Article first published online 19 May 2015.
Rheumatoid arthritis (RA) is an inflammatory joint disease leading to cartilage damage and ultimately impaired joint function. To gain new insight into the systemic immune manifestations of RA, we characterized the colon mucosa proteome from 11 RA-patients and 10 healthy controls. The biopsies were extracted by colonoscopy and analyzed by label-free quantitative proteomics, enabling the quantitation of 5366 proteins. The abundance of dihydrofolate reductase (DHFR) was statistically significantly increased in RA-patient biopsies compared with controls and correlated with the administered dosage of methotrexate (MTX), the most frequently prescribed immunosuppressive drug for RA. Additionally, our data suggest that treatment with Leflunomide, a common alternative to MTX, increases DHFR. The findings were supported by immunohistochemistry with confocal microscopy, which furthermore demonstrated that DHFR was located in the cytosol of the intestinal epithelial and interstitial cells. Finally, we identified 223 citrullinated peptides from 121 proteins. Three of the peptides were unique to RA. The list of citrullinated proteins was enriched in extracellular and membrane proteins and included known targets of anticitrullinated protein antibodies (ACPAs). Our findings support that the colon mucosa could trigger the production of ACPAs, which could contribute to the onset of RA. The MS data have been deposited to ProteomeXchange with identifiers PXD001608 and PXD003082.
Spondyloarthritis (SpA) comprises a heterogeneous group of inflammatory diseases, with strong association to human leukocyte antigen (HLA)-B27. A triggering bacterial infection has been considered as the cause of SpA, and bacterial heat shock protein (HSP) seems to be a strong T cell antigen. Since bacterial and human HSP60, also named HSPD1, are highly homologous, cross-reactivity has been suggested in disease initiation. In this study, levels of antibodies against bacterial and human HSP60 were analysed in SpA patients and healthy controls, and the association between such antibodies and disease severity in relation to HLA-B27 was evaluated.Serum samples from 82 patients and 50 controls were analysed by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin (Ig)G1, IgG2, IgG3 and IgG4 antibodies against human HSP60 and HSP60 from Chlamydia trachomatis, Salmonella enteritidis and Campylobacter jejuni. Disease severity was assessed by the clinical scorings Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI) and Bath Ankylosing Spondylitis Metrology Index (BASMI).Levels of IgG1 and IgG3 antibodies against human HSP60, but not antibodies against bacterial HSP60, were elevated in the SpA group compared with the control group. Association between IgG3 antibodies against human HSP60 and BASMI was shown in HLA-B27+ patients. Only weak correlation between antibodies against bacterial and human HSP60 was seen, and there was no indication of cross-reaction.These results suggest that antibodies against human HSP60 is associated with SpA, however, the theory that antibodies against human HSP60 is a specific part of the aetiology, through cross-reaction to bacterial HSP60, cannot be supported by results from this study. We suggest that the association between elevated levels of antibodies against human HSP60 and disease may reflect a general activation of the immune system and an increased expression of human HSP60 in the synovium of patients with SpA.
IntroductionSpondyloarthritis (SpA), an interrelated group of rheumatic diseases, has been suggested to be triggered by bacterial infections prior to the development of an autoimmune response that causes inflammation of the spinal and peripheral joints. Because human heat shock protein 60 (HSP60), recently renamed HSPD1, and bacterial HSP60 are highly homologous, immunological cross-reactivity has been proposed as a mechanism of disease initiation. However, previous investigations of the humoral immune response to HSP60 in SpA patients have lacked determination of immunoglobulin G (IgG) subclasses and patient follow-up. In this study, we have focused on these parameters in a cohort of axial SpA patients with a well-established set of clinical characteristics, including MRI changes and human leukocyte antigen B27.MethodsIgG subclass antibodies (IgG1, IgG2, IgG3 and IgG4) against recombinant HSP60 of three reactive arthritis-related bacteria; human HSP60; and the microorganisms Chlamydia trachomatis and C. pneumoniae were determined by ELISA. Serum samples collected from 2004 to 2006 and in 2010 and 2011 from 39 axial SpA patients were analyzed and compared with samples from 39 healthy controls. The Mann-Whitney U test and Wilcoxon matched pairs test were used to compare the antibody levels in different and paired groups, respectively. P < 0.01 was considered significant. The Spearman nonparametric correlation was used to determine correlation between antibody levels and between antibody levels and the disease parameters.ResultsElevated levels of IgG1 and IgG3 to human HSP60 and IgG1 to HSP60 of Salmonella enterica Enteritidis were observed in SpA patients compared with healthy controls at both time points. The antibody levels were almost constant over time for IgG1, whereas high levels of IgG3 to human HSP60 tended to decrease over time. The antibody response to human HSP60 was predominantly of the IgG3 subclass, and patients with high levels of IgG3 to this antigen had low levels of IgG1, indicating an inverse association. Different IgG subclasses were produced against bacterial and human HSP60 in the same serum sample, IgG1 and IgG3, respectively, indicating that there was no cross-reaction.ConclusionsA significant association was observed between axial SpA and the presence of IgG1/IgG3 antibodies to human HSP60 and of IgG1 to S. enterica Enteritidis and C. trachomatis. Generation of antibodies to human HSP60 was independent of the presence of antibodies to bacterial HSP60. No association was observed between clinical and MRI changes with antibodies over time. Altogether, such antibodies do not reflect the disease activity in these patients.This study has been approved by the Regional Research Ethics Committee of Central Jutland, Denmark. Trial registration numbers: 20050046 and 20100083
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