Response to abatacept is associated with the inhibition of proteasome β1i expression in T cells of patients with rheumatoid arthritis

Objective: Abatacept is a biological disease-modifying antirheumatic drug (DMARD) used for the treatment of rheumatoid arthritis (RA) and modulates the costimulatory signal by cluster of differentiation (CD)28:CD80/CD86 interaction required for T cell activation. Since CD28-mediated signalling regulates many T cell functions including cytokine production of, for example, interferons (IFNs), it is of interest to clarify, whether response to abatacept has an effect on the IFN inducible immunoproteasome, as a central regulator of the immune response. Methods: Effects of abatacept on the proteasome were investigated in 39 patients with RA over a period of 24 weeks. Using real-time PCR, transcript levels of constitutive and corresponding immunoproteasome catalytic subunits were investigated at baseline (T0), week 16 (T16) and week 24 (T24) in sorted blood cells. Proteasomal activity and induction of apoptosis after proteasome inhibition were also evaluated. Results: Abatacept achieved remission or low disease activity in 55% of patients at T16 and in 70% of patients at T24. By two-way analysis of variance (ANOVA), a significant reduction of proteasome immunosubunit β1i was shown only in CD4+ and CD8+ T cells of sustained responders at both T16 and T24. One-way ANOVA analysis for each response group confirmed the results and showed a significant reduction at T24 in CD4+ and CD8+ T cells of the same group. Abatacept did not influence chymotrypsin-like activity of proteasome and had no effect on induction of apoptosis under exposure to a proteasome inhibitor in vitro. Conclusion: The reduction of proteasome immunosubunit β1i in T cells of patients with RA with sustained response to abatacept suggests association of the immunoproteasome of T cells with RA disease activity

Upregulation of Immunoproteasome Subunits in Myositis Indicates Active Inflammation with Involvement of Antigen Presenting Cells, CD8 T-Cells and IFN gamma

Objective: In idiopathic inflammatory myopathies (IIM) infiltration of immune cells into muscle and upregulation of MHC-I expression implies increased antigen presentation and involvement of the proteasome system. To decipher the role of immunoproteasomes in myositis, we investigated individual cell types and muscle tissues and focused on possible immune triggers. Methods: Expression of constitutive (PSMB5,-6,-7) and corresponding immunoproteasomal subunits (PSMB8,-9,-10) was analyzed by real-time RT-PCR in muscle biopsies and sorted peripheral blood cells of patients with IIM, non-inflammatory myopathies (NIM) and healthy donors (HD). Protein analysis in muscle biopsies was performed by western blot. Affymetrix HG-U133 platform derived transcriptome data from biopsies of different muscle diseases and from immune cell types as well as monocyte stimulation experiments were used for validation, coregulation and coexpression analyses. Results: Real-time RT-PCR revealed significantly increased expression of immunoproteasomal subunits (PSMB8/-9/-10) in DC, monocytes and CD8+ T-cells in IIM. In muscle biopsies, the immunosubunits were elevated in IIM compared to NIM and exceeded levels of matched blood samples. Proteins of PSMB8 and -9 were found only in IIM but not NIM muscle biopsies. Reanalysis of 78 myositis and 20 healthy muscle transcriptomes confirmed these results and revealed involvement of the antigen processing and presentation pathway. Comparison with reference profiles of sorted immune cells and healthy muscle confirmed upregulation of PSMB8 and -9 in myositis biopsies beyond infiltration related changes. This upregulation correlated highest with STAT1, IRF1 and IFN gamma expression. Elevation of T-cell specific transcripts in active IIM muscles was accompanied by increased expression of DC and monocyte marker genes and thus reflects the cell type specific involvement observed in peripheral blood. Conclusions: Immunoproteasomes seem to indicate IIM activity and suggest that dominant involvement of antigen processing and presentation may qualify these diseases exemplarily for the evolving therapeutic concepts of immunoproteasome specific inhibition

Upregulation of Immunoproteasome Subunits in Myositis Indicates Active Inflammation with Involvement of Antigen Presenting Cells, CD8 T-Cells and IFN gamma

Objective: In idiopathic inflammatory myopathies (IIM) infiltration of immune cells into muscle and upregulation of MHC-I expression implies increased antigen presentation and involvement of the proteasome system. To decipher the role of immunoproteasomes in myositis, we investigated individual cell types and muscle tissues and focused on possible immune triggers. Methods: Expression of constitutive (PSMB5,-6,-7) and corresponding immunoproteasomal subunits (PSMB8,-9,-10) was analyzed by real-time RT-PCR in muscle biopsies and sorted peripheral blood cells of patients with IIM, non-inflammatory myopathies (NIM) and healthy donors (HD). Protein analysis in muscle biopsies was performed by western blot. Affymetrix HG-U133 platform derived transcriptome data from biopsies of different muscle diseases and from immune cell types as well as monocyte stimulation experiments were used for validation, coregulation and coexpression analyses. Results: Real-time RT-PCR revealed significantly increased expression of immunoproteasomal subunits (PSMB8/-9/-10) in DC, monocytes and CD8+ T-cells in IIM. In muscle biopsies, the immunosubunits were elevated in IIM compared to NIM and exceeded levels of matched blood samples. Proteins of PSMB8 and -9 were found only in IIM but not NIM muscle biopsies. Reanalysis of 78 myositis and 20 healthy muscle transcriptomes confirmed these results and revealed involvement of the antigen processing and presentation pathway. Comparison with reference profiles of sorted immune cells and healthy muscle confirmed upregulation of PSMB8 and -9 in myositis biopsies beyond infiltration related changes. This upregulation correlated highest with STAT1, IRF1 and IFN gamma expression. Elevation of T-cell specific transcripts in active IIM muscles was accompanied by increased expression of DC and monocyte marker genes and thus reflects the cell type specific involvement observed in peripheral blood. Conclusions: Immunoproteasomes seem to indicate IIM activity and suggest that dominant involvement of antigen processing and presentation may qualify these diseases exemplarily for the evolving therapeutic concepts of immunoproteasome specific inhibition

Differential Effects of Aging on Fore– and Hindpaw Maps of Rat Somatosensory Cortex

Getting older is associated with a decline of cognitive and sensorimotor abilities, but it remains elusive whether age-related changes are due to accumulating degenerational processes, rendering them largely irreversible, or whether they reflect plastic, adaptational and presumably compensatory changes. Using aged rats as a model we studied how aging affects neural processing in somatosensory cortex. By multi-unit recordings in the fore- and hindpaw cortical maps we compared the effects of aging on receptive field size and response latencies. While in aged animals response latencies of neurons of both cortical representations were lengthened by approximately the same amount, only RFs of hindpaw neurons showed severe expansion with only little changes of forepaw RFs. To obtain insight into parallel changes of walking behavior, we recorded footprints in young and old animals which revealed a general age-related impairment of walking. In addition we found evidence for a limb-specific deterioration of the hindlimbs that was not observed in the forelimbs. Our results show that age-related changes of somatosensory cortical neurons display a complex pattern of regional specificity and parameter-dependence indicating that aging acts rather selectively on cortical processing of sensory information. The fact that RFs of the fore- and hindpaws do not co-vary in aged animals argues against degenerational processes on a global scale. We therefore conclude that age-related alterations are composed of plastic-adaptive alterations in response to modified use and degenerational changes developing with age. As a consequence, age-related changes need not be irreversible but can be subject to amelioration through training and stimulation

Increased frequency of JC-polyomavirus detection in rheumatoid arthritis patients treated with multiple biologics

Progressive multifocal leukoencephalopathy (PML) represents a rare but potentially fatal reactivation of JC-polyomavirus (JCPyV) recently also reported in patients with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis (RA) treated with rituximab. The aim of the present study was to analyse the pattern of JCPyV infections in patients with RA undergoing treatment with biologic agents. Urine and blood samples were analysed from 80 patients for antibody levels and/or the presence of JCPyV DNA. Genotyping of the control region and VP1 was performed for all JCPyV DNA-positive specimens. Viremia of JCPyV was only temporarily detected in two patients, and these viruses did not carry any mutations associated with the occurrence of PML. JCPyV DNA was prevalent in initial urine samples of 33 % of all patients. RA patients who have consecutively been treated with two or more biologic agents revealed significantly higher prevalence of JCPyV DNA in the urine compared to RA patients treated with their first biologic agent. The presence of JCPyV DNA in the urine closely correlated to JCPyV antibody positivity, and therefore, antibody titres were higher in RA patients who had consecutively received two or more biologic agents over time. Therefore, the overall number of biologic agents had an impact on the pattern of JCPyV detection in this study. Hence, JCPyV antibody screening might be useful as part of the PML risk stratification for RA patients in the future

Correlation of immunoproteasome with IFN and IFNR expression in myositis muscle tissue:

<p>Transcriptome data referenced in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104048#pone.0104048.s007" target="_blank">table S2</a> were re-investigated. A) Correlation analysis was performed for 133P and 133A datasets independently. Only IFNγ revealed high correlation coefficients with PSMB8, PSMB9 and PSMB10. The corresponding constitutively expressed subunits PSMB5-7 were not or even negatively correlated. B) Comparing the association of IFNγ with PSMB8, -9 and -10 for each sample individually, the increase was much higher for PSMB8/-9 compared to PSMB10 as described before (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104048#pone-0104048-g006" target="_blank">figure 6</a>).</p

Expression of immunoproteasomal subunits in immune cells:

<p>Gene expression of immunoproteasomal subunits (PSMB8–10) in CD4+, CD8+, CD19+, CD14+ and DCs of patients with myopathies (PM, DM, OM, NIM) and controls (HD). Data are shown as relative expression normalized to beta actin. Box plots indicate percentiles 0, 25, 50, 75 and 100. Groups were compared by Mann-Whitney U test and statistical significance is indicated for p<0.05 (*) and p<0.01 (**). Significantly higher expression of PSMB8 was observed in CD14+ cells and DC of PM patients compared to HD or DM, NIM and HDs, respectively. PSMB9 was increased in CD8+ and CD14+ of DM and in DCs of PM and DM patients compared to NIM. PSMB10 was found increased in PM patients compared to DM, NIM and HD in CD8+, compared to HD in CD19+ and CD14+ cells and compared to OM, NIM, and HD in DCs.</p