IL-23 receptor deficiency results in lower bone mass via indirect regulation of bone formation.

The IL-23 receptor (IL-23R) signaling pathway has pleiotropic effects on the differentiation of osteoclasts and osteoblasts, since it can inhibit or stimulate these processes via different pathways. However, the potential role of this pathway in the regulation of bone homeostasis remains elusive. Therefore, we studied the role of IL-23R signaling in physiological bone remodeling using IL-23R deficient mice. Using µCT, we demonstrate that 7-week-old IL-23R-/- mice have similar bone mass as age matched littermate control mice. In contrast, 12-week-old IL-23R-/- mice have significantly lower trabecular and cortical bone mass, shorter femurs and more fragile bones. At the age of 26 weeks, there were no differences in trabecular bone mass and femur length, but most of cortical bone mass parameters remain significantly lower in IL-23R-/- mice. In vitro osteoclast differentiation and resorption capacity of 7- and 12-week-old IL-23R-/- mice are similar to WT. However, serum levels of the bone formation marker, PINP, are significantly lower in 12-week-old IL-23R-/- mice, but similar to WT at 7 and 26 weeks. Interestingly, Il23r gene expression was not detected in in vitro cultured osteoblasts, suggesting an indirect effect of IL-23R. In conclusion, IL-23R deficiency results in temporal and long-term changes in bone growth via regulation of bone formation.

Interleukin-17A Is Produced by CD4+ but Not CD8+ T Cells in Synovial Fluid Following T Cell Receptor Activation and Regulates Different Inflammatory Mediators Compared to Tumor Necrosis Factor in a Model of Psoriatic Arthritis Synovitis.

OBJECTIVE: Interleukin-17A (IL-17A) and tumor necrosis factor (TNF) contribute to the pathogenesis of psoriatic arthritis (PsA). However, their functional relationship in PsA synovitis has not been fully elucidated. Additionally, although CD8+ T cells in PsA have been recognized via flow cytometry as a source of IL-17A production, it is not clear whether CD8+ T cells secrete IL-17A under more physiologically relevant conditions in the context from PsA synovitis. This study was undertaken to clarify the roles of IL-17A and TNF in the synovial fluid (SF) from patients with PsA and investigate the impact of CD8+ T cells on IL-17A production. METHODS: IL-17A+ T cells were identified by flow cytometry in SF samples from 20 patients with active PsA, blood samples from 22 treatment-naive patients with PsA, and blood samples from 22 healthy donors. IL-17A+ T cells were sorted from 12 PsA SF samples and stimulated using anti-CD3/anti-CD28 or phorbol myristate acetate (PMA) and ionomycin ex vivo, alone (n = 3) or together with autologous monocytes (n = 3) or PsA fibroblast-like synoviocytes (FLS) (n = 5-6). To evaluate the differential allogeneic effects of neutralizing IL-17A and TNF, SF CD4+ T cells and PsA FLS cocultures were also used (n = 5-6). RESULTS: Flow cytometry analyses of SF samples from patients with PsA showed IL-17A positivity for CD4+ and CD8+ T cells (IL-17A, median 0.71% [interquartile range 0.35-1.50%] in CD4+ cells; median 0.44% [interquartile range 0.17-1.86%] in CD8+ T cells). However, only CD4+ T cells secreted IL-17A after anti-CD3/anti-CD28 activation, when cultured alone and in cocultures with PsA monocytes or PsA FLS (each P < 0.05). Remarkably, CD8+ T cells only secreted IL-17A after 4- or 72-hour stimulation with PMA/ionomycin. Anti-IL-17A and anti-TNF treatments both inhibited PsA synovitis ex vivo. Neutralizing IL-17A strongly inhibited IL-6 (P < 0.05) and IL-1ß (P < 0.01), while anti-TNF treatment was more potent in reducing matrix metalloproteinase 3 (MMP-3) (P < 0.05) and MMP-13. CONCLUSION: CD8+ T cells, in contrast to CD4+ T cells, in SF specimens obtained from PsA patients did not secrete IL-17A following T cell receptor activation. Overlapping, but distinct, effects at the level of inflammatory cytokines and MMPs were found after neutralizing IL-17A or TNF ex vivo in a human model of PsA synovitis.

CD4+ CCR6+ T cells, but not γδ T cells, are important for the IL-23R-dependent progression of antigen-induced inflammatory arthritis in mice.

IL-23 plays an important role in the development of arthritis and the IL-23 receptor (IL-23R) is expressed on different types of T cells. However, it is not fully clear which IL-23R+ T cells are critical in driving T cell-mediated synovitis. We demonstrate, using knock-in IL-23R-GFP reporter (IL-23RGFP/+ ) mice, that CD4+ CCR6+ T cells and γδ T cells, but not CD8+ T cells, express the IL-23R(GFP). During early arthritis, IL-23R(GFP)+ CD4+ CCR6+ T cells, but not IL-23R(GFP)+ γδ T cells, were present in the inflamed joints. IL-23RGFP/+ mice were bred as homozygotes to obtain IL-23RGFP/GFP (IL-23R deficient/IL-23R-/- ) mice, which express GFP under the IL-23R promotor. Arthritis progression and joint damage were significantly milder in IL-23R-/- mice, which revealed less IL-17A+ cells in their lymphoid tissues. Surprisingly, IL-23R-/- mice had increased numbers of IL-23R(GFP)+ CD4+ CCR6+ and CCR7+ CD4+ CCR6+ T cells in their spleen compared to WT, and IL-23 suppressed CCR7 expression in vitro. However, IL-23R(GFP)+ CD4+ CCR6+ T cells were present in the synovium of IL-23R-/- mice at day 4. Finally, adoptive transfer experiments revealed that CD4+ CCR6+ T cells and not γδ T cells drive arthritis progression. These data suggest that IL-23R-dependent T cell-mediated synovitis is dependent on CD4+ CCR6+ T cells and not on γδ T cells.

Allogeneic Chondrogenic Mesenchymal Stromal Cells Alter Helper T Cell Subsets in CD4+ Memory T Cells.

Implantation of chondrogenically differentiated mesenchymal stromal cells (MSCs) leads to bone formation in vivo through the process of endochondral ossification. The use of allogeneic MSCs for this purpose may be a promising new approach to replace the current gold standard of bone regeneration. However, the success of using allogeneic cells depends on the interaction between the implanted cells and the host's endogenous immune cells. Th17 T cells and other CD4 helper T cell subtypes have been shown to negatively impact chondrogenesis, however, it is unclear how the interaction between these cells affects bone regeneration mediated by these cells. The aim of the current work was to assess the effect of chondrogenic MSC pellets on Th1, Th2, Th17, and regulatory T cells in vitro. Human MSCs were nonchondrogenic (-TGFß3) and chondrogenically (+TGFß3) differentiated for 7 or 21 days. Memory T cells (sorted from the CD4 population of peripheral blood mononuclear cells [PBMCs]), as well as total PBMCs were cocultured with allogeneic nonchondrogenic and chondrogenic MSC pellets for 3 days. Seven-day differentiated allogeneic nonchondrogenic and chondrogenic MSC pellets that were cocultured with memory T cells resulted in a significant increase in Th2 and a decrease in Th1 T cells. Furthermore, the co-culture of 21-day differentiated nonchondrogenic and chondrogenic MSC pellets with memory T cells resulted in a significant increase in Th2 and Th17 T cells, as well as a decrease in Th1 and regulatory T cells. Interleukin (IL)-6 was identified as a predominant cytokine involved in this interaction between allogeneic chondrogenically differentiated MSC pellets and memory CD4 T cells, with high levels of IL-6 being secreted in the supernatants of this cocultured condition. The findings of this study highlight the potential of chondrogenically differentiated MSC pellets to alter the ratio of Th1 and Th2 as well as Th17 and regulatory T cell subsets. Additional analysis investigating bone formation by chondrogenically differentiated MSCs in an allogeneic setting may identify a novel role of these T cell subsets in bone regeneration processes mediated by chondrogenically differentiated MSCs. Impact statement Allogeneic mesenchymal stromal cells (MSCs) have the potential to be an off-the-shelf treatment for bone repair. However, the lack of knowledge of the immune cells involved in this process has hampered the progression to the clinic. The current study has shown that allogeneic chondrogenic MSCs have the potential to skew the ratio of specific helper CD4 T cell subsets in vitro. This has now provided insight for future in vivo experiments to investigate the role of these T cell subsets in the early stages of bone regeneration mediated by allogeneic chondrogenic MSCs.

Lack of IL-17 Receptor A signaling aggravates lymphoproliferation in C57BL/6 lpr mice.

Defects in Fas function correlate with susceptibility to systemic autoimmune diseases like autoimmune lymphoproliferative syndrome (ALPS) and systemic lupus erythematosus (SLE). C57BL/6 lpr (B6/lpr) mice are used as an animal model of ALPS and develop a mild SLE phenotype. Involvement of interleukin-17A (IL-17A) has been suggested in both phenotypes. Since IL-17 receptor A is part of the signaling pathway of many IL-17 family members we investigated the role of IL-17 receptor signaling in disease development in mice with a B6/lpr background. B6/lpr mice were crossed with IL-17 receptor A deficient (IL-17RA KO) mice and followed over time for disease development. IL-17RA KO/lpr mice presented with significantly enhanced lymphoproliferation compared with B6/lpr mice, which was characterized by dramatic lymphadenomegaly/splenomegaly and increased lymphocyte numbers, expansion of double-negative (DN) T-cells and enhanced plasma cell formation. However, the SLE phenotype was not enhanced, as anti-nuclear antibody (ANA) titers and induction of glomerulonephritis were not different. In contrast, levels of High Mobility Group Box 1 (HMGB1) and anti-HMGB1 autoantibodies were significantly increased in IL-17RA KO/lpr mice compared to B6/lpr mice. These data show that lack of IL-17RA signaling aggravates the lymphoproliferative phenotype in B6/lpr mice but does not affect the SLE phenotype.

1,25(OH)2D3 and dexamethasone additively suppress synovial fibroblast activation by CCR6+ T helper memory cells and enhance the effect of tumor necrosis factor alpha blockade.

BACKGROUND: Despite 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. METHODS: CCR6+ 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. RESULTS: 1,25(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. CONCLUSION: This 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.

JAK-inhibitor tofacitinib suppresses interferon alfa production by plasmacytoid dendritic cells and inhibits arthrogenic and antiviral effects of interferon alfa.

Tofacitinib is an oral Janus kinase inhibitor that is effective for the treatment of rheumatoid arthritis and shows encouraging therapeutic effects in several other autoimmune diseases. A prominent adverse effect of tofacitinib therapy is the increased risk of viral infections. Despite its advanced stage of clinical development, the modes of action that mediate the beneficial and adverse effects of tofacitinib in autoimmune diseases remain unclear. Interferon alfa (IFNα) produced by plasmacytoid dendritic cells (PDCs) is critically involved in the pathogenesis of many systemic autoimmune diseases and in immunity to viral infections. Using in vitro culture models with human cells, we studied the effects of tofacitinib on PDC survival and IFNα production, and on arthrogenic and antiviral effects of IFNα. Tofacitinib inhibited the expression of antiapoptotic BCL-A1 and BCL-XL in human PDC and induced PDC apoptosis. TLR7 stimulation upregulated the levels of antiapoptotic Bcl-2 family members and prevented the induction of PDC apoptosis by tofacitinib. However, tofacitinib robustly inhibited the production of IFNα by toll like receptor-stimulated PDC. In addition, tofacitinib profoundly suppressed IFNα-induced upregulation of TLR3 on synovial fibroblasts, thereby inhibiting their cytokine and protease production in response to TLR3 ligation. Finally, tofacitinib counteracted the suppressive effects of IFNα on viral replication. Tofacitinib inhibits PDC survival and IFNα production and suppresses arthrogenic and antiviral effects of IFNα signaling. Inhibition of the IFNα pathway at 2 levels may contribute to the beneficial effects of tofacitinib in autoimmune diseases and explain the increased viral infection rates observed during tofacitinib treatment.

Enhanced Expression of Bruton's Tyrosine Kinase in B Cells Drives Systemic Autoimmunity by Disrupting T Cell Homeostasis.

Upon BCR stimulation, naive B cells increase protein levels of the key downstream signaling molecule Bruton's tyrosine kinase (BTK). Transgenic CD19-hBtk mice with B cell-specific BTK overexpression show spontaneous germinal center formation, anti-nuclear autoantibodies, and systemic autoimmunity resembling lupus and Sjögren syndrome. However, it remains unknown how T cells are engaged in this pathology. In this study, we found that CD19-hBtk B cells were high in IL-6 and IL-10 and disrupted T cell homeostasis in vivo. CD19-hBtk B cells promoted IFN-γ production by T cells and expression of the immune-checkpoint protein ICOS on T cells and induced follicular Th cell differentiation. Crosses with CD40L-deficient mice revealed that increased IL-6 production and autoimmune pathology in CD19-hBtk mice was dependent on B-T cell interaction, whereas IL-10 production and IgM autoantibody formation were CD40L independent. Surprisingly, in Btk-overexpressing mice, naive B cells manifested increased CD86 expression, which was dependent on CD40L, suggesting that T cells interact with B cells in a very early stage of immune pathology. These findings indicate that increased BTK-mediated signaling in B cells involves a positive-feedback loop that establishes T cell-propagated autoimmune pathology, making BTK an attractive therapeutic target in autoimmune disease.

Loss of IL-22 inhibits autoantibody formation in collagen-induced arthritis in mice.

Interleukin 22 (IL-22) expression is associated with increased joint destruction and disease progression in rheumatoid arthritis (RA). Although IL-22 is considered a pro-inflammatory cytokine, its mechanism of action in RA remains incompletely understood. Here, we used the collagen-induced arthritis model in IL-22 deficient (IL-22(-/-) ) mice to study the role of IL-22 in RA. In spite of normal disease incidence, disease severity is significantly diminished in IL-22(-/-) mice. Moreover, pathogenicity of Th17 cells and development and function of B cells are unaffected. In contrast, splenic plasma cells, as well as serum autoantibody titers, are reduced in the absence of IL-22. At the peak of disease, germinal centers (GCs) are severely reduced in the spleens of IL-22(-/-) mice, correlating with a decline in GC B-cell numbers. Within the GC, we identified IL-22R1 expressing follicular dendritic cell-like stromal cells. Human lymphoid stromal cells respond to IL-22 ex vivo by inducing transcription of CXCL12 and CXCL13. We therefore postulate IL-22 as an important enhancer of the GC reaction, maintaining chemokine levels for the persistence of GC reactions, essential for the production of autoantibody-secreting plasma cells. Blocking IL-22 might therefore prevent immune-complex deposition and destruction of joints in RA patients.

Dendritic Cell-Specific Deletion of ß-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 ß-catenin pathway has been suggested to promote a regulatory DC phenotype. The aim of this study was to unravel the role of ß-catenin signalling to control DC function in the autoimmune collagen-induced arthritis model (CIA). Deletion of ß-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 ß-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 ß-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 ß-catenin expression in DCs did not alter the course and severity of CIA. We conclude that although deletion of ß-catenin resulted in a lower frequency of Tregs, this decrease was not sufficient to aggravate the onset and severity of CIA.

Surrogate light chain expression beyond the pre-B cell stage promotes tolerance in a dose-dependent fashion.

While surrogate light chain (SLC) expression is normally terminated in differentiating pre-B cells, co-expression of SLC and conventional light chains has been reported in a small population of autoreactive peripheral human B cells that accumulate in arthritic joints. Despite this association with autoimmunity the contribution of SLC expressing mature B cells to disease development is still unknown. We studied the pathogenicity of SLC(+) B cells in a panel of mice that transgenically express the SLC components VpreB and λ5 throughout B cell development. Here we report that although VpreB or λ5 expression mildly activated mature B cells, only moderate VpreB expression levels - in the absence of λ5 - enhanced IgG plasma cell formation. However, no autoantibody production was detectable in VpreB or λ5 transgenic mice and VpreB expression could not accelerate autoimmunity. Instead, moderate VpreB expression partially protected mice from induced autoimmune arthritis. In support of a tolerogenic role of SLC-transgenic B cells, we observed that in a dose-dependent manner SLC expression beyond the pre-B cell stage enhanced clonal deletion among immature and transitional B cells and rendered mature B cells anergic. These findings suggest that SLC expression does not propagate autoimmunity, but instead may impose tolerance.

A human vitamin D receptor mutation causes rickets and impaired Th1/Th17 responses.

We present a brother and sister with severe rickets, alopecia and highly elevated serum levels of 1,25-dihydroxyvitamin D (1,25-(OH)2D3). Genomic sequencing showed a homozygous point mutation (A133G) in the vitamin D receptor gene, leading to an amino acid change in the DNA binding domain (K45E), which was described previously. Hereditary vitamin D resistant rickets (HVDRR) was diagnosed. Functional studies in skin biopsy fibroblasts confirmed this. 1,25-(OH)2D3 reduced T helper (Th) cell population-specific cytokine expression of interferon γ (Th1), interleukins IL-17A (Th17) and IL-22 (Th17/Th22) in peripheral blood mononuclear cells (PBMCs) from the patient's parents, whereas IL-4 (Th2) levels were higher, reflecting an immunosuppressive condition. None of these factors were regulated by 1,25-(OH)2D3 in PBMCs from the boy. At present, both patients (boy is 23 years of age, girl is 7) have not experienced any major immune-related disorders. Although both children developed alopecia, the girl did so earlier than the boy. The boy showed complete recovery from the rickets at the age of 17 and does not require any vitamin D supplementations to date. In conclusion, we characterized two siblings with HVDRR, due to a mutation in the DNA binding domain of VDR. Despite a defective T cell response to vitamin D, no signs of any inflammatory-related abnormalities were seen, thus questioning an essential role of vitamin D in the immune system. Despite the fact that currently medicine is not required, close monitoring in the future of these patients is warranted for potential recurrence of vitamin D dependence and diagnosis of (chronic) inflammatory-related diseases.

Absence of interleukin-17 receptor a signaling prevents autoimmune inflammation of the joint and leads to a Th2-like phenotype in collagen-induced arthritis.

OBJECTIVE: 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. METHODS: Disease incidence and severity were scored in type II collagen-treated wild-type, IL-17RA-deficient, and IL-23p19-deficient mice. T helper cell profiles and humoral immune responses were analyzed at several time points. Pathogenicity of T cells and total splenocytes was determined by in vitro functional assay. IL-17RA signaling was blocked in vivo in mice with antigen-induced arthritis (AIA). RESULTS: Comparable to the findings in IL-23p19-deficient mice, IL-17RA-deficient mice were completely protected against the development of collagen-induced arthritis (CIA). However, IL-17RA-deficient mice exhibited an increased number of IL-4-producing CD4+ T cells, distinct from IL-17A+CD4+ T cells. This was associated with fewer plasma cells, lower production of pathogenic IgG2c antibody, and increased production of IgG1 antibody. Both isolated CD4+ T cells and total splenocytes from IL-17RA-deficient mice had a reduced ability to induce IL-6 production by synovial fibroblasts in the setting of CIA, in a functional in vitro assay. Furthermore, blocking of IL-17RA signaling in AIA reduced synovial inflammation. CONCLUSION: These results demonstrate that absence of IL-17RA leads to a Th2-like phenotype characterized by IL-4 production and suggest that IL-17RA signaling plays a critical role in the regulation of IL-4 in CIA and the development of autoimmune inflammation of the joint.

Synovial fibroblasts directly induce Th17 pathogenicity via the cyclooxygenase/prostaglandin E2 pathway, independent of IL-23.

Th17 cells are critically involved in autoimmune disease induction and severity. Recently, we showed that Th17 cells from patients with rheumatoid arthritis (RA) directly induced a proinflammatory loop upon interaction with RA synovial fibroblasts (RASF), including increased autocrine IL-17A production. To unravel the mechanism driving this IL-17A production, we obtained primary CD4(+)CD45RO(+)CCR6(+) (Th17) cells and CD4(+)CD45RO(+)CCR6(-) (CCR6(-)) T cells from RA patients or healthy individuals and cocultured these with RASF. IL-1ß, IL-6, IL-23p19, and cyclooxygenase (COX)-2 expression and PGE2 production in Th17-RASF cultures were higher than in CCR6(-) T cell-RASF cultures. Cytokine neutralization showed that IL-1ß and IL-6, but not IL-23, contributed to autocrine IL-17A induction. Importantly, treatment with celecoxib, a COX-2 inhibitor, resulted in significantly lower PGE2 and IL-17A, but not IFN-γ, production. Combined celecoxib and TNF-α blockade more effectively suppressed the proinflammatory loop than did single treatment, as shown by lower IL-6, IL-8, matrix metalloproteinase-1 and matrix metalloproteinase-3 production. These findings show a critical role for the COX-2/PGE2 pathway in driving Th17-mediated synovial inflammation in an IL-23- and monocyte-independent manner. Therefore, it would be important to control PGE2 in chronic inflammation in RA and potentially other Th17-mediated autoimmune disorders.

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.

IL-17/Th17 mediated synovial inflammation is IL-22 independent.

BACKGROUND: Interleukin (IL)-17A and Th17 cells are critically involved in T cell-mediated synovial inflammation. Besides IL-17A, Th17 cells produce IL-22. Recently, Th22 cells were discovered, which produce IL-22 in the absence of IL-17. However, it remains unclear whether IL-22 and Th22 cells contribute to T cell-mediated synovial inflammation. Therefore, we examined the potential of IL-22 and Th22 cells to induce synovial inflammation and whether IL-22 is required for T cell-mediated experimental arthritis. METHODS: Peripheral and synovial Th17 and Th22 cells were identified and sorted from patients with rheumatoid arthritis (RA). Co-culture experiments of these primary T cell populations with RA synovial fibroblasts (RASF) were performed. The in vivo IL-22 contribution to synovial inflammation was investigated by inducing T cell-mediated arthritis in IL-22 deficient mice and wild-type mice. RESULTS: Peripheral Th17 and Th22 cell populations were increased in patients with RA and present in RA synovial fluid. In T cell-RASF co-cultures, IL-22 in the presence of IL-17A had limited effects on IL-6, IL-8, matrix metalloproteinase-1 (MMP-1) and MMP-3 production. Furthermore, primary peripheral blood and synovial Th17 cells were more potent in the induction of these factors by RASF compared with Th22 cells. In line with this, similar synovial inflammation and disease severity was found between IL-22 deficient and wild-type mice in T cell-mediated experimental arthritis. CONCLUSIONS: These findings show that IL-17A/Th17 cell-mediated synovial inflammation is independent of IL-22 and Th22 cells. This implies that targeting IL-17A/Th17 cells, rather than IL-22/Th22 cells, should be the focus for treatment of T cell-mediated synovial inflammation.

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 D(3) (1,25(OH)(2)D(3)) 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)(2)D(3) 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)(2)D(3), but not TNFα blockade, significantly suppressed autocrine IL-17A production in Th17-RASF and synovial biopsy cultures. Combining 1,25(OH)(2)D(3) 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)(2)D(3). The combination of neutralising TNF activity and 1,25(OH)(2)D(3) controls human Th17 activity and additively inhibits synovial inflammation. This indicates more valuable therapeutic potential of activation of Vitamin D receptor signalling over current TNF neutralisation strategies in patients with RA and potentially other Th17-mediated inflammatory diseases.

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.

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

INTRODUCTION: Interleukin (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 (RORgammat) expression in CD4+ and TCRgammadelta+ T cells was evaluated systemically as well as at the site of inflammation. METHODS: Antigen-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)-gamma ex-vivo by flow cytometry. In splenic CD4+ and TCRgammadelta+ T cells gene expression was quantified by flow cytometry and quantitative PCR. RESULTS: IL-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-gamma 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 RORgammat but not IFN-gamma in TCRgammadelta+ 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. CONCLUSIONS: These 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-gamma-positive CD4+ T cells at the site of inflammation. Furthermore, IL-23 regulates IL-17A and RORgammat expression in TCRgammadelta T cells in arthritis. These findings indicate that regulating the IL-23 pathway may have therapeutic potential in non-autoimmune arthritis.