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Rheumatoid arthritis (RA) synovitis is dominated by monocytes/macrophages with inflammatory patterns resembling microbial stimulation. In search of triggers, we reduced the intestinal microbiome in 20 RA patients (open label study DRKS00014097) by bowel cleansing and 7-day fasting (≤250 kcal/day) and performed immune monitoring and microbiome sequencing. Patients with metabolic syndrome (n = 10) served as a non-inflammatory control group. Scores of disease activity (DAS28/SDAI) declined within a few days and were improved in 19 of 20 RA patients after breaking the fast (median ∆DAS28 = −1.23; ∆SDAI = −43%) or even achieved remission (DAS28 < 2.6/n = 6; SDAI < 3.3/n = 3). Cytometric profiling with 46 different surface markers revealed the most pronounced phenomenon in RA to be an initially increased monocyte turnover, which improved within a few days after microbiota reduction and fasting. Serum levels of IL-6 and zonulin, an indicator of mucosal barrier disruption, decreased significantly. Endogenous cortisol levels increased during fasting but were insufficient to explain the marked improvement. Sequencing of the intestinal microbiota indicated that fasting reduced potentially arthritogenic bacteria and changed the microbial composition to species with broader metabolic capabilities. More eukaryotic, predominantly fungal colonizers were observed in RA, suggesting possible involvement. This study demonstrates a direct link between the intestinal microbiota and RA-specific inflammation that could be etiologically relevant and would support targeted nutritional interventions against gut dysbiosis as a causal therapeutic approach.

ZusammenfassungDie idiopathischen inflammatorischen Myopathien (IIM) sind eine Gruppe entzündlicher Muskelerkrankungen für deren Diagnosestellung, Verlaufsbeurteilung, Prognoseabschätzung und Risikostratifizierung Biomarker eine jeweils essentielle Rolle spielen. Biomarker in diesem Kontext können sowohl „herkömmliche“ serologische Marker wie Muskelenzyme oder Autoantikörper, histologische Marker wie entitätsspezifische inflammatorische Muster, aber auch genomische und genetische Marker sein. Der vorliegende Artikel gibt einen Überblick über bewährte und innovative Marker.

Advances in microbiome research suggest involvement in chronic inflammatory diseases such as rheumatoid arthritis (RA). Searching for initial trigger(s) in RA, we compared transcriptome profiles of highly inflamed RA synovial tissue (RA-ST) and osteoarthritis (OA)-ST with 182 selected reference transcriptomes of defined cell types and their activation by exogenous (microbial) and endogenous inflammatory stimuli. Screening for dominant changes in RA-ST demonstrated activation of monocytes/macrophages with gene-patterns induced by bacterial and fungal triggers. Gene-patterns of activated B- or T-cells in RA-ST reflected a response to activated monocytes/macrophages rather than inducing their activation. In contrast, OA-ST was dominated by gene-patterns of non-activated macrophages and fibroblasts. The difference between RA and OA was more prominent in transcripts of secreted proteins and was confirmed by protein quantification in synovial fluid (SF) and serum. In total, 24 proteins of activated cells were confirmed in RA-SF compared to OA-SF and some like CXCL13, CCL18, S100A8/A9, sCD14, LBP reflected this increase even in RA serum. Consequently, pathogen-like response patterns in RA suggest that direct microbial influences exist. This challenges the current concept of autoimmunity and immunosuppressive treatment and advocates new diagnostic and therapeutic strategies that consider microbial persistence as important trigger(s) in the etiopathogenesis of RA.

Summary Background An acquired deficiency of interleukin-2 (IL-2) and related defects in regulatory T cell homeostasis are thought to play a crucial role in the pathogenesis of systemic lupus erythematosus. We hypothesised that reconstitution of regulatory T-cell homoeostasis with low doses of IL-2 would be beneficial to patients with systemic lupus erythematosus. Methods In this uncontrolled, phase 1 and 2a trial done in the Department of Rheumatology and Clinical Immunology at Charite—University Medicine Berlin (Berlin, Germany), we assessed the safety and tolerability of low-dose recombinant human IL-2 (aldesleukin) and its effects on regulatory T cells. We recruited patients aged 18–75 years with a confirmed diagnosis of systemic lupus erythematosus and moderate-to-severe disease activity despite previous treatment with at least two conventional therapies. Patients were given four cycles of low-dose aldesleukin daily for 5 days followed by a 9–16 day rest. The primary endpoints were safety and the number of patients who achieved at least a 100% increase in the proportion of CD25hi-expressing cells among circulating CD3 + CD4 + FOXP3 + CD127lo regulatory T cells at day 62 (ie, after four treatment cycles). Secondary endpoints included disease activity as measured by the Safety of Estrogens in Lupus National Assessment-Systemic Lupus Erythematosus Disease Activity Index (SELENA-SLEDAI) and the British Isles Lupus Assessment Group (BILAG) score, disease flares as measured by the SLEDAI flare index, auto-antibody and complement concentrations at day 62. Exploratory endpoints included various cellular and immunological parameters. The trial is registered with WHO/ICTRP, number DRKS00004858. Findings Between March 31, 2014, and May 27, 2016, 13 patients were screened, of whom ten met eligibility criteria and were enrolled in the trial. Two additional patients were treated between April 1, 2013, and March 11, 2014, in a compassionate use setting. Eleven (92%) of the 12 patients achieved the primary endpoint. 159 adverse events were recorded, 75 (47%) of which were treatment related. Most treatment-related adverse events were transient and mild to moderate (grade 1–2). The most common adverse event was injection-site reaction (20%). No serious adverse events occurred during the treatment period. In ten (83%) of 12 patients, SELENA-SLEDAI scores were lower at day 62 than at baseline, and no severe disease flares were observed during the treatment period. Decreased disease activity correlated with the magnitude of increase in the proportion of activated regulatory T cells. IL-2 treatment resulted in a preferential proliferation of regulatory T cells that retained suppressive capacity. We observed decreases in cells that are involved in the regulation of germinal-centre reactions. Interpretation Low-dose IL-2 therapy is safe and well tolerated and selectively promotes the expansion of functional regulatory T cells in patients with moderate-to-severe systemic lupus erythematosus. Low-dose IL-2 treatment might also be beneficial in reducing disease activity, although larger trials are needed to address efficacy. Funding German Research Foundation.

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.

Rheumatische Erkrankungen gehören zu den häufigsten chronisch entzündlichen Krankheiten. Neben ausgeprägter Schmerzhaftigkeit und progredienter Gelenkzerstörung reduzieren die rheumatoide Arthritis (RA), die Spondyloarthritiden (SpA) und die Psoriasisarthritis (PsA) die Arbeitsfähigkeit, die Lebensqualität und bei unzureichender Behandlung auch die Lebenserwartung. Seit der Einführung der Biologika zur Therapie dieser Erkrankungen hat die Suche nach geeigneten Biomarkern zur Frühdiagnostik und Vorhersage des Therapieerfolgs zunehmend an Bedeutung gewonnen. Das Hauptziel des Verbundes ArthroMark ist, neue Biomarker zu identifizieren und moderne Bildgebungsverfahren einzusetzen mit dem Ziel, die Diagnose, die Verlaufskontrolle und die Stratifizierung von Patienten mit RA, SpA und PsA zu verbessern. Mit der Entwicklung geeigneter Biomarker für diese Erkrankungen trägt dieses Vorhaben zur Gesundheitsforschung im Bereich chronischer Erkrankungen des Bewegungsapparates bei. Durch die Zusammenarbeit verschiedener nationaler Zentren sollen standortspezifische Ressourcen wie Probenbanken und klinische Studien gemeinsam nutzbar gemacht werden und individuelle Schwerpunkte in der Biomarkeranalyse mit einem entsprechenden Mehrwert vernetzt werden. Gemeinsames Datenmanagement und Vereinheitlichung der Datenerhebung sowie bestmögliche Charakterisierung der Patienten durch neue Bildgebungsmethoden sollen die Qualität der Markerprüfung optimieren.

Big Data wird mit der Vision verknupft, aus unuberschaubaren Datenmengen durch computerbasierte Analysemethoden neue Erkenntnisse zu gewinnen. Welche Analysekonzepte stehen hinter dieser Big-Data-Diskussion? Im Grunde sind mit dem Einsatz von Hochdurchsatztechnologien in der molekularen Forschung in der Rheumatologie schon seit ca. 15 Jahren Big Data vorhanden und Analysen in Entwicklung bzw. im Gebrauch. Hierzu gehoren insbesondere die Omics-Technologien, wie z. B. Genomics, Transcriptomics oder Cytomics. Einige Basismethoden der Datenanalyse werden mit der Technologie geliefert. Dagegen mussen fur die funktionelle Auswertung und Interpretation Softwarelosungen entwickelt bzw. an die Fragestellungen angepasst werden. Dabei sind die Strukturierung und Auswertung nach biologischen Zusammenhangen enorm wichtig und kein alleiniges mathematisches Problem. Dieser Aspekt muss bei molekularen Big Data noch weit mehr berucksichtigt werden als z. B. bei Big Data aus Gesundheitsokonomie und Epidemiologie. Molekulare Daten sind durch die Technologie der Erhebung in sich strukturiert und folgen in ihrer quantitativen Auspragung biologischen Gesetzmasigkeiten. Diese Zusammenhange mussen in Softwarelosungen abgebildet werden, zum Teil auch durch die Vernetzung zwischen molekularen Big Data der gleichen oder auch unterschiedlichen Technologien, um technologieubergreifende Bestatigung zu erzielen. Mit immer umfangreicherer Erfassung molekularer Vorgange in individuellen Patienten entstehen Big Data auch personenbezogen und stellen neue Anforderungen an die Bearbeitung, um datengetriebene individualisierte Losungskonzepte zu entwickeln. Damit sind fur die Umsetzung von Informationen aus Big Data im molekularen Bereich auch neue Anforderungen an Ausbildung und Berufskompetenzen zu erwarten.

Career situation of first and presenting author Instructor. Objectives An acquired deficiency of interleukin-2 (IL-2) and related defects in regulatory T cell (Treg) homeostasis play a crucial role in the pathogenesis of SLE. Here, we report the responses of Treg and other lymphocyte subsets to low-dose IL-2 therapy observed during an open-label, uncontrolled, dose adaption, phase 1/2a single-center clinical trial in patients with active and refractory SLE. Methods 12 patients with active and refractory SLE (SLEDAI ≥6) were treated at our site with a low-dose IL-2 regimen consisting of four separate treatment cycles each with daily subcutaneous injections of recombinant human IL-2 (aldesleukin) at single daily doses of either 0.75, 1.5 or 3.0 million IU for five consecutive days. Cells from peripheral blood were analyzed by flow cytometry before and one day after each treatment cycle. Results All patients showed highly significant cycle- and dose-dependent increases in the proportions and absolute numbers of CD3+CD4+FoxP3+CD127 lo Treg and of CD25hi expressing cells among Treg. By contrast, we observed no relevant changes in the absolute numbers of CD3+CD4+FoxP3 conventional T cells (Tcon), of CD3+CD8+ T cells, of CD3+CD56+NK T cells and of CD3-CD56+NK cells. The IL-2 expanded Treg population displayed a preserved suppressive capacity and expressed high levels of the Treg-associated molecules Helios, CD39 and CD137. In addition, we noted robust and dose-dependent increases in the proportions of Treg expressing the proliferation marker Ki67. Although significant increases in the proportions of Ki67 +cells among Tcon and also among other lymphocyte subsets were observed at the end of each treatment cycle, the calculated ratio between Ki67+ Treg and Ki67+ Tcon continuously increased and was significantly higher at the end of the treatment phase, suggesting a preferential targeting of the Treg population. Conclusions Low-dose IL-2 therapy promotes the selective expansion of a functionally competent and thymic-derived Treg population in patients with refractory SLE. This study also provides novel insights into the pharmacodynamics and the broad biologic effects of low-dose IL-2 therapy. Disclosure of Interest J. Humrich Consultant for: ILTOO Pharma, C. von Spee-Mayer: None declared, E. Siegert: None declared, M. Bertolo: None declared, A. Rose: None declared, D. Abdirama: None declared, P. Enghard: None declared, B. Stuhlmuller: None declared, F. Hiepe: None declared, T. Alexander: None declared, E. Feist: None declared, A. Radbruch: None declared, G.-R. Burmester: None declared, G. Riemekasten Consultant for: ILTOO Pharma.

Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent, acute, and self-limiting attacks of fever. Mutations in MEFV gene encoding pyrin account for FMF, but the high number of heterozygote patients with typical symptoms of the disease has driven a number of alternative aetiopathogenic hypotheses. The MEFV gene was knocked down in human myelomonocytic cells that express endogenous pyrin to identify deregulated microRNAs (miRNAs). Microarray analyses revealed 29 significantly differentially expressed miRNAs implicated in pathways associated with cellular integrity and survival. Implementation of in silico gene network prediction algorithms and bioinformatics analyses showed that miR-4520a is predicted to target genes implicated in autophagy through regulation of RHEB/mTOR signaling. Differential expression levels of RHEB were confirmed by luciferase reporter gene assays providing further evidence that is directly targeted by miR-4520a. Although the relative expression levels of miR-4520a were variable among FMF patients, the statistical expression of miR-4520a was different between FMF mutation carriers and controls (P = 0.0061), indicating an association between miR-4520a expression and MEFV mutations. Comparison between FMF patients bearing the M694V mutation, associated with severe disease, and healthy controls showed a significant increase in miR-4520a expression levels (P = 0.00545). These data suggest that RHEB, the main activator of mTOR signaling, is a valid target of miR-4520a with the relative expression levels of the latter being significantly deregulated in FMF patients and highly dependent on the presence of pyrin mutations, especially of the M694V type. These results suggest a role of deregulated autophagy in the pathogenesis of FMF. J. Cell. Physiol. 232: 1326-1336, 2017. © 2016 Wiley Periodicals, Inc.

Background: Fasting can improve clinical disease activity in rheumatoid arthritis (RA) [1], but mechanism involved are not clear. Recently, we demonstrated that monocytes in RA express transcriptome patterns of increased myelopoiesis, premature egress from bone marrow and reduced circulation time as indicators of permanent activation of the innate immune response [2]. Objectives: We investigated the influence of bowel cleanse and fasting on monocyte subpopulations in the blood to determine the extent of microbiota and gut immunity related triggering of chronic inflammation in RA. Methods: RA patients (n=22) and controls (n=12, metabolic syndrome), who presented for fasting according to the Buchinger procedure (bowel cleanse with colonoscopy fluid), were analyzed for DAS28, CrP, differential blood count and high resolution cytometric phenotyping at baseline, day 3, day 7 (end of fasting) and day 10. ImmunoClust was applied for automated cell clustering [3]. Results: Disease activity was strikingly decreased after fasting in virtually all RA patients (DAS28 from 4.24 to 3.17, p + CD16 + ) and intermediate (CD14 ++ CD16 + ) monocytes prior to fasting in RA compared to controls and confirmed previous results [2]. Bowel cleanse and fasting induced a significant increase of these two monocyte subpopulations by absolute counts and even more by percentage of total monocytes. This indicates reduced recruitment to inflamed tissue and prolonged circulation with more cells differentiating from classical to non-classical monocytes in the blood [4]. The decrease of lymphocytes in RA patients after fasting was characterized by a dominant reduction of naive T-, B-cells and CD16 - NK-cells along with a relative increase in memory lymphocytes and CD16 + NK-cells. These effects were also observed but less pronounced in controls. Conclusions: Bowel cleanse and fasting in RA induces a reduction of inflammation related to monocyte activation and turnover immediately within few days. Changes in the monocyte compartment were specific for RA compared to controls and dominated the immunological changes, suggesting that innate triggering mechanisms from gut and its microbiota are etiologically relevant in RA. References [1] Kjeldsen-Kragh J, et al. Lancet1991;338(8772):899. [2] Smiljanovic B, et al. Ann Rheum Dis2018;77(2):300. [3] Sorensen T, et al. Cytometry A2015;87(7):603. [4] Tak T, et al. Blood2017;130(12):1474. Acknowledgements: Technical assitance: Silvia Pade, Barbara Walewska Funding: German Federal Ministry of Education and Research grant ArthoMark (01EC1009A), Corona-Stiftung grant BioFast (S199/10063/2016). Disclosure of Interest: None declared

ObjectiveRheumatoid arthritis (RA) accompanies infiltration and activation of monocytes in inflamed joints. We investigated dominant alterations of RA monocytes in bone marrow (BM), blood and inflamed joints.MethodsCD14+ cells from BM and peripheral blood (PB) of patients with RA and osteoarthritis (OA) were profiled with GeneChip microarrays. Detailed functional analysis was performed with reference transcriptomes of BM precursors, monocyte blood subsets, monocyte activation and mobilisation. Cytometric profiling determined monocyte subsets of CD14++CD16−, CD14++CD16+ and CD14+CD16+ cells in BM, PB and synovial fluid (SF) and ELISAs quantified the release of activation markers into SF and serum.ResultsInvestigation of genes differentially expressed between RA and OA monocytes with reference transcriptomes revealed gene patterns of early myeloid precursors in RA-BM and late myeloid precursors along with reduced terminal differentiation to CD14+CD16+monocytes in RA-PB. Patterns associated with tumor necrosis factor/lipopolysaccharide (TNF/LPS) stimulation were weak and more pronounced in RA-PB than RA-BM. Cytometric phenotyping of cells in BM, blood and SF disclosed differences related to monocyte subsets and confirmed the reduced frequency of terminally differentiated CD14+CD16+monocytes in RA-PB. Monocyte activation in SF was characterised by the predominance of CD14++CD16++CD163+HLA-DR+ cells and elevated concentrations of sCD14, sCD163 and S100P.ConclusionPatterns of less mature and less differentiated RA-BM and RA-PB monocytes suggest increased turnover with accelerated monocytopoiesis, BM egress and migration into inflamed joints. Predominant activation in the joint indicates the action of local and primary stimuli, which may also promote adaptive immune triggering through monocytes, potentially leading to new diagnostic and therapeutic strategies.

Background Targeting molecules involved in monocyte activation is an important treatment strategy for RA. In this study we aimed to determine monocyte maturation and activation from bone marrow (BM) via blood into synovial fluid (SF) by investigating monocytes transcriptomes and by cytometric profiling of classical (CD14++CD16-), intermediated (CD14++CD16+) and non-classical (CD14+CD16+) monocytes. Materials and methods CD14+ cells from BM and blood of RA and osteoarthritis (OA) patients were profiled with Affymetrix microarrays. A detailed functional analysis was performed with reference transcriptomes of BM precursors, monocyte blood subsets, monocyte activation and egress from BM induced by G-CSF (granulocyte colony-stimulating factor). Cytometric profiling of CD14, CD16, HLA-DR and CD163 expression were used to determine monocyte subsets and to follow their activation and differentiation in BM, blood and SF. Results Transcriptomes of RA-BM monocytes exhibited i) pronounce gene pattern of early myeloid precursors from BM and ii) weak gene pattern of late myeloid precursors from BM. Transcriptomes of RA blood monocytes demonstrated i) pattern of late myeloid precursors from BM and ii) reduced pattern of terminally differentiated CD14+CD16+ monocytes from blood. Cytometric profiling of BM, blood and SF monocytes in RA and OA showed that all three body compartments have their own distribution of monocyte subsets. BM was characterised with classical and intermediate subsets and both subsets showed decreased CD16 expression in RA when compared to OA. As expected, blood was characterised with three subsets, and RA blood showed decreased CD14 and HLA-DR expression on classical monocytes and reduced frequency of non-classical subset. In RA-SF, classical monocytes were absent, intermediate were most dominant and cell-phenotype with low CD16 expression but similar to non-classical monocytes was related to macrophages. Cell frequency of intermediate subset in SF positively correlated with inflammation (ESR; R>0.85) and showed the highest expression of HLA-DR, CD14, CD163. Conclusions Monocyte turnover is increased in RA and characterised with accelerated monocytopoiesis, faster BM egress and migration into inflamed joints. Permanent monocyte activation in the joint and their role in linking innate and adaptive immunity, which is targeted by biologics, emphasises their high diagnostic value and relevance for therapeutic stratification.

In vitro tissue models are useful tools for the development of novel therapy strategies in cartilage repair and care. The limited availability of human primary tissue and high costs of animal models hamper preclinical tests of innovative substances and techniques. In this study we tested the potential of porcine chondrocyte micromass cultures to mimic human articular cartilage and essential aspects of osteoarthritis (OA) in vitro. Primary chondrocytes were enzymatically isolated from porcine femoral condyles and were maintained in 96-multiwell format to establish micromass cultures in a high-throughput scale. Recombinant porcine tumor necrosis factor alpha (TNF-α) was used to induce OA-like changes documented on histological (Safranin O, collagen type II staining), biochemical (hydroxyproline assay, dimethylmethylene blue method), and gene expression level (Affymetrix porcine microarray, real time PCR) and were compared with published data from human articular cartilage and human micromass cultures. After 14 days in micromass culture, porcine primary chondrocytes produced ECM rich in proteoglycans and collagens. On gene expression level, significant correlations of detected genes with porcine cartilage (r = 0.90), human cartilage (r = 0.71), and human micromass culture (r = 0.75) were observed including 34 cartilage markers such as COL2A1, COMP, and aggrecan. TNF-α stimulation led to significant proteoglycan (-75%) and collagen depletion (-50%). Comparative expression pattern analysis revealed the involvement of catabolic enzymes (MMP1, -2, -13, ADAM10), chemokines (IL8, CCL2, CXCL2, CXCL12, CCXL14), and genes associated with cell death (TNFSF10, PMAIPI, AHR) and skeletal development (GPNMB, FRZB) including transcription factors (WIF1, DLX5, TWIST1) and growth factors (IGFBP1, -3, TGFB1) consistent with published data from human OA cartilage. Expression of genes related to cartilage ECM formation (COL2A1, COL9A1, COMP, aggrecan) as well as hypertrophic bone formation (COL1A1, COL10A1) was predominantly found decreased. These findings indicating significant parallels between human articular cartilage and the presented porcine micromass model and vice versa confirm the applicability of known cartilage marker and their characteristics in the porcine micromass model. TNF-α treatment enabled the initiation of typical OA reaction patterns in terms of extensive ECM loss, cell death, formation of an inflammatory environment through the induction of genes coding for chemokines and enzymes, and the modulation of genes involved in skeletal development such as growth factors, transcription factors, and cartilage ECM-forming genes. In conclusion, the porcine micromass model represents an alternative tissue platform for the evaluation of innovative substances and techniques for the treatment of OA.

Idiopathische inflammatorische Myopathien (IIM) sind entzundliche Erkrankungen des Muskelapparates mit chronischem Verlauf und Muskelschwache. Hierbei kommt es bei den 3 Haupterkrankungen, der Dermatomyositis, der Polymyositis und der Einschlusskorperchenmyositis, zu immunologischen Angriffsprozessen auf das Muskelgewebe durch das humorale antikorperbildende Immunsystem (B-Zellen), wie auch durch das zellulare Immunsystem (dendritische Zellen, Monozyten/Makrophagen, CD4+- und CD8+-T-Zellen und naturliche Killerzellen). Letzteres wirkt dabei nicht nur autoaggressiv zytotoxisch, sondern unterhalt auch die Entzundung. Uber die Pathogenese der IIM ist bislang noch wenig bekannt. Dennoch scheinen genetische Pradispositionen und Umweltfaktoren eine unterstutzende Rolle bei der Krankheitsinitiierung und im Verlauf zu spielen. Insbesondere scheinen auch Infektionen mit Pilzen, Bakterien und/oder Viren atiologisch bedeutsam. Bis dato existieren nur wenige Merkmale zur Beurteilung der individuellen Prognose oder zur Abschatzung des therapiespezifischen Behandlungserfolgs. Konventionelle und neuere Behandlungsstrategien sind kritisch unter Berucksichtigung der Vergleichbarkeit der Studien zu bewerten. Zur Entwicklung neuer und zielgerichteter Therapien wird zunehmend die Einbeziehung von genomweiten Untersuchungsmethoden relevant, um molekulare Pathomechanismen systematisch zu erfassen und die Atiopathogenese bei IIM besser zu verstehen. Diese Methoden wecken auch die Hoffnung, geeignete Biomarker fur die Klassifikation, therapeutische Stratifizierung, Aktivitatsbestimmung, Prognose und die krankheitsspezifischen Subgruppen patientenspezifisch zu identifizieren.

Effective drug selection is the current challenge in rheumatoid arthritis (RA). Treatment failure may follow different pathomechanisms and therefore require investigation of molecularly defined subgroups. In this exploratory study, whole blood transcriptomes of 68 treatment-naive early RA patients were analyzed before initiating MTX. Subgroups were defined by serologic and genetic markers. Response related signatures were interpreted using reference transcriptomes of various cell types, cytokine stimulated conditions and bone marrow precursors. HLA-DRB4-negative patients exhibited most distinctive transcriptional differences. Preponderance of transcripts associated with phagocytes and bone marrow activation indicated response and transcripts of T- and B-lymphocytes non-response. HLA-DRB4-positive patients were more heterogeneous, but also linked failure to increased adaptive immune response. RT-qPCR confirmed reliable candidate selection and independent samples of responders and non-responders the functional patterning. In summary, genomic stratification identified different molecular pathomechanisms in early RA and preponderance of innate but not adaptive immune activation suggested response to MTX therapy.

Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent, acute, and self-limiting attacks of fever. Mutations in MEFV gene encoding pyrin account for FMF, but the high number of heterozygote patients with typical symptoms of the disease has driven a number of alternative aetiopathogenic hypotheses. The MEFV gene was knocked down in human myelomonocytic cells that express endogenous pyrin to identify deregulated microRNAs (miRNAs). Microarray analyses revealed 29 significantly differentially expressed miRNAs implicated in pathways associated with cellular integrity and survival. Implementation of in silico gene network prediction algorithms and bioinformatics analyses showed that miR-4520a is predicted to target genes implicated in autophagy through regulation of RHEB/mTOR signaling. Differential expression levels of RHEB were confirmed by luciferase reporter gene assays providing further evidence that is directly targeted by miR-4520a. Although the relative expression levels of miR-4520a were variable among FMF patients, the statistical expression of miR-4520a was different between FMF mutation carriers and controls (P = 0.0061), indicating an association between miR-4520a expression and MEFV mutations. Comparison between FMF patients bearing the M694V mutation, associated with severe disease, and healthy controls showed a significant increase in miR-4520a expression levels (P = 0.00545). These data suggest that RHEB, the main activator of mTOR signaling, is a valid target of miR-4520a with the relative expression levels of the latter being significantly deregulated in FMF patients and highly dependent on the presence of pyrin mutations, especially of the M694V type. These results suggest a role of deregulated autophagy in the pathogenesis of FMF. J. Cell. Physiol. 232: 1326-1336, 2017. © 2016 Wiley Periodicals, Inc.

Many cytokines are involved in the pathogenesis of autoimmune diseases and are recognized as relevant therapeutic targets to attenuate inflammation, such as tumor necrosis factor (TNF)-α in rheumatoid arthritis (RA) and interferon (IFN)-α/γ in systemic lupus erythematosus (SLE). To relate the transcriptional imprinting of cytokines in a cell type- and disease-specific manner, we generated gene expression profiles from peripheral monocytes of SLE and RA patients and compared them to in vitro-generated signatures induced by TNF-α, IFN-α2a, and IFN-γ. Monocytes from SLE and RA patients revealed disease-specific gene expression profiles. In vitro-generated signatures induced by IFN-α2a and IFN-γ showed similar profiles that only partially overlapped with those induced by TNF-α. Comparisons between disease-specific and in vitro-generated signatures identified cytokine-regulated genes in SLE and RA with qualitative and quantitative differences. The IFN responses in SLE and RA were found to be regulated in a STAT1-dependent and STAT1-independent manner, respectively. Similarly, genes recognized as TNF-α regulated were clearly distinguishable between RA and SLE patients. While the activity of SLE monocytes was mainly driven by IFN, the activity from RA monocytes showed a dominance of TNF-α that was characterized by STAT1 down-regulation. The responses to specific cytokines were revealed to be disease-dependent and reflected the interplay of cytokines within various inflammatory milieus. This study has demonstrated that monocytes from RA and SLE patients exhibit disease-specific gene expression profiles, which can be molecularly dissected when compared with in vitro-generated cytokine signatures. The results suggest that an assessment of cytokine-response status in monocytes may be helpful for improvement of diagnosis and selection of the best cytokine target for therapeutic intervention.

Seit Einfuhrung der Biologika sind Biomarker zur Fruhdiagnostik und Therapieentscheidung von zunehmender Bedeutung. Sie sollen im BMBF-geforderten Forschungsverbund ArthroMark fur die rheumatoide Arthritis und die Spondylarthritis etabliert werden. Biobanken und Vorarbeiten zu Genotypen, Genexpressions- und Autoantigenprofilen dienen als Grundlage. Bioinformatische Vernetzung soll die Untersuchungen aufeinander abstimmen und eine Studie mit modernen Bildgebungsverfahren die funktionelle Bedeutung der neuen Biomarker bestmoglich charakterisieren. Zur Validierung der diagnostischen Marker wird eine schrittweise Erweiterung des Netzwerkes angestrebt.

Die zunehmende Verfugbarkeit der Hochdurchsatz-Technologien, die exponentiell anwachsenden Informationen zum humanen Genom und der Genexpression, sowie die globale Vernetzung von Datenbanken mit strukturierten biomedizinischen Informationen wird die Betrachtungsweise entzundlich-rheumatischer Krankheitsbilder grundlegend verandern. Es stehen bereits heute in mehreren Forschungslaboren erste Datensatze zu umfangreichen Expressionsanalysen zur Verfugung. Der Umgang mit diesen Techniken lehrt, dass neben einer gewissenhaften Auswertung, Prufung und Validierung der Ergebnisse auch auf Zellpopulationsebene eine anspruchsvolle Charakterisierung der Patienten nach besten konventionellen klinischen, labordiagnostischen, bildgebenden und insbesondere auch histologischen Methoden unabdingbar ist. Zur funktionellen Beurteilung werden invitro-Testsysteme, Tiermodelle und Medikamentenwirkungsstudien weiteren Aufschluss geben. Entwicklungsbedarf hat auch die bioinformatische Aufarbeitung der sich entwickelnden immensen Datenmengen. Das gesteckte Ziel ist, nach initialer genomweiter Betrachtung diejenigen Gene zu identifizieren, die zur Charakterisierung der Erkrankung, zur Einteilung nach molekular pathophysiologischen Gesichtspunkten, zur Beurteilung der Prognose und zur Entscheidungsfindung fur das richtige therapeutische Vorgehen beitragen. Aus dem verbesserten Verstandnis uber die molekularen Ablaufe werden sich weitere und – so die Erwartung – entscheidende Ansatze fur eine effektive Therapie der chronischen Entzundung, Organ-Destruktion und pathologischen Immunantwort rheumatischer Erkrankungen ableiten lassen.

Abstract —The purpose of this study was to investigate the possible involvement of human peripheral blood monocytes in the pathology of hypertensive disease. We determined the in vitro secretion patterns of proinflammatory cytokines obtained from isolated peripheral monocytes from normal controls and from hypertensive patients either after in vitro stimulation with angiotensin II (Ang II) with or without preincubation with an Ang II type 1 receptor antagonist (losartan) or after stimulation with lipopolysaccharide. Blood samples were obtained from 22 patients with essential hypertension (before any drug administration or after interruption of antihypertensive therapy) and from 24 normotensive healthy individuals used as a control group. Peripheral blood monocytes were isolated by density gradient centrifugation and plastic adherence. The state of monocyte activity was determined by the capacity to secrete tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6, (IL-6) either spontaneously or after stimulation. Cytokine concentrations were determined in culture supernatants by specific ELISA. Proinflammatory cytokine levels were assessed by semiquantitative reverse transcribed polymerase chain reaction. After stimulation with Ang II, the IL-1β secretion of peripheral blood monocytes was significantly increased in hypertensive patients versus healthy individuals ( P P P

Objective. The p205 autoantigen and interleukin-2 (IL-2) function synergistically to stimulate T lymphocytes from patients with rheumatoid arthritis (RA), and a p205-derived amino acid sequence is identical to an immunoglobulin sequence located within a domain that is reactive with rheumatoid factors (RF). This study was conducted to analyze in detail the T cell immune response against p205 and to investigate whether immunity to p205 may play a role in T cell-mediated immunopathology in active RA. Methods. Cibachron blue, protein A-Sepharose, and gel filtration on Sephacryl were used successively to enrich p205 from synovial fluid (SF). T lymphocytes from RA patients were isolated from the peripheral blood (PB), lymph nodes, and SF, and p205 and peptides derived from known sequences were assessed by T cell proliferation assays in the presence of IL-2. Results. P205-specific proliferation of T cells was observed in PB as well as in SF. When p205 was isolated from RA SF, proliferation of RA T cells peaked on day 3. With p205 purified from SF from trauma patients, there was a significant shift of the maximum T cell proliferation to day 8. T cells were of CD4 or CD8 phenotype, and B cells did not proliferate to a significant degree. The T cell response to p205 was always higher for SF mononuclear cells (SFMC) compared with PBMC (P < 0.001). In 1 RA patient who underwent repeated leukapheresis, this led to a reproducible decline in p205-specific T cell proliferation to control levels. PB T cells specifically proliferating in response to p205 were detected in 20 of 32 RA patients (63%). Of 26 patients with other inflammatory rheumatic diseases, only 1 showed a minor response to p205, while normal donors did not demonstrate a significant T cell proliferation. A synthetic p205-derived peptide, with an amino acid sequence identical to an immunoglobulin sequence located in the area where RF binds, was reactive with T cells from RA patients. Conclusion. P205 appears to be a major target of autoreactive T cells in RA. P205-specific T cells are primed and more abundant at the site of inflammation. As a T cell target in RA, p205 may well be an antigen involved in the initiation of RF production.

Background and objectives Clinical signs and symptoms, radiographic changes and routine laboratory tests have indispensable roles in diagnosis of rheumatoid arthritis (RA). Nevertheless, a high degree of heterogeneity between RA patients and increasing options of treatment require the identification of objective criteria relevant for diagnosis and therapeutic stratification of patients. This study focused on global approaches in dissecting inflammation in RA including transcriptome analyses of synovial tissue, blood and bone marrow monocytes and proteome analyses of selected molecules in serum from long-lasting and early RA. Materials and methods Gene-expression profiling of synovial tissues, blood and bone marrow monocytes of RA and osteoarthritis (OA) patients were performed by Affymetrix microarrays. Based on transcriptome data, 28 molecules were selected for protein analyses by ELISA and multiplex immunoassays in sera from patients with long-lasting RA (n = 17) and OA (n = 16), early RA (n = 10) and healthy donors (n = 14). Results Transcriptome analyses of synovial tissues from RA and OA patients showed the most prominent differences between these two diseases and identified more than 1000 differentially expressed genes. More subtle differences were disclosed by gene-expression profiling of blood and bone marrow monocytes from RA and OA with 300 and 150 differentially expressed genes, respectively. From RA tissue- and cell-specific transcriptomes 28 genes were selected for protein analyses in serum from RA and OA patients including: chemokines (CXCL13, CCL18), adhesion molecules (VCAM1, ICAM1, E- and P-Selectins), enzymes (MMP3, A1AT), alarmins (S100P and S1008/9) and the soluble form of cell surface molecules (CD14, CD163). Out of 28 markers only 16 reached statistical significance to discriminate long-lasting RA from OA. A combination of 5 markers was able to correctly classify long-lasting RA. However, the same combination of markers identified only one-third of early RA patients. Conclusions Tissue- and cell-specific transcriptomes demonstrated the systemic nature of RA. Proteome analyses of serum from long-lasting RA patients confirmed transcriptome data and showed that molecular patterns determined by the combination of inflammatory and cell-specific markers are required for disease stratification. In early RA transcriptome data outperformed proteome data suggesting that focus on transcriptional alterations is more sensitive approach for disease management of early RA.

Background Microfracture is a first-line treatment option for cartilage repair. In microfracture, subchondral mesenchymal cortico-spongious progenitor cells (CSP) enter the defect and form cartilage repair tissue. The aim of our study was to investigate the effects of joint disease conditions on the in vitro chondrogenesis of human CSP. Methods CSP were harvested from the subchondral bone marrow. CSP characterization was performed by analysis of cell surface antigen pattern and by assessing the chondrogenic, osteogenic and adipogenic differentiation potential, histologically. To assess the effect of synovial fluid (SF) on chondrogenesis of CSP, micro-masses were stimulated with SF from healthy (ND), osteoarthritis (OA) and rheumatoid arthritis donors (RA) without transforming growth factor beta 3. Results CSP showed the typical cell surface antigen pattern known from mesenchymal stem cells and were capable of osteogenic, adipogenic and chondrogenic differentiation. In micro-masses stimulated with SF, histological staining as well as gene expression analysis of typical chondrogenic marker genes showed that SF from ND and OA induced the chondrogenic marker genes aggrecan, types II and IX collagen, cartilage oligomeric matrix protein (COMP) and link protein, compared to controls not treated with SF. In contrast, the supplementation with SF from RA donors decreased the expression of aggrecan, type II collagen, COMP and link protein, compared to CSP treated with SF from ND or OA. Conclusion These results suggest that in RA, SF may impair cartilage repair by subchondral mesenchymal progenitor cells in microfracture, while in OA, SF may has no negative, but a delaying effect on the cartilage matrix formation.

We performed transcription profiling using monocytes to identify predictive markers of response to anti-tumor necrosis factor (anti-TNF) therapy in patients with rheumatoid arthritis (RA). Several potential predictors of response were identified, including CD11c. Validation in samples from independent cohorts (total of n = 27 patients) using reverse transcription-PCR confirmed increased expression of CD11c in responders to adalimumab (100% sensitivity; 91.7% specificity, power 99.6%; alpha = 0.01). Pretherapy CD11c levels significantly correlated with the response criteria as defined by the American College of Rheumatology (ACR) (r = 0.656, P < 0.0001). However, CD11c was neither predictive of response to methotrexate (MTX) alone (n = 34) nor to MTX in combination with adalimumab (n = 16). Clinical responders revealed a reset to a normal expression pattern of resident/inflammatory monocyte markers, which was absent in nonresponders. Therefore, an analysis of key cell types identifies potentially predictive biomarkers that may help to restrict the use of adalimumab to therapy responders. Larger studies, including studies of monotherapy with other drugs, are now needed to confirm and validate the specificity of CD11c for anti-TNF biologics.

Several genome-wide transcriptome studies have shown that chronic inflammatory responses generally taking place in the inflamed tissue are also reflected at the level of peripheral blood leukocytes. Blood monocytes are highly sensitized cell type continuously activated under inflammatory conditions. For a better understanding of the transcriptional imprinting influenced by a multitude of pro- and anti-inflammatory mediators, we established a whole blood in vitro system to explore cell- and stimulus-specific gene expression signatures in peripheral monocytes. In an explorative study, whole blood from healthy donors was stimulated with tumour necrosis factor-alpha (TNF-α) or lipopolysaccharide (LPS) for 1.5 h. Subsequently, monocytes were isolated with a purity of >99% by high-speed fluorescence activated cell sorting. Transcriptional changes were explored by whole genome Affymetrix arrays using highly validated filtering algorithm to identify differentially expressed genes. In vitro stimulation of whole blood samples with TNF-α and LPS resulted in 4,529 and 5,036 differentially expressed genes, respectively. Although both stimuli induced similar inflammatory profiles in monocytes, TNF-α- or LPS-specific gene signatures were characterized. Functional classification identified significant numbers of differentially expressed cytokines, cytokine receptors and apoptosis-associated genes. To our knowledge, this is the first study presenting cell- and stimulus-specific gene expression signatures that can be used to decipher complex disease specific profiles of acute and chronic inflammation. Once a library of signatures from the most important inflammatory mediators is defined, it can be helpful to identify those signatures, which are predominantly driving the disease pathogenesis and which are of potential interest for a therapeutical intervention.

Transcription profiling has become a standard technology in research. It is mainly applied in the search for biomarkers to improve diagnostic and prognostic classification, to quantify disease activity and to predict or indicate response to therapy. This review will focus on rheumatoid arthritis and discuss considerations for sample selection, prerequisites for functional interpretation of data and the current status of information deduced in the field of biomarkers for the various clinical questions. In the next few years, prediction of response to treatment is the most important aim of biomarker research. With the growing number of new biological agents, there is increasing pressure to identify molecular parameters that will not only guide the therapeutic decision but also help to define the most important targets for which new biological agents should be tested in clinical studies.

The multitude and abundance of macrophage-derived mediators in rheumatoid arthritis and their paracrine/autocrine effects identify macrophages as local and systemic amplifiers of disease. Although uncovering the etiology of rheumatoid arthritis remains the ultimate means to silence the pathogenetic process, efforts in understanding how activated macrophages influence disease have led to optimization strategies to selectively target macrophages by agents tailored to specific features of macrophage activation. This approach has two advantages: (a) striking the cell population that mediates/amplifies most of the irreversible tissue destruction and (b) sparing other cells that have no (or only marginal) effects on joint damage.

Chronic inflammation is the central pathophysiology in rheumatic diseases. Blood contains most types of immune cells involved in this inflammation. Investigation of purified cell types has various advantages but also technical limitations compared to whole blood. Infiltration of immune cells into involved organs is a leading phenomenon and molecular processes in the inflamed tissue seem to change and increase by number and intensity compared to those in the blood. Many genes found differentially expressed in tissues are elevated as part of the infiltration, while only a smaller fraction becomes regulated upon inflammation. Gene products as biomarkers, which are affected by disease-specific pathomechanisms or by treatment, are of diagnostic importance. With the growing number of different biologics, markers indicative of different pathomechanisms are important for predicting drug responsiveness and treatment stratification. Finally, a comparison of pathological signatures between different types of diseases will contribute to identifying both similarities and differences in molecular processes and thus may provide insight into the driving mechanisms and etiology of rheumatic diseases.

Objective Sjogren's syndrome (SS) is characterized by autoimmune infiltration and focal accumulation of lymphocytes in the exocrine glands, with a predominance of CD4-positive T cells. Since these histologic findings are nonspecific, determination of clinical and serologic abnormalities contribute to the diagnosis. The aim of this study was to identify a novel, disease-specific, immunologically relevant marker for SS. Methods To analyze disease-related and tissue-specific expression of candidate markers, we examined biopsied minor salivary glands and peripheral blood mononuclear cells from patients with primary and secondary SS (n = 26) as well as from patients with sicca symptoms without autoimmune sialadenitis (n = 15). Expression of the Th1/Th2-related chemokines CCL3 (macrophage inflammatory protein 1α) and CCL2 (monocyte chemoattractant protein 1), CXCL7 (neutrophil-activating peptide 2 [NAP-2]), interleukin-1β, inducible costimulator, and the proteasome subunits α3 (C9) and β5i (LMP7) was analyzed at the messenger RNA (mRNA) level using real-time polymerase chain reaction techniques. Immunohistochemical analysis was used to identify the β5i (LMP7)–expressing cell populations in minor salivary glands. Results The expression profiles revealed a significant up-regulation of β5i (LMP7) exclusively in the salivary glands of SS patients. Immunohistochemistry confirmed expression of the immunoproteasome subunit β5i (LMP7) within the acinar and ductal epithelial cells. No significant difference in the distinct histologic focus scores was evident for the expression of the markers investigated. In the peripheral blood compartment, the expression of CXCL7 was up-regulated both in primary and in secondary SS. Conclusion Tissue-specific up-regulation of β5i (LMP7) mRNA was shown to be characteristic of SS, indicating a disease-specific modulation of the proteasome system. Expression of β5i (LMP7) represents an independent parameter that can be used in addition to the focus score to distinguish SS in biopsied labial salivary glands.

Objective Tumor necrosis factor (TNF)–neutralizing agents are the most successful means of ameliorating systemic autoimmune inflammation. Neutralization of TNF, however, is often associated with the development of autoantibodies, particularly to nuclear antigens, and the mechanisms of this are unknown. We undertook this study to analyze the effect of TNF and its neutralization on the expression of major histocompatibility complex class II molecules and on the function of antigen-presenting myeloid cells in rheumatoid arthritis (RA). Methods Monocytes were isolated from the peripheral blood of RA patients before and after anti-TNF monoclonal antibody (mAb) treatment and from the peripheral blood of controls by negative selection, differentiated in vitro to macrophages, and analyzed by flow cytometry for HLA–DR expression. T cell responses to activation by myeloid cells were assessed in proliferation assays, and messenger RNA (mRNA) levels of the class II transactivator (CIITA) were determined by semiquantitative reverse transcriptase–polymerase chain reaction. Results HLA–DR expression was significantly reduced on myeloid cells from RA patients with active disease, but was increased to normal levels after anti-TNF mAb treatment. Concordantly, in vitro application of TNF to monocytes from healthy individuals reduced their ability to up-regulate HLA–DR during differentiation to macrophages and, importantly, inhibited their ability to stimulate T cells in mixed lymphocyte reactions. Molecular analysis revealed that the effect of TNF on HLA–DR expression was mediated via suppression of the transcription factor CIITA. Conclusion The data indicate that TNF decreases HLA–DR expression by reducing CIITA mRNA levels in myeloid cells, functionally resulting in a decreased capacity of myeloid cells to stimulate T cells. Concordantly, ameliorating disease activity in chronic inflammatory diseases by neutralizing TNF restores expression of HLA–DR on myeloid cells as well as the ability of myeloid cells to stimulate T cells. Thus, anti-TNF treatment might lead to augmented T cell activation by myeloid cells, thereby promoting immune responses to (auto)antigens and the development of antinuclear antibodies that are frequently associated with anti-TNF therapy.

The deciphering of the sequence of the human genome has raised the expectation of unravelling the specific role of each gene in physiology and pathology. High-throughput technologies for gene expression profiling provide the first practical basis for applying this information. In rheumatology, with its many diseases of unknown pathogenesis and puzzling inflammatory aspects, these advances appear to promise a significant advance towards the identification of leading mechanisms of pathology. Expression patterns reflect the complexity of the molecular processes and are expected to provide the molecular basis for specific diagnosis, therapeutic stratification, long-term monitoring and prognostic evaluation. Identification of the molecular networks will help in the discovery of appropriate drug targets, and permit focusing on the most effective and least toxic compounds. Current limitations in screening technologies, experimental strategies and bioinformatic interpretation will shortly be overcome by the rapid development in this field. However, gene expression profiling, by its nature, will not provide biochemical information on functional activities of proteins and might only in part reflect underlying genetic dysfunction. Genomic and proteomic technologies will therefore be complementary in their scientific and clinical application.

Background and Objectives A main challenge in disease-management of rheumatoid arthritis (RA) is to establish objective criteria relevant for diagnosis and therapeutic stratification of patients. Clinical signs and symptoms, radiographic changes and routine laboratory tests have indispensable roles in diagnosis of RA. Nevertheless, a high degree of heterogeneity between RA patients and an increasing numbers of treatment opportunities, demands identification of personalised profiles that might indicate on and/or predict response to particular treatment. In this study we applied global gene-expression profiling of cells from various body locations, including synovial tissue as a principal place of destruction and inflammation, and blood and bone marrow monocytes, as a cell type that acts as biosensor for revealing on-going alterations in the body. Based on transcriptome data candidate markers were selected and validated at the protein level. Materials and Methods Affymetrix arrays were used for gene-expression profiles of synovial tissues, blood and bone marrow monocytes from RA and osteoarthritis (OA) patients. ELISA and multiplex immunoassays were used for validation of 28 markers in synovial fluid (SF) and matched serum samples from RA and OA patients. Results Transcriptome analyses of synovial tissues from RA and OA indicated on infiltration and activation of various cell types in synovial tissue including monocytes/macrophages, T-, B-, NK-cells, fibroblast. Monocyte transcriptomes from blood and bone marrow showed a minor overlap, indicating that pathophysiological changes in RA were disease- and tissue-specific. Candidate markers from these 3 transcripomtes were selected for measurement at the protein level in SF and serum. Out of 28 markers, 23 reached statistical significance when measured in SF, while 16 were significant when measured in serum. ROC analyses of individual markers from SF and serum enabled ranking for the best candidates, whose combinations allowed almost perfect classification of RA. Conclusions RA transcriptome was the most robust in synovial tissue. Nevertheless, systemic nature of RA was also demonstrated by transcriptomes from blood and bone marrow monocytes. All transcriptomes demonstrated a great potential in identifying biomarkers whose concentrations were strongly diluted and neutralised in serum. Interestingly, none of the measured markers in serum perform well enough to stratify all RA patients. However, combination of markers as a disease-pattern allowed correct stratification of RA patients and demonstrated their heterogeneity. Further prediction analyses should indicate on a minimal number of top markers relevant for disease and therapeutic stratification, and this would be an indispensable step for establishment of personalised medicine in RA.

Background and Objectives An early prediction of the outcome of particular DMARD therapies is necessary to treat the patients individually. This study aimed at defining predictive mRNA biomarkers in rheumatoid arthritis (RA) patients for the response to future treatment with methotrexate (MTX). Materials and Methods For this purpose whole blood samples from 52 active RA patients before treatment were collected in PAXgene® Blood RNA Tubes (PreAnalytiX). Extraction of intracellular RNA including miRNA was performed according to PAXgene® Blood miRNA. Kit protocol. Extracted RNA samples were amplified employing the GeneChip® 3′IVT Express Kit and hybridised onto Affymetrix GeneChip® 133 Plus 2.0 Arrays. Labelling was carried out with the GeneChip® Hybridisation, Wash and Stain Kit in a GeneChip® Fluidics Station 450. Generated signal data was normalised and evaluated using the Affymetrix Expression Console (MAS5.0) and the BioRetis online database for candidate gene selection (BioRetis GmbH, Berlin). Classification of RA patients into good, moderate and non-responders was performed based on the DAS28 and EULAR response criteria after 3 months of MTX treatment. Prior to further expression analysis all patients were divided into two genetically more homogeneous subsets – one group of 29 donors expressing a specific HLA allele mRNA and one group of 23 not expressing this specific HLA mRNA. Hierarchical clustering of discriminating candidate genes was performed using the Genesis software v1.7.6 (IGB-TU, Graz) including and excluding 9 medium responders in the HLA positive cluster and 4 medium responders in the HLA negative cluster. Results Gene comparison analysis was improved by separation of the patient collective into positive (n = 29) or negative (n = 23) groups expressing a specific HLA mRNA. Differential mRNA expression before treatment was determined between 14 good responders and 6 non-responders in the HLA positive and between 12 good responders and 7 non-responders in the HLA negative group, calculating expression change calls and fold changes. A clear discrimination between responders and non-responders to future MTX treatment was achieved with 16 distinct candidate genes for each group. These mRNA candidates will be validated in independent qPCR analyses and with further statistical cross validation algorithms. Conclusions Early prediction of response to MTX monotherapy using microarray analyses is an opportunity for effective individual medication and therefore allows preventing side effects. Employing specific response marker genes such as CD11c for anti-TNF monotherapy it is also of interest to define predictive biomarkers for the commonly used anti-TNF/MTX combination therapy not only preventing side-effects but also reducing costs.

Background and Objectives Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with joint destruction. Joint inflammation is characterised by infiltration and activation of various immune cells. To avoid difficulties in sampling synovial tissue and to avoid fluctuation in cellular composition of leukocytes, which accompanies inflammation both in synovial tissue and blood, this study was focused on transcriptome analyses of synovial tissues, blood and bone marrow monocytes. The main aim was to analyse potential of blood monocytes in dissecting inflammation in RA and in reflecting inflammation that is evident in synovial tissue. Osteoarthritis (OA), which represents a non-inflammatory disease was used as control in this study. Materials and Methods Synovial tissues from 10 RA and 10 OA patients were used for gene-expression profiling by Affymetrix HG-U133A arrays. Blood and bone marrow monocytes, obtained from 8 RA and 8 OA patients undergoing hip replacement surgery, were utilised for gene-expression profiling by Affymetrix HG-U133Plus arrays. The BioRetis database was used for microarray analyses and generation of RA profiles. Results Transcriptome analyses of synovial tissues from RA and OA patients revealed more than 1000 differentially expressed genes. Increased expression of genes involved in chemotaxis (CCL13, CCL18, CXCL9, CXCL10, CXCL13), cell adhesion and activation (ICAM1, PECAM1, ITGAL, ITGB2, CD40, CD86) indicate to inflammation but also to infiltration of various cell types like monocytes/macrophages, NK, T- and B-cells. By comparing transcriptome of RA and OA monocytes, both from blood and bone marrow, it was obvious that monocytes were able to disclose differences between these two diseases. The RA disease-specific gene-expression profile was evident both in blood and bone marrow and it demonstrated only a minor overlap between these two bodies compartments. Altogether, a typical RA inflammatory profile disclosed in synovial tissues was greatly silenced in blood monocytes, and almost completely absent in bone marrow derived monocytes. Conclusions The RA gene-expression profile was the most specific and robust in synovial tissue, demonstrating the dominance of the inflammatory process in the joints. Nevertheless, the systemic nature of RA was also evident at the level of blood and bone marrow monocytes. Concerning that blood is a favourable and easily accessible material for diagnosis and that monocytes are able to exhibit disease-specific alterations, understanding monocyte response in different rheumatic diseases seems to be advantageous approach for biomarkers discovery. This approach should be essential for identifying the objective criteria relevant for disease and therapeutic stratification of patients with RA.

Background and objectives The type 1 interferon signature (IFN1) has been reported in systemic lupus erythematosus (SLE) and subsequently in many other diseases. It is deduced from the expression of a group of genes known as downstream events after IFN1 stimulation. Involving certain toll-like receptors and being related especially to an antiviral immune response, interpretation of these findings is difficult, in particular if such signatures are reported in bacterial infections as well. Therefore, the authors aimed for a better interpretation by comparing qualities and quantities of the IFN1 signature after stimulation and in various diseases. Materials and methods Transcriptomes of chronic inflammatory diseases, infections and defined stimulation experiments with IFN1 and other cytokines, which were generated by the authors or retrieved from the Gene Expression Omnibus were systematically investigated using the BioRetis analysis platform for primary analysis and Genesis for comparative visualisation. Results A typical IFN1 signature was identified by screening for differentially expressed genes before and after IFN1 treatment in patients or in vitro stimulation of immune cells with IFN1 compared to other cytokines. The pattern of these genes was compared with the pattern in transcriptomes from patients with different rheumatic diseases including rheumatoid arthritis (RA), spondyloarthritis, SLE, Sjogren9s Syndrome, Myositis, Scleroderma and juvenile rheumatic diseases as well as with infectious diseases. Leading candidates related to IFN1 were IFIT3, IFI44L, IFI35, IFI44, MX1, MX2, OAS1, OAS2, OAS3 or SIGLEC1 already known from previous studies. These genes were characteristic for viral infections, SLE and other connective tissue diseases. Bacterial infections were positive only in few exceptions, suggesting co-infection with viruses. When observed in arthritic conditions, the diagnosis of RA was challenged. When comparing signatures induced in vitro, a much broader range of immune reactions was observed in early stages with substantial overlap between different cytokines. However, magnitude of change was still an indicator for specificity, suggesting that specific triggers and responses are always associated with additional more or less unspecific activation patterns. This may be a general rule to enable subsequently the development of a higher complexity of the immune response whenever necessary. Conclusions In summary, the IFN1 signature is certainly a prominent observation in the current literature. However, definitions for the IFN1 response have to be applied carefully in order to avoid misinterpretations by overestimation of minor or negligible amounts. Therefore, analysis should include comparisons with typical positive IFN1 signatures as reference and if possible with changes in other pathways relevant for differential diagnosis.

Rheumatoid arthritis (RA) is a chronic inflammatory and destructive joint disease characterized by overexpression of pro-inflammatory/pro-destructive mediators, whose regulation has been the focus of our previous studies. Since the expression of these proteins commonly depends on AP-1, the expression of the AP-1-forming subunits cJun, JunB, JunD, and cFos was assessed in synovial membrane (SM) samples of RA, osteoarthritis (OA), joint trauma (JT), and normal controls (NC) using ELISA and qRT-PCR. With respect to an observed discrepancy between mRNA and protein levels, the expression of the mRNA stability-modifying factors AU-rich element RNA-binding protein (AUF)-1, tristetraprolin (TTP), and human antigen R (HuR) was measured. JunB and JunD protein expression was significantly higher in RA-SM compared to OA and/or NC. By contrast, jun/fos mRNA expression was significantly (cjun) or numerically decreased (junB, junD, cfos) in RA and OA compared to JT and/or NC. Remarkably, TTP and HuR were also affected by discrepancies between their mRNA and protein levels, since they were significantly decreased at the mRNA level in RA versus NC, but significantly or numerically increased at the protein level when compared to JT and NC. Discrepancies between the mRNA and protein expression for Jun/Fos and TTP/HuR suggest broad alterations of post-transcriptional processes in the RA-SM. In this context, increased levels of mRNA-destabilizing TTP may contribute to the low levels of jun/fos and ttp/hur mRNA, whereas abundant mRNA-stabilizing HuR may augment translation of the remaining mRNA into protein with potential consequences for the composition of the resulting AP-1 complexes and the expression of AP-1-dependent genes in RA.

In the pathogenesis of rheumatoid arthritis (RA), macrophages (Mφ) play a pivotal role in synovitis, especially at the cartilage–pannus junction. Both Mφ and peripheral blood monocytes (MO) are activated and release proinflammatory cytokines (IL-1β, tumour necrosis factor [TNF] alpha). Neutralization of IL1-β and TNF-α leads to clinical improvement in RA.

Rheumatoid arthritis (RA) is a chronic inflammatory and destructive joint disease characterized by overexpression of pro-inflammatory/pro-destructive mediators, whose regulation has been the focus of our previous studies. Since the expression of these proteins commonly depends on AP-1, the expression of the AP-1-forming subunits cJun, JunB, JunD, and cFos was assessed in synovial membrane (SM) samples of RA, osteoarthritis (OA), joint trauma (JT), and normal controls (NC) using ELISA and qRT-PCR. With respect to an observed discrepancy between mRNA and protein levels, the expression of the mRNA stability-modifying factors AU-rich element RNA-binding protein (AUF)-1, tristetraprolin (TTP), and human antigen R (HuR) was measured. JunB and JunD protein expression was significantly higher in RA-SM compared to OA and/or NC. By contrast, jun/fos mRNA expression was significantly (cjun) or numerically decreased (junB, junD, cfos) in RA and OA compared to JT and/or NC. Remarkably, TTP and HuR were also affected by discrepancies between their mRNA and protein levels, since they were significantly decreased at the mRNA level in RA versus NC, but significantly or numerically increased at the protein level when compared to JT and NC. Discrepancies between the mRNA and protein expression for Jun/Fos and TTP/HuR suggest broad alterations of post-transcriptional processes in the RA-SM. In this context, increased levels of mRNA-destabilizing TTP may contribute to the low levels of jun/fos and ttp/hur mRNA, whereas abundant mRNA-stabilizing HuR may augment translation of the remaining mRNA into protein with potential consequences for the composition of the resulting AP-1 complexes and the expression of AP-1-dependent genes in RA.

Neutralizing agents to tumor necrosis factor (TNF) are the most successful means to ameliorate systemic autoimmune inflammation. Neutralization of TNF, however, is often associated with the development of autoantibodies, in particular to nuclear antigens, the mechanisms of which are unknown. Here, we analyzed the effect of TNF and its neutralization on MHC class II expression and function of antigen-presenting myeloid cells in rheumatoid arthritis (RA). Monocytes were isolated from the peripheral blood of RA patients before and after anti-TNF mAb treatment and from controls by negative selection, differentiated in vitro into macrophages and analyzed by flow cytometry for HLA-DR expression. T-cell responses to activation by myeloid cells were assessed in proliferation assays, and mRNA levels of the class II transactivator (CIITA) were determined by semi-quantitative RT-PCR. HLA-DR expression was significantly reduced on myeloid cells from RA patients with active disease, but was increased to normal levels after TNF mAb treatment. Concordantly, in vitro application of TNF to monocytes from healthy individuals reduced their ability to upregulate HLA-DR during differentiation to macrophages and, importantly, inhibited their ability to stimulate T cells in mixed lymphocyte reactions. Molecular analysis revealed that the effect of TNF on HLA-DR expression was mediated via suppression of the transcription factor CIITA. The data indicate that TNF decreases HLA-DR expression by reducing CIITA mRNA levels in myeloid cells, functionally resulting in a decreased stimulatory capacity of myeloid cells for T cells. Concordantly, ameliorating disease activity in chronic inflammatory diseases by neutralizing TNF restores HLA-DR expression of myeloid cells and their ability to stimulate T cells. Thus, anti-TNF treatment might lead to augmented T-cell activation by myeloid cells, thereby promoting immune responses to (auto)antigens and the development of anti-nuclear antibodies that are frequently associated with anti-TNF therapy.

The deciphering of the sequence of the human genome has raised the expectation of unravelling the specific role of each gene in physiology and pathology. High-throughput technologies for gene expression profiling provide the first practical basis for applying this information. In rheumatology, with its many diseases of unknown pathogenesis and puzzling inflammatory aspects, these advances appear to promise a significant advance towards the identification of leading mechanisms of pathology. Expression patterns reflect the complexity of the molecular processes and are expected to provide the molecular basis for specific diagnosis, therapeutic stratification, long-term monitoring and prognostic evaluation. Identification of the molecular networks will help in the discovery of appropriate drug targets, and permit focusing on the most effective and least toxic compounds. Current limitations in screening technologies, experimental strategies and bioinformatic interpretation will shortly be overcome by the rapid development in this field. However, gene expression profiling, by its nature, will not provide biochemical information on functional activities of proteins and might only in part reflect underlying genetic dysfunction. Genomic and proteomic technologies will therefore be complementary in their scientific and clinical application.