot pain, left equinovarus foot, and numbness in both hands and feet. Laboratory data showed elevated levels of c-reactive protein (CRP). The patient was positive for HLA B51; pathergy testing was also positive. Nerve conduction velocity and electromyography were normal. MRI showed swelling of the left crural muscle group. PET/CT showed intense FDG uptake in the left popliteal artery, demonstrating active vasculitis. The patient was diagnosed with vasculo-Behçet's disease and treated with corticosteroids, colchicine, and infliximab, which led to obvious improvement of the MRI findings and reduction in CRP. However, left equinovarus foot and numbness in the extremities persisted. She also developed aphonia. They were attributed to psychogenic dystonia and conversion disorder, and psychiatric treatment was effective in relieving those symptoms. We suggest that PET/CT may be useful for the early diagnosis of medium-sized vessel vasculitis in patients with Behçet's disease.

Clinical significance of myositis-specific autoantibody profiles in Japanese patients with polymyositis/dermatomyositis.

Myositis-specific autoantibodies, such as anti-melanoma differentiation associated gene 5 (MDA5) and anti-anti-amino acyl-tRNA synthetases (ARS) antibodies, are associated with interstitial lung diseases (ILD), which determine the prognosis of polymyositis/dermatomyositis (PM/DM) patients. However, there is a paucity of data on the clinical correlation between anti-Sjögren syndrome-related antigen A (anti-SSA)/Ro52 antibodies in PM/DM. We investigated the prevalence of myositis-specific autoantibodies including anti-SSA/Ro52 antibody and assessed the clinical significance of these antibodies in patients with PM/DM.We retrospectively reviewed demographic data and clinical outcomes in patients with PM/DM. The study population comprised 24 patients with PM and 60 patients with DM. The presence of anti-myositis-specific antibodies (MDA5, ARS, Jo-1, SSA/Ro52) was determined by immunosorbent assay (ELISA).Anti-MDA5 antibody was detected in 18 patients with DM (n = 60). Anti-ARS/anti-SSA/Ro52 antibodies were detected in 31 and 39 patients with PM/DM (n = 84). Rapidly progressive ILD patients were mainly found in the anti-MDA5 antibody-positive DM group. During the follow-up period, 9 patients died. Kaplan-Meier analysis demonstrated that survival rates seem to be lower in DM patients with anti-MDA5 antibodies compared with those without anti-MDA5 antibodies. Furthermore, dual positivity for anti-SSA/Ro52 and anti-MDA5 antibodies was significantly higher in nonsurviving DM patients compared with survivors.Although the presence of anti-ARS or anti-MDA5 antibodies is a prognostic marker in patients with PM/DM, combined presence of anti-SSA/Ro52 and anti-MDA5 antibodies represent another marker for clinical outcome in DM patients. Our results suggest that anti-SSA/Ro52 antibody positivity in DM patients with anti-MDA5 antibody reveals a subgroup of DM patients with poor prognosis.

Coexistence of Mixed Connective Tissue Disease and Familial Mediterranean Fever in a Japanese Patient.

We herein report a Japanese patient with familial Mediterranean fever (FMF) who developed the clinical manifestations of mixed connective tissue disease (MCTD) and Sjögren's syndrome. The patient was a 36-year-old woman presenting with a periodic short-duration (2-3 days) fever and pleural pain. An Mediterranean fever (MEFV) gene analysis detected a complex allele mutation (P369S/R408Q) in exon 3 of the MEFV gene. Serological and clinical data showed the coexistence of MCTD and Sjögren's syndrome. Treatment with colchicine (1.0 mg/day) successfully eliminated febrile attack and pleuritis, leading to the diagnosis of FMF. Four months after the initiation of colchicine treatment, she presented with MCTD-related pulmonary artery hypertension. This is the first report of FMF coexisting with MCTD.

Diagnostic value of ultrasonography in synovitis-acne-pustulosis-hyperostosis-osteitis (SAPHO) syndrome: A case report.

RATIONALE: Synovitis-acne-pustulosis-hyperostosis-osteitis (SAPHO) syndrome is a rare condition that affects the skin, bones, and joints. Diagnosis of SAPHO syndrome is established based on clinical manifestations and imaging features on radiography or magnetic resonance imaging. PATIENT CONCERNS: We report a 44-year-old male with a 20-year history of pustulosis who presented with pain in the lower extremities. Plain radiography demonstrated hyperostosis with subperiosteal erosions in the right tibia. Magnetic resonance imaging and computed tomography showed inflammatory accumulation, whereas musculoskeletal ultrasonography clearly depicted a periosteal reaction, osteitis, and enthesitis with abnormal blood flow in the surface of the right tibia. DIAGNOSES: A diagnosis of SAPHO syndrome was made. INTERVENTIONS: The patient was treated with combination therapy comprising prednisolone, methotrexate, and infliximab, which resulted in clinical improvement. OUTCOMES: The elevated levels of C-reactive protein and matrix metalloproteinase-3 normalized, and the abnormal ultrasonographic findings disappeared. LESSONS: The present case report demonstrates that multiple imaging modalities are important for the definitive diagnosis of SAPHO syndrome. Ultrasonography might be a useful tool for evaluating local musculoskeletal inflammation in patients with SAPHO syndrome.

Tofacitinib inhibits granulocyte-macrophage colony-stimulating factor-induced NLRP3 inflammasome activation in human neutrophils.

BACKGROUND: Granulocyte-macrophage colony-stimulating factor (GM-CSF) has emerged as a crucial cytokine that activates myeloid cells to initiate tissue inflammation. However, the molecular actions of GM-CSF against innate immunity are still poorly characterized. Here, we investigated the in vitro effects of GM-CSF on the activation of human myeloid lineages, neutrophils, and the underlying intracellular signaling mechanism, including inflammasome activation. METHODS: Human neutrophils were stimulated with GM-CSF in the presence or absence of tofacitinib. The cellular supernatants were analyzed for interleukin-1 beta (IL-1ß) and caspase-1 by enzyme-linked immunosorbent assay (ELISA) methods. Pro-IL-1ß mRNA expressions in human neutrophils were analyzed by real-time polymerase chain reaction. Protein phosphorylation of neutrophils was assessed by Western blot using phospho-specific antibodies. RESULTS: Stimulation with GM-CSF alone, but not tumor necrosis factor-alpha, was shown to increase the release of IL-1ß and cleaved caspase-1 (p20) from human neutrophils. Tofacitinib, which inhibits GM-CSF-induced Janus kinase 2 (Jak2)-mediated signal transduction, completely abrogated GM-CSF-induced IL-1ß and caspase-1 (p20) secretion from neutrophils. GM-CSF stimulation also induced pro-IL-1ß mRNA expression in neutrophils and induced NLR family pyrin domain-containing 3 (NLRP3) protein expression. Although tofacitinib pretreatment marginally inhibited GM-CSF-induced pro-IL-1ß mRNA expression, tofacitinib completely abrogated NLRP3 protein expression in neutrophils. CONCLUSIONS: These results indicate that GM-CSF signaling induces NLRP3 expression and subsequent IL-1ß production by affecting neutrophils, which may cause the activation of innate immunity. Therefore, GM-CSF is a key regulator of the NLRP3 inflammasome and IL-1ß production by activating innate immune cells. This process can be blocked by tofacitinib, which interferes with JAK/STAT signaling pathways.

Adding colchicine to immunosuppressive treatments; a potential option for biologics-refractory adult-onset Still's disease.

BACKGROUND: Adult-onset Still's disease (AOSD) is a rare inflammatory disorder characterized by the classical triad of daily spiking fever, arthritis, and typical salmon-colored rash. Resistance to first-line corticosteroids and second-line disease modified anti-rheumatic-drugs defines refractory AOSD, which mostly includes the polycyclic or chronic courses of the disease. Anti-cytokine therapies are recommended in AOSD patients who are refractory to traditional treatments. This is the first report on the efficacy of colchicine in a patient with AOSD which was refractory to immunosuppressive treatments including biologics. CASE PRESENTATION: A 24-years Japanese female patient was referred to our hospital for the flare-up of AOSD under the combined treatments with steroid, immunosuppressants, and biologics. She was diagnosed with AOSD according to the Yamaguchi criteria, based on the presence of spiking fever, polyarthralgia, skin rash, and hyperferritinemia. Interleukin-6 or tumor necrosis factor-α blockade treatments were not effective, the oral administration of colchicine was stared under the immunosuppressive treatments with steroid and cyclosporine A (CyA). Colchicine treatment silenced the disease activity of AOSD. The dose of prednisolone was successfully tapered, and the elevated levels of C-reactive protein were normalized. Remission has been maintained for 13 months with the start of oral administration of colchicine. CONCLUSION: We concluded that colchicine is an alternative treatment in patients with refractory AOSD, particularly in those with impaired therapeutic effects against anti-cytokines therapies.