Dynamics of the Type I Interferon Response During Immunosuppressive Therapy in Rheumatoid Arthritis.

Objective: The type I interferon (IFN) response in rheumatoid arthritis (RA) has been extensively studied in relation to therapy with biological DMARDs (bDMARDs). However, the effect of conventional synthetic (cs)DMARDs and glucocorticoids (GCs) on IFN response gene (IRG) expression remains largely unknown, even though csDMARDS are used throughout all disease phases, including simultaneously with biologic therapy. This study was aimed to determine the dynamics of IFN response upon immunosuppressive treatment. Methods: Whole blood was collected in PAXgene tubes from 35 RA patients who received either COBRA therapy (combination of prednisone, initially 60 mg, methotrexate and sulfasalazine) (n = 14) or COBRA-light therapy (prednisone, initially 30 mg, and methotrexate) (n = 21). Expression of 10 IRGs was determined by real-time PCR at baseline (T0), after 4 weeks (T4), and 13 weeks (T13) of treatment. IRG selection was based on the differential presence of transcription factor binding sites (TFBS), in order to study the therapy effect on different pathway components involved in IFN signaling. Results: Seven of the 10 IRGs displayed significant changes during treatment (p ≤ 0.016). These 7 IRGs all displayed a particularly pronounced decrease between T0 and T4 (≥1.6-fold, p ≤ 0.0059). The differences between IRG sensitivity to the treatment appeared related to the presence of TFBS for STAT1 and IRF proteins within the genes. The extent of the decreases between T0 and T4 was similar for the COBRA- and COBRA-light-treated group, despite the differences in drug combination and doses in those groups. Between T4 and T13, however, IRG expression in the COBRA-light-treated group displayed a significant increase, whereas it remained stable or decreased even further in most COBRA-treated patients (comparison of mean fold changes, p = 0.011). A significant association between IRG dynamics and clinical response to therapy was not detected. Conclusions: Immunosuppressive treatment with csDMARDs, in this case a combination of prednisolone, methotrexate and sulfasalazine, substantially downregulates the IFN response in RA patients. The dynamics of this downregulation were partly dependent on the presence of TFBS within the IRGs and the combination and dosages of agents, but they were irrespective of the clinical response to therapy.

Imaging and Methotrexate Response Monitoring of Systemic Inflammation in Arthritic Rats Employing the Macrophage PET Tracer [18F]Fluoro-PEG-Folate.

Background: In rheumatoid arthritis, articular inflammation is a hallmark of disease, while the involvement of extra-articular tissues is less well defined. Here, we examined the feasibility of PET imaging with the macrophage tracer [18F]fluoro-PEG-folate, targeting folate receptor ß (FRß), to monitor systemic inflammatory disease in liver and spleen of arthritic rats before and after methotrexate (MTX) treatment. Methods: [18F]Fluoro-PEG-folate PET scans (60 min) were acquired in saline- and MTX-treated (1 mg/kg, 4x) arthritic rats, followed by tissue resection and radiotracer distribution analysis. Liver and spleen tissues were stained for ED1/ED2-macrophage markers and FRß expression. Results: [18F]Fluoro-PEG-folate PET and ex vivo tissue distribution studies revealed a significant (p < 0.01) 2-fold lower tracer uptake in both liver and spleen of MTX-treated arthritic rats. Consistently, ED1- and ED2-positive macrophages were significantly (p < 0.01) decreased in liver (4-fold) and spleen (3-fold) of MTX-treated compared with saline-treated rats. Additionally, FRß-positive macrophages were also significantly reduced in liver (5-fold, p < 0.005) and spleen (3-fold, p < 0.01) of MTX- versus saline-treated rats. Conclusions: MTX treatment reduced activated macrophages in liver and spleen, as markers for systemic inflammation in these organs. Macrophage PET imaging with [18F]fluoro-PEG-folate holds promise for detection of systemic inflammation in RA as well as therapy (MTX) response monitoring.

A multi-parameter response prediction model for rituximab in rheumatoid arthritis.

OBJECTIVES: To validate the IFN response gene (IRG) set for the prediction of non-response to rituximab in rheumatoid arthritis (RA) and assess the predictive performance upon combination of this gene set with clinical parameters. METHODS: In two independent cohorts of 93 (cohort I) and 133 (cohort II) rituximab-starting RA patients, baseline peripheral blood expression of eight IRGs was determined, and averaged into an IFN score. Predictive performance of IFN score and clinical parameters was assessed by logistic regression. A multivariate prediction model was developed using a forward stepwise selection procedure. Patients with a decrease in disease activity score (ΔDAS28)≥1.8 after 6 months of therapy were considered responders. RESULTS: The mean IFN score was higher in non-responders compared to responders in both cohorts, but this difference was most pronounced in patients who did not use prednisone, as described before. Univariate analysis in cohort I showed that baseline DAS28, IFN score, DMARD use and negativity for IgM-RF and/or ACPA were associated with rituximab non-response. The multivariate model consisted of DAS28, IFN score and DMARD use, which showed an area under the curve (AUC) of 0.82. In cohort II, this model revealed a comparable AUC in PREDN-negative patients (0.78), but AUC in PREDN-positive patients was significantly lower (0.63), which seemed due to effect modification of the IFN score by prednisone. CONCLUSIONS: Combination of predictive parameters provided a promising model for the prediction of non-response to rituximab, with possibilities for optimization via definition of the exact interfering effect of prednisone on IFN score. TRIAL REGISTRATION (COHORT II, SMART TRIAL): NCT01126541, registered 18 May 2010.

Changes in peripheral blood lymphocyte subsets during arthritis development in arthralgia patients.

BACKGROUND: Multiple lymphocyte subsets like T and B cells have been connected to joint infiltration and inflammation in rheumatoid arthritis (RA). Identification of leucocyte subsets that are dysregulated in arthritis development could provide insight into the aetiology of RA. This study aimed to investigate the composition of the peripheral blood components, i.e. CD14(+) monocytes, CD4(+) and CD8(+) T lymphocytes (CD3(+)), CD80(+), C-X-C chemokine receptor 3 (CXCR3)(+) and CD27(+) B lymphocytes (CD19(+)), CD16(+)CD56(+)CD3(-) natural killer (NK) cells and activated CD56(+)CD3(+) T cells, for association with arthritis development in patients with arthralgia. METHODS: Peripheral blood was collected from 89 patients with early RA (disease duration <6 months), 37 healthy controls (HC) and 113 patients with arthralgia (22 developed arthritis within ≤1 year, 18 developed arthritis after >1 year and 73 did not develop arthritis). Absolute numbers of monocytes and lymphocyte subsets in whole heparinized blood were determined with flow cytometry using quantification beads in combination with fluorescent labelled antibodies for T cells, B cells, monocytes, NK cells and activated T cells. RESULTS: In patients with early RA, significant decreases in numbers of (activated) T cells, CD80(+) and memory B cells and a trend towards smaller numbers of CD8(+) T cells was observed compared to HC. Similar differences were seen in patients with arthralgia who developed or did not develop arthritis (non-converters), with significantly decreased CD8(+) T cells and memory B cells. Patients with arthralgia who developed arthritis were split into groups that developed arthritis within 1 year (early converters) or after 1 year (late converters). Late converters had a significantly decreased number of CD8(+) T cells compared to non-converters; early converters had a decreased number of memory B cells. Longitudinal analysis of converters showed a significant relative increase in CD80(+) B cells towards the conversion time point compared to 24 months prior to conversion. CONCLUSIONS: This study revealed that patients with arthralgia who develop arthritis demonstrate a change in cellular immune parameters apparent in the periphery, starting with a decrease in cytotoxic T cells 24 months prior to arthritis development, followed by a decrease in the number of memory B cells 12 months prior to disease onset.

The type I interferon signature in leukocyte subsets from peripheral blood of patients with early arthritis: a major contribution by granulocytes.

BACKGROUND: The type I interferon (IFN) signature in rheumatoid arthritis (RA) has shown clinical relevance in relation to disease onset and therapeutic response. Identification of the cell type(s) contributing to this IFN signature could provide insight into the signature's functional consequences. The aim of this study was to investigate the contribution of peripheral leukocyte subsets to the IFN signature in early arthritis. METHODS: Blood was collected from 26 patients with early arthritis and lysed directly or separated into peripheral blood mononuclear cells (PBMCs) and polymorphonuclear granulocytes (PMNs). PBMCs were sorted into CD4(+) T cells, CD8(+) T cells, CD19(+) B cells, and CD14(+) monocytes by flow cytometry. Messenger RNA expression of three interferon response genes (IRGs RSAD2, IFI44L, and MX1) and type I interferon receptors (IFNAR1 and IFNAR2) was determined in whole blood and blood cell subsets by quantitative polymerase chain reaction. IRG expression was averaged to calculate an IFN score for each sample. RESULTS: Patients were designated "IFN(high)" (n = 8) or "IFN(low)" (n = 18) on the basis of an IFN score cutoff in whole peripheral blood from healthy control subjects. The difference in IFN score between IFN(high) and IFN(low) patients was remarkably large for the PMN fraction (mean 25-fold) compared with the other subsets (mean 6- to 9-fold), indicating that PMNs are the main inducers of IRGs. Moreover, the relative contribution of the PMN fraction to the whole-blood IFN score was threefold higher than expected from its abundance in blood (p = 0.008), whereas it was three- to sixfold lower for the other subsets (p ≤ 0.063), implying that the PMNs are most sensitive to IFN signaling. Concordantly, IFNAR1 and IFNAR2 were upregulated compared with healthy controls selectively in patient PMNs (p ≤ 0.0077) but not in PBMCs. CONCLUSIONS: PMNs are the main contributors to the whole-blood type I IFN signature in patients with early arthritis, which seems due to increased sensitivity of these cells to type I IFN signaling. Considering the well-established role of neutrophils in the pathology of arthritis, this suggests a role of type I IFN activity in the disease as well.

Physiological evidence for diversification of IFNα- and IFNß-mediated response programs in different autoimmune diseases.

BACKGROUND: Activation of the type I interferon (IFN) response program is described for several autoimmune diseases, including systemic lupus erythematosus (SLE), multiple sclerosis (MS), myositis (IIM) and rheumatoid arthritis (RA). While IFNα contributes to SLE pathology, IFNß therapy is often beneficial in MS, implying different immunoregulatory roles for these IFNs. This study was aimed to investigate potential diversification of IFNα-and IFNß-mediated response programs in autoimmune diseases. METHODS: Peripheral blood gene expression of 23 prototypical type I IFN response genes (IRGs) was determined in 54 healthy controls (HCs), 69 SLE (47 test, 22 validation), 149 IFNß-treated MS (71 test, 78 validation), 160 untreated MS, 78 IIM and 76 RA patients. Patients with a type I IFN signature were selected for analysis. RESULTS: We identified IFNα- and IFNß-specific response programs (GC-A and GC-B, respectively) in SLE and IFNß-treated MS patients. Concordantly, the GC-A/GC-B log-ratio was positive for all SLE patients and negative for virtually all IFNß-treated MS patients, which was confirmed in additional cohorts. Applying this information to other autoimmune diseases, IIM patients displayed positive GC-A/GC-B log-ratios, indicating predominant IFNα activity. The GC-A/GC-B log-ratio in RA was lower and approached zero in part of the patients, implying relative importance of both clusters. Remarkably, GC-A/GC-B log-ratios appeared most heterogeneous in untreated MS; half of the patients displayed GC-A dominance, whereas others showed GC-B dominance or log-ratios near zero. CONCLUSIONS: Our findings show diversification of the type I IFN response in autoimmune diseases, suggesting different pathogenic roles of the type I IFNs.