Ubiquitin Proteasom System und Myopathien

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 IFNγ 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.Ziel Idiopathische inflammatorische Myopathien (IIM) sind gekennzeichnet durch die Infiltration von Immunzellen in den Muskel und eine erhöhte MHC-I-Expression. Dies lässt auf eine erhöhte Antigenpräsentation und Beteiligung des Proteasom-Systems schließen. Um die Rolle von Immunoproteasomen bei der Myositis zu ergründen, wurden einzelne Zelltypen sowie Muskelgewebe betrachtet und nach möglichen Auslösern der Immunantwort gesucht. Methoden Die Expression von konstitutiven (PSMB5, -6, -7) und korrespondierenden Immunoproteasom- Untereinheiten (PSMB8, -9, -10) wurde mittels real-time RT-PCR in Muskelbiopsien und sortierten peripheren Blutzellen von Patienten mit IIM im Vergleich zu nicht-inflammatorischen Myopathien (NIM) und gesunden Spendern (HD) analysiert. Die Proteinanalyse in Muskelbiopsien wurde über Western-Blot-Verfahren durchgeführt. Affymetrix HG-U133 Transkriptom Daten aus Biopsien von verschiedenen Muskelerkrankungen, Immunzelltypen und spezifisch stimulierten Monozyten dienten zur Validierung und Analyse der Co-Regulierung bzw. Co-Expression von Genen. Ergebnisse Die Real-time RT-PCR Untersuchung ergab eine signifikant erhöhte Expression der Immunoproteasom-Untereinheiten (PSMB8 / -9 / -10) in dendritischen Zellen (DC), Monozyten und CD8 + T-Zellen bei IIM. In Muskelbiopsien der IIM lagen die Messwerte für die Immunproteasom-Untereinheiten höher als in NIM und übertrafen auch die Spiegel aus den parallel erhobenen Blutproben. Die Proteine PSMB8 und -9 wurden nur in IIM aber nicht in NIM Muskelbiopsien gefunden. Die Nachuntersuchung von bereits publizierten Muskel- Transkriptomen von 78 Myositisbiopsien und 20 gesunden Kontrollgeweben bestätigte diese Ergebnisse und zeigte eine Beteiligung von Antigenprozessierung und -präsentation. Der Vergleich mit den Referenzprofilen aus sortierten Immunzellen und gesundem Muskel bestätigte die Hochregulation von PSMB8 und -9 in Myositisbiopsien über eine durch Zellinfiltration bedingte Veränderung hinaus. Diese Hochregulation korrelierte am stärksten mit der Expression von STAT1, IRF1 und IFNγ. Eine Erhöhung der T-Zell-spezifischen Transkripte in aktivem IIM Muskelgewebe wurde von einem Anstieg der DC und Monozyten Markergene begleitet und spiegelte damit die Zelltyp-spezifische Beteiligung aus dem peripheren Blut wider. Schlussfolgerungen Das Proteasom-System ist bei aktiver IIM aktiviert, wobei die Hochregulation von Immunoproteasomen auf eine Involvierung in Antigenprozessierung und -präsentation bei diesen Erkrankungen hinweist. Dies könnte einen therapeutischen Ansatzpunkt durch spezifische Hemmung von Immunoproteasomen darstellen

Distinct Effects of Interleukin-1β Inhibition upon Cytokine Profile in Patients with Adult-Onset Still’s Disease and Active Articular Manifestation Responding to Canakinumab

Adult-onset Still’s disease (AOSD) is a systemic auto-inflammatory disease characterized by the presence of immunologically mediated inflammation and deficient resolution of inflammation. Canakinumab is an approved IL-1β inhibitor in the treatment of AOSD with a balanced efficacy and safety profile. Since inflammatory cytokines play a major role in the pathogenesis of AOSD, we investigated the effects of canakinumab on the cytokine profile of AOSD patients from a randomized controlled trial. Multiplex analysis and ELISA were used to test the concentrations of several cytokines at three time points—week 0 (baseline), week 1 and week 4—in two patient groups—placebo and canakinumab. Two-way repeated-measures analysis of variance revealed a significant temporal effect on the concentrations of MRP 8/14, S100A12, IL-6 and IL-18 with a significant decrease at week 4 in the canakinumab group exclusively. Comparing responders with non-responders to canakinumab showed a significant decrease in MRP 8/14, IL-1RA, IL-18 and IL-6 in responders at week 4, while S100A12 levels decreased significantly in responders and non-responders. In summary, canakinumab showed a striking effect on the cytokine profile in patients with AOSD, exhibiting a clear association with clinical response

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

Carbamylation of vimentin is inducible by smoking and represents an independent autoantigen in rheumatoid arthritis

OBJECTIVES: Smoking has been connected to citrullination of antigens and formation of anti-citrullinated peptide antibodies (ACPAs) in rheumatoid arthritis (RA). Since smoking can modify proteins by carbamylation (formation of homocitrulline), this study was conducted to investigate these effects on vimentin in animal models and RA. METHODS: The efficiency of enzymatic carbamylation of vimentin was characterised. B-cell response was investigated after immunisation of rabbits with different vimentin isoforms. Effects of tobacco smoke exposure on carbamylation of vimentin and formation of autoantibodies were analysed in mice. The antibody responses against isoforms of vimentin were characterised with respect to disease duration and smoking status of patients with RA. RESULTS: Enzymatic carbamylation of vimentin was efficiently achieved. Subsequent citrullination of vimentin was not disturbed by homocitrullination. Sera from rabbits immunised with carbamylated vimentin (carbVim), in addition to carbVim also recognised human IgG-Fc showing rheumatoid factor-like reactivity. Smoke-exposed mice contained detectable amounts of carbVim and developed a broad immune response against carbamylated antigens. Although the prevalence of anti-carbamylated antibodies in smokers and non-smokers was similar, the titres of carbamylated antibodies were significantly increased in sera of smoking compared with non-smoking RA. CarbVim antibodies were observed independently of ACPAs in early phases of disease and double-positive patients for anti-mutated citrullinated vimentin (MCV) and anti-carbVim antibodies showed an extended epitope recognition pattern towards MCV. CONCLUSIONS: Carbamylation of vimentin is inducible by cigarette smoke exposure. The polyclonal immune response against modified antigens in patients with RA is not exclusively citrulline-specific and carbamylation of antigens could be involved in the pathogenesis of disease. TRIAL REGISTRATION NUMBER: ISRCTN36745608; EudraCT Number: 2006-003146-41

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

Correlation of proteasome subunit expression with myositis signatures and overlap with immune cell infiltration:

<p>Expression of all proteasome units were investigated in transcriptome data of muscle biopsies referenced in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104048#pone.0104048.s007" target="_blank">table S2</a> and were correlated with genes increased in myositis muscle tissue. The heatmap of the probeset signal intensities (A) is opposed to the heatmap of the gene by gene correlation matrix (B) in 133P array experiments. Identical analysis in 133A arrays are shown in D and E. Expression of the 1209 probesets in reference transcriptomes of purified immune cells from the blood of healthy donors served as basis for clustering by probesets and cell types (C). Identification of cell type specific transcriptional patterns in the 1209 probesets demonstrates the extent of overlap of these probesets with immune cell related transcripts. Myositis samples include IBM (red), PM (orange), DM (yellow), NM (necrotizing myopathy)/IM (inflammatory myopathy) (light green) and HD (green). Immune cell types include neutrophils (dark blue), monocytes (blue), dendritic cells (light blue), CD4+ T-cells (dark violet), CD8+ T-cells (violet), NK-cells (light violet), B-cells (cyan), and healthy muscle (moss-green). A detailed graph with all probesets labelled is presented in supplemental material (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104048#pone.0104048.s004" target="_blank">figure S4</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

Comparison of immunoproteosomal subunit expression between paired samples from isolated cells and muscle tissue:

<p>Gene expression analysis of immunoproteasomal subunits (PSMB8–10) in muscle biopsies vs. CD4+, CD8+, CD14+, CD19+, and DCs from patients with inflammatory myopathies (IM) and patients with non-inflammatory myopathies (NIM). 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 (**).</p