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Various autoimmune diseases are characterized by distinct cell subset distributions and activation profiles of peripheral blood mononuclear cells (PBMCs). PBMCs can therefore serve as an ideal biomarker material, which is easily accessible and allows for screening of multiple cell types. A detailed understanding of the immune landscape is critical for the diagnosis of patients with autoimmune diseases, as well as for a personalized treatment approach. In our study, we investigate the potential of multi-parameter spectral flow cytometry for the identification of patients suffering from autoimmune diseases and its power as an evaluation tool for in vitro drug screening approaches (advanced immunophenotyping). We designed a combination of two 22-color immunophenotyping panels for profiling cell subset distribution and cell activation. Downstream bioinformatics analyses included percentages of individual cell populations and median fluorescent intensity of defined markers which were then visualized as heatmaps and in dimensionality reduction approaches. In vitro testing of epigenetic immunomodulatory drugs revealed an altered activation status upon treatment, which supports the use of spectral flow cytometry as a high-throughput drug screening tool. Advanced immunophenotyping might support the exploration of novel therapeutic drugs and contribute to future personalized treatment approaches in autoimmune diseases and beyond.

ObjectivesAnti-citrullinated peptide antibodies (ACPA) are specific markers for rheumatoid arthritis (RA) and typically measured by assays employing a cyclic citrullinated peptide (CCP) as antigen. This study was aimed at investigating the diagnostic performance of anti-CCP2 and anti-CCP3 IgG and IgA assays in patients with early RA with a particular focus on the potential prognostic value of IgA ACPA.MethodsThe anti-CCP3.1 assay (Inova Diagnostics) measuring IgG and IgA antibodies simultaneously was compared to anti-CCP2 IgG and IgA assays (Thermo Fisher Scientific) employing sera of 184 early RA patients, 360 disease controls and 98 healthy subjects.ResultsAnti-CCP2 IgG and IgA assays showed high specificity versus disease controls (98.9%; 99.4%). Sensitivity was 52.2% (IgG) and 28.8% (IgA), resulting in positive likelihood ratios (LR+) of 47.5 (IgG) and 48.0 (IgA). The anti-CCP3.1 assay proved slightly more sensitive than the anti-CCP2 IgG assay (56%) but specificity was markedly lower (90.8% versus disease controls). However, when using a threefold higher cut-off specificity of the anti-CCP3.1 assay increased (97.5%) while sensitivity (52.7%) became comparable to the anti-CCP2 IgG assay resulting in a LR+ of 21.5. Anti-CCP2 IgA antibodies did not increase the diagnostic sensitivity of ACPA testing, but IgA positive patients showed diminished responses to treatment with anti-TNF biologicals compared to patients who had only IgG antibodies.ConclusionSpecificity of ACPA assays should be adjusted to reduce the risk of misclassification and a false positive diagnosis. Determination of ACPA IgA might provide important prognostic information concerning therapeutic responses.

Abstract 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.

Rheumatoid arthritis (RA) is the most common systemic autoimmune disease and also the most severe arthritic disorder. The measurement of rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA) in serum supports the diagnosis of RA, which gained increasing significance over the last 65 years. However, a high variability between RF and ACPA methods has been described, impacting the diagnostic performance of the current ACR/EULAR RA classification criteria.The great number of commercially available assays, often lacking traceability to an international standard, is a major factor attributing to this in-between assay variability. The adoption of an international standard for ACPA, as is since long available for rheumatoid factor, is therefore highly desirable.Further harmonization in clinical interpretation of RF/ACPA assays could be obtained by harmonization of the cut-offs, for both the low and high antibody levels, based on predefined specificity in disease controls. Reporting test result specific likelihood ratios (LR) adds value in the interpretation of autoantibody tests. However, a good understanding of the control population used to define antibody test result interval-associated LRs is crucial in defining the diagnostic performance characteristics of antibody serology.Finally, specificity in RA classification can be improved by refining serological weight scoring taking into account the nature of the antibody, the antibody level and double RF + ACPA positivity.

Aims: To determine the diagnostic value of anti-acetylated peptide antibodies (AAPA) in patients with rheumatoid arthritis (RA). Methods: Three acetylated peptides (ac-lysine, ac-lysine.inv and ac-ornithine) derived from vimentin were employed to measure AAPA by enzyme-linked immunosorbent assay (ELISA) in sera of 120 patients with early RA (eRA), 195 patients with established RA (est RA), 99 healthy controls (HC), and 216 patients with other inflammatory rheumatic diseases. A carbamylated and a citrullinated version of the vimentin peptide were used additionally. Receiver operating characteristics and logistic regression analyses were used to assess the discriminative capacity of AAPA. Results: AAPA were detected in 60% of eRA and 68.7% of estRA patients, 22.2% of HC, and 7.1– 30.6% of patients with other rheumatic diseases. Importantly, AAPA were also present in 40% of seronegative RA patients, while antibodies to the carbamylated peptide were detected less frequently. Diagnostic sensitivity of individual peptides for eRA was 28.3%, 35.8%, and 34% for ac-lysine, ac-ornithine, and ac-lysine.inv, respectively. Positive likelihood ratios (LR+) for eRA versus HC were 14.0, 7.1, and 2.1. While the presence of a single AAPA showed varying specificity (range: 84–98%), the presence of two AAPA increased specificity considerably since 26.7% of eRA, as compared with 6% of disease controls, were double positive. Thus, double positivity discriminated eRA from axial spondyloarthritis with a LR+ of 18.3. Remarkably, triple positivity was 100% specific for RA, being observed in 10% of eRA and 21.5% of estRA patients, even in the absence of RF and ACPA. Conclusion: AAPA are highly prevalent in early RA and occur also independently of RF and ACPA, thereby reducing the gap of seronegativity. Furthermore, multiple AAPA reactivity increased the specificity for RA, suggesting high diagnostic value of AAPA testing.

IRF-1: a key regulator in rheumatoid Arthritis Thomas Karonitsch at the Medical University of Vienna, Austria, and colleagues showed that activation of the protein interferon regulatory factor 1 (IRF-1) in human synoviocytes promoted the expression of genes that contribute to sustained synovial inflammation in rheumatoid Arthritis (RA). They found that the Januse kinase (JAK) inhibitors baricitinb and tofacitinib, which are both approved for the treatment of RA, prevented the activation of IRF1 regulated proinflammatory genes. Their findings unvover a potential role for IRF1 activated genes as a biomarker to predict the treatment response to RA therapies and moreover highlight the benefit of targeting inflammatory pathways in synoviocytes for the treatment of RA.

For the identification of antigenic protein biomarkers for rheumatoid arthritis (RA), we conducted IgG profiling on high density protein microarrays. Plasma IgG of 96 human samples (healthy controls, osteoarthritis, seropositive and seronegative RA, n = 24 each) and time-series plasma of a pristane-induced arthritis (PIA) rat model (n = 24 total) were probed on AIT’s 16k protein microarray. To investigate the analogy of underlying disease pathways, differential reactivity analysis was conducted. A total of n = 602 differentially reactive antigens (DIRAGs) at a significance cutoff of p < 0.05 were identified between seropositive and seronegative RA for the human samples. Correlation with the clinical disease activity index revealed an inverse correlation of antibodies against self-proteins found in pathways relevant for antigen presentation and immune regulation. The PIA model showed n = 1291 significant DIRAGs within acute disease. Significant DIRAGs for (I) seropositive, (II) seronegative and (III) PIA were subjected to the Reactome pathway browser which also revealed pathways relevant for antigen presentation and immune regulation; of these, seven overlapping pathways had high significance. We therefore conclude that the PIA model reflects the biological similarities of the disease pathogenesis. Our data show that protein array analysis can elucidate biological differences and pathways relevant in disease as well be a useful additional layer of omics information.

MicroRNA (miR) 155 has been implicated in the regulation of innate and adaptive immunity as well as autoimmune processes. Importantly, it has been shown to regulate several antiviral responses, but its contribution to the immune response against cytopathic viruses such as vesicular stomatitis virus (VSV) infections is not known. Using transgenic/recombinant VSV expressing ovalbumin, we show that miR-155 is crucially involved in regulating the T helper cell response against this virus. Our experiments indicate that miR-155 in CD4+ T cells controls their activation, proliferation, and cytokine production in vitro and in vivo upon immunization with OVA as well as during VSV viral infection. Using intravital multiphoton microscopy we analyzed the interaction of antigen presenting cells (APCs) and T cells after OVA immunization and found impaired complex formation when using miR-155 deficient CD4+ T cells compared to wildtype CD4+ T cells ex vivo. In contrast, miR-155 was dispensable for the maturation of myeloid APCs and for their T cell stimulatory capacity. Our data provide the first evidence that miR-155 is required for efficient CD4+ T cell activation during anti-viral defense by allowing robust APC-T cell interaction required for activation and cytokine production of virus specific T cells.

Anti-citrullinated protein antibodies (ACPA) and rheumatoid factor (RF) are the most commonly used diagnostic markers of rheumatoid arthritis (RA). These antibodies are predominantly of the immunoglobulin (Ig) M (RF) or IgG (ACPA) isotype. Other subtypes of both antibodies—particularly IgA isotypes and other autoantibodies—such as RA33 antibodies—have been repeatedly reported but their diagnostic value has still not been fully elucidated. Here, we investigated the prevalence of IgA, IgG, and IgM subtypes of RF, ACPA, and RA33 antibodies in patients with RA. To determine the diagnostic specificity and sensitivity sera from 290 RA patients (165 early and 125 established disease), 261 disease controls and 100 healthy subjects were tested for the presence of IgA, IgG, and IgM isotypes of RF, ACPA, and RA33 by EliA™ platform (Phadia AB, Uppsala, Sweden). The most specific antibodies were IgG-ACPA, IgA-ACPA, and IgG-RF showing specificities >98%, closely followed by IgG- and IgA-RA33 while IgM subtypes were somewhat less specific, ranging from 95.8% (RA33) to 90% (RF). On the other hand, IgM-RF was the most sensitive subtype (65%) followed by IgG-ACPA (59.5%) and IgA-RF (50.7%). Other subtypes were less sensitive ranging from 35 (IgA-ACPA) to 6% (IgA-RA33). RA33 antibodies as well as IgA-RF and IgA-ACPA were found to increase the diagnostic sensitivity of serological testing since they were detected also in seronegative patients reducing their number from 109 to 85. Moreover, analyzing IgM-RF by EliA™ proved more sensitive than measuring RF by nephelometry and further reduced the number of seronegative patients to 76 individuals. Importantly, among antibody positive individuals, RA patients were found having significantly more antibodies (≥3) than disease controls which generally showed one or two antibody species. Thus, increasing the number of autoantibodies in serological routine testing provides valuable additional information allowing to better distinguish between RA and other rheumatic disorders, also in patients not showing antibodies in current routine diagnostics. In conclusion, testing for multiple autoantibody specificities increases the diagnostic power of autoimmune diagnostics and could further support physicians in clinical decision-making.

BACKGROUND:To facilitate the development of therapies for rheumatoid arthritis (RA), the Innovative Medicines Initiative BTCure has combined the experience from several laboratories worldwide to establish a series of protocols for different animal models of arthritis that reflect the pathogenesis of RA. Here, we describe chronic pristane-induced arthritis (PIA) model in DA rats, and provide detailed instructions to set up and evaluate the model and for reporting data. METHODS:We optimized dose of pristane and immunization procedures and determined the effect of age, gender, and housing conditions. We further assessed cage-effects, reproducibility, and frequency of chronic arthritis, disease markers, and efficacy of standard and novel therapies. RESULTS:Out of 271 rats, 99.6% developed arthritis after pristane-administration. Mean values for day of onset, day of maximum arthritis severity and maximum clinical scores were 11.8±2.0 days, 20.3±5.1 days and 34.2±11 points on a 60-point scale, respectively. The mean frequency of chronic arthritis was 86% but approached 100% in long-term experiments over 110 days. Pristane was arthritogenic even at 5 microliters dose but needed to be administrated intradermally to induce robust disease with minimal variation. The development of arthritis was age-dependent but independent of gender and whether the rats were housed in conventional or barrier facilities. PIA correlated well with weight loss and acute phase reactants, and was ameliorated by etanercept, dexamethasone, cyclosporine A and fingolimod treatment. CONCLUSIONS:PIA has high incidence and excellent reproducibility. The chronic relapsing-remitting disease and limited systemic manifestations make it more suitable than adjuvant arthritis for long-term studies of joint-inflammation and screening and validation of new therapeutics.

Reversible lysine acetylation plays an important role in the regulation of T cell responses. HDAC1 has been shown to control peripheral T helper cells, however the role of HDAC1 in CD8+ T cell function remains elusive. By using conditional gene targeting approaches, we show that LckCre-mediated deletion of HDAC1 led to reduced numbers of thymocytes as well as peripheral T cells, and to an increased fraction of CD8+CD4- cells within the CD3/TCRβlo population, indicating that HDAC1 is essential for the efficient progression of immature CD8+CD4- cells to the DP stage. Moreover, CD44hi effector CD8+ T cells were enhanced in mice with a T cell-specific deletion of HDAC1 under homeostatic conditions and HDAC1-deficient CD44hi CD8+ T cells produced more IFNγ upon ex vivo PMA/ionomycin stimulation in comparison to wild-type cells. Naïve (CD44l°CD62L+) HDAC1-null CD8+ T cells displayed a normal proliferative response, produced similar amounts of IL-2 and TNFα, slightly enhanced amounts of IFNγ, and their in vivo cytotoxicity was normal in the absence of HDAC1. However, T cell-specific loss of HDAC1 led to a reduced anti-viral CD8+ T cell response upon LCMV infection and impaired expansion of virus-specific CD8+ T cells. Taken together, our data indicate that HDAC1 is required for the efficient generation of thymocytes and peripheral T cells, for proper CD8+ T cell homeostasis and for an efficient in vivo expansion and activation of CD8+ T cells in response to LCMV infection.

To analyze changes in autoantibodies occurring in patients with systemic lupus erythematosus (SLE) treated with 4 infusions of the chimeric anti–tumor necrosis factor α (TNFα) antibody infliximab.In an open‐label safety study, 7 patients with SLE were treated with infliximab at weeks 0, 2, 6, and 10 in combination with azathioprine or methotrexate. Antibodies to double‐stranded DNA (dsDNA) were determined by radioimmunoassay and the Crithidia luciliae indirect immunofluorescence assay; anticardiolipin antibodies (aCL) and antibodies to histone and chromatin were measured by enzyme‐linked immunosorbent assay. Antihistone antibodies were also analyzed by immunoblotting. Peripheral blood mononuclear cells from healthy individuals and SLE patients were incubated for 2 weeks with or without TNFα. TNFα was removed by washing and by the addition of infliximab. Apoptotic cells were stained with annexin V and analyzed by flow cytometry.Autoantibodies to dsDNA increased in 5 of 7 patients. Histone, chromatin, and IgM aCL levels were increased in 4 of 7, 6 of 7, and 4 of 7 patients, respectively, peaking 4–10 weeks after the last infliximab infusion, but falling to baseline levels or lower thereafter. In the in vitro experiments, TNF withdrawal after long‐term incubation with recombinant human TNF led to increased percentages of apoptotic cells.While TNF blockade was clinically effective, the majority of SLE patients treated with infliximab showed an increase in autoantibodies to nuclear antigens and phospholipids. These increases were transient and were not associated with disease flares. Increased availability of apoptotic antigens after TNF blockade may play a role in the autoantibody formation induced by TNF blockade. [ABSTRACT FROM AUTHOR]

To investigate the safety of therapeutic tumor necrosis factor α (TNFα) blockade in patients with systemic lupus erythematosus (SLE), in whom this proinflammatory cytokine is significantly increased and may be involved in the disease pathogenesis.In an open‐label study, 6 patients with moderately active SLE (4 with nephritis and 3 with arthritis refractory to other therapies) were given 4 300‐mg doses of infliximab, a chimeric anti‐TNFα antibody, in addition to immunosuppression with azathioprine or methotrexate. The only significant adverse events observed were urinary tract infection in 3 patients, 1 of which was accompanied by Escherichia coli bacteremia, and a prolonged febrile episode of putatively viral origin in 1 of them. These patients had similar infectious conditions in the past. In none of the patients was it necessary to terminate the treatment prematurely. Levels of antibodies to double‐stranded DNA and cardiolipin increased in 4 patients each, but this was not associated with a decrease in serum complement levels, with vascular events, or with flares. In contrast, disease activity declined during therapy. All 3 patients with joint involvement experienced remission of arthritis, which relapsed 8–11 weeks after the last infliximab infusion. In the 4 patients with lupus nephritis, proteinuria decreased significantly within 1 week after initiation of therapy and was diminished by ≥60% within 8 weeks, remaining at low levels until the end of the observation period (at least several months). Infliximab did not lead to adverse events related to an increase in SLE activity, although autoantibodies to double‐stranded DNA and cardiolipin increased, as expected. This finding, coupled with the clinical improvement in the inflammatory manifestations of the disease, indicates that further study in larger controlled trials is warranted. [ABSTRACT FROM AUTHOR]

To investigate the efficacy of single and combined blockade of tumor necrosis factor (TNF), interleukin-1 (IL-1), and RANKL pathways on synovial inflammation, bone erosion, and cartilage destruction in a TNF-driven arthritis model. Human TNF–transgenic (hTNFtg) mice were treated with anti-TNF (infliximab), IL-1 receptor antagonist (IL-1Ra; anakinra), or osteoprotegerin (OPG; an OPG-Fc fusion protein), either alone or in combinations of 2 agents or all 3 agents. Synovial inflammation, bone erosion, and cartilage damage were evaluated histologically. Synovial inflammation was inhibited by anti-TNF (-51%), but not by IL-1Ra or OPG monotherapy. The combination of anti-TNF with either IL-1Ra (-91%) or OPG (-81%) was additive and almost completely blocked inflammation. Bone erosion was effectively blocked by anti-TNF (-79%) and OPG (-60%), but not by IL-1Ra monotherapy. The combination of anti-TNF with IL-1Ra, however, completely blocked bone erosion (-98%). Inhibition of bone erosion was accompanied by a reduction of osteoclast numbers in synovial tissue. Cartilage destruction was inhibited by anti-TNF (-43%) and was weakly, but not significantly, inhibited by IL-1Ra, but was not inhibited by OPG monotherapy. The combination of anti-TNF with IL-1Ra was the most effective double combination therapy in preventing cartilage destruction (-80%). In all analyses, the triple combination of anti-TNF, IL-1Ra, and OPG was not superior to the double combination of anti-TNF and IL-1Ra. Articular changes caused by chronic overexpression of TNF are not completely blockable by monotherapies that target TNF, IL-1, or RANKL. However, combined approaches, especially the combined blockade of TNF and IL-1 and, to a lesser extent, TNF and RANKL, lead to almost complete remission of disease. Differences in abilities to block synovial inflammation, bone erosion, and cartilage destruction further strengthen the rationale for using combined blockade of more than one proinflammatory pathway. [ABSTRACT FROM AUTHOR]