Increased soluble CD4 in serum of rheumatoid arthritis patients is generated by matrix metalloproteinase (MMP)-like proteinases.
ABSTRACT: Higher soluble CD4 (sCD4) levels in serum have been detected in patients of infectious and chronic inflammatory diseases. However, how and why sCD4 is produced remains poorly understood. We establish sensitive ELISA and WB assays for sCD4 detection in conditioned medium of in vitro cell culture system and serum of chronic inflammatory patients. Serum samples from patients with systemic lupus erythematosus (SLE) (n?=?79), rheumatoid arthritis (RA) (n?=?59), ankylosing spondylitis (AS) (n?=?25), gout (n?=?31), and normal controls (n?=?99) were analyzed using ELISA for sCD4 detection. Results from each assay were analyzed by the Kruskal-Wallis test. Dunn's multiple comparison post-test was then applied between groups. We confirm that cells expressing exogenous CD4 produce sCD4 in a constitutive and PMA-induced manner. Importantly, sCD4 production in a heterologous expression system is inhibited by GM6001 and TAPI-0, suggesting receptor shedding by matrix metalloproteinase (MMP)-like proteinases. Moreover, similar findings are recapitulated in human primary CD4(+) T cells. Finally, we show that serum sCD4 levels are increased in patients of chronic inflammatory diseases including RA and SLE, but not in those with gout. Intriguingly, sCD4 levels in RA patients are correlated positively with the disease activities and higher sCD4 levels seem to associate with poor prognosis. Taken together, we conclude that CD4 is shed from cell surface by a MMP-like sheddase and sCD4 level is closely related with the inflammatory condition in certain chronic diseases. Hence, sCD4 might be considered an important parameter for RA disease progression with potential diagnostic importance.
Project description:Elevated concentrations of inflammatory mediators are characteristic of autoimmune disease accompanied by chronic or recurrent inflammation. We examined the hypothesis that mediators of inflammation known to be elevated in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) are associated with genetic polymorphism previously identified in studies of inflammatory disease. Serum interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF?) concentrations in patients with SLE (n?=?117) or RA (n?=?164) and in inflammatory disease-free control subjects (n?=?172) were measured by multiplex ELISA. Candidate genes were chosen from studies of autoimmune and inflammatory disease. Genotypes were determined for 345 SNP markers in 75 genes. Association between serum analytes and single alleles was tested by linear regression. Polymorphisms in several genes were associated with IL-6 levels (including IL10, TYK2, and CD40L in SLE and DRB1, NOD2, and CSF1 in RA) or with TNF? levels (including TNFSF4 and CSF2 in SLE and PTPN2, DRB1, and NOD2 in RA). Some associations were shared between disease and control groups or between IL-6 and TNF? within a group. In conclusion, variation in genes implicated in disease pathology is associated with serum IL-6 or TNF? concentration. Some genetic associations are more apparent in healthy controls than in SLE or RA, suggesting dysregulation of the principal mediators of chronic inflammation in disease. Susceptibility genes may affect inflammatory response with variable effect on disease etiology.
Project description:The herpes zoster (HZ) vaccine is recommended for adults in the US ages ?60 years who do not have weakened immune systems. It is unclear how the risk of HZ varies according to age and disease conditions in younger patients with autoimmune or inflammatory (AI) diseases. This study was undertaken to evaluate the age-stratified incidence of HZ in patients with AI diseases as compared to older adults for whom the HZ vaccine is currently recommended by the US Centers for Disease Control and Prevention.Using linked data obtained from patients who were insured by US commercial and government health care plans during the period 2007-2010, 7 cohorts of patients with AI diseases were assembled: systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), rheumatoid arthritis (RA), psoriatic arthritis (PsA), psoriasis (PsO), ankylosing spondylitis (AS), and gout. Two comparator cohorts were also assembled as controls: adult patients with diabetes and adult subjects without AI diseases or diabetic conditions. HZ was identified using diagnostic codes. Age-specific incidence rates (IRs) of HZ were calculated and compared to the IRs of HZ in control subjects ages 61-70 years who were without AI diseases or diabetic conditions.After review of the linked data, the following number of enrollment periods were identified: 8,395 for patients with SLE, 7,916 for patients with IBD, 50,646 for patients with RA, 2,629 for patients with PsA, 4,299 for patients with PsO, 1,019 for patients with AS, 58,934 for patients with gout, 214,631 for control patients with diabetes, and 330,727 for control subjects without AI diseases and diabetic conditions. The respective highest and lowest IRs of HZ during the study were 19.9 per 1,000 person-years in the SLE cohort and 6.8 per 1,000 person-years in the gout cohort, as compared to an IR of 5.3 per 1,000 person-years in control subjects without AI diseases or diabetic conditions. The age-specific IRs of HZ in patients with RA and those with SLE ages ?40 years were 1.5-2 times greater than those observed in older healthy adults (IR 8.5 per 1,000 person-years), for whom the vaccine is currently recommended.SLE, IBD, and RA are AI diseases associated with a higher risk of HZ compared to that in older adults for whom vaccination is currently recommended, suggesting that individuals with these conditions who are as young as age 40 years could potentially benefit from the HZ vaccine.
Project description:BACKGROUND: Matrix metalloproteinase-3 (MMP-3) plays an important role in the pathology of rheumatoid arthritis (RA) and ankylosing spondylitis (AS). Measurement of active MMP-3 in clinical samples could provide information about progression of rheumatoid diseases, and potentially response to treatment. Hence, we aimed to develop a sensitive assay specifically measuring the active form of MMP-3 (act-MMP-3) both in ex vivo models and in human sera. METHODS: A monoclonal antibody against the first 6 amino acids of act-MMP-3 was developed, and the specificity was carefully tested by comparing total and active MMP-3. A technically robust act-MMP-3 ELISA was produced. For biological validation, human synovial membrane and human cartilage explant (HEX) culture models were measured and compared by ELISA and immunoblots. For clinical relevance, the serum levels of act-MMP-3 in AS and RA patients before and after anti-TNF-? treatment were evaluated. RESULTS: A highly specific and technically robust ELISA detecting act-MMP-3 in serum was developed. The lower limit of detection was 33.7 pg/mL. The dilution and spiking recovery of human serum was within 100 ± 20%. The average intra- and inter-assay variations were 3.1% and 13.5% respectively.High levels of act-MMP-3 expression were observed in human synovial membrane culture and oncostatin M and TNF-? stimulated human cartilage. In a cross-sectional study of both AS and RA patients, serum act-MMP-3 level was correlated with C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). In addition, in patients receiving anti-TNF-? treatment, the serum level of act-MMP-3 was significantly reduced compared to baseline level reflecting the anti-inflammatory effects of the treatment. CONCLUSION: We have successfully developed an assay measuring act-MMP-3 in human serum showing correlation to inflammatory markers. Further studies are required to clarify, whether act-MMP-3 can serve as a predictive marker for outcome in chronic rheumatoid disorders.
Project description:This translational multi-centre study explored early changes in serologic variables following B lymphocyte depletion by rituximab (RTX) treatment in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients and investigated in vitro effects on the activity of other immune cells and the vascular endothelium. Eighty-five SLE patients, seventy-five RA patients and ninety healthy donors were enrolled. Two additional cohorts of selected SLE and RA patients were treated with RTX for 3 months. Changes in circulating levels of inflammatory mediators, oxidative stress markers and NETosis-derived bioproducts were evaluated. Serum miRNomes were identified by next-generation sequencing, and RTX-induced changes were delineated. Mechanistic in vitro studies were performed to assess activity profiles. Altered inflammatory, oxidative and NETosis-derived biomolecules were found in SLE and RA patients, closely interconnected and associated to specific miRNA profiles. RTX treatment reduced SLE and RA patients' disease activity, linked to a prominent alteration in those biomolecules and the reversal of altered regulating miRNAs. In vitro studies showed inhibition of NETosis and decline of pro-inflammatory profiles of leucocytes and human umbilical vein endothelial cells (HUVECs) after B cell depletion. This study provides evidence supporting an early RTX-induced re-setting of the pro-inflammatory status in SLE and RA, involving a re-establishment of the homeostatic equilibrium in immune system and the vascular wall.
Project description:Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disorder. MASP2 is a mediator that plays an important role in complement system. As dysregulation of the complement system has been demonstrated to correlate with SLE pathogenesis, the role of MASP2 in lupus has not been widely discussed. In the present study, serum levels of MASP2 were evaluated in 61 lupus patients and 98 healthy controls by training cohort, and then a validation cohort including 100 lupus, 100 rheumatoid arthritis, 100 osteoarthritis, 100 gout, 44 Sjogren's syndrome, 41 ankylosing spondylitis patients confirmed the findings. Receiver operating characteristic (ROC) curve analysis determined the discriminatory capacity for serum MASP2. PCR methods tested the association of MASP2 gene polymorphisms (rs7548659, rs17409276, rs2273346, rs1782455 and rs6695096) and SLE risk. Impact of polymorphism on MASP2 serum levels was evaluated as well. Results showed that serum levels of MASP2 were significantly higher in lupus patients and correlated with some clinical, laboratory characteristics in the training cohort, and were much higher as compared to that in different rheumatic diseases patients in the validation cohort. Serum MASP2 showed a good diagnostic ability for lupus. Genotype frequencies and allele frequency of polymorphisms rs7548659, rs2273346 were strongly related to SLE risk, and genotypes of rs17409276, rs1782455, rs76695096 were significantly correlated with lupus genetic susceptibility. Interestingly, patients carrying GA genotype of rs17409276, TT, TC genotype of rs6695096 showed higher levels of serum MASP2. The findings suggested that MASP2 may be a potential disease marker for lupus, and correlate with SLE pathogenesis.
Project description:Objective: Gene expression studies performed on PBMC from systemic lupus erythematosus (SLE) patients provided strong evidence of a type I interferon signature, underscoring the potential role of these cytokines in the physiopathology of SLE. In this work, we performed microarray analyses of differential gene expression using purified CD4 T and B cells sorted from SLE PBMC. In order to discriminate genes specific to SLE from those induced by inflammatory responses in general, control samples were obtained not only from healthy individuals but also from rheumatoid arthritis (RA) patients. Results: A strong interferon signature was found both in the CD4 T and the B lymphocytes from SLE patients, thereby confirming the results obtained on total PBMC. Interestingly, many interferon-induced genes were also over-expressed in CD4 and B cells from RA patients. Some genes were more specifically over-expressed in SLE lymphocytes, and 3 of them, SLAMF1, BRDG1 and RASGRP1, were exclusively up-regulated in SLE B cells. SLAMF1 and BRDG1 are localized in disease-associated loci, thereby suggesting that they might play a role in the physiopathology of the disease. Experiment Overall Design: CD4 T and B cells were sorted by flow cytometry from PBMC of patients with SLE, RA and healthy controls. GeneChip® Human genome U133 Plus 2.0 arrays were hybridized in monoplicates and the genes differentially expressed among the three groups of patients were identified using ANOVA tests with corrections for multiple comparisons.
Project description:INTRODUCTION: Anti-citrullinated protein/peptide antibodies (ACPAs) are highly specific to rheumatoid arthritis (RA) patients and are thought to have a close relationship with the pathogenesis of arthritis. Several proteins, including fibrinogen, vimentin, and alpha-enolase, were reported as ACPA-target antigens, and their importance in RA pathogenesis was widely proposed. We identified citrullinated immunoglobulin binding protein (citBiP) as another ACPA target in RA patients and examined its pro-inflammatory role in arthritis. METHODS: We measured the levels of anti-citBiP, anti-BiP, and anti-cyclic citrullinated peptide (CCP) antibodies in the serum of RA patients (n = 100), systemic lupus erythematosus (SLE) patients (n = 60), and healthy controls (n = 30) using ELISA and immunoblotting. Epitope mapping was performed using 27 citBiP-derived peptides. In the mouse study, after DBA/1J mice were immunized with BiP or citBiP, serum titers of ACPAs were measured by ELISA and immunohistochemistry. The development of collagen-induced arthritis (CIA) was observed in BiP- or citBiP-pre-immunized mice. RESULTS: The serum levels of anti-BiP and anti-citBiP antibodies were significantly increased in RA patients, although only anti-BiP antibodies were slightly increased in SLE patients. Interestingly, anti-citBiP antibody levels were higher than anti-BiP antibody levels in 72% of RA patients, whereas no significant increase in anti-citBiP antibody levels was detected in SLE patients and healthy controls. The serum levels of anti-CCP antibodies were correlated with those of anti-citBiP antibodies in RA patients (R2 = 0.41). Several citrulline residues of citBiP were determined to be major epitopes of anti-citBiP antibodies, one of which showed cross-reactivity with CCP. Immunization of DBA/1J mice with citBiP induced several kinds of ACPAs, including anti-CCP and anti-citrullinated fibrinogen antibodies. Pre-immunization with citBiP exacerbated CIA, and anti-CCP antibody levels were increased in citBiP-pre-immunized CIA mice. CONCLUSIONS: CitBiP is a newly described ACPA target that may play a pro-inflammatory role in arthritis.
Project description:BACKGROUD:This study is to explore the prevalence of different stages of bone loss and the potential risk factors in rheumatic patients. METHOD:A cross-sectional study recruits 1398 rheumatic patients and 302 healthy subjects. Demographic data, blood, and bone mineral density (BMD) tests are collected. Risk factors for bone loss in rheumatic patients are analyzed by logistic regression. RESULTS:(1) Rheumatic patients are consisted of 40.0% rheumatoid arthritis (RA), 14.7% systemic lupus erythematosus (SLE), 14.2% osteoarthritis (OA), 9.2% ankylosing spondylosis (AS), 7.9% gout, 7.0% primary Sjogren syndrome (pSS), 3.8% systemic sclerosis (SSc), and 3.2% mixed connective tissue disease (MCTD). (2) In male patients aged under 50 and premenopausal female patients, the bone mineral density score of AS (53.9%, P?< 0.001) and SLE (39.6%, P =?0.034) patients is lower than the healthy controls (18.2%). (3) Osteopenia and osteoporosis are more prevailing in male patients aged or older than 50 and postmenopausal female patients with RA (P <?0.001), OA (P?= 0.02) and SLE (P =?0.011) than healthy counterparts. (4) Those with SLE, RA and AS gain the highest odd ratio of 'score below the expected range for age', osteopenia and osteoporosis, respectively. (5) Age, female, low BMI and hypovitaminosis D are found negatively associated with bone loss. Dyslipidemia and hyperuricemia could be protective factors. CONCLUSION:Young patients with AS and SLE have a significant higher occurrence of bone loss, and older patients with RA, OA and SLE had higher prevalence than healthy counterparts. SLE, RA, SSc and AS were founded significant higher risks to develop into bone loss after adjustment. Age, BMI and gender were commonly-associated with bone loss in all age-stratified rheumatic patients. These findings were not markedly different from those of previous studies.
Project description:Introduction:Matrix metalloproteinase (MMP) is an emerging disease marker in rheumatic diseases. This is a meta-analysis aimed at systematically reviewing association between serum MMP-3 levels and systematic lupus erythematosus (SLE) activity, which sought to raise interest in MMP-3 as a putative biomarker. Methods:We conducted a meta-analysis of serum MMP-3 levels in patients with SLE and controls. We performed a PubMed search, EMBASE search, and forward search of the retrieved articles published until Oct. 1, 2018. In addition to this, we included data from a case-control study on a national pediatric SLE cohort, in which serum MMP-3 levels were measured in 11 SLE patients and 9 controls (unpublished). Subgroup analyses based on gender and disease activity were performed. Results:A total of 662 cases and 771 controls including 651 patients and 762 controls from 11 publications were studied. We observed significantly higher MMP-3 levels in SLE patients compared to healthy controls (P < 0.001, Hedges' g: 2.104, 95% CI 1.426-2.782). In subgroup analyses, we found a significant elevation of MMP-3 in the patients with nephritis compared to those without (P = 0.006, Hedges' g: 0.611, 95% CI 0.611-1.704). This finding was consistent between patients with persistent proteinuria and those without (P = 0.023, Hedges' g: 1.535, 95% CI 0.207-2.862). Meta-analysis showed no association between MMP-3 levels and gender or anti-double strand DNA antibody titer. Conclusions:Our meta-analysis demonstrated significantly higher MMP-3 levels in SLE patients than in controls and in patients with renal involvement than in those without.
Project description:Single nucleotide polymorphisms (SNPs) in microRNA-target sites influence an individual's risk and prognosis for autoimmune diseases. Myotubularin-related protein 3 (MTMR3), an autophagy-related gene, is a direct target of miR-181a. We investigated whether MTMR3 SNP rs12537 in the miR-181a-binding site is associated with the susceptibility and progression of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Overall, 94 patients with RA, 80 patients with SLE, and 104 healthy volunteers were recruited. Genotyping and expression analysis of circulating MTMR3 and miR-181a were performed by qPCR. The autophagic marker MAP1LC3B was measured by ELISA. The rs12537 minor homozygote (TT) genotype was a candidate risk factor of both RA and SLE. rs12537TT was associated with lower serum MTMR3 expression and higher LC3B levels than other genotypes in patients with both diseases. Serum miR-181a expression was higher in rs12537TT carriers than in other genotypes among SLE patients. Serum miR-181a and MTMR3 levels were inversely correlated in SLE but not in RA patients. rs12537TT and serum miR-181a were positively associated with disease severity in both diseases. Our results identify a novel role of rs12537 in the susceptibility and progression of RA and SLE, possibly through impacting the interaction between miR-181a and MTMR3 leading to increased autophagy.