Annemieke M. H. Boots

Human CD25highFoxp3pos regulatory T cells differentiate into IL-17-producing cells.

The effector T-cell lineage shows great plasticity. Th17 cells are acknowledged to be instrumental in the response against microbial infection, but are also associated with autoimmune inflammatory processes. Here, we report that human regulatory T cells (CD4(pos)CD25(high)Foxp3(pos)CD127(neg)CD27(pos)) can differentiate into IL-17-producing cells, when stimulated by allogeneic antigen-presenting cells, especially monocytes, in the presence of rhIL-2/rhIL-15. These regulatory T cell (Treg)-derived IL-17-producing cells showed high expression of the Th17-related transcription factor RORgammat and were positively identified by CCR6 expression. This differentiation process was enhanced by exogenous IL-1beta, IL-23, and IL-21, whereas IL-6 or TGFbeta did not affect the emergence of IL-17-producing cells. The addition of IL-1 receptor antagonist (IL-1Ra), but not anti-IL-23 antibody, reduced IL-17-producing cell numbers. When an histone deacetylase (HDAC) inhibitor trichostatin A (TSA) was evaluated, we found a profound negative effect on the emergence of IL-17-producing cells from Tregs, implying that Treg differentiation into IL-17-producing cells depends on histone/protein deacetylase activity. Thus, the data suggest that epigenetic modification underlies the phenomenon of Treg plasticity here described.

Blocking of Interleukin-17 during Reactivation of Experimental Arthritis Prevents Joint Inflammation and Bone Erosion by Decreasing RANKL and Interleukin-1

Rheumatoid arthritis is characterized by an intermittent course of disease with alternate periods of remission and relapse. T cells, and in particular the T-cell cytokine interleukin-17 (IL-17), are expected to be involved in arthritic flares. Here, we report that neutralizing endogenous IL-17 during reactivation of antigen-induced arthritis prevents joint inflammation and bone erosion. Synovial IL-17 mRNA expression was clearly up-regulated during primary arthritis and was further enhanced after antigen rechallenge. Neutralization of IL-17 significantly prevented joint swelling at day 1 of flare and significantly suppressed joint inflammation and cartilage proteoglycan depletion at day 4, as assessed by histology. Blocking IL-17 also clearly reduced bone erosions. Cathepsin K, a marker of osteoclast-like activity, and synovial RANKL mRNA expression were both suppressed. The degree of bone erosions strongly correlated with the severity of joint inflammation, suggesting that anti-IL-17 treatment reduced bone erosion by suppressing joint inflammation. Interestingly, blocking IL-17 suppressed synovial expression of both IL-1beta and tumor necrosis factor-alpha, whereas blocking IL-1 did not affect tumor necrosis factor-alpha levels. These data indicate that IL-17 is an important upstream mediator in joint pathology during flare-up of experimental arthritis.

Synovial histopathology of psoriatic arthritis, both oligo- and polyarticular, resembles spondyloarthropathy more than it does rheumatoid arthritis

At present only few biological data are available to indicate whether psoriatic arthritis (PsA) is part of the spondyloarthropathy (SpA) concept, whether it is a separate disease entity or a heterogeneous disease group with oligoarticular/axial forms belonging to SpA and polyarticular forms resembling rheumatoid arthritis (RA). To address this issue with regard to peripheral synovitis, we compared the synovial characteristics of PsA with those of ankylosing spondylitis (AS)/undifferentiated SpA (USpA) and RA, and compared the synovium of oligoarticular versus polyarticular PsA. Synovial biopsies were obtained from patients with RA, nonpsoriatic SpA (AS + USpA), and oligoarticular and polyarticular PsA. The histological analysis included examination(s) of the lining layer thickness, vascularity, cellular infiltration, lymphoid aggregates, plasma cells and neutrophils. Also, we performed immunohistochemical assessments of CD3, CD4, CD8, CD20, CD38, CD138, CD68, CD163, CD83, CD1a, CD146, αVβ3, E-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, S100A12, intracellular citrullinated proteins and major histocompatibility complex (MHC)–human cartilage (HC) gp39 peptide complexes. Comparing SpA (PsA + AS + USpA) with RA, vascularity, and neutrophil and CD163+ macrophage counts were greater in SpA (P < 0.05), whereas lining layer thickness and the number of CD83+ dendritic cells were greater in RA (P < 0.05). In RA, 44% of samples exhibited positive staining for intracellular citrullinated proteins and 46% for MHC–HC gp39 peptide complexes, whereas no staining for these markers was observed in SpA samples. We excluded influences of disease-modifying antirheumatic drug and/or corticosteroid treatment by conducting systematic analyses of treated and untreated subgroups. Focusing on PsA, no significant differences were observed between PsA and nonpsoriatic SpA. In contrast, vascularity (P < 0.001) and neutrophils were increased in PsA as compared with RA (P = 0.010), whereas staining for intracellular citrullinated proteins and MHC–HC gp39 peptide complexes was exclusively observed in RA (both P = 0.001), indicating that the same discriminating features are found in PsA and other SpA subtypes compared with RA. Exploring synovial histopathology between oligoarticular and polyarticular PsA, no significant differences were noted. Moreover, intracellular citrullinated proteins and MHC–HC gp39 peptide complexes, which are specific markers for RA, were observed in neither oligoarticular nor polyarticular PsA. Taken together, these data indicate that the synovial histopathology of PsA, either oligoarticular or polyarticular, resembles that of other SpA subtypes, whereas both groups can be differentiated from RA on the basis of these same synovial features, suggesting that peripheral synovitis in PsA belongs to the SpA concept.

Human cartilage gp-39+,CD16+ monocytes in peripheral blood and synovium: correlation with joint destruction in rheumatoid arthritis.

OBJECTIVE To investigate the expression of human cartilage (HC) gp-39, a possible autoantigen in rheumatoid arthritis (RA), in peripheral blood and synovium, to characterize its cellular source, and to analyze correlations with clinical features. METHODS The expression of HC gp-39 in synovium and peripheral blood mononuclear cells (PBMC) was assessed by immunohistochemistry and flow cytometry. Synthesis and secretion were investigated by both reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS PBMC expressing HC gp-39 were increased in RA patients compared with spondylarthropathy patients (P = 0.0029) and with healthy control subjects (P = 0.0013). HC gp-39+ cells were also slightly overrepresented in RA synovium (P = 0.01). In both blood and synovium, HC gp-39+ cells were identified as CD14dim,CD16+ monocytes, a phenotype which can differentiate from classic CD14++ monocytes by maturation in vitro. HC gp-39 messenger RNA was detected in RA synovium and PBMC, and PBMC secreted HC gp-39 in vitro. The number of HC gp-39+ PBMC correlated with serum levels of C-reactive protein (r = 0.39, P = 0.003) and HC gp-39 (r = 0.52, P = 0.014). HC gp-39 expression in RA synovial lining correlated with joint destruction (r = 0.77, P < 0.001). CONCLUSION CD16+ monocytes, a cellular source of HC gp-39 in vivo, are overrepresented in both RA peripheral blood and synovial tissue. The presence of HC gp-39+ cells in RA synovium is correlated with the degree of joint destruction. These data support a role of these cells in the local autoimmune response that leads to chronic inflammation and joint destruction.

Interleukin-1 drives pathogenic Th17 cells during spontaneous arthritis in interleukin-1 receptor antagonist–deficient mice

OBJECTIVE Interleukin-1 receptor antagonist-deficient (IL-1Ra-/-) mice spontaneously develop an inflammatory and destructive arthritis due to unopposed excess IL-1 signaling. In this study, the role of Th17 cells and the effect of neutralization of IL-17, IL-1, and tumor necrosis factor alpha (TNFalpha) were investigated in this IL-1-driven murine arthritis model. METHODS T cells isolated from IL-1Ra-/- and wild-type (WT) mice were stained for IL-17 and interferon-gamma, with results assessed by fluorescence-activated cell sorting analysis. To investigate the contribution of IL-1 and IL-17 in further progression of arthritis in this model, mice were treated with neutralizing antibodies after the onset of arthritis. RESULTS Compared with WT mice, IL-1Ra-/- mice had similar levels of Th1 cells but clearly enhanced levels of Th17 cells; this increase in the number of Th17 cells was evident even before the onset of arthritis, in young, nonarthritic IL-1Ra-/- mice. The percentage of Th17 cells increased even more after the onset of arthritis and, similar to the serum levels and local messenger RNA levels of IL-17, the percentage of IL-17+ Th17 cells clearly correlated with the severity of arthritis. Anti-IL-17 treatment prevented any further increase in inflammation and bone erosion, whereas blocking of TNFalpha after the onset of arthritis had no effect. In contrast, neutralization of IL-1 resulted in a complete suppression of arthritis. Interestingly, this anti-IL-1 treatment also significantly reduced the percentage of IL-17+ Th17 cells in the draining lymph nodes of these arthritic mice. CONCLUSION Increased levels of Th17 cells can be detected in IL-1Ra-/- mice even preceding the onset of arthritis. In addition, the results of cytokine-blocking studies demonstrated that IL-17 contributes to the inflammation and bone erosion in this model, which suggests that IL-1 is the driving force behind the IL-17-producing Th17 cells.

Raised human cartilage glycoprotein-39 plasma levels in patients with rheumatoid arthritis and other inflammatory conditions

OBJECTIVE To evaluate plasma human cartilage glycoprotein (HC gp-39) as a possible marker for the presence and/or activity of rheumatoid arthritis (RA) and other inflammatory conditions. BACKGROUND HC gp-39 is a secretory product of chondrocytes, synovial cells, macrophages, and neutrophils. HC gp-39, also described as YKL-40, was found to be a marker of joint disease and tissue injury in RA and various other diseases. METHODS Levels of HC gp-39 were determined by a sandwich enzyme linked immunosorbent assay (ELISA) in 47 patients with RA, 47 with osteoarthritis (OA), 24 with systemic lupus erythematosus (SLE), 24 with inflammatory bowel disease (IBD), and in 47 healthy controls. A disease activity score was assessed in the patients with RA, SLE, and IBD. RESULTS The plasma level of HC gp-39 in the RA patient group was significantly higher than in the other patient groups and healthy controls. The level in patients with OA, SLE, and IBD was also significantly higher than the HC gp-39 level found in the healthy control group. HC gp-39 levels in patients with RA correlated positively with the ESR and IgM rheumatoid factor level but not with other variables of disease activity. In the patients with SLE and IBD no correlation was found with the disease activity score. CONCLUSION The plasma level of HC gp-39 is increased in inflammatory conditions with and without joint disease (SLE, IBD, OA, and RA). Thus increased levels of HC gp-39 do not only reflect joint disease but also reflect inflammation or tissue degradation in various conditions. Notably, the highest level of HC gp-39 was found in patients with RA. Only in the RA patient group was a correlation between HC gp-39 plasma levels and some laboratory variables of disease activity found.

Immuno-miRs: critical regulators of T-cell development, function and ageing

MicroRNAs (miRNAs) are instrumental to many aspects of immunity, including various levels of T‐cell immunity. Over the last decade, crucial immune functions were shown to be regulated by specific miRNAs. These ‘immuno‐miRs’ regulate generic cell biological processes in T cells, such as proliferation and apoptosis, as well as a number of T‐cell‐specific features that are fundamental to the development, differentiation and function of T cells. In this review, we give an overview of the current literature with respect to the role of miRNAs at various stages of T‐cell development, maturation, differentiation, activation and ageing. Little is known about the involvement of miRNAs in thymic T‐cell development, although miR‐181a and miR‐150 have been implicated herein. In contrast, several broadly expressed miRNAs including miR‐21, miR‐155 and miR‐17~92, have now been shown to regulate T‐cell activation. Other miRNAs, including miR‐146a, show a more T‐cell‐subset‐specific expression pattern and are involved in the regulation of processes unique to that specific T‐cell subset. Importantly, differences in the miRNA target gene repertoires of different T‐cell subsets allow similar miRNAs to control different T‐cell‐subset‐specific functions. Interestingly, several of the here described immuno‐miRs have also been implicated in T‐cell ageing and there are clear indications for causal involvement of miRNAs in immunosenescence. It is concluded that immuno‐miRs have a dynamic regulatory role in many aspects of T‐cell differentiation, activation, function and ageing. An important notion when studying miRNAs in relation to T‐cell biology is that specific immuno‐miRs may have quite unrelated functions in closely related T‐cell subsets.

Comprehensive analysis of miRNA expression in T-cell subsets of rheumatoid arthritis patients reveals defined signatures of naive and memory Tregs

Disturbed expression of microRNAs (miRNAs) in regulatory T cells (Tregs) leads to development of autoimmunity in experimental mouse models. However, the miRNA expression signature characterizing Tregs of autoimmune diseases, such as rheumatoid arthritis (RA) has not been determined yet. In this study, we have used a microarray approach to comprehensively analyze miRNA expression signatures of both naive Tregs (CD4+CD45RO-CD25++) and memory Tregs (CD4+CD45RO+CD25+++), as well as conventional naive (CD4+CD45RO−CD25−) and memory (CD4+CD45RO+CD25−) T cells (Tconvs) derived from peripheral blood of RA patients and matched healthy controls. Differential expression of selected miRNAs was validated by TaqMan-based quantitative reverse transcription-PCR. We found a positive correlation between increased expression of miR-451 in T cells of RA patients and disease activity score (DAS28), erythrocyte sedimentation rate levels and serum levels of interleukin-6. Moreover, we found characteristic, disease- and treatment-independent, global miRNA expression signatures defining naive Tregs, memory Tregs, naive Tconvs and memory Tconvs. The analysis allowed us to define miRNAs characteristic for a general naive phenotype (for example, miR-92a) and a general memory phenotype (for example, miR-21, miR-155). Importantly, the analysis allowed us to define miRNAs that are specifically expressed in both naive and memory Tregs, defining as such miRNA signature characterizing the Treg phenotype (that is, miR-146a, miR-3162, miR-1202, miR-1246 and miR-4281).

Increased expression of interleukin-22 by synovial Th17 cells during late stages of murine experimental arthritis is controlled by interleukin-1 and enhances bone degradation

OBJECTIVE Interleukin-22 (IL-22) is a mediator in antimicrobial responses and inflammatory autoimmune diseases. Although IL-22 and its receptor, IL-22R, have been identified in the synovium of rheumatoid arthritis patients, the source of IL-22 and its contribution to disease pathogenicity remain to be established. This study was undertaken to investigate the regulation of IL-22 by Th17 cells in vitro and to evaluate the potential for IL-22 depletion in an experimental arthritis model using mice deficient in the IL-1 receptor antagonist (IL-1Ra-/-). METHODS Naive murine T cells were cultured under conditions leading to polarization of the cells into subsets of Th1, Th2, induced Treg, and Th17. Cytokines were measured in the culture supernatants, and the cells were analyzed by fluorescence-activated cell sorting. Tissue samples from the inflamed ankle synovium of IL-1Ra-/- mice were isolated, and messenger RNA levels of marker genes were quantified. IL-1Ra-/- mice were treated with neutralizing anti-IL-22 antibodies. Synovial cells were isolated from the inflamed tissue and sorted into fractions for analysis of cytokine production. RESULTS In vitro tests showed that Th17 cells produced high levels of IL-22 after stimulation with IL-1 or IL-23. Interestingly, a synergistic increase in the production of IL-22 was observed after combining IL-1 and IL-23. In vivo, IL-1Ra-/- mice displayed a progressive erosive arthritis, characterized by up-regulation of IL-17 in mildly and severely inflamed tissue, whereas the levels of IL-22 and IL-22R were increased only in severely inflamed synovia. Anti-IL-22 treatment of IL-1Ra-/- mice significantly reduced the inflammation and bone erosion. Analysis of isolated single cells from the inflamed synovia revealed that IL-22 was mainly produced by IL-17-expressing T cells. CONCLUSION These findings suggest that IL-22 plays an important role in IL-1-driven chronic joint destruction.

A potent and selective p38 inhibitor protects against bone damage in murine collagen-induced arthritis: a comparison with neutralization of mouse TNFα

Background and purpose: The p38 kinase regulates the release of proinflammatory cytokines including tumour‐necrosis factor‐α (TNFα) and is regarded as a potential therapeutic target in rheumatoid arthritis (RA). Using the novel p38 inhibitor Org 48762‐0, we investigated the therapeutic potential of p38 inhibition and compared this to anti‐mouse (m)TNFα antibody treatment in murine collagen‐induced arthritis (CIA).