Identification of gene expression biomarkers to predict clinical response to methotrexate in patients with rheumatoid arthritis.

OBJECTIVES: To identify biomarkers at the gene expression level to predict response to methotrexate (MTX) in patients with rheumatoid arthritis (RA). METHODS: MTX-naïve patients with RA were started on MTX and followed up over three months. The disease activity score 28 (DAS28) was used to classify patients into responders and non-responders. Genome-wide gene expression analysis was performed in CD4 + and CD14 + mononuclear cells sampled from whole blood at baseline to identify differentially expressed genes in responders versus non-responders. Gene selection methods and prediction modelling obtained the most relevant differentially expressed genes. A logistic regression prediction model was subsequently constructed and validated via bootstrapping. The area under the receiver operating characteristic (AUC) curve was calculated to judge model quality. RESULTS: Seventy-nine patients with RA (53.4 ± 13.9 years, 74.7% females) were enrolled, and 70 finished the study with a documented treatment EULAR response (77.1% responders). Forty-six differentially expressed genes were found. The most promising genes were KRTAP4-11, LOC101927584, and PECAM1 in CD4 + cells and PSMD5 and ID1 in CD14 + cells. The final prediction model using these genes reached an AUC of 90%; the validation set's AUC was 82%. CONCLUSIONS: Our prediction model constructed via genome-wide gene expression analysis in CD4 + and CD14 + mononuclear cells yielded excellent predictions. Our findings necessitate confirmation in other cohorts of MTX-naïve RA patients. Especially if used in conjunction with previously identified clinical and laboratory (bio)markers, our results could help predict response to MTX in RA to guide treatment decisions. Key Points • Patients with rheumatoid arthritis may or may not respond to treatment with methotrexate, which is the recommended first-line drug in guidelines around the world. • In non-responders, valuable time is lost until second-line treatments are started. • This study aimed at predicting response to methotrexate by identifying differentially expressed genes from peripheral blood samples. • The final prediction model yielded excellent prognostic values, but validation in other cohorts is necessary to corroborate these findings.

Intestinal Microbiota Reduction Followed by Fasting Discloses Microbial Triggering of Inflammation in Rheumatoid Arthritis.

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.

To eat or not to eat-an exploratory randomized controlled trial on fasting and plant-based diet in rheumatoid arthritis (NutriFast-Study).

Background: Fasting is beneficial in many diseases, including rheumatoid arthritis (RA), with lasting effects for up to 1 year. However, existing data dates back several decades before the introduction of modern therapeutic modalities. Objective: This exploratory RCT compares the effects of a 7-day fast followed by a plant-based diet (PBD) to the effects of the dietary recommendations of the German society for nutrition (Deutsche Gesellschaft für Ernährung, DGE) on RA disease activity, cardiovascular (CV) risk factors, and well-being. Methods: In this RCT we randomly assigned 53 RA patients to either a 7-day fast followed by an 11-week PBD or a 12-week standard DGE diet. The primary endpoint was the group change from baseline to 12 weeks on the Health Assessment Questionnaire Disability Index (HAQ-DI). Further outcomes included other disease activity scores, body composition, and quality of life. Results: Of 53 RA patients enrolled, 50 participants (25 per group) completed the trial and were included into the per-protocol analysis. The primary endpoint was not statistically significant. However, HAQ-DI improved rapidly in the fasting group by day 7 and remained stable over 12 weeks (Δ-0.29, p = 0.001), while the DGE group improved later at 6 and 12 weeks (Δ-0.23, p = 0.032). DAS28 ameliorated in both groups by week 12 (Δ-0.97, p < 0.001 and Δ-1.14, p < 0.001; respectively), with 9 patients in the fasting but only 3 in the DGE group achieving ACR50 or higher. CV risk factors including weight improved stronger in the fasting group than in the DGE group (Δ-3.9 kg, p < 0.001 and Δ-0.7 kg, p = 0.146). Conclusions: Compared with a guideline-based anti-inflammatory diet, fasting followed by a plant-based diet showed no benefit in terms of function and disability after 12 weeks. Both dietary approaches had a positive effect on RA disease activity and cardiovascular risk factors in patients with RA. Clinical trial registration: https://clinicaltrials.gov/ct2/show/NCT03856190, identifier: NCT03856190.

Monocyte transcriptomes from patients with axial spondyloarthritis reveal dysregulated monocytopoiesis and a distinct inflammatory imprint.

BACKGROUND: In patients with axial spondyloarthritis (axSpA), monocytes show a pre-activated phenotype. Gut inflammation is a trigger of monocyte activation and may also affect their development in the bone marrow (BM). As gut inflammation is commonly observed in axSpA patients, we performed a detailed analysis of monocyte transcriptomes of axSpA patients in two cohorts and searched for signs of activation and developmental adaptations as putative imprints of gut inflammation. METHODS: Transcriptomes of blood CD14+ monocytes of HLA-B27+ axSpA patients and healthy controls (HC) were generated by microarrays from cohort 1 and by RNA-sequencing from cohort 2. Differentially expressed genes from both analyses were subjected to gene set enrichment analysis (GSEA) and to co-expression analysis in reference transcriptomes from BM cells, blood cells and activated monocytes. As serological markers of translocation, 1,3 beta-glycan, intestinal fatty acid binding protein, and lipopolysaccharide binding protein (LBP) were determined by LAL and ELISA. RESULTS: Transcriptome analysis identified axSpA-specific monocyte signatures showing an imprint of LPS/cytokine-activated monocytes, late granulopoietic BM cells, blood neutrophils, and G-CSF-mobilized blood cells, which suggests LPS/TNF activation and more prominent BM adaptation promoting a neutrophil-like phenotype. GSEA mapped axSpA upregulated genes to inflammatory responses and TNFα signaling and downregulated probe-sets to metabolic pathways. Among translocation markers, LBP levels were significantly increased in axSpA patients vs. HC (p < 0.001). Stratified analysis by disease activity and stage identified an "active disease signature" (BASDAI ≥ 4) with an imprint of LPS/cytokine-activated monocytes and CD16+ monocyte subsets. The "AS signature" (vs. non-radiographic axSpA) showed a reinforced neutrophil-like phenotype due to deprivation of dendritic cell transcripts. CONCLUSIONS: The neutrophil-like phenotype of axSpA monocytes points towards a biased monocytopoiesis from granulocyte-monocyte progenitors. This shift in monocytopoiesis and the LPS/cytokine imprint as well as the elevated LBP levels are indicators of systemic inflammation, which may result from bacterial translocation. The BM adaptation is most prominent in AS patients while disease activity appears to be linked to activation and trafficking of monocytes.

The citrullinated/native index of autoantibodies against hnRNP-DL predicts an individual "window of treatment success" in RA patients.

BACKGROUND: There is a need for biomarker to identify patients "at risk" for rheumatoid arthritis (risk-RA) and to better predict the therapeutic response and in this study we tested the hypothesis that novel native and citrullinated heterogeneous nuclear ribonucleoprotein (hnRNP)-DL autoantibodies could be possible biomarkers. METHODS: Using protein macroarray and ELISA, epitope recognition against hnRNP-DL was analysed in sera from different developed RA disease and diagnosed SLE patients. Toll-like receptor (TLR) 7/9 and myeloid differentiation primary response gene 88 (MyD88)-dependency were studied in sera from murine disease models. HnRNP-DL expression in cultivated cells and synovial tissue was analysed by indirect immunofluorescence, immunoblot and immunohistochemistry. RESULTS: HnRNP-DL was highly expressed in stress granules, citrullinated in the rheumatoid joint and targeted by autoantibodies either as native or citrullinated proteins in patient subsets with different developed RA disease. Structural citrullination dependent epitopes (SCEs) of hnRNP-DL were detected in 58% of the SLE patients although 98% of these sera were α-CCP-2-negative. To obtain a specific citrullinated signal value, we subtracted the native antibody value from the citrullinated signal. The citrullinated/native index of autoantibodies against hnRNP-DL (CNDL-Index) was identified as a new value for an "individual window of treatment success" in early RA and for the detection of RF IgM/α-CCP-2 seronegative RA patients (24-46%). Negative CNDL-index was found in SLE patients, risk-RA and early RA cohorts such as EIRA where the majority of these patients are DAS28-responders to methotrexate (MTX) treatment (87%). High positive CNDL-values were associated with more severe RA, shared epitope and parenchymal changes in the lung. Specifically, native α-hnRNP-DL is TLR7/9-dependent, associated with pain and ROC analysis revealed an association to initial MTX or etanercept treatment response, especially in seronegative RA patients. CONCLUSION: CNDL-index defines people at risk to develop RA and the "window of treatment success" thereby closing the sensitivity gap in RA.

Efficacy of therapeutic fasting and plant-based diet in patients with rheumatoid arthritis (NutriFast): study protocol for a randomised controlled clinical trial.

BACKGROUND: Previous studies have shown beneficial effects of therapeutic fasting and plant-based dietary interventions on disease activity in patients with rheumatoid arthritis (RA) for a duration of up to 1 year. To date, the effects of such interventions on the gut microbiome and on modern diagnostic markers in patients with RA have not been studied. This trial aims to investigate the clinical effects of therapeutic fasting and a plant-based diet in patients with RA, additionally considering current immunological diagnostic tools and microbiome analyses. METHODS/DESIGN: This trial is an open-label, single-centre, randomised, controlled, parallel-group clinical trial. We will randomly assign 84 patients with RA under a stable standard therapy to either (1) therapeutic fasting followed by a plant-based dietary intervention or (2) to a conventional nutritional counselling focusing on an anti-inflammatory dietary pattern according to the recommendations of the Deutsche Gesellschaft für Ernährung (German society for nutrition). Primary outcome parameter is the group difference from baseline to 12 weeks on the Health Assessment Questionnaire (HAQ). Other secondary outcomes include established clinical criteria for disease activity and treatment response in RA (Disease Activity Score 28, Simple Disease Activity Index, ACR-Response Criteria), changes in self-reported health and physical functional ability, mood, stress, quality of life, dietary behaviour via 3-day food records and a modified Food Frequency Questionnaire, body composition, changes in the gut microbiome, metabolomics and cytometric parameters. Outcomes will be assessed at baseline and day 7, after 6 weeks, 12 weeks and after 6 months. ETHICS AND DISSEMINATION: Ethical approval to process and analyse data, and to publish the results was obtained through the institutional review board of Charité-Universitätsmedizin Berlin. Results of this trial will be disseminated through peer-reviewed publications and scientific presentations. TRIAL REGISTRATION NUMBER: NCT03856190.

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.

The Relevance of Complementary and Integrative Medicine in the COVID-19 Pandemic: A Qualitative Review of the Literature.

Background: During the COVID-19 pandemic people are facing risks of adverse health effects due to the restrictions implemented such as quarantine measures, reduced social contact, and self-isolation. In this qualitative review, we collected data on potential preventive and therapeutic health benefits of Complementary and Integrative Medicine (CIM) that might be useful during the COVID-19 pandemic. We have reviewed the scientific literature to summarize CIM practices that could be beneficial for improving physical and mental health and well-being of the population under the current pandemic circumstances. It must be noted that this review is not SARS-CoV-2 specific and we explicitly do not intend to make any SARS-CoV-2 specific health claims in this article. Methods and Findings: A qualitative, non-systematic literature review was conducted in Medline to identify literature describing preventive and therapeutic CIM approaches for strengthening mental and physical health. For a variety of CIM approaches clinical evidence was identified, indicating beneficial effects. CIM approaches include specific dietary measures and selected micronutrients, physical activity, techniques from Mind-Body Medicine, single botanicals or botanical compounds, and spending time in nature among others. The effects of CIM measures on conditions like obesity and hypertension are of special relevance here, as these conditions are considered as risk factors for a severe course of COVID-19. Moreover, a possibly direct effect of CIM approaches on immune functions and clinical parameters in respiratory tract infections, such as influenza, were identified. The findings of this review could be helpful for clinicians, patients, and the general population during the current pandemic when discussing and/or considering CIM options. Conclusions: CIM offers a variety of preventive and therapeutic options for strengthening physical and mental resilience, which could also be useful in the current COVID-19 pandemic. The evidence of CIM approaches with a potential benefit in the COVID-19 pandemic in different areas is worth to be analyzed. While this qualitative review has several obvious limitations, it might serve as useful starting point for further research on this topic.

Response to abatacept is associated with the inhibition of proteasome ß1i 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 ß1i 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 ß1i in T cells of patients with RA with sustained response to abatacept suggests association of the immunoproteasome of T cells with RA disease activity.

Bacterial citrullinated epitopes generated by Porphyromonas gingivalis infection-a missing link for ACPA production.

OBJECTIVES: Porphyromonas gingivalis (P.g.) is discussed to be involved in triggering self-reactive immune responses. The aim of this study was to investigate the autocitrullinated prokaryotic peptidylarginine deiminase (PPAD) from P.g. CH2007 (RACH2007-PPAD) from a rheumatoid arthritis (RA) patient and a synthetic citrullinated PPAD peptide (CPP) containing the main autocitrullination site as potential targets for antibody reactivity in RA and to analyse the possibility of citrullinating native human proteins by PPAD in the context of RA. METHODS: Recombinant RACH2007-PPAD was cloned and expressed in Escherichia coli. Purified RACH2007-PPAD and its enzymatic activity was analysed using two-dimensional electrophoresis, mass spectrometry, immunoblot and ELISA. Autoantibody response to different modified proteins and peptides was recorded and bioinformatically evaluated. RESULTS: RACH2007-PPAD was capable to citrullinate major RA autoantigens, such as fibrinogen, vimentin, hnRNP-A2/B1, histone H1 and multiple peptides, which identify a common RG/RGG consensus motif. 33% of RA patients (n=30) revealed increased reactivity for α-cit-RACH2007-PPAD before RA onset. 77% of RA patients (n=99) presented α-cit-specific signals to CPP amino acids 57-71 which were positively correlated to α-CCP2 antibody levels. Interestingly, 48% of the α-CPP-positives were rheumatoidfactor IgM/anti-citrullinated peptide/protein antibodies (ACPA)-negative. Anti-CPP and α-RACH2007-PPAD antibody levels increase with age. Protein macroarrays that were citrullinated by RACH2007-PPAD and screened with RA patient sera (n=6) and controls (n=4) uncovered 16 RACH2007-PPAD citrullinated RA autoantigens and 9 autoantigens associated with lung diseases. We showed that the α-CPP response could be an important determinant in parenchymal changes in the lung at the time of RA diagnosis (n=106; p=0.018). CONCLUSIONS: RACH2007-PPAD induced internal citrullination of major RA autoantigens. Anti-RACH2007-PPAD correlates with ACPA levels and interstitial lung disease autoantigen reactivity, supporting an infection-based concept for induction of ACPAs via enzymatic mimicry.

Synovial tissue transcriptomes of long-standing rheumatoid arthritis are dominated by activated macrophages that reflect microbial stimulation.

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.

Fibrinogen, factor XIII and α2-antiplasmin genotypes are associated with inflammatory activity and anti-citrullinated protein antibodies.

BACKGROUND: Fibrin(ogen) derivatives, crosslinked fibrin and fibrinolysis play important roles in inflammation and are involved in pathogenesis of rheumatoid arthritis (RA). About 2/3 of RA patients exhibit anti-citrullinated protein antibodies (ACPA) that target deiminated fibrinogen. Genetic variants of ß-fibrinogen (FGB) (rs1800790G>A) and factor XIII A-subunit (F13A) Val34Leu (rs5985) are known to influence interactively inflammatory processes. It is hypothesized that predisposition for dense fibrin clots is related to better inflammation control. METHODS: To test this hypothetical model a cohort of 924 patients (288 RA and 636 non-RA patients) (3545 observations) was genotyped for FGB (rs1800790G>A, rs1800788C>T), α-fibrinogen (FGA) (rs6050A>G, rs2070006G>A, rs2070016T>C), γ-fibrinogen (FGG) (rs1049636T>C), F13A Val34Leu (rs5985) and α2-antiplasmin (A2AP) Arg6Trp (rs2070863). Genotype constellations potentially predisposing for dense fibrin clots were defined and their relation to inflammatory activity as measured by C-reactive protein (CRP) and disease activity score of 28 joints (DAS28) was assessed in univariate and multivariate analyses. The relation of these genotype constellations with presence of ACPA was tested. RESULTS: Genotype constellations involving FGB rs1800790G>A and FGA rs2070016T>C were inversely associated with CRP levels (≥10 mg/L) (OR: 0.49, P < 10-8/7adj = 0.0001; OR: 0.52, P < 0.0005/Padj = 0.01). In RA, both genotype constellations were observed with higher frequencies of low disease activity (DAS28 ≤ 3.2) (OR: 2.66, P = .009; OR 2.78, P = .01) and lower frequencies of high disease activity (DAS28>5.1) (OR: 0.52, P < .03, OR: 0.42, P = .01). Associations with CRP depended on A2AP 6Arg/Arg genotype known to be necessary for optimal anti-fibrinolytic capacity (P = .001). Finally, Genotype constellations involving FGB rs1800790G>A and FGA rs2070016T>C were found to be associated with ACPA-positivity in RA (OR: 2.18, P < .03; OR: 1.95, P = .09). CONCLUSIONS: These results support the hypothesis that genotypes, which increase fibrin clot density and anti-fibrinolytic capacity, reduce inflammatory activity and are related to humoral autoimmunity in RA.

The etiology of rheumatoid arthritis.

Rheumatoid arthritis is a heterogeneous disease, which can be, based on data combining genetic risk factors and autoantibodies, sub-classified into ACPA-positive and -negative RA. Presence of ACPA and RF as well as rising CRP-levels in some patients years before onset of clinical symptoms indicate that relevant immune responses for RA development are initiated very early. ACPA are highly specific for RA, whereas RF can also be found among healthy (elderly) individuals and patients with other autoimmune diseases or infection. The most important genetic risk factor for RA development, the shared epitope alleles, resides in the MHC class II region. Shared epitope alleles, however, only predispose to the development of ACPA-positive RA. Smoking is thus far the most important environmental risk factor associated with the development of RA. Studies on synovitis have shown the importance not only of adaptive but also of innate immune responses. In summary of the various results from immunological changes in blood and synovial tissue, the extension of the immune response from a diffuse myeloid to a lympho-myeloid inflammation appears to be associated with a more successful therapeutic response to biologics. With respect to advances in synovitis research, new targets for treatment against pathological subsets of immune cells or fibroblasts are already on the horizon. However, alternative strategies involving the microbiome may play an important role as well and research in this field is growing rapidly.

Fluorescence optical imaging for treatment monitoring in patients with early and active rheumatoid arthritis in a 1-year follow-up period.

BACKGROUND: Fluorescence optical imaging (FOI) enables visualization of inflammation in the hands in rheumatic joint diseases with currently a lack of long-term follow-up studies. OBJECTIVE: To investigate FOI for treatment monitoring in a homogenous cohort of patients with early (disease duration < 2 years) and active (DAS28 > 3.2) RA over a period of 12 months. METHODS: Thirty-five RA patients (24 (68.6%) females, mean age 53.3 years (SD 13.6)) were investigated clinically by DAS28, tender joint count (TJC) and swollen joint count (SJC) and by FOI in phases 1-3 and PrimaVistaMode (PVM) before therapy change and after 12 months. The FOI activity score (FOIAS) was calculated based on individual joint scores from 0 to 3 in 30 joints per patient, adding up to a sum score (0-90). RESULTS: We found a statistically significant reduction of FOIAS in phase 1 from baseline (median 5.0, IQR 24.96) to follow-up (median 1.0, IQR 4.0) in all patients (p = 0.0045), both in responders and non-responders according to EULAR response criteria by DAS28. Statistically significant reductions over 12 months were found for median DAS28(ESR) 5.61 to 3.31, TJC 7.0 to 1.0, and SJC 5.0 to 1.0 (each p <  0.001). No statistically significant correlations were detected between the FOIAS change in phase 1 and DAS28(ESR), TJC, or SJC. Correlations between the other phases and clinical outcomes were weak to moderate. CONCLUSION: Reduced early enhancement in FOI phase 1 can be observed in clinically responding and non-responding early RA patients under treatment. Regarding potential marker performance, FOI probably shows a reduction of inflammation more objectively.

Peripheral Lymphocyte Multidrug Resistance Activity as a Predictive Tool of Biological Therapeutic Response in Rheumatoid Arthritis.

OBJECTIVE: Multidrug resistance (MDR) transporters may be used as biomarkers to monitor disease progression in RA and as a predictive tool to establish responsiveness to biological therapy. In this multicenter clinical trial, we aimed to assess the predictive value of activity measurement of transporters MDR1, MD resistance protein (MRP)1, and breast cancer resistance protein (BCRP) for biological therapeutic response in RA before the initiation of biological therapy as well as 4 to 6 and 12 weeks after. METHODS: Peripheral blood samples were collected from 27 responders and 12 nonresponders to biological disease-modifying antirheumatic drugs (bDMARD) at the indicated timepoints as well as from 35 healthy controls. MDR activity factor (MAF) of MDR1, MRP1, and BCRP was measured in CD3+ and CD19+ cells using the Solvo MDQ Kit and cell surface staining by flow cytometry following peripheral blood mononuclear cells isolation. RESULTS: At the start of therapy, MAFC (composite MAF of MRP1 and MDR1) and MAFMDR values, and at 4 to 6 weeks of treatment, MAFC, MAFMRP, and MAFMDR values of CD3 cells were higher in nonresponders compared to responders. Receiver-operation characteristic curve analysis revealed that RA patients with MAFC values above 21.3 in CD3 cells at the start of bDMARD therapy are likely to be nonresponders. At 4 to 6 weeks of treatment, these also predict unfavorable response: MAFC values above 20.3, MAFMRP values above 6.0, and MAFMDR values above 13.9 in CD3 cells. CONCLUSION: Our results indicate that the determination of MAFC values in CD3 cells of patients with RA may be of predictive value prior to the initiation of biological therapy, to establish whether the patient will demonstrate sufficient therapeutic response.

An explorative study on deep profiling of peripheral leukocytes to identify predictors for responsiveness to anti-tumour necrosis factor alpha therapies in ankylosing spondylitis: natural killer cells in focus.

BACKGROUND: Therapeutic targeting of tumour necrosis factor (TNF)-α is highly effective in ankylosing spondylitis (AS) patients. However, since one-third of anti-TNF-treated AS patients do not show an adequate clinical response there is an urgent need for new biomarkers that would aid clinicians in their decision-making to select appropriate therapeutic options. Thus, the aim of this explorative study was to identify cell-based biomarkers in peripheral blood that could be used for a pre-treatment stratification of AS patients. METHODS: A high-dimensional, multi-parametric flow cytometric approach was applied to identify baseline predictors in 31 AS patients before treatment with the TNF blockers adalimumab (TNF-neutralisation) and etanercept (soluble TNF receptor). RESULTS: As the major result, the frequencies of natural killer (NK) cells, and in particular CD8-positive (CD8+) NK cell subsets, were most predictive for therapeutic outcome in AS patients. While an inverse correlation between classical CD56+/CD16+ NK cells and reduction of disease activity was observed, the CD8+ NK cell subset behaved in the opposite direction. At baseline, responders showed significantly increased frequencies of CD8+ NK cells compared with non-responders. CONCLUSIONS: This is the first study demonstrating that the composition of the NK cell compartment has predictive power for prediction of therapeutic outcome for anti-TNF-α blockers, and we identified CD8+ NK cells as a potential new player in the TNF-α-driven chronic inflammatory immune response of AS.

Hypogalactosylation of immunoglobulin G in rheumatoid arthritis: relationship to HLA-DRB1 shared epitope, anticitrullinated protein antibodies, rheumatoid factor, and correlation with inflammatory activity.

BACKGROUND: Galactosylation of immunoglobulin G (IgG) is reduced in rheumatoid arthritis (RA) and assumed to correlate with inflammation and altered humoral immunity. IgG hypogalactosylation also increases with age. To investigate dependencies in more detail, we compared IgG hypogalactosylation between patients with RA, patients with axial spondyloarthritis (axSpA), and healthy control subjects (HC), and we studied it in RA on the background of HLA-DRB1 shared epitope (SE), anticitrullinated protein antibodies (ACPA), and/or rheumatoid factor (RF) status. METHODS: Patients with RA (n = 178), patients with axSpA (n = 126), and HC (n = 119) were characterized clinically, and serum IgG galactosylation was determined by capillary electrophoresis. Markers of disease activity, genetic susceptibility, and serologic response included C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), DAS28, SE, HLA-B27, ACPA, and RF. Expression of glycosylation enzymes, including beta 1-4 galactosyltransferase (B4GALT3) activity, were estimated from transcriptome data for B-cell development (GSE19599) and differentiation to plasma cells (GSE12366). RESULTS: IgG hypogalactosylation was restricted to RA and associated with increasing CRP levels (p < 0.0001). In axSpA, IgG hypogalactosylation was comparable to HC and only marginally increased upon elevated CRP. Restriction to RA was maintained after correction for CRP and age. Treatment with sulfasalazine resulted in significantly reduced IgG hypogalactosylation (p = 0.003) even after adjusting for age, sex, and CRP (p = 0.009). SE-negative/ACPA-negative RA exhibited significantly less IgG hypogalactosylation than all other strata (vs SE-negative/ACPA-positive, p = 0.009; vs SE-positive/ACPA-negative, p = 0.04; vs SE-positive/ACPA-positive, p < 0.02); however, this indicated a trend only after Bonferroni correction for multiple testing. In SE-positive/ACPA-negative RA IgG hypogalactosylation was comparable to ACPA-positive subsets. The relationship between IgG hypogalactosylation and disease activity was significantly different between strata defined by SE (CRP, p = 0.0003, pBonferroni = 0.0036) and RF (CRP, p < 0.0001, pBonferroni < 0.0012), whereas ACPA strata revealed only a nonsignificant trend (p = 0.15). Gene expression data indicated that the key enzyme for galactosylation of immunoglobulins, B4GALT3, is expressed at lower levels in B cells than in plasma cells. CONCLUSIONS: Increased IgG hypogalactosylation in RA but not in axSpA points to humoral immune response as a precondition. Reduced B4GALT3 expression in B cells compared with plasma cells supports relatedness to early B-cell triggering. The differential influence of RA treatment on IgG hypogalactosylation renders it a potential diagnostic target for further studies.

Delivery of miR-146a to Ly6Chigh Monocytes Inhibits Pathogenic Bone Erosion in Inflammatory Arthritis.

Rationale: Monocytes play critical roles in the pathogenesis of arthritis by contributing to the inflammatory response and bone erosion. Among genes involved in regulating monocyte functions, miR-146a negatively regulates the inflammatory response and osteoclast differentiation of monocytes. It is also the only miRNA reported to differentially regulate the cytokine response of the two classical Ly6Chigh and non-classical Ly6Clow monocyte subsets upon bacterial challenge. Although miR-146a is overexpressed in many tissues of arthritic patients, its specific role in monocyte subsets under arthritic conditions remains to be explored. Methods: We analyzed the monocyte subsets during collagen-induced arthritis (CIA) development by flow cytometry. We quantified the expression of miR-146a in classical and non-classical monocytes sorted from healthy and CIA mice, as well as patients with rheumatoid arthritis (RA). We monitored arthritis features in miR-146a-/- mice and assessed in vivo the therapeutic potential of miR-146a mimics delivery to Ly6Chigh monocytes. We performed transcriptomic and pathway enrichment analyses on both monocyte subsets sorted from wild type and miR-146a-/- mice. Results: We showed that the expression of miR-146a is reduced in the Ly6Chigh subset of CIA mice and in the analogous monocyte subset (CD14+CD16-) in humans with RA as compared with healthy controls. The ablation of miR-146a in mice worsened arthritis severity, increased osteoclast differentiation in vitro and bone erosion in vivo. In vivo delivery of miR-146a to Ly6Chigh monocytes, and not to Ly6Clow monocytes, rescues bone erosion in miR-146a-/- arthritic mice and reduces osteoclast differentiation and pathogenic bone erosion in CIA joints of miR-146a+/+ mice, with no effect on inflammation. Silencing of the non-canonical NF-κB family member RelB in miR-146a-/- Ly6Chigh monocytes uncovers a role for miR-146a as a key regulator of the differentiation of Ly6Chigh, and not Ly6Clow, monocytes into osteoclasts under arthritic conditions. Conclusion: Our results show that classical monocytes play a critical role in arthritis bone erosion. They demonstrate the theranostics potential of manipulating miR-146a expression in Ly6Chigh monocytes to prevent joint destruction while sparing inflammation in arthritis.