Yujin Ye

Metabolic Disturbances Associated with Systemic Lupus Erythematosus

The metabolic disturbances that underlie systemic lupus erythematosus are currently unknown. A metabolomic study was executed, comparing the sera of 20 SLE patients against that of healthy controls, using LC/MS and GC/MS platforms. Validation of key differences was performed using an independent cohort of 38 SLE patients and orthogonal assays. SLE sera showed evidence of profoundly dampened glycolysis, Krebs cycle, fatty acid β oxidation and amino acid metabolism, alluding to reduced energy biogenesis from all sources. Whereas long-chain fatty acids, including the n3 and n6 essential fatty acids, were significantly reduced, medium chain fatty acids and serum free fatty acids were elevated. The SLE metabolome exhibited profound lipid peroxidation, reflective of oxidative damage. Deficiencies were noted in the cellular anti-oxidant, glutathione, and all methyl group donors, including cysteine, methionine, and choline, as well as phosphocholines. The best discriminators of SLE included elevated lipid peroxidation products, MDA, gamma-glutamyl peptides, GGT, leukotriene B4 and 5-HETE. Importantly, similar elevations were not observed in another chronic inflammatory autoimmune disease, rheumatoid arthritis. To sum, comprehensive profiling of the SLE metabolome reveals evidence of heightened oxidative stress, inflammation, reduced energy generation, altered lipid profiles and a pro-thrombotic state. Resetting the SLE metabolome, either by targeting selected molecules or by supplementing the diet with essential fatty acids, vitamins and methyl group donors offers novel opportunities for disease modulation in this disabling systemic autoimmune ailment.

Antiinflammatory effect of Rho kinase blockade via inhibition of NF‐κB activation in rheumatoid arthritis

OBJECTIVE There is increasing evidence that the RhoA signaling pathway may play a critical role in the inflammatory response. This study was undertaken to examine the effects of RhoA and its downstream effector Rho kinase (ROK) in synovial inflammation in rheumatoid arthritis (RA). METHODS RhoA activity was assessed by pull-down assay. Fasudil and Y27632, both specific inhibitors of ROK, were used to examine the role of ROK in inflammatory responses in vivo and in vitro. Nuclear translocation of NF-kappaB was measured by confocal fluorescence microscopy, and DNA binding activity was assessed with a sensitive multiwell colorimetric assay. Enzyme-linked immunosorbent assay was used to detect cytokine production. RESULTS Increased activation of RhoA was found in inflamed synovial membrane cells isolated from patients with RA and from rats with collagen-induced arthritis (CIA). Intraperitoneal administration of fasudil in rats with CIA significantly reduced synovial inflammation and ROK activity. In vitro, treatment with fasudil or Y27632 decreased production of tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and IL-6 by synovial membrane cells, peripheral blood mononuclear cells, and fibroblast-like synoviocytes from patients with active RA. Inhibition of ROK by specific inhibitors or ROK small interfering RNA suppressed lipopolysaccharide- or TNFalpha-induced NF-kappaB nuclear translocation, DNA binding activity, luciferase reporter gene expression, and IkappaBalpha degradation. CONCLUSION The results of this study provide new evidence that blockade of ROK inhibits activation of NF-kappaB and production of proinflammatory cytokines, suggesting a critical role of ROK in the synovial inflammation of RA. Specific inhibition of ROK may be a novel therapeutic approach in RA.

The anti-malaria agent artesunate inhibits expression of vascular endothelial growth factor and hypoxia-inducible factor-1α in human rheumatoid arthritis fibroblast-like synoviocyte

Increasing evidence indicates that the anti-malarial agent artemisinin and its derivatives may exert anti-angiogenic effect. In the present study, we explored the effect of artesunate, a artemisinin derivative, on TNFα- and hypoxia-induced expression of hypoxia inducible factor-1α (HIF-1α) and secretion of vascular endothelial growth factor (VEGF) and inteleukin-8 (IL-8) in human rheumatoid arthritis fibroblast-like synoviocytes (RA FLS), and further investigated the signal mechanism by which this compound modulates HIF-1α, VEGF and IL-8 expression. RA FLS obtained from patients with active rheumatoid arthritis were pretreated with artesunate, and then stimulated with TNFα and hypoxia. Production of VEGF and IL-8 was measured by ELISA. Nuclear location of HIF-1α was measured by confocal fluorescence microscopy. HIF-1α and other signal transduction proteins expression was measured by Western blot. Artesunate decreased the secretion of VEGF and IL-8 from TNFα- or hypoxia-stimulated RA FLS in a dose-dependent manner. Artesunate also inhibited TNFα- or hypoxia-induced nuclear expression and translocation of HIF-1α. We also showed that artesunate prevented Akt phosphorylation, but did not find evidence that phosphorylation of p38 and ERK was affected. TNFα- or hypoxia-induced secretion of VEGF and IL-8 and expression of HIF-1α were hampered by treatment with the PI3 kinase inhibitor LY294002, suggesting that inhibition of PI3 kinase/Akt activation might inhibit VEGF and IL-8 secretion and HIF-1α expression induced by TNFα or hypoxia. Our results suggest that artesunate inhibits angiogenic factor expression in RA FLS, and provide novel evidence that, as a low-cost agent, artesunate may have therapeutic potential for RA.

Clinical features and independent predictors of pulmonary arterial hypertension in systemic lupus erythematosus

Pulmonary arterial hypertension (PAH) is a devastating complication of systemic lupus erythematosus (SLE). We aim to estimate the putative predictors contributing to early identification of PAH, thus improve appropriate medical intervention and a better prognosis. A retrospective case–control study was conducted. Forty-one SLE patients with PAH and 106 SLE patients without PAH were enrolled. Demographic variables, clinical features, and laboratory data were compared between the two groups. Univariate and multivariate logistic regression models were used to examine the predictors contributing to PAH in SLE. Serositis, Raynaud’s phenomenon, high disease activity, anticardiolipin antibodies, and anti-U1RNP were significantly associated with SLE-PAH. Univariate and multivariate analysis showed that Raynaud’s phenomenon, anticardiolipin antibodies, and anti-U1RNP were independent predictors of PAH in SLE. This study highlighted the clinical pattern of SLE-PAH patients, and underlined the leading predictors of PAH development among patients with SLE. Routine echocardiography is recommended in SLE patients with the independent predictors mentioned above.

Inflammation associated anemia and ferritin as disease markers in SLE

IntroductionIn a recent screening to detect biomarkers in systemic lupus erythematosus (SLE), expression of the iron storage protein, ferritin, was increased. Given that proteins that regulate the storage, transfer and release of iron play an important role in inflammation, this study aims to determine the serum and urine levels of ferritin and of the iron transfer protein, transferrin, in lupus patients and to correlate these levels with disease activity, inflammatory cytokine levels and markers of anemia.MethodsA protein array was utilized to measure ferritin expression in the urine and serum of SLE patients and healthy controls. To confirm these results as well as the role of the iron transfer pathway in SLE, ELISAs were performed to measure ferritin and transferrin levels in inactive or active SLE patients and healthy controls. The relationship between ferritin/transferrin levels and inflammatory markers and anemia was next analyzed.ResultsProtein array results showed elevated ferritin levels in the serum and urine of lupus patients as compared to controls, which were further validated by ELISA. Increased ferritin levels correlated with measures of disease activity and anemia as well as inflammatory cytokine titers. Though active SLE patients had elevated urine transferrin, serum transferrin was reduced.ConclusionUrine ferritin and transferrin levels are elevated significantly in SLE patients and correlate with disease activity, bolstering previous reports. Most importantly, these changes correlated with the inflammatory state of the patients and anemia of chronic disease. Taken together, altered iron handling, inflammation and anemia of chronic disease constitute an ominous triad in SLE.

Bromodomain and extra-terminal domain bromodomain inhibition prevents synovial inflammation via blocking IκB kinase–dependent NF-κB activation in rheumatoid fibroblast-like synoviocytes

OBJECTIVE To explore the roles of the bromodomain (Brd) and extra-terminal domain (BET) of chromatin adaptors in regulating synovial inflammation in RA. METHODS Fibroblast-like synoviocytes (FLSs) were isolated from synovial tissue from RA patients. A specific BET inhibitor, JQ1, and short hairpin RNA (shRNA) for Brd2 or Brd4 were used to evaluate the role of the BET Brd in inflammatory responses. Protein expression was measured by western blot or immunofluorescence staining. Nuclear factor kappa B (NF-κB) gene activity was detected by luciferase assay. The secretion and gene expression of cytokines and MMPs were evaluated by ELISA and real-time PCR, respectively. FLS proliferation was detected by BrdU incorporation. RESULTS Four Brd proteins, including Brd2, Brd3, Brd4 and Brdt, were expressed in FLSs from patients with RA and OA; however, the expression of Brd2 and Brd4 was increased in RA compared with that in OA. Treatment with JQ1, Brd2 shRNA or Brd4 shRNA decreased the production of pro-inflammatory cytokines (TNFα, IL-1β, IL-6 and IL-8), MMPs expression (MMP-1, MMP-3 and MMP-13) and proliferation by RA FLSs. BET inhibition downregulated TNFα-induced NF-κB-dependent transcription and expression of the NF-κB target genes. JQ1 suppressed the phosphorylation of IκB kinaseβ and IκBα, and nuclear translocation of p65. Intraperitoneal injection of JQ1 in mice with collagen-induced arthritis reduced synovial inflammation, joint destruction and serum levels of the anti-CII antibodies TNFα and IL-6. CONCLUSION This study implicates BET Brds as important regulators of IκB kinase/NF-κB-mediated synovial inflammation of RA and identifies BET proteins as novel therapeutic targets in inflammatory arthritis.

Lupus mesenteric vasculitis: Clinical features and associated factors for the recurrence and prognosis of disease

OBJECTIVE To evaluate the clinical characteristics of lupus mesenteric vasculitis (LMV) and identify the potential factors and appropriate treatments that are associated with disease relapse and prognosis in LMV. METHODS A retrospective cohort study was performed among patients admitted to the First Affiliated Hospital of Sun Yet-sen University between 2002 and 2011. Demographic information, clinical symptoms, laboratory findings, imaging characteristics like abdominal CT scan, ultrasonography, medications including corticosteroid, cyclophosphamide, and other immunosuppressive agents, and outcomes were documented. The endpoints of the study were defined as occurrence of severe complications that needed surgical intervention, disease recurrence, or death. RESULTS Out of 3823 systemic lupus erythematosus (SLE) patients, 97 were diagnosed with mesenteric vasculitis with the overall prevalence of 2.5%. Among these 97 LMV patients, 13 died because of serious complications (13/97, 13.4%) and 2 presented intestinal perforation during the induction therapy stage. The logistic regression multivariate analysis indicated that leukopenia [peripheral WBC, odds ratio (OR) = 0.640, 95% confidence interval (CI): 0.456-0.896, P = 0.009], hypoalbuminemia (serum albumin, OR = 0.891, 95% CI: 0.798-0.994, P = 0.039) and elevated serum amylase (OR = 7.719, 95% CI: 1.795-33.185, P = 0.006) were positively associated with the occurrence of serious complications, while intravenous cyclophosphamide (CYC) therapy inhibited the occurrence of serious complications (OR = 0.220, 95% CI: 0.053-0.903, P = 0.036). A total of 79 patients who achieved remission were followed-up for 2-96 months and 18 cases experienced disease relapse (18/79, 22.8%). The statistical analysis adjusted by Cox proportional hazards models indicated that high-dose CYC therapy (≥ 1.0 g/m(2)/month) was a protective factor for disease relapse and led to better outcomes [hazard ratio (HR) = 0.209, 95% CI: 0.049-0.887, P = 0.034], while the severe thickness of the bowel wall (>8mm) was a risk factor (HR = 7.308, 95% CI: 1.740-30.696, P = 0.007). LMV and lupus cystitis occurred concurrently in 22 (22/97, 22.7%) patients, and the symptoms of urinary tract resolved after treatment with corticosteroid and immunosupressants. CONCLUSION LMV is one of the serious complications of SLE with high mortality. The current study demonstrated that leukopenia, hypoalbuminemia, and elevated serum amylase were associated with severe adverse events, while CYC therapy led to better outcomes during remission-induction stage. Severe thickness of the bowel was a risk factor while high-dose CYC therapy was a protective factor for disease relapse in intensification therapy stage. It is necessary to evaluate the urinary tract involvement once LMV is diagnosed due to the frequent coexistence of these 2 diseases.

Protein Inhibitor of Activated STAT3 Regulates Migration, Invasion, and Activation of Fibroblast-like Synoviocytes in Rheumatoid Arthritis

The aggressive phenotype displayed by fibroblast-like synoviocytes (FLSs) is a critical factor of cartilage destruction in rheumatoid arthritis (RA). Increased FLSs migration and subsequent degradation of the extracellular matrix are essential to the pathology of RA. Protein inhibitor of activated STAT (PIAS), whose family members include PIAS1, PIAS2 (PIASx), PIAS3, and PIAS4 (PIASy), play important roles in regulating various cellular events, such as cell survival, migration, and signal transduction in many cell types. However, whether PIAS proteins have a role in the pathogenesis of RA is unclear. In this study, we evaluated the role of PIAS proteins in FLSs migration, invasion, and matrix metalloproteinases (MMPs) expression in RA. We observed increased expression of PIAS3, but not PIAS1, PIAS2, or PIAS4, in FLSs and synovial tissues from patients with RA. We found that PIAS3 knockdown by short hairpin RNA reduced migration, invasion, and MMP-3, MMP-9, and MMP-13 expression in FLSs. In addition, we demonstrated that PIAS3 regulated lamellipodium formation during cell migration. To gain insight into molecular mechanisms, we evaluated the effect of PIAS3 knockdown on Rac1/PAK1 and JNK activation. Our results indicated that PIAS3-mediated SUMOylation of Rac1 controlled its activation and modulated the Rac1 downstream activity of PAK1 and JNK. Furthermore, inhibition of Rac1, PAK1, or JNK decreased migration and invasion of RA FLSs. Thus, our observations suggest that PIAS3 suppression may be protective against joint destruction in RA by regulating synoviocyte migration, invasion, and activation.

HMG-CoA reductase inhibitor simvastatin suppresses Toll-like receptor 2 ligand-induced activation of nuclear factor kappa B by preventing RhoA activation in monocytes from rheumatoid arthritis patients

To investigate whether anti-inflammatory effects of HMG-CoA reductase inhibitor simvastatin (SMV) in rheumatoid arthritis (RA) is mediated by Toll-like receptor-2 (TLR-2) signal via inhibiting activation of RhoA, a small Rho GTPase that plays an important role in inflammatory responses. Peripheral blood monocytes from active RA patients were treated with Staphylococcus aureus peptidoglycan (PG), a ligand of TLR-2, in the presence or absence of SMV. RhoA activity was assessed by a pull-down assay. DNA-binding activity was measured by a sensitive multi-well colorimetric assay. Cytokine secretion was measured by ELISA. PG stimulation increased the level of active GTP-bound RhoA compared with unstimulated monocytes, and the effect of PG on RhoA activity was suppressed with anti-TLR-2 monoclonal antibody. RhoA inhibition either with a specific inhibitor or by siRNA transfection inhibited activation of NF-κB and secretion of TNFα and IL-1β in PG-induced RA monocytes. SMV mitigated PG-induced increase in RhoA activity and NF-κB activation as well as secretion of TNFα and IL-1β. The inhibitory effects of SMV were completely reversed by mevalonate and geranylgeranyl pyrophosphate. Our results indicate the modulation of RhoA on TLR-2-mediated inflammatory signaling in RA and provide a novel evidence for anti-inflammatory effects of statins through influencing TLR-2 signaling via RhoA in RA.

Resistin as a potential marker of renal disease in lupus nephritis.

Systemic lupus erythematosus (SLE) and lupus nephritis (LN) have strong concomitance with cardiovascular disease that cannot be explained fully by typical risk factors. We examined the possibility that serum or urine expression of adipokines may act as biomarkers for LN, as these proteins have been associated previously with cardiovascular disease as well as SLE. Antibody arrays were performed on serum and urine from lupus patients and matched controls using a cross‐sectional study design. From the initial array‐based screening data of 15 adipokines, adiponectin, leptin and resistin were selected for validation by enzyme‐linked immunosorbent assay (ELISA). Correlations were determined between adipokine expression levels and measures of disease activity or lupus nephritis. The expression of adiponectin and resistin was increased in both sera and urine from LN patients, while leptin was increased in LN patient sera, compared to matched controls. Serum resistin, but not urine resistin, was correlated with measures of renal dysfunction in LN. Serum resistin expression may be useful as a marker of renal dysfunction in patients with LN, although longitudinal studies are warranted. Further studies are necessary to determine if resistin has functional consequences in LN.