Adriana Mc Mus

Interleukin-23 promotes Th17 differentiation by inhibiting T-bet and FoxP3 and is required for elevation of interleukin-22, but not interleukin-21, in autoimmune experimental arthritis.

OBJECTIVE To examine the role of interleukin-23 (IL-23) in subgroup polarization of IL-17A-positive and/or interferon-gamma (IFNgamma)-positive T cells in autoimmune disease-prone DBA/1 mice with and without collagen-induced arthritis. METHODS A magnetic-activated cell sorting system was used to isolate CD4+ T cells from the spleen of naive and type II collagen (CII)-immunized DBA/1 mice. These CD4+ T cells were stimulated in vitro under Th0, Th1, or different Th17 culture conditions. Intracellular staining for IL-17A and IFNgamma was evaluated by flow cytometry. In addition, Th17 cytokines and T helper-specific transcription factors were analyzed by enzyme-linked immunosorbent assay and/or quantitative polymerase chain reaction. RESULTS In CD4+ T cells from naive DBA/1 mice, IL-23 alone hardly induced retinoic acid-related orphan receptor gammat (RORgammat), Th17 polarization, and Th17 cytokines, but it inhibited T-bet expression. In contrast, transforming growth factor beta1 (TGFbeta1)/IL-6 was a potent inducer of RORgammat, RORalpha, IL-17A, IL-17F, IL-21, and FoxP3 in these cells. In contrast to TGFbeta1/IL-6, IL-23 was critical for the induction of IL-22 in CD4+ T cells from both naive and CII-immunized DBA/1 mice. Consistent with these findings, IL-23 showed a more pronounced induction of the IL-17A+IFNgamma- subset in CD4+ T cells from CII-immunized mice. However, in CD4+ T cells from naive mice, IL-23 significantly increased the TGFbeta1/IL-6-induced Th17 polarization, including elevated levels of IL-17A and IL-17F and decreased expression of T-bet and FoxP3. Of note, the IL-23-induced increase in IL-17A and IL-17F levels was prevented in T-bet-deficient mice. CONCLUSION IL-23 promotes Th17 differentiation by inhibiting T-bet and FoxP3 and is required for elevation of IL-22, but not IL-21, levels in autoimmune arthritis. These data indicate different mechanisms for IL-23 and TGFbeta1/IL-6 at the transcription factor level during Th17 differentiation in autoimmune experimental arthritis.

TNF blockade requires 1,25(OH)2D3 to control human Th17-mediated synovial inflammation

Objectives T helper 17 (Th17) cells from patients with early rheumatoid arthritis (RA) induce a proinflammatory feedback loop upon RA synovial fibroblast (RASF) interaction, including autocrine interleukin (IL)-17A production. A major challenge in medicine is how to control the pathogenic Th17 cell activity in human inflammatory autoimmune diseases. The objective of this study was to examine whether tumour necrosis factor (TNF) blockade and/or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) controls Th17-mediated synovial inflammation. Methods Peripheral CD4+CD45RO+CCR6+ Th17 cells of patients with early RA, Th17–RASF cocultures and synovial biopsy specimens were cultured with or without 1,25(OH)2D3 and/or TNFα blockade. Intracellular cytokine expression was detected by flow cytometry. Cytokine and matrix metalloprotease (MMP) production was determined by ELISA. Results The authors show that the 1,25(OH)2D3, but not TNFα blockade, significantly suppressed autocrine IL-17A production in Th17–RASF and synovial biopsy cultures. Combining 1,25(OH)2D3 and TNFα blockade had a significant additive effect compared with single treatment in controlling synovial inflammation, indicated by a further reduction in IL-6, IL-8, MMP-1 and MMP-3 in Th17–RASF cocultures and IL-6 and IL-8 expression in cultures of RA synovial tissue. Conclusions These data show that TNF blockade does not suppress IL-17A and IL-22, which can be overcome by 1,25(OH)2D3. The combination of neutralising TNF activity and 1,25(OH)2D3 controls human Th17 activity and additively inhibits synovial inflammation. This indicates more valuable therapeutic potential of activation of Vitamin D receptorsignalling over current TNF neutralisation strategies in patients with RA and potentially other Th17-mediated inflammatory diseases.

Interleukin-23 is critical for full-blown expression of a non-autoimmune destructive arthritis and regulates interleukin-17A and RORγt in γδ T cells

IntroductionInterleukin (IL)-23 is essential for the development of various experimental autoimmune models. However, the role of IL-23 in non-autoimmune experimental arthritis remains unclear. Here, we examined the role of IL-23 in the non-autoimmune antigen-induced arthritis (AIA) model. In addition, the regulatory potential of IL-23 in IL-17A and retinoic acid-related orphan receptor gamma t (RORγt) expression in CD4+ and TCRγδ+ T cells was evaluated systemically as well as at the site of inflammation.MethodsAntigen-induced arthritis was induced in wild-type, IL-23p19-deficient and IL-17 Receptor A - knockout mice. At different time points, synovial cytokine and chemokine expression was measured. At days 1 and 7 of AIA, splenocytes and joint-infiltrating cells were isolated and analyzed for intracellular IL-17A and interferon (IFN)-γ ex-vivo by flow cytometry. In splenic CD4+ and TCRγδ+ T cells gene expression was quantified by flow cytometry and quantitative PCR.ResultsIL-23 was critical for full-blown AIA. Lack of IL-23 did not prevent the onset of joint inflammation but stopped the progression to a destructive synovitis. IL-23 regulated IL-17A expression in CD4+ T cells in the spleen. Of note, IL-17A and IFN-γ expression was reduced in CD4+ T cells in the inflamed joints of IL-23p19-deficient mice. Interestingly, IL-23 was also critical for the induction of IL-17A and RORγt but not IFN-γ in TCRγδ+ T cells in the inflamed joints. The importance of the IL-23/IL-17 axis was further confirmed using IL-17 Receptor A knockout mice showing significantly milder AIA compared to control mice, with a disease course comparable to that of IL-23p19-deficient mice.ConclusionsThese data show that IL-23 is critical for full-blown expression of a non-autoimmune destructive arthritis and regulates the proportion of IL-17A and IFN-γ-positive CD4+ T cells at the site of inflammation. Furthermore, IL-23 regulates IL-17A and RORγt expression in TCRγδ T cells in arthritis. These findings indicate that regulating the IL-23 pathway may have therapeutic potential in non-autoimmune arthritis.

Absence of Interleukin-17 Receptor A Signaling Prevents Autoimmune Inflammation of the Joint and Leads to a Th2-like Phenotype in Collagen-Induced Arthritis

Interleukin‐17A (IL‐17A) signals through the IL‐17 receptor (IL‐17R) A/C heterodimer. IL‐17RA serves as a common receptor subunit for several IL‐17 cytokine family members. Lack of IL‐17RA signaling may therefore have additional effects beyond those of lack of IL‐17A alone. The present study was undertaken to determine the role of IL‐17RA signaling in autoimmune arthritis.

IL-23 dependent and independent stages of experimental arthritis: no clinical effect of therapeutic IL-23p19 inhibition in collagen-induced arthritis.

IL-23p19 deficient mice have revealed a critical role of IL-23 in the development of experimental autoimmune diseases, such as collagen-induced arthritis (CIA). Neutralizing IL-23 after onset of CIA in rats has been shown to reduce paw volume, but the effect on synovial inflammation and the immunological autoimmune response is not clear. In this study, we examined the role of IL-23 at different stages of CIA and during T cell memory mediated flare-up arthritis with focus on changes in B cell activity and Th1/Th17 modulation. Anti-IL-23p19 antibody (anti-IL23p19) treatment, starting 15 days after the type II collagen (CII)-immunization but before clinical signs of disease onset, significantly suppressed the severity of CIA. This was accompanied with significantly lower CII-specific IgG1 levels and lower IgG2a levels in the anti-IL-23p19 treated mice compared to the control group. Importantly, neutralizing IL-23 after the first signs of CIA did not ameliorate the disease. This was in contrast to arthritic mice that underwent an arthritis flare-up since a significantly lower disease score was observed in the IL-23p19 treated mice compared to the control group, accompanied by lower synovial IL-17A and IL-22 expression in the knee joints of these mice. These data show IL-23-dependent and IL-23-independent stages during autoimmune CIA. Furthermore, the memory T cell mediated flare-up arthritis is IL-23-mediated. These data suggest that specific neutralization of IL-23p19 after onset of autoimmune arthritis may not be beneficial as a therapeutic therapy for patients with rheumatoid arthritis (RA). However, T cell mediated arthritis relapses in patients with RA might be controlled by anti-IL-23p19 treatment.

Dendritic Cell-Specific Deletion of β-Catenin Results in Fewer Regulatory T-Cells without Exacerbating Autoimmune Collagen-Induced Arthritis

Dendritic cells (DCs) are professional antigen presenting cells that have the dual ability to stimulate immunity and maintain tolerance. However, the signalling pathways mediating tolerogenic DC function in vivo remain largely unknown. The β-catenin pathway has been suggested to promote a regulatory DC phenotype. The aim of this study was to unravel the role of β-catenin signalling to control DC function in the autoimmune collagen-induced arthritis model (CIA). Deletion of β-catenin specifically in DCs was achieved by crossing conditional knockout mice with a CD11c-Cre transgenic mouse line. Bone marrow-derived DCs (BMDCs) were generated and used to study the maturation profile of these cells in response to a TLR2 or TLR4 ligand stimulation. CIA was induced by intra-dermal immunization with 100 μg chicken type II collagen in complete Freund’s adjuvant on days 0 and 21. CIA incidence and severity was monitored macroscopically and by histology. The T cell profile as well as their cytokine production were analysed by flow cytometry. Lack of β-catenin specifically in DCs did not affect the spontaneous, TLR2- or TLR4-induced maturation and activation of BMDCs or their cytokine production. Moreover, no effect on the incidence and severity of CIA was observed in mice lacking β-catenin in CD11c+ cells. A decreased frequency of splenic CD3+CD8+ T cells and of regulatory T cells (Tregs) (CD4+CD25highFoxP3+), but no changes in the frequency of splenic Th17 (CCR6+CXCR3-CCR4+), Th2 (CCR6-CXCR3-CCR4+) and Th1 (CCR6-CXCR3+CCR4-) cells were observed in these mice under CIA condition. Furthermore, the expression of IL-17A, IL-17F, IL-22, IL-4 or IFNγ was also not affected. Our data indicate that ablation of β-catenin expression in DCs did not alter the course and severity of CIA. We conclude that although deletion of β-catenin resulted in a lower frequency of Tregs, this decrease was not sufficient to aggravate the onset and severity of CIA.

Vitamin D suppresses the pathogenic behaviour of primary Th17 cells from patients with early rheumatoid arthritis

Background and objectives Recently, the authors showed that CCR6+ Th17 cells from early rheumatoid arthritis (RA) patients are potent inducers of a pro-inflammatory feed-back loop upon RA synovial fibroblast (RASF) interaction, including autocrine interleukin (IL)-17A production. In this study, the effect of vitamin D on this pathogenic behaviour of Th17 cells was investigated. Materials and methods Peripheral CCR6+ Th17 cells of patients with early RA, CCR6+ Th17/RASF co-cultures and synovial biopsies of patients with established RA, were cultured in the absence or presence of 1,25(OH)2D3, the active vitamin D metabolite and/or Etanercept. Intracellular cytokine expression was detected by flow cytometry. Cytokine and matrix metalloprotease (MMP) expression was determined by ELISA. Transcription of factors involved in Th differentiation and function was analysed by quantitative PCR analysis. Results In Th17 cultures and Th17/RASF co-cultures vitamin D suppressed the expression of IL-17A, IL-22 and IFN-γ. Furthermore, in Th17/RASF co-cultures, vitamin D induced IL-4 and IL-10 expression, which was accompanied with reduced Rorγt and induced Gata3 transcription. Moreover, vitamin D suppressed IL-6, IL-8, MMP-1 and MMP-3 expression in the Th17/RASF co-cultures and synovial biopsies. Interestingly, the enhanced autocrine production of IL-17A in Th17/RASF co-cultures was inhibited by vitamin D, but not with tumour necrosis factor (TNF)-α neutralisation. Vitamin D in combination with TNF-α neutralisation has an additive effect on the suppression of Th17 activity as indicated by a significant further reduction in IL-6, IL-8, MMP-1 and MMP-3 expression in the co-cultures and IL-6 and IL-8 in the synovial biopsy cultures. Conclusions These data show that vitamin D modulates the pathogenic behaviour of Th17 cells and may have additional therapeutic potential when used in combination with TNF treatment in RA and potentially other Th17-mediated inflammatory diseases.

Experimental Arthritis Mouse Models Driven by Adaptive and/or Innate Inflammation.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease mainly affecting synovial joints. The clinical presentation of RA shows the heterogeneity of this disease with its underlying complex interactions between the innate and adaptive immune system and flare-ups of disease. Different disease models such as collagen induced arthritis, antigen induced arthritis, and Streptococcal cell wall induced arthritis can be exploited to investigate different aspects of the pathogenesis of arthritis. The disease can be monitored macroscopically over time via scoring systems. For histological examination, paraffin embedded knee sections can be used for hematoxylin and eosin staining to visualize cellular infiltration as well as for tartrate-resistant acid phosphatase (TRAP) staining to identify osteoclast-like cells. Cellular infiltration of the synovium by different myeloid cells such as tissue resident macrophages, dendritic cells and neutrophils can be monitored using flow cytometry. Here, we describe the methods for inducing the different mouse models for arthritis, including scoring systems per model, histological and flow cytometric analysis.

1,25(OH)2D3 modulates gene expression involved in cytokine production, proliferation, survival and migration of TH17 cells from patients with rheumatoid arthritis

Background Vitamin D has suppressive effects on autoimmune diseases, such as rheumatoid arthritis (RA). Within these diseases, T-helper-17 (Th17) cells have been implicated to play a crucial role in the development and progression of chronic inflammation. Recently, the authors have found that the active vitamin D compound, 1,25(OH)2D3, has direct suppressive effects on both human and mouse Th17 cytokine expression and activity. Using gene-expression profiling, the authors aim to identify molecular targets of 1,25(OH)2D3 signaling underlying this suppressive action of 1,25(OH)2D3 in Th17 cells. Materials and methods Primary Th17 cells were sorted from peripheral blood of treatment naïve patients with early RA and cultured with or without 1,25(OH)2D3. From these cultures gene-expression profiles were generated. Expression of genes of interest was confirmed by Q-PCR and/or specific ELISA. Results In the presence of 1,25(OH)2D3, protein expression of Th17 associated cytokines IL-17A and IL-22 was inhibited, while in contrast the anti-inflammatory cytokine IL-10 was induced. These findings were supported by the gene-expression profiles from these cultures. Furthermore, 1,25(OH)2D3 inhibited transcription of the cytokine receptors IL-23R and IL-7R, which are involved in Th17 survival and proliferation. Chemokines CCL20 and CXCL10 were down-regulated and chemokine receptors CCR2, CXCR6, CXCR3 and CCR10 were up-regulated. Importantly, Rorγt, which is critically involved in Th17 differentiation and function and the cell-size regulator and oncogene, c-Myc were down-regulated by 1,25(OH)2D3. Conclusions From these findings, the authors concluded that 1,25(OH)2D3 modulates the expression of genes involved in cytokine production, proliferation, survival and migration of Th17 cells. These data indicate that 1,25(OH)2D3 not only suppresses Th17 cell activity but also regulates migration of these cells to sites of tissue inflammation in RA.

1,25(OH) 2 D 3 and dexamethasone additively suppress synovial fibroblast activation by CCR6 + T helper memory cells and enhance the effect of tumor necrosis factor alpha blockade

BackgroundDespite recent improvements in the treatment of rheumatoid arthritis (RA), an insufficient treatment response and the development of treatment resistance in many patients illustrates the need for new therapeutic strategies. Chronic synovial inflammation could be suppressed by targeting RA synovial fibroblast (RASF) activation by, for example, interleukin (IL)-17A-producing CCR6+ T helper memory (memTh) cells. Here, we modulated this interaction by combining the active vitamin D metabolite 1,25(OH)2D3 with dexamethasone (DEX) and explored the potential therapeutic applications.MethodsCCR6+ memTh cells from peripheral blood mononuclear cells (PBMCs) of healthy donors or treatment-naive early RA patients were cultured alone or with RASF from established RA patients for 3 days and treated with or without 1,25(OH)2D3, DEX, or etanercept. Treatment effects were assessed using enzyme-linked immunosorbent assay (ELISA) and flow cytometry.Results1,25(OH)2D3, and to lesser extent DEX, reduced production of the pro-inflammatory cytokines IL-17A, IL-22, and interferon (IFN)γ in CCR6+ memTh cells. Tumor necrosis factor (TNF)α was only inhibited by the combination of 1,25(OH)2D3 and DEX. In contrast, DEX was the strongest inhibitor of IL-6, IL-8, and tissue-destructive enzymes in RASF. As a result, 1,25(OH)2D3 and DEX additively inhibited inflammatory mediators in CCR6+ memTh-RASF cocultures. Interestingly, low doses of mainly DEX, but also 1,25(OH)2D3, combined with etanercept better suppressed synovial inflammation in this coculture model compared with etanercept alone.ConclusionThis study suggests that 1,25(OH)2D3 and DEX additively inhibit synovial inflammation through targeting predominantly CCR6+ memTh cells and RASF, respectively. Furthermore, low doses of DEX and 1,25(OH)2D3 enhance the effect of TNFα blockade in inhibiting RASF activation, thus providing a basis to improve RA treatment.