Itaconate reduces proliferation and migration of fibroblast-like synoviocytes and ameliorates arthritis models.

Fibroblast-like synoviocytes (FLS) play critical roles in rheumatoid arthritis (RA). Itaconate (ITA), an endogenous metabolite derived from the tricarboxylic acid (TCA) cycle, has attracted attention because of its anti-inflammatory, antiviral, and antimicrobial effects. This study evaluated the effect of ITA on FLS and its potential to treat RA. ITA significantly decreased FLS proliferation and migration in vitro, as well as mitochondrial oxidative phosphorylation and glycolysis measured by an extracellular flux analyzer. ITA accumulates metabolites including succinate and citrate in the TCA cycle. In rats with type II collagen-induced arthritis (CIA), intra-articular injection of ITA reduced arthritis and bone erosion. Irg1-deficient mice lacking the ability to produce ITA had more severe arthritis than control mice in the collagen antibody-induced arthritis. ITA ameliorated CIA by inhibiting FLS proliferation and migration. Thus, ITA may be a novel therapeutic agent for RA.

Itaconate ameliorates autoimmunity by modulating T cell imbalance via metabolic and epigenetic reprogramming.

Dysregulation of Th17 and Treg cells contributes to the pathophysiology of many autoimmune diseases. Herein, we show that itaconate, an immunomodulatory metabolite, inhibits Th17 cell differentiation and promotes Treg cell differentiation by orchestrating metabolic and epigenetic reprogramming. Mechanistically, itaconate suppresses glycolysis and oxidative phosphorylation in Th17- and Treg-polarizing T cells. Following treatment with itaconate, the S-adenosyl-L-methionine/S-adenosylhomocysteine ratio and 2-hydroxyglutarate levels are decreased by inhibiting the synthetic enzyme activities in Th17 and Treg cells, respectively. Consequently, these metabolic changes are associated with altered chromatin accessibility of essential transcription factors and key gene expression in Th17 and Treg cell differentiation, including decreased RORγt binding at the Il17a promoter. The adoptive transfer of itaconate-treated Th17-polarizing T cells ameliorates experimental autoimmune encephalomyelitis. These results indicate that itaconate is a crucial metabolic regulator for Th17/Treg cell balance and could be a potential therapeutic agent for autoimmune diseases.

Inhibitor of NF-κB Kinase Subunit ε Contributes to Neuropsychiatric Manifestations in Lupus-Prone Mice Through Microglial Activation.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by multiorgan dysfunction. Neuropsychiatric SLE (NPSLE) occurs in 30-40% of lupus patients and is the most severe presentation of SLE, frequently resulting in limitation of daily life. Recent studies have shown that microglia, tissue-resident macrophages in the central nervous system, are involved in the pathogenesis of NPSLE. This study was undertaken to explore new therapeutic targets for NPSLE focusing on microglia. METHODS: RNA sequencing of microglia in MRL/lpr, lupus-prone mice, as well as that of microglia cultured in vitro with cytokines were performed. A candidate gene, which could be a therapeutic target for NPSLE, was identified, and its role in microglial activation and phagocytosis was investigated using specific inhibitors and small interfering RNA. The effect of intracerebroventricular administration of the inhibitor on the behavioral abnormalities of MRL/lpr was also evaluated. RESULTS: Transcriptome analysis revealed the up-regulation of Ikbke, which encodes the inhibitor of NF-κB kinase subunit ɛ (IKBKε) in both microglia from MRL/lpr mice and cytokine-stimulated microglia in vitro. Intracerebroventricular administration of an IKBKε inhibitor ameliorated cognitive function and suppressed microglial activation in MRL/lpr mice. Mechanistically, IKBKε inhibition reduced glycolysis, which dampened microglial activation and phagocytosis. CONCLUSION: These findings suggest that IKBKε plays a vital role in the pathogenesis of NPSLE via microglial activation, and it could serve as a therapeutic target for NPSLE.

Acquired factor V inhibitor with erythema and eosinophilia in a patient with end-stage renal disease.

Autoimmune factor V deficiency (AiFVD) is a rare bleeding disorder caused by factor V inhibitors. In this report, we present the case of an 89-year-old man who developed bleeding tendency during surgery to create arteriovenous fistula for hemodialysis. The bleeding tendency developed with prolongation of activated partial thromboplastin and prothrombin time, following drug-induced eruption and eosinophilia. Significant reduction in coagulation factor activity and inhibitory pattern in cross-mixing tests suggested the presence of inhibitors to coagulation factors. Subsequently, we detected a factor V inhibitor and anti-factor V autoantibodies was confirmed using enzyme-linked immunosorbent assay with purified human plasma factor V. Thus, the patient was 'definitely diagnosed' with AiFVD in accordance with the diagnostic criteria enacted by the Japanese Ministry of Health, Labor, and Welfare. The bleeding tendency improved after initiating oral prednisolone 50 mg (1 mg/kg) followed by normalization of activated partial thromboplastin time and prothrombin time at the 34th day. After improving the coagulation system prolongation, the inhibitor and autoantibodies has been eradicated. Since it is suggested that drug-induced immune response can cause AiFVD, AiFVD should be considered in patients who undergo hemodialysis and develop failure of hemostasis and drug-induced eruption.

Acute compartment syndrome of the lower limb after revision anterior cruciate ligament reconstruction: a case report.

Introduction: Acute compartment syndrome (ACS) is one of the most serious orthopedic diseases causing complications and requiring emergency surgery. Most cases of ACS are associated with fractures and crush injuries. However, surgical procedures can also cause ACS. Case presentation: We herein describe a 41-year-old man who underwent arthroscopic surgery for revision anterior cruciate ligament reconstruction with the semitendinosus tendon. Two days after the operation, the patient developed ACS of the left lower leg. This patient had undergone anterior cruciate ligament(ACL) reconstruction previously, and we assumed that the cause of the ACS was damage to the small blood vessels from the collateral circulation during hamstring tendon harvesting. Fasciotomy was performed under general anesthesia, and his postoperative course was uneventful. Conclusion: ACS after revision anterior cruciate ligament reconstruction is rare. We should keep in mind that patients who have undergone previous surgery may develop ACS due to damage to the collateral circulation. To avoid ACS, harvesting tendon from the healthy side can avoid damage to the collateral circulation.

Changes in patellar baja progress until 3 months after medial open-wedge high tibial osteotomy.

PURPOSE: The purpose of this study was to evaluate sequential patellar height changes as well as the factors leading to these changes after medial open-wedge high tibial osteotomy (MOWHTO). METHODS: The study cohort constituted 37 knees from 36 patients who underwent MOWHTO for varus knee. The Caton-Deschamps index (CDI) for patellar height was measured preoperatively and at 2 weeks and 3, 6, and 12 months postoperatively. The factors related to sequential changes in patellar height were evaluated. RESULTS: Significant differences were observed between preoperative CDI and postoperative CDI at all time points (p < .05). Two-week postoperative CDI and postoperative CDI at 3,6,12-months was also significantly different (p < .05). The only significant factor for the change in patellar height between preoperative CDI and postoperative CDI at 2-weeks and 12-months was the change in the Δ medial proximal tibial angle (ΔMPTA) (postoperative MPTA-preoperative MPTA). We could not identify the factor that affected the change in patellar height change from 2-weeks postoperatively. CONCLUSION: The change in patellar height continued sequentially until at least 3 months postoperatively. ΔMPTA was associated with the change in patellar height at 2 weeks postoperatively compared with preoperatively; however, no factors associated with the change in patellar height from 2 weeks postoperatively to 3, 6, and 12-months postoperatively were identified.

Moyamoya Disease Associated with a Deficiency of Complement Component 6.

OBJECTIVES: Complement component 6 (C6) deficiency is a very rare genetic defect that leads to significantly diminished synthesis, secretion, or function of C6. In the current report, we demonstrate a previously undescribed, homozygous missense mutation in exon 17 of the C6 gene (c.2545A>G p.Arg849Gly) in a 35-year-old Japanese woman with moyamoya disease and extremely low levels of CH50 (<7.0 U/mL). MATERIALS AND METHODS: The complement gene analysis using hybridization capture-based next generation sequencing was performed. CH50 was determined in patient's plasma mixed with plasma from a healthy donor or purified human C6 protein. Western blot was performed on patient's plasma using polyclonal antibodies against C6, with healthy donor's plasma and purified human C6 protein as positive controls while C6-depleted human serum as a negative control. The carriage of ring finger protein 213 variant (c.14576G>A p.Arg4859Lys), a susceptibility gene for moyamoya disease, was examined by direct sequencing. RESULTS: CH50 mixing test clearly showed a deficiency pattern, being rescued by addition of only 1% healthy donor's plasma or 1 µg/mL purified human C6 protein (1/50-1/100 of physiological concentration). Western blot revealed the absence of C6 protein in the patient's plasma, confirming a quantitative deficiency of C6. The ring finger protein 213 variant was not detected. CONCLUSIONS: Our data implies that unrecognized complement deficiencies would be harbored in cerebrovascular diseases with unknown etiologies.

Protective Role of Optineurin Against Joint Destruction in Rheumatoid Arthritis Synovial Fibroblasts.

OBJECTIVE: Optineurin (OPTN) is an autophagy adaptor/receptor that acts as an intrinsic negative regulator of osteoclast differentiation. RANKL expressed by rheumatoid arthritis synovial fibroblasts (RASFs) is primarily responsible for the development of bone erosions in patients with RA. The aim of the present study was to explore the role of OPTN in the pathogenesis of joint destruction in RA. METHODS: RASFs were left untreated or incubated with tumor necrosis factor (TNF) or interferon-γ (IFNγ), and expression of OPTN by RASFs was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Expression of RANKL and osteoprotegerin (OPG) was evaluated in cultures of OPTN-reduced RASFs with or without TNF or IFNγ treatment. OPTN-reduced RASFs were cocultured with monocytes and stained for tartrate-resistant acid phosphatase (TRAP). IκBα, NF-κB1, and RelA protein levels were measured to evaluate NF-κB signaling. Expression of messenger RNA (mRNA) for matrix metalloproteinase 3 (MMP3), interleukin-6 (IL6), GATA binding protein 3 (GATA3), carbohydrate sulfotransferase 15 (CHST15), hyaluronan synthase 1 (HAS1), and GATA1 was analyzed by RT-qPCR. RESULTS: In RASFs incubated with TNF or IFNγ, OPTN expression was up-regulated and RANKL expression was increased, and these effects were further pronounced in OPTN-reduced RASFs (all P < 0.05 versus controls). OPG mRNA levels remained unchanged. Monocytes cocultured with OPTN-reduced RASFs differentiated to a greater extent into TRAP+ multinucleated cells compared to monocytes cocultured with control RASFs (P < 0.05). IκBα degradation and nuclear NF-κB1 expression following TNF treatment were both prolonged in OPTN-reduced RASFs (each P < 0.05 versus controls). MMP3 mRNA levels were up-regulated, while GATA3, CHST15, and HAS1 mRNA levels were down-regulated in OPTN-reduced RASFs (each P < 0.05 versus controls). CONCLUSION: OPTN plays a protective role in RA when it is up-regulated in RASFs in the presence of proinflammatory cytokines. Absence of OPTN might worsen RA by generating a joint-destructive state, as indicated by evidence of increased RANKL expression on RASFs and subsequent osteoclast differentiation.

MicroRNA-9 ameliorates destructive arthritis through down-regulation of NF-κB1-RANKL pathway in fibroblast-like synoviocytes.

We investigated the effect of miR-9 on fibroblast-like synoviocytes (FLS) from RA patients and animal arthritis model. The binding of miR-9 to NF-κB1 3'UTR was analyzed by luciferase reporter assay and immunoprecipitation. ChIP assay and luciferase promoter assay were performed to identify the binding of NF-κB1 to RANKL promoter and its activity. FLS were treated with miR-9/anti-miR-9 to evaluate cell proliferation and the expression of RANKL. Therapeutic effect of intra-articular miR-9 was evaluated in type-II collagen-induced arthritis in rats. miR-9 bound to the 3'-UTR of NF-κB1 and downregulated NF-κB1. NF-κB1 bound to RANKL promoter and increased the promoter activity of RANKL. RANKL was downregulated by miR-9. Proliferation of FLS was increased by miR-9 inhibitor. miR-9 dampened experimental arthritis by lowering inflammatory state, reducing RANKL and osteoclasts formation. Our findings revealed miR-9-NF-κB1-RANKL pathway in RA-FLS, further, miR-9 ameliorated inflammatory arthritis in vivo which propose therapeutic implications of miR- 9 in RA.

Autophagy promotes citrullination of VIM (vimentin) and its interaction with major histocompatibility complex class II in synovial fibroblasts.

We aimed to investigate the involvement of macroautophagy/autophagy in autoimmunity in rheumatoid arthritis (RA) through citrullination of VIM (vimentin) and its interaction with MHC class II in synovial fibroblasts (SFs). The cell surface expression of MHC class II and B7 costimulatory molecules on SFs was analyzed by flow cytometry after treatment with IFNG/IFN-γ (interferon gamma). Intracellular citrullinated autoantigens in SFs were analyzed by immunoblotting using serum from anti-citrullinated peptide antibodies (ACPA)-positive patient as a primary antibody. SFs were incubated in serum-free medium or treated with proteasome inhibitor MG132 to induce autophagy. An autophagy inhibitor 3-methyladenin (3-MA) was used. Intracellular citrullinated VIM (cVIM) was evaluated by immunoblotting and immunocytochemistry. The interaction between MHC class II and cVIM was evaluated with co-immunoprecipitation and proximity ligation assay (PLA). We demonstrated that MHC class II, CD274/B7-H1 and PDCD1LG2/B7-DC were expressed on SFs following treatment with IFNG whereas CD276/B7-H3 was detected on SFs regardless of the presence of IFNG. ACPA-positive sera recognized a 54 kDa protein in SFs. By immunoprecipitation, the 54 kDa protein recognized by RA sera was revealed to be cVIM. Following induction of autophagy, intracellular cVIM was increased in SFs but the effect was canceled by 3-MA. The interaction between MHC class II and cVIM was demonstrated by co-immunoprecipitation. Furthermore, PLA revealed the significant increase of MHC class II-cVIM interaction following induction of autophagy. Our findings suggest that SFs may contribute to the autoimmunity in RA through citrullination of VIM and its interaction with MHC class II promoted by autophagy.Abbreviations: 3-MA: 3-methyladenine; ACPA: anti-citrullinated peptide antibodies; anti-CCP: anti-cyclic citrullinated peptide antibody; cVIM: citrullinated VIM; BECN1: beclin1; DAPI: 4',6-diamidino-2-phenylindole; FBS: fetal bovine serum; HLA: human leukocyte antigen; IFNG/IFN-γ: interferon gamma; IL6: interleukin 6; IP: immunoprecipitation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFI: mean fluorescence index; MHC: major histocompatibility complex; OA: osteoarthritis; PADI: peptidyl arginine deiminase; PepA: pepstatin A; PBS: phosphate-buffered saline; PtdIns3K: phosphatidylinositol 3-kinase; RA: rheumatoid arthritis; SFs: synovial fibroblasts; siRNA: small interfering RNA; VIM: vimentin.

Draft Genome Sequence of the Phenol-Degrading Bacterium Cupriavidus sp. Strain P-10, Isolated from Trichloroethene-Contaminated Aquifer Soil.

A batch culture was enriched on phenol with trichloroethene-contaminated aquifer soil as an inoculum. Cupriavidus sp. strain P-10 was isolated from the culture using a diluted plating method. Here, we report the draft genome sequence and annotation of strain P-10, which provides insights into the metabolic processes of phenol degradation.

CHK1 cleavage in programmed cell death is intricately regulated by both caspase and non-caspase family proteases.

BACKGROUND: CHK1 is an important effector kinase that regulates the cell cycle checkpoint. Previously, we showed that CHK1 is cleaved in a caspase (CASP)-dependent manner during DNA damage-induced programmed cell death (PCD) and have examined its physiological roles. METHODS AND RESULTS: In this study, we investigated the behavior of CHK1 in PCD. Firstly, we found that CHK1 is cleaved at three sites in PCD, and all cleavages were inhibited by the co-treatment of a pan-CASP inhibitor or serine protease inhibitors. We also showed that CHK1 is cleaved by CASP3 and/or CASP7 recognizing at (296)SNLD(299) and (348)TCPD(351), and that the cleavage results in the enhancement of CHK1 kinase activity. Furthermore, as a result of the characterization of cleavage sites by site-directed mutagenesis and an analysis performed using deletion mutants, we identified (320)EPRT(323) as an additional cleavage recognition sequence. Considering the consensus sequence cleaved by CASP, it is likely that CHK1 is cleaved by non-CASP family protease(s) recognizing at (320)EPRT(323). Additionally, the cleavage catalyzed by the (320)EPRT(323) protease(s) markedly and specifically increased when U2OS cells synchronized into G1 phase were induced to PCD by cisplatin treatment. CONCLUSION: CHK1 cleavage is directly and indirectly regulated by CASP and non-CASP family proteases including serine protease(s) and the "(320)EPRT(323) protease(s)." Furthermore, (320)EPRT(323) cleavage of CHK1 occurs efficiently in PCD which is induced at the G1 phase by DNA damage. GENERAL SIGNIFICANCE: CASP and non-CASP family proteases intricately regulate cleavage for up-regulation of CHK1 kinase activity during PCD.

DCK is frequently inactivated in acquired gemcitabine-resistant human cancer cells.

Although gemcitabine is the most effective chemotherapeutic agent against pancreatic cancer, a growing concern is that a substantial number of patients acquire gemcitabine chemoresistance. To elucidate the mechanisms of acquisition of gemcitabine resistance, we developed gemcitabine-resistant cell lines from six human cancer cell lines; three pancreatic, one gastric, one colon, and one bile duct cancer. We first analyzed gemcitabine uptake using three paired parental and gemcitabine resistant pancreatic cancer cell lines (PK-1 and RPK-1, PK-9 and RPK-9, PK-59 and RPK-59) and found that uptake of gemcitabine was rapid. However, no DNA damage was induced in resistant cells. We further examined the microarray-based expression profiles of the cells to identify genes associated with gemcitabine resistance and found a remarkable reduction in the expression of deoxycytidine kinase (DCK). DCK is a key enzyme that activates gemcitabine by phosphorylation. Genetic alterations and expression of DCK were studied in these paired parental and derived gemcitabine-resistant cell lines, and inactivating mutations were found only in gemcitabine-resistant cell lines. Furthermore, siRNA-mediated knockdown of DCK in the parental cell lines yielded gemcitabine resistance, and introduction of DCK into gemcitabine-resistant cell lines invariably restored gemcitabine sensitivities. Mutation analyses were expanded to three other different paired cell lines, DLD-1 and RDLD-1 (colon cancer cell line), MKN-28 and RMKN-28 (gastric cancer cell line), and TFK-1 and RTFK -1 (cholangiocarcinoma cell line). We found inactivating mutations in RDLD-1 and RTFK-1 and decreased expression of DCK in RMKN-28. These results indicate that the inactivation of DCK is one of the crucial mechanisms in acquisition of gemcitabine resistance.

Preparation of two-dimensionally ordered microbeads structure dispensed with an ink-jet and its application to ELISA.

In order to the increase sensitivity of enzyme-linked immunosorbent assay (ELISA) using micro-droplet reaction system formed by ink-jet, we employed small dots of two-dimensionally well-ordered structure of polystyrene (PS) microbeads (710 +/- 25 microm in diameter) formed on the surface of polydimethyl siloxane (PDMS). An aqueous suspension of polystyrene microbeads (5 microm) was ejected on the PDMS plate with ink-jet. The PS beads were automatically assembled by capillary force accompanied with solvent evaporation. The evaporation rate was controlled by ambient relative humidity. The assembled beads were solidly immobilized on the surface of PDMS surface. The dots of well-ordered structure were stable against vigorous washing. ELISA using the structure as a reaction location was three times more sensitive than the material without the structure due to the increase of the surface area and consequent increase of the amount of antibody.

Checkpoint kinase 1 is cleaved in a caspase-dependent pathway during genotoxic stress-induced apoptosis.

Checkpoint kinase 1 (Chk1) plays important roles in genotoxic stress-induced cell cycle checkpoint and in normal cell cycle progression. Here, we show that Chk1 is cleaved in the treatment of apoptotic dose of etoposide (ETP) or cisplatin (CIS) but not of viable dose in HeLa S3 cells. The cleavage of Chk1 was completely inhibited by an irreversible and cell-permeable pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (z-VAD-fmk). These results identify Chk1 as a novel substrate that is cleaved by a caspase-dependent manner during genotoxic stress-induced apoptosis. Our data may also indicate the existence of a novel Chk1-regulated apoptotic pathway.

Development of a surface-reaction system in a nanoliter droplet made by an ink-jet microchip.

A surface-reaction system in a nanoliter water pool using an ink-jet microchip was developed. The reaction system in the nanodroplets formed on a poly(dimethylsiloxane) (PDMS) coated glass slide increased the diffusion-controlled reaction without using a nano-pump, specialized connector or highly sensitive detector. When nanoliter droplets were placed on the PDMS surface with a distance of 100 microm between them by the ink-jet microchip, the repeatabilities of the fluorescence intensity were 2.9% RSD (n = 7). The used ink-jet microchip had 4 different injection ports, and the distance between the ports was 0.995 mm. It was necessary to correct the distance in order to mix or dilute samples in a small droplet. The correction was successfully performed by moving the X-Y stage using inhouse-made software. A linear relationship was obtained between the Resorufin concentrations and the fluorescence intensity. We applied this system to an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin A (IgA), and observed a difference in the fluorescence intensity derived from the amount of IgA (blank, 6.25 ng/mL, 12.5 ng/mL). These results show the usefulness of the open-type micro-analytical systems proposed by us.

A flow-based enzyme-linked immunosorbent assay on a polydimethylsiloxane microchip for the rapid determination of immunoglobulin A.

A flow-based enzyme-linked immunosorbent assay (ELISA) on a polydimethylsiloxane (PDMS) microchip has been developed for the rapid determination of immunoglobulin A (IgA). The analytical principle of this integrated method is the same as the conventional sandwich-type ELISA. A primary antibody (anti-IgA) was adsorbed on the surface of a PDMS microchannel, and then an antigen (IgA) and a secondary antibody (anti-IgA HRP labeled) were reacted successively. The resulting antigen-antibody complex, fixed on the surface of the microchannel, was detected using Amplex((R)) Red and a fluorescent imaging system. The calibration curve of the IgA standard solution was linear in the range of 0-50ng/mL at the flow rate of 10muL/min. This flow rate corresponds to the reaction time of 4.8s. Compared to the conventional assay on a 96-well microtiter plate, the present assay on the microchip dramatically shortened the reaction time necessary for the enzyme-substrate reaction from 30min to 4.8s, i.e., to 1/375. The amounts of the reagent and sample were also reduced to 1/100 compared to the 96-well microtiter plate.