Promotion of macrophage activation by Tie2 in the context of the inflamed synovia of rheumatoid arthritis and psoriatic arthritis patients.

OBJECTIVE: To examine the role of Tie2 signalling in macrophage activation within the context of the inflammatory synovial microenvironment present in patients with RA and PsA. METHODS: Clinical responses and macrophage function were examined in wild-type and Tie2-overexpressing (Tie2-TG) mice in the K/BxN serum transfer model of arthritis. Macrophages derived from peripheral blood monocytes from healthy donors, RA and PsA patients, and RA and PsA synovial tissue explants were stimulated with TNF (10 ng/ml), angiopoietin (Ang)-1 or Ang-2 (200 ng/ml), or incubated with an anti-Ang2 neutralizing antibody. mRNA and protein expression of inflammatory mediators was analysed by quantitative PCR, ELISA and Luminex. RESULTS: Tie2-TG mice displayed more clinically severe arthritis than wild-type mice, accompanied by enhanced joint expression of IL6, IL12B, NOS2, CCL2 and CXCL10, and activation of bone marrow-derived macrophages in response to Ang-2 stimulation. Ang-1 and Ang-2 significantly enhanced TNF-induced expression of pro-inflammatory cytokines and chemokines in macrophages from healthy donors differentiated with RA and PsA SF and peripheral blood-derived macrophages from RA and PsA patients. Both Ang-1 and Ang-2 induced the production of IL-6, IL-12p40, IL-8 and CCL-3 in synovial tissue explants of RA and PsA patients, and Ang-2 neutralization suppressed the production of IL-6 and IL-8 in the synovial tissue of RA patients. CONCLUSION: Tie2 signalling enhances TNF-dependent activation of macrophages within the context of ongoing synovial inflammation in RA and PsA, and neutralization of Tie2 ligands might be a promising therapeutic target in the treatment of these diseases.

Dysregulated miRNome of plasmacytoid dendritic cells from patients with Sjögren's syndrome is associated with processes at the centre of their function.

OBJECTIVE: A considerable body of evidence supports a role for type-I IFN in the pathogenesis of primary SS (pSS). As plasmacytoid dendritic cells (pDCs) are a major source of type-I IFN, we investigated their molecular regulation by measuring expression of a large set of miRNAs. METHODS: pDCs were isolated from peripheral blood of pSS patients (n = 30) and healthy controls (n = 16) divided into two independent cohorts (discovery and replication). Screening of 758 miRNAs was assessed by an OpenArray quantitative PCR-based technique; replication of a set of identified miRNAs was performed by custom array. Functional annotation of miRNA targets was performed using pathway enrichment. Novel targets of miR-29a and miR-29c were identified using a proteomic approach (stable isotope labelling with amino acids in cell culture). RESULTS: In the discovery cohort, 20 miRNAs were differentially expressed in pSS pDCs compared with healthy control pDCs. Of these, differential expression of 10 miRNAs was confirmed in the replication cohort. The dysregulated miRNAs were involved in phosphoinositide 3-kinase-Ak strain transforming and mammalian target of rapamycin signalling, as well as regulation of cell death. In addition, a set of novel protein targets of miR-29a and miR-29c were identified, including five targets that were regulated by both miRs. CONCLUSION: The dysregulated miRNome in pDCs of patients with pSS is associated with aberrant regulation of processes at the centre of pDC function, including type-I IFN production and cell death. As miR-29a and miR-29c are pro-apoptotic factors and several of the novel targets identified here are regulators of apoptosis, their downregulation in patients with pSS is associated with enhanced pDC survival.

Increased expression of Fas on group 2 and 3 innate lymphoid cells is associated with an interferon signature in systemic lupus erythematosus and Sjögren's syndrome.

OBJECTIVE: The role of innate lymphoid cells (ILCs) in the pathophysiology of rheumatic diseases is emerging. Evidence from animal studies implicate type I IFN, produced by plasmacytoid dendritic cells, to be involved in regulating the survival of group 2 and group 3 ILCs (ILC2s and ILC3s) via the upregulation of Fas (CD95) expression. For the first time, we explored the frequency and phenotype of circulating ILCs in SLE and primary Sjögren's syndrome (pSS) in relationship to the IFN signature. METHODS: Frequencies and phenotypes of ILC subsets and plasmacytoid dendritic cells were assessed by flow cytometry in peripheral blood of patients with SLE (n = 20), pSS (n = 20) and healthy controls (n = 17). Patients were stratified by the presence or absence of an IFN signature as assessed by RT-qPCR on circulating mononuclear cells. RESULTS: ILC1 frequencies were increased in peripheral blood of patients with SLE as compared with healthy controls and correlate with disease activity in pSS patients. Overall, the frequencies of ILC2s or ILC3s did not differ between patients with SLE, pSS and healthy controls. However, patients with a high type I IFN signature expressed elevated levels of Fas on ILC2s and ILC3s, which coincided with decreased frequencies of these cells in blood. CONCLUSION: The presence of a type I IFN signature is related to Fas expression and frequencies of circulating ILC2s and ILC3s in patients with SLE and pSS, potentially altering the homeostatic balance of ILCs.

IL4-10 Fusion Protein Is a Novel Drug to Treat Persistent Inflammatory Pain.

UNLABELLED: Chronic pain is a major clinical problem that is difficult to treat and requires novel therapies. Although most pain therapies primarily target neurons, neuroinflammatory processes characterized by spinal cord and dorsal root ganglion production of proinflammatory cytokines play an important role in persistent pain states and represent potential therapeutic targets. Anti-inflammatory cytokines are attractive candidates to regulate aberrant neuroinflammatory processes, but the therapeutic potential of these cytokines as stand-alone drugs is limited. Their optimal function requires concerted actions with other regulatory cytokines, and their relatively small size causes rapid clearance. To overcome these limitations, we developed a fusion protein of the anti-inflammatory cytokines interleukin 4 (IL4) and IL10. The IL4-10 fusion protein is a 70 kDa glycosylated dimeric protein that retains the functional activity of both cytokine moieties. Intrathecal administration of IL4-10 dose-dependently inhibited persistent inflammatory pain in mice: three IL4-10 injections induced full resolution of inflammatory pain in two different mouse models of persistent inflammatory pain. Both cytokine moieties were required for optimal effects. The IL4-10 fusion protein was more effective than the individual cytokines or IL4 plus IL10 combination therapy and also inhibited allodynia in a mouse model of neuropathic pain. Mechanistically, IL4-10 inhibited the activity of glial cells and reduced spinal cord and dorsal root ganglion cytokine levels without affecting paw inflammation. In conclusion, we developed a novel fusion protein with improved efficacy to treat pain, compared with wild-type anti-inflammatory cytokines. The IL4-10 fusion protein has potential as a treatment for persistent inflammatory pain. SIGNIFICANCE STATEMENT: The treatment of chronic pain is a major clinical and societal challenge. Current therapies to treat persistent pain states are limited and often cause major side effects. Therefore, novel analgesic treatments are urgently needed. In search of a novel drug to treat chronic pain, we developed a fusion protein consisting of two prototypic regulatory cytokines, interleukin 4 (IL4) and IL10. The work presented in this manuscript shows that this IL4-10 fusion protein overcomes some major therapeutic limitations of pain treatment with individual cytokines. The IL4-10 fusion protein induces full resolution of persistent inflammatory pain in two different mouse models. These novel findings are significant, as they highlight the IL4-10 fusion protein as a long-needed potential new drug to stop persistent pain states.

TOFA-PREDICT study protocol: a stratification trial to determine key immunological factors predicting tofacitinib efficacy and drug-free remission in psoriatic arthritis (PsA).

INTRODUCTION: Psoriatic arthritis (PsA) is a chronic, inflammatory, musculoskeletal disease that affects up to 30% of patients with psoriasis. Current challenges in clinical care and research include personalised treatment, understanding the divergence of therapy response and unravelling the multifactorial pathophysiology of this complex disease. Moreover, there is an urgent clinical need to predict, assess and understand the cellular and molecular pathways underlying the response to disease-modifying antirheumatic drugs (DMARDs). The TOFA-PREDICT clinical trial addresses this need. Our primary objective is to determine key immunological factors predicting tofacitinib efficacy and drug-free remission in PsA. METHODS AND ANALYSIS: In this investigator-initiated, phase III, multicentre, open-label, four-arm randomised controlled trial, we plan to integrate clinical, molecular and imaging parameters of 160 patients with PsA. DMARD-naïve patients are randomised to methotrexate or tofacitinib. Additionally, patients who are non-responsive to conventional synthetic (cs)DMARDs continue their current csDMARD and are randomised to etanercept or tofacitinib. This results in four arms each with 40 patients. Patients are followed for 1 year. Treatment response is defined as minimal disease activity at week 16. Clinical data, biosamples and images are collected at baseline, 4 weeks and 16 weeks; at treatment failure (treatment switch) and 52 weeks. For the first 80 patients, we will use a systems medicine approach to assess multiomics biomarkers and develop a prediction model for treatment response. Subsequently, data from the second 80 patients will be used for validation. ETHICS AND DISSEMINATION: The study was approved by the Medical Research Ethics Committee in Utrecht, Netherlands, is registered in the European Clinical Trials Database and is carried out in accordance with the Declaration of Helsinki. The study's progress is monitored by Julius Clinical, a science-driven contract research organisation. TRIAL REGISTRATION NUMBER: EudraCT: 2017-003900-28.

Blockade of the interleukin-7 receptor inhibits collagen-induced arthritis and is associated with reduction of T cell activity and proinflammatory mediators.

OBJECTIVE: To study the effects of interleukin-7 receptor α-chain (IL-7Rα) blockade on collagen-induced arthritis (CIA) and to investigate the effects on T cell numbers, T cell activity, and levels of proinflammatory mediators. METHODS: We studied the effect of anti-IL-7Rα antibody treatment on inflammation and joint destruction in CIA in mice. Numbers of thymocytes, splenocytes, T cell subsets, B cells, macrophages, and dendritic cells were assessed. Cytokines indicative of Th1, Th2, and Th17 activity and several proinflammatory mediators were assessed by multianalyte profiling in paw lysates. In addition, T cell-associated cytokines were measured in supernatants of lymph node cell cultures. RESULTS: Anti-IL-7Rα treatment significantly reduced clinical arthritis severity in association with reduced radiographic joint damage. Both thymic and splenic cellularity were reduced after treatment with anti-IL-7Rα. IL-7Rα blockade specifically reduced the total number of cells as well as numbers of naive, memory, CD4+, and CD8+ T cells from the spleen and significantly reduced T cell-associated cytokines (interferon-γ, IL-5, and IL-17). IL-7Rα blockade also decreased local levels of proinflammatory cytokines and factors associated with tissue destruction, including tumor necrosis factor α, IL-1ß, IL-6, matrix metalloproteinase 9, and RANKL. IL-7Rα blockade did not significantly affect B cells, macrophages, and dendritic cells. B cell activity, indicated by serum anticollagen IgG antibodies, was not significantly altered. CONCLUSION: Blockade of IL-7Rα potently inhibited joint inflammation and destruction in association with specific reductions of T cell numbers, T cell-associated cytokines, and numerous mediators that induce inflammation and tissue destruction. This study demonstrates an important role of IL-7R-driven immunity in experimental arthritis and indicates the therapeutic potential of IL-7Rα blockade in human arthritic conditions.

Numbers of CD25+Foxp3+ T cells that lack the IL-7 receptor are increased intra-articularly and have impaired suppressive function in RA patients.

OBJECTIVES: To investigate numbers and function of CD25(+) regulatory T cells (Tregs) that lack IL-7 receptor (CD127) expression in RA. METHODS: Numbers of CD4 T cells expressing either CD25 or CD127, and those co-expressing or lacking both CD25 and CD127 were assessed in peripheral blood (PB) of RA patients and healthy controls, and in paired samples of SF and PB from RA patients. All T-cell subsets were analysed for FoxP3 expression. The anergic state and the capacity to suppress CD127(+) proliferating responder T cells were determined. RESULTS: Numbers of CD127(-) T cells and CD25(+) Tregs in PB of RA patients were not different from controls but significantly increased in SF compared with PB. CD25(+) and CD127(-) T cells showed comparable FoxP3 expression. CD127(+) T cells hardly expressed FoxP3. PB CD25(+)CD127(-) T cells identified a subset that consisted for 75% of FoxP3(+) cells. SF CD25(+)CD127(-) T-cell number was increased; however, in SF fewer of these cells were FoxP3(+). CD25(+)CD127(-) T cells were anergic, and in controls potent suppressors of CD127(+) proliferating T cells, but in RA patients these cells showed impaired suppression. In line with this, IL-7 had an increased capacity to activate total CD4 T cells from SF as compared with PB despite increased numbers of CD25(+)CD127(-) in SF. CONCLUSIONS: These data demonstrate improved identification of FoxP3(+) T cells in RA patients by the absence of CD127 in addition to CD25 expression. Increased numbers of CD25(+)CD127(-) T cells are found in inflamed RA joints, but they have an impaired suppressive function, which could contribute to the persistent arthritis in these patients.

Plasmacytoid DCs From Patients With Sjögren's Syndrome Are Transcriptionally Primed for Enhanced Pro-inflammatory Cytokine Production.

Primary Sjögren's syndrome (pSS) is a systemic auto-immune disease typified by dryness of the mouth and eyes. A majority of patients with pSS have a type-I interferon (IFN)-signature, which is defined as the increased expression of IFN-induced genes in circulating immune cells and is associated with increased disease activity. As plasmacytoid dendritic cells (pDC) are the premier type-I IFN-producing cells and are present at the site of inflammation, they are thought to play a significant role in pSS pathogenesis. Considering the lack of data on pDC regulation and function in pSS patients, we here provided the first in-depth molecular characterization of pSS pDCs. In addition, a group of patients with non-Sjögren's sicca (nSS) was included; these poorly studied patients suffer from complaints similar to pSS patients, but are not diagnosed with Sjögren's syndrome. We isolated circulating pDCs from two independent cohorts of patients and controls (each n = 31) and performed RNA-sequencing, after which data-driven networks and modular analysis were used to identify robustly reproducible transcriptional "signatures" of differential and co-expressed genes. Four signatures were identified, including an IFN-induced gene signature and a ribosomal protein gene-signature, that indicated pDC activation. Comparison with a dataset of in vitro activated pDCs showed that pSS pDCs have higher expression of many genes also upregulated upon pDC activation. Corroborating this transcriptional profile, pSS pDCs produced higher levels of pro-inflammatory cytokines, including type-I IFN, upon in vitro stimulation with endosomal Toll-like receptor ligands. In this setting, cytokine production was associated with expression of hub-genes from the IFN-induced and ribosomal protein gene-signatures, indicating that the transcriptional profile of pSS pDCs underlies their enhanced cytokine production. In all transcriptional analyses, nSS patients formed an intermediate group in which some patients were molecularly similar to pSS patients. Furthermore, we used the identified transcriptional signatures to develop a discriminative classifier for molecular stratification of patients with sicca. Altogether, our data provide in-depth characterization of the aberrant regulation of pDCs from patients with nSS and pSS and substantiate their perceived role in the immunopathology of pSS, supporting studies that target pDCs, type-I IFNs, or IFN-signaling in pSS.

MicroRNA-130a Contributes to Type-2 Classical DC-activation in Sjögren's Syndrome by Targeting Mitogen- and Stress-Activated Protein Kinase-1.

Objectives: Considering the critical role of microRNAs (miRNAs) in regulation of cell activation, we investigated their role in circulating type-2 conventional dendritic cells (cDC2s) of patients with primary Sjögren's syndrome (pSS) compared to healthy controls (HC). Methods: CD1c-expressing cDC2s were isolated from peripheral blood. A discovery cohort (15 pSS, 6 HC) was used to screen the expression of 758 miRNAs and a replication cohort (15 pSS, 11 HC) was used to confirm differential expression of 18 identified targets. Novel targets for two replicated miRNAs were identified by SILAC in HEK-293T cells and validated in primary cDC2s. Differences in cytokine production between pSS and HC cDC2s were evaluated by intracellular flow-cytometry. cDC2s were cultured in the presence of MSK1-inhibitors to investigate their effect on cytokine production. Results: Expression of miR-130a and miR-708 was significantly decreased in cDC2s from pSS patients compared to HC in both cohorts, and both miRNAs were downregulated upon stimulation via endosomal TLRs. Upstream mediator of cytokine production MSK1 was identified as a novel target of miR-130a and overexpression of miR-130a reduced MSK1 expression in cDC2s. pSS cDC2s showed higher MSK1 expression and an increased fraction of IL-12 and TNF-α-producing cells. MSK1-inhibition reduced cDC2 activation and production of IL-12, TNF-α, and IL-6. Conclusions: The decreased expression of miR-130a and miR-708 in pSS cDC2s seems to reflect cell activation. miR-130a targets MSK1, which regulates pro-inflammatory cytokine production, and we provide proof-of-concept for MSK1-inhibition as a therapeutic avenue to impede cDC2 activity in pSS.

The Additive Inflammatory In Vivo and In Vitro Effects of IL-7 and TSLP in Arthritis Underscore the Therapeutic Rationale for Dual Blockade.

INTRODUCTION: The cytokines interleukin (IL)-7 and thymic stromal lymphopoietin (TSLP) signal through the IL-7R subunit and play proinflammatory roles in experimental arthritis and rheumatoid arthritis (RA). We evaluated the effect of inhibition of IL-7R- and TSLPR-signalling as well as simultaneous inhibition of IL-7R- and TSLPR-signalling in murine experimental arthritis. In addition, the effects of IL-7 and TSLP in human RA dendritic cell (DC)/T-cell co-cultures were studied. METHODS: Arthritis was induced with proteoglycan in wildtype mice (WT) and in mice deficient for the TSLP receptor subunit (TSLPR-/-). Both mice genotypes were treated with anti-IL-7R or phosphate buffered saline. Arthritis severity was assessed and local and circulating cytokines were measured. Autologous CD1c-positive DCs and CD4 T-cells were isolated from peripheral blood of RA patients and were co-cultured in the presence of IL-7, TSLP or both and proliferation and cytokine production were assessed. RESULTS: Arthritis severity and immunopathology were decreased in WT mice treated with anti-IL-7R, in TSLPR-/- mice, and the most robustly in TSLPR-/- mice treated with anti-IL-7R. This was associated with strongly decreased levels of IL-17, IL-6 and CD40L. In human DC/T-cell co-cultures, TSLP and IL-7 additively increased T-cell proliferation and production of Th17-associated cytokines, chemokines and tissue destruction factors. CONCLUSION: TSLP and IL-7 have an additive effect on the production of Th17-cytokines in a human in vitro model, and enhance arthritis in mice linked with enhanced inflammation and immunopathology. As both cytokines signal via the IL-7R, these data urge for IL-7R-targeting to prevent the activity of both cytokines in RA.

Brief report: enrichment of activated group 3 innate lymphoid cells in psoriatic arthritis synovial fluid.

OBJECTIVE: Innate lymphoid cells (ILCs) are a recently discovered group of cells that are essential to epithelial homeostasis and are implicated in psoriasis pathogenesis, yet they have never been reported in psoriatic arthritis (PsA). METHODS: ILC classes and subsets were characterized in the peripheral blood (PB) of healthy controls, patients with psoriasis, and patients with PsA and in the synovial fluid (SF) of patients with PsA and patients with rheumatoid arthritis (RA). Cell surface marker expression and intracellular cytokine production following stimulation were analyzed using flow cytometry. RESULTS: ILCs were identified in the SF and were 4-fold more abundant in PsA SF than in PsA PB. Fewer CCR6+ ILCs were found in PsA PB than in healthy control PB, while PsA SF was enriched for CCR6+ ILCs compared to PsA PB and RA SF. Natural cytotoxicity receptor NKp44+ group 3 ILCs were rare in PB and RA SF, but abundant in PsA SF. Increased numbers of interleukin-17A (IL-17A)-producing ILCs were present in PsA SF compared to RA SF. CCR6, NKp44, and melanoma cell adhesion molecule (MCAM) were expressed on the cell surface of SF ILCs that produced IL-17A. The number of circulating NKp44+, CCR6+, and MCAM+ ILCs in blood was inversely correlated with PsA disease activity. CONCLUSION: Our findings indicate that PsA SF is enriched for group 3 ILCs that express CCR6 and NKp44, which distinguishes the synovial compartment from RA. The increased IL-17A production by SF ILCs indicates a novel role for ILCs in PsA.

Interleukin-7-aggravated joint inflammation and tissue destruction in collagen-induced arthritis is associated with T-cell and B-cell activation.

INTRODUCTION: We sought to investigate the capacity of interleukin (IL)-7 to enhance collagen-induced arthritis and to study by what mechanisms this is achieved. METHODS: Mice received multiple injections with IL-7 or phosphate-buffered saline (PBS) as a control. Arthritis severity and incidence were determined by visual examination of the paws. Joint destruction was determined by assessing radiographs and immunohistochemistry of the ankle joints. Total cellularity and numbers of T-cell and B-cell subsets were assessed, as well as ex vivo production of interferon-γ (IFN-γ), IL-17, and IL-4. Proinflammatory mediators were measured in serum with multianalyte profiling. RESULTS: IL-7 increased arthritis severity and radiology-assessed joint destruction. This was consistent with IL-7-increased intensity of cell infiltrates, bone erosions, and cartilage damage. Splenic CD19+ B cells and CD19+/GL7+ germinal center B cells, as well as CD4 and CD8 numbers, were increased by IL-7. IL-7 expanded memory T cells, associated with increased percentages of IFN-γ-, IL-4-, and IL-17-producing CD4+ T cells. On antigen restimulation of draining lymph node cells in vitro IL-7 treatment was found to increase IFN-γ and IL-17 production, whereas IL-4 was reduced. IL-7 also increased concentrations of proinflammatory mediators, indicative of T-cell activation (sCD40L), vascular activation (VCAM-1, VEGF), tissue destruction (fibroblast growth factor-basic (FGF-b), LIF), and chemotaxis (MIP-1γ, MIP-3ß, lymphotactin, MDC, and MCP-5). CONCLUSIONS: In arthritic mice, IL-7 causes expansion of T and B cells, associated with increased levels of proinflammatory mediators. IL-7 intensifies arthritis severity and joint destruction, accompanied by increased Th1 and Th17 activity. These data indicate that IL-7 could be an important mediator in arthritic conditions and that targeting IL-7 or its receptor represent novel therapeutic strategies.

Elevated expression of interleukin-7 receptor in inflamed joints mediates interleukin-7-induced immune activation in rheumatoid arthritis.

OBJECTIVE: To evaluate the expression and functional ability of the high-affinity interleukin-7 receptor (IL-7Ralpha) in patients with rheumatoid arthritis (RA). METHODS: Expression of IL-7Ralpha and IL-7 was determined in synovial tissue from RA patients and was compared with that in synovial tissue from patients with undifferentiated arthritis (UA) and osteoarthritis (OA). IL-7Ralpha expression on CD4 T cells, CD19 B cells, and CD14 monocyte/macrophages from RA synovial tissue, synovial fluid, and peripheral blood was also assessed. The proliferative capacity of IL-7Ralpha(bright) and IL-7Ralpha(dim/-) T cells was measured. In addition, we examined IL-7R blockade with soluble human IL-7Ralpha (hIL-7Ralpha) in the prevention of immune activation of peripheral blood mononuclear cells. RESULTS: We found significantly higher IL-7Ralpha expression in RA and UA synovial tissue than in OA synovial tissue, and the level of IL-7Ralpha expression correlated significantly with the levels of CD3 and IL-7 expression. CD4 T cells from RA synovial fluid and synovial tissue strongly expressed IL-7Ralpha. A substantial percentage of B cells and macrophages from RA synovial fluid and synovial tissue also expressed IL-7Ralpha, although less prominently than T cells. We found that peripheral blood IL-7Ralpha(bright) T cells that did not express FoxP3 were highly proliferative as compared with IL-7Ralpha(dim/-) T cells that did express high levels of FoxP3. Soluble hIL-7Ralpha inhibited IL-7-induced proliferation and interferon-gamma production by mononuclear cells from RA patients. CONCLUSION: Our data suggest that enhanced expression of IL-7Ralpha and IL-7 in RA patients contributes significantly to the joint inflammation by activating T cells, B cells, and macrophages. The inhibition of IL-7R-mediated immune activation by soluble hIL-7Ralpha further indicates an important role of IL-7Ralpha in inflammatory responses in RA, suggesting IL-7Ralpha as a therapeutic target for immunotherapy in RA.

Persistence of interleukin 7 activity and levels on tumour necrosis factor alpha blockade in patients with rheumatoid arthritis.

OBJECTIVES: To identify the mechanism of interleukin (IL)7-stimulated tumour necrosis factor alpha (TNFalpha) production and to determine the relationship between intra-articular IL7 and TNFalpha expression levels in patients with rheumatoid arthritis (RA). In addition, the effect of TNFalpha blockade on IL7 activity and on IL7 levels was studied. METHODS: The effect of IL7 on isolated CD4 T cells and CD14 monocytes/macrophages was studied. IL7 and TNFalpha levels were measured in the synovial fluid of patients with RA. In RA synovial tissue, IL7 and TNFalpha expression was assessed in addition to IL1beta, numbers of inflammatory cells and adhesion molecule expression. The extent to which TNFalpha blockade could prevent IL7-induced lymphocyte responses was studied in vitro. In addition, regulation of serum IL7 levels on anti-TNFalpha therapy (adalimumab) was studied. RESULTS: IL7 induced cell contact-dependent TNFalpha production by cocultures of T cells and monocytes, but not by T cells and monocytes cultured separately. IL7 and TNFalpha levels in RA synovial fluid and synovial tissue significantly correlated. IL7-stimulated lymphocyte responses were not inhibited by TNFalpha blockade. Circulating IL7 levels were significantly reduced in patients who successfully responded to anti-TNFalpha treatment. However, IL7 levels persisted in non-responders. CONCLUSION: The present data suggest that IL7 is an important inducer of T cell-dependent TNFalpha production in RA joints. This may contribute to the correlation of intra-articular IL7 and TNFalpha in these joints. Furthermore, the persistence of IL7-induced inflammatory activity on TNFalpha blockade in vitro and persistence of IL7 levels and disease activity in anti-TNFalpha non-responders suggest that IL7 might additionally promote TNFalpha-independent inflammation.