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

Causal Modeling Using Network Ensemble Simulations of Genetic and Gene Expression Data Predicts Genes Involved in Rheumatoid Arthritis

Tumor necrosis factor α (TNF-α) is a key regulator of inflammation and rheumatoid arthritis (RA). TNF-α blocker therapies can be very effective for a substantial number of patients, but fail to work in one third of patients who show no or minimal response. It is therefore necessary to discover new molecular intervention points involved in TNF-α blocker treatment of rheumatoid arthritis patients. We describe a data analysis strategy for predicting gene expression measures that are critical for rheumatoid arthritis using a combination of comprehensive genotyping, whole blood gene expression profiles and the component clinical measures of the arthritis Disease Activity Score 28 (DAS28) score. Two separate network ensembles, each comprised of 1024 networks, were built from molecular measures from subjects before and 14 weeks after treatment with TNF-α blocker. The network ensemble built from pre-treated data captures TNF-α dependent mechanistic information, while the ensemble built from data collected under TNF-α blocker treatment captures TNF-α independent mechanisms. In silico simulations of targeted, personalized perturbations of gene expression measures from both network ensembles identify transcripts in three broad categories. Firstly, 22 transcripts are identified to have new roles in modulating the DAS28 score; secondly, there are 6 transcripts that could be alternative targets to TNF-α blocker therapies, including CD86 - a component of the signaling axis targeted by Abatacept (CTLA4-Ig), and finally, 59 transcripts that are predicted to modulate the count of tender or swollen joints but not sufficiently enough to have a significant impact on DAS28

wie Monozyten den Einfluss von Zytokinen modulieren

Chronic rheumatic diseases like systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and ankylosing spondylitis (AS) define a group of disorders with unknown aetiology. There are several lines of evidence suggesting that cytokines play an important role in their pathogenesis and in the maintenance of chronic inflammation, including TNFα, type I IFN, IFNγ, IL1, IL6, IL17 and BAFF. To estimate the role of cytokines in chronic rheumatic diseases is intriguing considering that they act together within complex cytokine networks. The role of cytokines has been emphasised by the fact that the increasing numbers of anti-cytokine drugs have been approved for clinical applications. However, a significant proportion of patients showed only a partial response or failed to respond to this type of treatment. Thus, to estimate the response to various cytokines in chronic rheumatic diseases would be essential for a better understanding of disease pathogenesis and for the identification of the most adequate target(s) for therapeutic intervention. The aim of this study was to determine the role of pro-inflammatory cytokines in the pathogenesis of SLE, RA and AS. Global gene-expression profiling has been considered as a strategy that provides a comprehensive insight into transcriptional alterations that characterize various diseases. Gene- expression profiles generated in monocytes from SLE, RA and AS patients were found to be disease-specific. Functional annotation of disease-specific profiles identified the effects induced by various cytokines, including TNFα, type I IFN, IFNγ, IL1 and IL8. However, the response to these cytokines was disclosed to be different in SLE, RA and AS. To address this question in more detail, the cytokine-specific gene expression profiles were generated by stimulating monocytes in vitro with TNFα, IFNα2a and IFNγ (IFNs). Comparisons between disease-specific and the in vitro generated reference signatures showed that the SLE profile was predominantly driven by IFNs, while the RA profile was primarily influenced by TNFα. The IFNs response in SLE was characterized by an activation of the transcription factor STAT1. Interestingly, the activation of STAT1 was found to be silenced by TNFα in patients with RA. However, the IFN imprints were also identified in RA and the TNFα imprint was evident in SLE. It was obvious that the responses to the same cytokines in SLE and RA were identified to be qualitatively and quantitatively different. Unlike SLE and RA, monocytes from AS showed weak changes in gene expression. The responses induced by IFNs and TNFα in AS were disclosed as rather vain imprints, and the dominance of a particular cytokine was less obvious. Altogether, this study has demonstrated that monocytes from SLE, RA and AS exhibit disease-specific gene-expression profiles, which can be molecularly dissected when compared to the in vitro generated cytokine- specific signatures. The IFNs and TNFα imprints were identified to be disease- dependent and principally they reflected the interplay of cytokines within various inflammatory milieus. The results from this study suggest that estimating the imprints of cytokines in rheumatic diseases would be indispensible for an improvement of diagnosis, proper selection of particular cytokine target(s) for therapeutic intervention and for following up and predicting the response to anti-cytokine drug(s). Ultimately these results should help clinicians to personalize treatment for each rheumatic patient.Chronisch-rheumatische Erkrankungen, wie der systemische Lupus Erythematodes (SLE), die rheumatoide Arthritis (RA) und die ankylosierende Spondylitis (AS) sind durch das Auftreten von Autoimmunreaktionen gekennzeichnet, deren Ätiologie bisher weitgehend unbekannt ist. Obwohl in zahlreichen Untersuchungen klar gezeigt werden konnte, dass an der Pathogenese und der Chronifizierung dieser Erkrankungen massgeblich pro-inflammatorische Zytokine beteiligt sind, so wie TNFα, type I IFN, IFNγ, IL1, IL6, IL17 und BAFF, bleibt bisher weitgehend unverstanden, wie diese Mediatoren innerhalb eines komplexen Zytokin-Netzwerks miteinander interagieren. Die pathophysiologische Bedeutung von Zytokinen in SLE, RA und AS wird durch die Tatsache hervorgehoben, dass in den letzten 10 Jahren eine zunehmende Anzahl von Antikörper-basierten anti- Zytokin Medikamenten für klinische Anwendungen zugelassen worden sind. Ziel dieser Studie ist es, die pathophysiologische Rolle von pro-inflammatorischen Zytokinen im Krankheitsverlauf von SLE, RA und AS zu bestimmen und zu vergleichen. Hierzu wurden zunächst globale Genexpressionsprofile von peripheren Blutmonozyten generiert, die aus dem Blut von Rheumapatienten und gesunden Spendern isoliert worden sind. Mit Hilfe dieses experimentellen Ansatzes ist es möglich, die Genaktivitäten sämtlicher bekannten Gene gleichzeitig quantitativ zu erfassen. Durch den Vergleich der Krankheiten untereinander und dem Vergleich zu den gesunden Spendern konnte erstmals gezeigt werden, dass periphere Monozyten krankheitsspezifische Transkriptionsmuster aufweisen. Nach funktioneller Annotation der krankheits- assoziierten Gene konnte eindeutig die Beteiligung verschiedener Zytokine nachgewiesen werden, allen voraus TNFα, type I IFN, IFNγ, IL1 und IL8. Interessanterweise konnte für diese Zytokine gezeigt werden, dass die durch diese Mediatoren induzierten zellulären Antworten in Abhängigkeit von der jeweiligen Erkrankung unterschiedlich moduliert worden sind. Um diese Beobachtung molekular weiter aufschlüsseln zu können, wurden zusätzlich Zytokin-spezifische Genexpressionsprofile durch in vitro Stimulation von Monozyten mit TNFα, IFNα2a und IFNγ (IFN) erstellt. Vergleiche zwischen den Krankheits-spezifischen und den in vitro-generierten Referenzsignaturen zeigten, dass das Transkriptom von Monozyten beim SLE durch IFN-induzierte Gene dominiert ist, während das RA Genexpressionsprofil hauptsächlich durch TNFα beeinflusst ist. Die IFN-Effekte beim SLE wurden ganz offensichtlich primär durch den überexprimierten Transkriptionsfaktor STAT1 vermittelt. Interessanterweise konnte gezeigt werden, dass in Monozyten von RA Patienten die Aktivierung von STAT1 durch die Überexpression von TNFα inhibiert wurde. Allerdings liessen sich auch IFN-induzierte Signaturen bei RA und TNFα- induzierte Signaturen in SLE Monozyten nachweisen. Diese Ergebnisse lassen die Schlussfolgerung zu, dass die transkriptionellen TNFα und IFN-Antworten auf unterschiedliche Weise krankheitsabhängig bei SLE und RA moduliert werden. Im Gegensatz zu SLE und RA, zeigten Monozyten von AS Patienten generell nur schwache Veränderungen in der Genexpression, und obwohl TNFα und IFN- induzierte Gensignaturen nachweisbar waren, konnte aufgrund der niedrigen Expressionsstärken kein dominierendes Zytokin nachgewiesen werden. Zusammenfassend konnte diese Studie zeigen, dass Monozyten von SLE, RA und AS Patienten Krankheits-spezifische Genexpressionsprofile aufwiesen, die durch den Vergleich mit in vitro-generierten, Zytokin-spezifischen Gensignaturen im Hinblick auf dominierende und interagierende Zytokine wesentlich detaillierter analysiert werden konnten. Die IFN- und TNFα-vermittelten Gensignaturen zeigten krankheitsabhängige Modulationsmuster und spiegelten das Zusammenspiel von Zytokinen innerhalb verschiedener inflammatorischer Milieus wieder. Die Ergebnisse dieser Studie legen den Schluss nahe, dass die Erhebung eines molekularen Zytokinstatus bei Rheumapatienten von grosser Bedeutung für eine verbesserte Differentialdiagnose und eine gezielte Therapieempfehlung sein kann. Hierdurch würde man dem generell in der modernen Medizin angestrebten Grundsatz der personalisierten Medizin einen entscheidenden Schritt näher gekommen

Peripheral blood mononuclear cells are hypomethylated in active rheumatoid arthritis and methylation correlates with disease activity

OBJECTIVE: Epigenetic modifications are dynamic and influence cellular disease activity. The aim of this study was to investigate global DNA methylation in peripheral blood mononuclear cells (PBMCs) of RA patients to clarify whether global DNA methylation pattern testing might be useful in monitoring disease activity as well as the response to therapeutics. METHODS: Flow cytometric measurement of 5-methyl-cytosine (5'-mC) was established using the cell line U937. In the subsequent prospective study, 62 blood samples were investigated, including 17 healthy donors and 45 RA patients at baseline and after 3 months of treatment with methotrexate, the IL-6 receptor inhibitor sarilumab, and Janus kinase inhibitors. Methylation status was assessed with an anti-5'-mC antibody and analysed in PBMCs and CD4+, CD8+, CD14+ and CD19+ subsets. Signal intensities of 5'-mC were correlated with 28-joint DASs with ESR and CRP (DAS28-ESR and DAS28-CRP). RESULTS: Compared with healthy individuals, PBMCs of RA patients showed a significant global DNA hypomethylation. Signal intensities of 5'-mC correlated with transcription levels of DNMT1, DNMT3B and MTR genes involved in methylation processes. Using flow cytometry, significant good correlations and linear regression values were achieved in RA patients between global methylation levels and DAS28-ESR values for PBMCs (r = -0.55, P = 0.002), lymphocytes (r = -0.57, P = 0.001), CD4+ (r = -0.57, P = 0.001), CD8+ (r = -0.54, P = 0.001), CD14+ (r = -0.49, P = 0.008) and CD19+ (r = -0.52, P = 0.004) cells. CONCLUSIONS: The degree of global DNA methylation was found to be associated with disease activity. Based on this novel approach, the degree of global methylation is a promising biomarker for therapy monitoring and the prediction of therapy outcome in inflammatory diseases