Identification of early risk factors for anti-citrullinated-protein-antibody positive rheumatoid arthritis-a prospective cohort study.

OBJECTIVE: Individuals positive for anti-cyclic-peptide-antibodies (anti-CCP) and musculoskeletal complaints (MSK-C) are at risk for developing rheumatoid arthritis (RA). In this study we aimed to investigate factors involved in arthritis progression. METHODS: Anti-CCP2-positive individuals with MSK-C referred to a rheumatologist were recruited. Individuals lacked arthritis at clinical and ultrasound examination and were followed for ≥three years or until clinical arthritis diagnosis. Blood samples from inclusion were analyzed for; nine anti-citrullinated-protein-antibody (ACPA) reactivities (citrullinated α-1-enolase, fibrinogen, filaggrin, histone, vimentin and tenascin peptides); 92 inflammation-associated proteins; and HLA-shared epitope alleles. Cox regression was applied to the data to identify independent predictors in a model. RESULTS: 267 individuals were included with median follow up of 49 months (IQR: 22-60). 101 (38%) developed arthritis after median 14 months (IQR: 6-27). The analysis identified that presence of at least one ACPA reactivity (HR 8.0, 95% CI 2.9-22), ultrasound detected tenosynovitis (HR 3.4, 95% CI 2.0-6.0), IL6 levels (HR 1.5, 95% CI 1.2-1.8) and IL15-Rα levels (HR 0.6, 95% CI 0.4-0.9) are significant independent predictors for arthritis progression in a prediction model (Harrell's C 0.76 [SE 0.02], AUC 0.82 [95% CI 0.76-0.89], cross-validated AUC 0.70 [95% CI 0.56-0.85]). CONCLUSION: We propose a high-Risk-RA phase characterized by presence of ACPA reactivity, tenosynovitis, IL6, and IL15-Rα and suggest that these factors need to be further investigated for their biological effects and clinical values, to identify individuals at particular low risk and high risk for arthritis progression.

Early prediction of clinical response to anti-TNF treatment using multi-omics and machine learning in rheumatoid arthritis.

OBJECTIVES: Advances in immunotherapy by blocking TNF have remarkably improved treatment outcomes for Rheumatoid arthritis (RA) patients. Although treatment specifically targets TNF, the downstream mechanisms of immune suppression are not completely understood. The aim of this study was to detect biomarkers and expression signatures of treatment response to TNF inhibition. METHODS: Peripheral blood mononuclear cells (PBMCs) from 39 female patients were collected before anti-TNF treatment initiation (day 0) and after 3 months. The study cohort included patients previously treated with MTX who failed to respond adequately. Response to treatment was defined based on the EULAR criteria and classified 23 patients as responders and 16 as non-responders. We investigated differences in gene expression in PBMCs, the proportion of cell types and cell phenotypes in peripheral blood using flow cytometry and the level of proteins in plasma. Finally, we used machine learning models to predict non-response to anti-TNF treatment. RESULTS: The gene expression analysis in baseline samples revealed notably higher expression of the gene EPPK1 in future responders. We detected the suppression of genes and proteins following treatment, including suppressed expression of the T cell inhibitor gene CHI3L1 and its protein YKL-40. The gene expression results were replicated in an independent cohort. Finally, machine learning models mainly based on transcriptomic data showed high predictive utility in classifying non-response to anti-TNF treatment in RA. CONCLUSIONS: Our integrative multi-omics analyses identified new biomarkers for the prediction of response, found pathways influenced by treatment and suggested new predictive models of anti-TNF treatment in RA patients.

Integration of known DNA, RNA and protein biomarkers provides prediction of anti-TNF response in rheumatoid arthritis: results from the COMBINE study.

OBJECTIVE: In rheumatoid arthritis (RA) several recent efforts have sought to discover means of predicting which patients would benefit from treatment. However, results have been discrepant with few successful replications. Our objective was to build a biobank with DNA, RNA and protein measurements to test the claim that the current state-of-the-art precision medicine will benefit RA patients. METHODS: We collected 451 blood samples from 61 healthy individuals and 185 RA patients initiating treatment, before treatment initiation and at a 3 month follow-up time. All samples were subjected to high-throughput RNA sequencing, DNA genotyping, extensive proteomics and flow cytometry measurements, as well as comprehensive clinical phenotyping. Literature review identified 2 proteins, 52 single-nucleotide polymorphisms (SNPs) and 72 gene-expression biomarkers that had previously been proposed as predictors of TNF inhibitor response (∆DAS28-CRP). RESULTS: From these published TNFi biomarkers we found that 2 protein, 2 SNP and 8 mRNA biomarkers could be replicated in the 59 TNF initiating patients. Combining these replicated biomarkers into a single signature we found that we could explain 51% of the variation in ∆DAS28-CRP. This corresponds to a sensitivity of 0.73 and specificity of 0.78 for the prediction of three month ∆DAS28-CRP better than -1.2. CONCLUSIONS: The COMBINE biobank is currently the largest collection of multi-omics data from RA patients with high potential for discovery and replication. Taking advantage of this we surveyed the current state-of-the-art of drug-response stratification in RA, and identified a small set of previously published biomarkers available in peripheral blood which predicts clinical response to TNF blockade in this independent cohort.

Infliximab Versus Conventional Combination Treatment and Seven-Year Work Loss in Early Rheumatoid Arthritis: Results of a Randomized Swedish Trial.

OBJECTIVE: To compare long-term work loss in methotrexate-refractory early rheumatoid arthritis (RA) patients randomized to the addition of infliximab or conventional combination treatment. METHODS: This study was a multicenter, 2-arm, parallel, randomized, active-controlled, open-label trial. RA patients with <1-year symptom duration were recruited from 15 rheumatology clinics in Sweden between 2002-2005. Patients who did not achieve low disease activity after 3-4 months of methotrexate therapy were randomized to the addition of infliximab or conventional combination treatment with sulfasalazine plus hydroxychloroquine. Yearly sick leave and disability pension days >7 years after randomization were retrieved from nationwide registers kept by the Swedish Social Insurance Agency. RESULTS: Of 210 working-age patients, 109 were randomized to infliximab (mean age 48.4 years, 73% women) and 101 to conventional treatment (mean age 48.7 years, 77% women). The year before randomization, the mean number of annual work days lost was 127 in the infliximab arm and 118 in the conventional treatment group (mean difference 9 [95% confidence interval (95% CI) -23, 39]). Compared to the year before randomization, the mean changes at 7 years were -25 days in the infliximab and -26 days in the conventional treatment group (adjusted mean difference 10 [95% CI -25, 46]). The cumulative mean for work-loss days was 846 in the infliximab group and 701 in the conventional treatment group (adjusted mean difference 104 [95% CI -56, 284]). CONCLUSION: Long-term work loss improved significantly in early RA patients randomized to infliximab plus methotrexate or conventional combination therapy. No difference was detected between strategies, and the level of work-loss days remained twice that observed in the general population.

Identification of a novel chemokine-dependent molecular mechanism underlying rheumatoid arthritis-associated autoantibody-mediated bone loss.

OBJECTIVES: Rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) appear before disease onset and are associated with bone destruction. We aimed to dissect the role of ACPAs in osteoclast (OC) activation and to identify key cellular mediators in this process. METHODS: Polyclonal ACPA were isolated from the synovial fluid (SF) and peripheral blood of patients with RA. Monoclonal ACPAs were isolated from single SF B-cells of patients with RA. OCs were developed from blood cell precursors with or without ACPAs. We analysed expression of citrullinated targets and peptidylarginine deiminases (PAD) enzymes by immunohistochemistry and cell supernatants by cytometric bead array. The effect of an anti-interleukin (IL)-8 neutralising antibody and a pan-PAD inhibitor was tested in the OC cultures. Monoclonal ACPAs were injected into mice and bone structure was analysed by micro-CT before and after CXCR1/2 blocking with reparixin. RESULTS: Protein citrullination by PADs is essential for OC differentiation. Polyclonal ACPAs enhance OC differentiation through a PAD-dependent IL-8-mediated autocrine loop that is completely abolished by IL-8 neutralisation. Some, but not all, human monoclonal ACPAs derived from single SF B-cells of patients with RA and exhibiting distinct epitope specificities promote OC differentiation in cell cultures. Transfer of the monoclonal ACPAs into mice induced bone loss that was completely reversed by the IL-8 antagonist reparixin. CONCLUSIONS: We provide novel insights into the key role of citrullination and PAD enzymes during OC differentiation and ACPA-induced OC activation. Our findings suggest that IL8-dependent OC activation may constitute an early event in the initiation of the joint specific inflammation in ACPA-positive RA.

Advances in the treatment of rheumatoid arthritis.

The intense pursuit of novel therapies in rheumatoid arthritis has provided physicians with an assorted set of biologic drugs to treat patients with moderate to severe disease activity. Nine different biologic therapies are currently available: seven inhibitors of pro-inflammatory cytokines (five targeting tumor necrosis factor [TNF], one interleukin [IL]-1 and one IL-6), as well as a T- and a B-lymphocyte targeting agent. All these drugs have roughly similar efficacy profiles and are approved as first- or second-line therapy in patients who failed to respond to conventional disease-modifying anti-rheumatic drugs (DMARDs) and in most cases for first line use in rheumatoid arthritis as well. Despite the irrefutable clinical and radiological benefits of biologic therapies, there are still low rates of patients achieving stable remission. Therefore, the quest for new and more effective biologic therapies continues and every year new drugs are tested. Simultaneously, optimal use of established agents is being studied in different ways. Recently, the approval of the first small molecule targeting intracellular pathways has opened a new chapter in the treatment of rheumatoid arthritis. Other emerging treatment strategies include the activation of regulatory T cells as well as new cytokine-targeting therapies.

Dendritic cell reprogramming by endogenously produced lactic acid.

The demand for controlling T cell responses via dendritic cell (DC) vaccines initiated a quest for reliable and feasible DC modulatory strategies that would facilitate cytotoxicity against tumors or tolerance in autoimmunity. We studied endogenous mechanisms in developing monocyte-derived DCs (MoDCs) that can induce inflammatory or suppressor programs during differentiation, and we identified a powerful autocrine pathway that, in a cell concentration-dependent manner, strongly interferes with inflammatory DC differentiation. MoDCs developing at low cell culture density have superior ability to produce inflammatory cytokines, to induce Th1 polarization, and to migrate toward the lymphoid tissue chemokine CCL19. On the contrary, MoDCs originated from dense cultures produce IL-10 but no inflammatory cytokines upon activation. DCs from high-density cultures maintained more differentiation plasticity and can develop to osteoclasts. The cell concentration-dependent pathway was independent of peroxisome proliferator-activated receptor γ (PPARγ), a known endogenous regulator of MoDC differentiation. Instead, it acted through lactic acid, which accumulated in dense cultures and induced an early and long-lasting reprogramming of MoDC differentiation. Our results suggest that the lactic acid-mediated inhibitory pathway could be efficiently manipulated in developing MoDCs to influence the immunogenicity of DC vaccines.

IL-7 modulates B cells survival and activation by inducing BAFF and CD70 expression in T cells.

Interleukin-7 (IL-7) promotes the maintenance and activation of peripheral T cells, whereas it does not act directly on mature B cells due to the lack of IL-7Rα expression on these. We report here that, in spite of the insensitivity of B cells to IL-7, high concentration of IL-7 can lead to increased B cell survival and antibody production in the presence of T cells, without the use of any further B cell stimulatory signal. IL-7 promoted B cell activation through inducing CD70 expression on resting T cells, particularly on CD4+ memory cells. The interaction of CD70 molecules with the B cell costimulatory receptor CD27 led to B cell proliferation, the accumulation of CD38 + CD20- plasmablasts and antibody production. In addition, IL-7 treatment induced BAFF secretion from resting peripheral T cells thereby promoting B cell survival. IL-7 levels can increase in lymphopenic conditions, in autoimmune diseases or in patients receiving T cell regenerative IL-7 therapy. Based on our findings high IL-7 levels can lead to increased B cell activation by inducing the B cell regulatory proteins CD70 and BAFF in resting T cells. Such activity might be beneficial in short term immune-stimulatory IL-7 therapies; permanently increased IL-7 levels, on the other hand, can contribute to impaired B cell tolerance.

Constraints for monocyte-derived dendritic cell functions under inflammatory conditions.

The activation of TLRs expressed by macrophages or DCs, in the long run, leads to persistently impaired functionality. TLR signals activate a wide range of negative feedback mechanisms; it is not known, however, which of these can lead to long-lasting tolerance for further stimulatory signals. In addition, it is not yet understood how the functionality of monocyte-derived DCs (MoDCs) is influenced in inflamed tissues by the continuous presence of stimulatory signals during their differentiation. Here we studied the role of a wide range of DC-inhibitory mechanisms in a simple and robust model of MoDC inactivation induced by early TLR signals during differentiation. We show that the activation-induced suppressor of cytokine signaling 1 (SOCS1), IL-10, STAT3, miR146a and CD150 (SLAM) molecules possessed short-term inhibitory effects on cytokine production but did not induce persistent DC inactivation. On the contrary, the LPS-induced IRAK-1 downregulation could alone lead to persistent MoDC inactivation. Studying cellular functions in line with the activation-induced negative feedback mechanisms, we show that early activation of developing MoDCs allowed only a transient cytokine production that was followed by the downregulation of effector functions and the preservation of a tissue-resident non-migratory phenotype.

IL-7 promotes CD95-induced apoptosis in B cells via the IFN-γ/STAT1 pathway.

Interleukin-7 (IL-7) concentrations are increased in the blood of CD4+ T cell depleted individuals, including HIV-1 infected patients. High IL-7 levels might stimulate T cell activation and, as we have shown earlier, IL-7 can prime resting T cell to CD95 induced apoptosis as well. HIV-1 infection leads to B cell abnormalities including increased apoptosis via the CD95 (Fas) death receptor pathway and loss of memory B cells. Peripheral B cells are not sensitive for IL-7, due to the lack of IL-7Ra expression on their surface; however, here we demonstrate that high IL-7 concentration can prime resting B cells to CD95-mediated apoptosis via an indirect mechanism. T cells cultured with IL-7 induced high CD95 expression on resting B cells together with an increased sensitivity to CD95 mediated apoptosis. As the mediator molecule responsible for B cell priming to CD95 mediated apoptosis we identified the cytokine IFN-γ that T cells secreted in high amounts in response to IL-7. These results suggest that the lymphopenia induced cytokine IL-7 can contribute to the increased B cell apoptosis observed in HIV-1 infected individuals.

Survival and proliferation of CD28- T cells during HIV-1 infection relate to the amplitude of viral replication.

BACKGROUND: CD28(-) T lymphocytes progressively increase during aging, autoimmunity, and HIV-1 infection. Expansion of these cells stands in contrast with their senescent phenotype described by several studies. Understanding the functional properties and phenotype of CD28(-) T cell during HIV-1 infection is important, because this subset incorporates T cells specific for HIV-1 and other chronic pathogens. METHODS: Blood samples were obtained from 23 healthy and 43 HIV-1-infected individuals: 26 receiving antiretroviral therapy and 17 naive to treatment. The phenotype of CD28(-) and CD28(+) T cells was determined by flow cytometry. T cells were activated through T-cell receptor before apoptosis and proliferation measurements. Interleukin (IL)-2, tumor-necrosis factor, interferon-γ, and perforin production were analyzed using enzyme-linked immunosorbent assay. RESULTS: CD28(-) T cells from patients receiving antiretroviral therapy exhibited a low sensitivity to apoptosis and enhanced proliferation after TCR stimulation, compared with T cells of uninfected individuals. On the contrary, CD28(-) T cells from viremic patients showed a decreased Bcl-2 expression, a high sensitivity to apoptosis, and poor proliferative ability, compared with treated patients and control subjects. T cells from untreated patients produced less IL-2, possibly underlying their decreased proliferative abilities. CONCLUSIONS: The level of HIV-1 replication and associated immunoactivation represent a critical factor in regulating survival and activation of CD28(-) T cells.

Potential role of CD8+CD28- T lymphocytes in immune activation during HIV-1 infection.

As CD8+CD28- T cells have been associated with dendritic and T cell suppression, we analyzed whether an increase in CD8+CD28- T cell numbers during HIV-1 infection could lead to impaired T cell responses. In contrast to the in-vitro generated CD8+CD28- suppressors, peripheral blood CD8+CD28- T cells of both HIV-infected and noninfected individuals promoted dendritic cell activation. The CD8+CD28- T cell accumulation during HIV-1 infection may thus contribute to accelerated inflammatory reactions and immune activation.

Priming of T cells to Fas-mediated proliferative signals by interleukin-7.

T-cell depletion associated with HIV infection or cytoreductive therapies triggers potential T-cell regenerative mechanisms such as peripheral T-lymphocyte expansion to weak antigenic stimuli and the increased availability of interleukin-7 (IL-7), a cytokine with potent antiapoptotic and proliferative activities. Deleterious mechanisms also associated with lymphopenia, such as increased Fas expression and apoptosis of T cell, however, may result in opposing effects. In this study, we show that Fas molecules, primarily associated with T-cell depletion in lymphopenic settings, may also contribute to compensatory T-cell expansion through transmitting costimulatory signals to suboptimally activated T cells. Proliferation of T lymphocytes in response to concomitant Fas and T-cell receptor (TCR) triggering was shown to be increased in HIV-infected individuals compared with noninfected controls. As IL-7 levels are often elevated in lymphopenic individuals in association with increased Fas expression, we analyzed whether IL-7 would influence Fas-mediated proliferative signals in T cells. We show that IL-7 is able to increase the efficacy of Fas to induce proliferation of suboptimally activated T cells. Thus, high IL-7 levels associated with lymphopenic conditions may simultaneously induce sensitivity to Fas-mediated apoptosis in nonactivated T cells and increase Fas-induced costimulatory signals in T cells recognizing low-affinity antigens.

Potential role for IL-7 in Fas-mediated T cell apoptosis during HIV infection.

IL-7 promotes survival of resting T lymphocytes and induces T cell proliferation in lymphopenic conditions. As elevated IL-7 levels occur in HIV-infected individuals in addition to high Fas expression on T cells and increased sensitivity to Fas-induced apoptosis, we analyzed whether IL-7 has a regulatory role in Fas-mediated T cell apoptosis. We show that IL-7 up-regulates Fas expression on naive and memory T cells through a mechanism that involves translocation of Fas molecules from intracellular compartments to the cell membrane. IL-7 induced the association of Fas with the cytoskeletal component ezrin and a polarized Fas expression on the cell surface. The potential role of IL-7 in Fas up-regulation in vivo was verified in IL-7-treated macaques and in HIV-infected or chemotherapy treated patients by the correlation between serum IL-7 levels and Fas expression on T cells. IL-7 treatment primed T cells for Fas-induced apoptosis in vitro and serum IL-7 levels correlated with the sensitivity of T cells to Fas-induced apoptosis in HIV-infected individuals. Our data suggest an important role for IL-7 in Fas-mediated regulation of T cell homeostasis. Elevated IL-7 levels associated with lymphopenic conditions, including HIV-infection, might participate in the increased sensitivity of T cells for activation-induced apoptosis.

Relationship between serum IL-7 concentrations and lymphopenia upon different levels of HIV immune control.

Serum IL-7 levels correlate with T-cell depletion in HIV-infected individuals. In some patients, we observed that serum IL-7 decreases upon progression to AIDS, suggesting a role for IL-7 in T-cell maintenance in sporadic cases. Interestingly, IL-7 levels were significantly lower in stable long-term non-progressors (LTNP) than in patients who lost the LTNP status in a 3-year follow-up (P < 0.001), indicating that the serum IL-7 concentration might be a valuable marker for maintenance of the LTNP state.