Anne-Marie Mus

Imiquimod-Induced Psoriasis-Like Skin Inflammation in Mice Is Mediated via the IL-23/IL-17 Axis

Topical application of imiquimod (IMQ), a TLR7/8 ligand and potent immune activator, can induce and exacerbate psoriasis, a chronic inflammatory skin disorder. Recently, a crucial role was proposed for the IL-23/IL-17 axis in psoriasis. We hypothesized that IMQ-induced dermatitis in mice can serve as a model for the analysis of pathogenic mechanisms in psoriasis-like dermatitis and assessed its IL-23/IL-17 axis dependency. Daily application of IMQ on mouse back skin induced inflamed scaly skin lesions resembling plaque type psoriasis. These lesions showed increased epidermal proliferation, abnormal differentiation, epidermal accumulation of neutrophils in microabcesses, neoangiogenesis, and infiltrates consisting of CD4+ T cells, CD11c+ dendritic cells, and plasmacytoid dendritic cells. IMQ induced epidermal expression of IL-23, IL-17A, and IL-17F, as well as an increase in splenic Th17 cells. IMQ-induced dermatitis was partially dependent on the presence of T cells, whereas disease development was almost completely blocked in mice deficient for IL-23 or the IL-17 receptor, demonstrating a pivotal role of the IL-23/IL-17 axis. In conclusion, the sole application of the innate TLR7/8 ligand IMQ rapidly induces a dermatitis closely resembling human psoriasis, critically dependent on the IL-23/IL-17 axis. This rapid and convenient model allows further elucidation of pathogenic mechanisms and evaluation of new therapies in psoriasis.

Th17 cells, but not Th1 cells, from patients with early rheumatoid arthritis are potent inducers of matrix metalloproteinases and proinflammatory cytokines upon synovial fibroblast interaction, including autocrine interleukin‐17A production

OBJECTIVE Both Th1 cells and Th17 cells have been recognized in rheumatoid arthritis (RA); however, it remains unclear whether Th1 cells and/or Th17 cells are involved in driving disease chronicity and destructiveness. The aim of this study was to identify and characterize the functional role of Th17 cells in early RA. METHODS Flow cytometry analysis was performed on peripheral blood mononuclear cells (PBMCs) from treatment-naive patients with early RA and age-matched healthy volunteers. PBMCs from these patients, naive T cells, and primary CCR6- Th1 cells and CCR6+ Th17 cells were sorted and cultured in the absence or presence of synovial fibroblasts from patients with early RA (RASFs), and cytokine expression and gene transcription were analyzed. In addition, tumor necrosis factor α (TNFα)- and interleukin-17A (IL-17A)-blocking experiments were performed. RESULTS In the PBMCs of treatment-naive patients with early RA, an increased fraction of IL-17A-and TNFα-producing CCR6+ Th17 cells was observed. When cocultured with RASFs, these primary Th17 cells were potent inducers of IL-6 and IL-8 and the tissue-destructive enzymes matrix metalloproteinase 1 (MMP-1) and MMP-3, whereas primary Th1 cells or naive T cells were not. Importantly, specific up-regulation of IL-17A but not TNFα or interferon-γ was observed in RASF/Th17 cell cocultures. In addition to TNFα blocking, IL-17A neutralization was required to further down-regulate Th17 activity in RASF/Th17 cell cocultures. CONCLUSION Th17 cells, but not Th1 cells, cooperated with RASFs in a proinflammatory feedback loop, revealing a potential mechanism by which human Th17 cells drive chronic destructive disease in patients with RA. Furthermore, the neutralization of IL-17A activity is essential in current anti-TNF therapies to suppress Th17 cell activity in patients with early RA and potentially other Th17 cell-mediated disorders.

1,25-Dihydroxyvitamin D3 modulates Th17 polarization and interleukin-22 expression by memory T cells from patients with early rheumatoid arthritis

OBJECTIVE To examine the immunologic mechanism by which 1,25-dihydroxyvitamin D(3) (1,25[OH](2)D(3)) may prevent corticosteroid-induced osteoporosis in patients with early rheumatoid arthritis (RA), with a focus on T cell biology. METHODS Peripheral blood mononuclear cells (PBMCs) and CD4+CD45RO+ (memory) and CD4+CD45RO- (non-memory) T cells separated by fluorescence-activated cell sorting (FACS) from treatment-naive patients with early RA were stimulated with anti-CD3/anti-CD28 in the absence or presence of various concentrations of 1,25(OH)(2)D(3), dexamethasone (DEX), and 1,25(OH)(2)D(3) and DEX combined. Levels of T cell cytokines were determined by enzyme-linked immunosorbent assay and flow cytometry. RESULTS The presence of 1,25(OH)(2)D(3) reduced interleukin-17A (IL-17A) and interferon-gamma levels and increased IL-4 levels in stimulated PBMCs from treatment-naive patients with early RA. In addition, 1,25(OH)(2)D(3) had favorable effects on tumor necrosis factor alpha (TNFalpha):IL-4 and IL-17A:IL-4 ratios and prevented the unfavorable effects of DEX on these ratios. Enhanced percentages of IL-17A- and IL-22-expressing CD4+ T cells and IL-17A-expressing memory T cells were observed in PBMCs from treatment-naive patients with early RA as compared with healthy controls. Of note, we found no difference in the percentage of CD45RO+ and CD45RO- cells between these 2 groups. Interestingly, 1,25(OH)(2)D(3), in contrast to DEX, directly modulated human Th17 polarization, accompanied by suppression of IL-17A, IL-17F, TNFalpha, and IL-22 production by memory T cells sorted by FACS from patients with early RA. CONCLUSION These data indicate that 1,25(OH)(2)D(3) may contribute its bone-sparing effects in RA patients taking corticosteroids by the modulation of Th17 polarization, inhibition of Th17 cytokines, and stimulation of IL-4.

GATA-3 protects against severe joint inflammation and bone erosion and reduces differentiation of Th17 cells during experimental arthritis

OBJECTIVE Rheumatoid arthritis is associated with the infiltration of T helper cells into the joints. It is unclear whether interferon-gamma (IFNgamma)-producing Th1 cells or the novel T helper subset, interleukin-17 (IL-17)-producing Th17 cells, are the pathogenic mediators of joint inflammation in chronic nonautoimmune arthritis. Therefore, this study was aimed at examining whether the Th2-specific transcription factor GATA-3 can regulate arthritis, in an experimental murine model, by modulating Th1 and/or Th17 cell polarization. METHODS Arthritis was induced with methylated bovine serum albumin (mBSA) in both wild-type and CD2 T cell-specific GATA-3 (CD2-GATA-3)-transgenic mice. At days 1 and 7 after the induction of arthritis, knee joints were scored macroscopically for arthritis severity and for histologic changes. Single-cell suspensions were generated from the spleens, lymph nodes, and inflamed knee joints. Cytokine expression by CD4+ T cells was determined using flow cytometry, and IL-17 expression in the inflamed knee joints was determined by enzyme-linked immunosorbent assay. Analyses of gene expression were performed for Th17-associated factors. RESULTS Wild-type mice developed severe joint inflammation, including massive inflammatory cell infiltration and bone erosion that increased significantly over time, reaching maximal arthritis scores at day 7. In contrast, only mild joint inflammation was observed in CD2-GATA-3-transgenic mice. This mild effect was further accompanied by systemic and local reductions in the numbers of IL-17+IFNgamma- and IL-17+IFNgamma+, but not IL-17-IFNgamma+, CD4+ T cells, and by induction of Th2 cytokine expression. Moreover, GATA-3 overexpression resulted in reduced gene expression of the Th17-associated transcription factor retinoic acid-related orphan receptor gammat. CONCLUSION These results indicate that enforced GATA-3 expression protects against severe joint inflammation and bone erosion in mice, accompanied by reduced differentiation of Th17 cells, but not Th1 cells, during mBSA-induced arthritis.

FcγRIIb on Myeloid Cells Rather than on B Cells Protects from Collagen-Induced Arthritis

Extensive analysis of a variety of arthritis models in germline KO mice has revealed that all four receptors for the Fc part of IgG (FcγR) play a role in the disease process. However, their precise cell type–specific contribution is still unclear. In this study, we analyzed the specific role of the inhibiting FcγRIIb on B lymphocytes (using CD19Cre mice) and in the myeloid cell compartment (using C/EBPαCre mice) in the development of arthritis induced by immunization with either bovine or chicken collagen type II. Despite their comparable anti-mouse collagen autoantibody titers, full FcγRIIb knockout (KO), but not B cell–specific FcγRIIb KO, mice showed a significantly increased incidence and severity of disease compared with wild-type control mice when immunized with bovine collagen. When immunized with chicken collagen, disease incidence was significantly increased in pan-myeloid and full FcγRIIb KO mice, but not in B cell–specific KO mice, whereas disease severity was only significantly increased in full FcγRIIb KO mice compared with incidence and severity in wild-type control mice. We conclude that, although anti-mouse collagen autoantibodies are a prerequisite for the development of collagen-induced arthritis, their presence is insufficient for disease development. FcγRIIb on myeloid effector cells, as a modulator of the threshold for downstream Ab effector pathways, plays a dominant role in the susceptibility to collagen-induced arthritis, whereas FcγRIIb on B cells, as a regulator of Ab production, has a minor effect on disease susceptibility.

Impaired B cell immunity in IL-22 knock-out mice in collagen induced arthritis

Background The role of IL-22, a cytokine produced by Th17 cells, in autoimmunity is not fully understood. Previous research has shown that IL-22 is important in the development of collagen induced arthritis (CIA). However, the mechanism behind the apparent protection in IL-22 knock-out mice against CIA remains unclear. Objective To investigate the mechanisms by which IL-22 knock-out mice are protected against collagen induced arthritis. Materials and methods For CIA, IL-22 knock-out and wild type mice were immunised with chicken collagen type II and complete Freuds adjuvant (CFA) and boosted 21 days later. Antigen specific serum IgG levels were measured by ELISA. Mice were sacrificed 50 days after immunisation. Splenocytes were analysed by flow cytometry and immunohistochemistry. Functional assays were performed with splenic Th17 cells sorted from mice ten days after immunisation. For antigen induced arthritis (AIA), mice were immunised with methylated bovine serum albumin (mBSA) and CFA and triggered seven days later by intra-articular mBSA injection. Results In IL-22 knock-out mice, the severity, but not the incidence, of CIA was lower compared to wild type controls. This prompted us to study the pathogenicity of Th17 cells from these mice. Synovial fibroblasts produced higher levels of IL-6 when co-cultured with Th17 cells from IL-22 knock-out mice compared to wild type Th17 cells. To study the pathogenicity in vivo, we made use of the Th17 mediated AIA model. IL-22 knock-out mice developed AIA similarly to wild type controls, showing that Th17 cells without IL-22 function normally in vivo. As CIA is strongly driven by B cells and immune complexes, we investigated whether B cell immunity is normal in IL-22 knock-out mice. Antigen specific serum IgG2a levels were significantly lower in IL-22 knock-out mice at early onset of disease. We observed no significant differences in splenic B cell numbers or activation. However, spleens from IL-22 knock-out mice show no or very small germinal centers and fewer IgG2a plasma cells compared to wild type controls. Discussion Here we show that lack of IL-22 production affects B cell immunity in CIA. However, Th17 cells in IL-22 knock-out mice function normally in vitro and in vivo, indicating that lack of IL-22 production does not affect the pathogenicity of Th17 cells. Importantly, germinal center formation, plasma cell formation and IgG2a antibody production are lower in IL-22 knock-out mice in CIA, suggesting that IL-22 has a role in further differentiation of B cells in autoimmunity.

03.22 Vitamin d utilises different pathways to inhibit th17 differentiation dependent on the maturation stage of the cd4+t cell

Background In many human autoimmune diseases, including rheumatoid arthritis (RA), vitamin D is associated with disease suppression. Furthermore, experimental collagen-induced arthritis (CIA) is prevented by 1,25(OH)2D3 supplementation, the active form of vitamin D. Since high doses of 1,25(OH)2D3 in humans can cause serious side effects, understanding the mechanism behind the suppression may lead to new therapeutic targets. Although currently this mechanism is not known, it is hypothesised that Th17 cells play an important role in the immunomodulatory effect of vitamin D. Here we study the effect of 1,25(OH)2D3 on the differentiation of Th17 cells and the underlying mechanism in the context of the autoimmune-mediated CIA model. Materials and methods DBA/1 mice were immunised with type II collagen (CII) in complete Freund’s adjuvant. 10 days after immunisation the mice were sacrificed and spleens were harvested. Total CD4+, naïve CD4+ or effector CD4+ T cells were isolated and cultured for 3–7 days under Th17 polarising conditions with or without 1,25(OH)2D3. Results 1,25(OH)2D3 inhibits Th17 differentiation in total CD4+ T cells in CII-immunised mice. Interestingly, 1,25(OH)2D3 induces IL-4 and IL-10 in these cells. Therefore we investigated whether the effect of 1,25(OH)2D3 on Th17 differentiation was dependent on IL-4 or IL-10. In both naïve and effector CD4+ T cells from CII-immunised mice, 1,25(OH)2D3 inhibited Th17 differentiation as demonstrated by decreased IL-17A, IL-17F and IL-22. Blocking IL-10 did not prevent this effect in either subsets. However, in the effector cells, but not the naïve cells, blocking IL-4 partially reduced the effect of 1,25(OH)2D3 on Th17 differentiation. Conclusions 1,25(OH)2D3 similarly inhibits Th17 differentiation of naïve and effector CD4+ T cells from CII-immunised mice. However, only the inhibition in effector cells, not in naïve cells, is partially dependent on IL-4. These data show that 1,25(OH)2D3utilises different pathways to inhibit Th17 differentiation, depending on the maturation stage of the CD4+ T cell.

A5.21 Lack of IL-27R Signaling Leads to an Intrinsic B Cell Defect and Protection against CIA

Introduction The IL-27 receptor (IL-27R) is expressed on naïve T helper cells. Signaling induces Th1 differentiation and IL-10 production and inhibits Th17 differentiation. In addition, the IL-27R is also expressed on B cells, where it is linked to B cell proliferation and IgG2a class switch recombination. However, its role is not fully understood. We therefore aim to determine the role of IL-27R signalling on B cells, both in T cell independent and in T cell dependent immune responses. Methods To determine the effect of IL-27 on B cells, we isolated naïve B cells from wild type mice and cultured them in the absence or presence of rIL-27. T cell dependent responses were investigated using the collagen induced arthritis (CIA) model in wild type and IL-27R deficient (WSX-1 deficient) mice. T cell independent B cell responses were investigated using the TNP-Ficoll immunisation model. Results Naïve wild type B cells cultured in the presence of rIL-27 showed increased proliferation and expression of activation markers. Next, we investigated the role of IL-27R signalling in vivo in CIA. Both the incidence and severity of CIA were significantly lower in IL-27R deficient mice. Splenic germinal centre B cells were decreased in these mice. In addition, both auto-reactive IgM and IgG2a antibody levels were significantly decreased. These data suggest an impairment of B cell immunity in IL-27R deficient mice. However, CIA also depends on T cells. To determine whether the impairment of B cell immunity in IL-27R deficient mice was B cell intrinsic, we used the T cell independent TNP-Ficoll immunisation model. Wild type and IL-27R deficient mice were immunised with TNP-Ficoll i.p. and sacrificed seven days later. IL-27R deficient mice had fewer splenic IgM and IgG3 plasma cells and lower serum TNP specific IgM and IgG3 antibody levels. Conclusions Here we show that IL-27R signalling on B cells is essential for proliferation and activation of B cells. Impaired B cell immunity in IL-27R deficient mice is caused by an intrinsic B cell defect, leading to impaired plasma cell formation and antibody production in both T cell independent as well as T cell dependent immune responses.

FRI0016 Critical role for the IL-23/TNF-alpha axis during TLR2/NOD2 mediated acute joint inflammation

Background IL-23 is essential in the development of chronic autoimmune diseases and supports the maturation of pathogenic Th17 cells. Although the role of IL-23 during adaptive immunity in arthritis has been studied extensively, the role of IL-23 during acute joint inflammation is unknown. Objectives To investigate the role of IL-23 in the development of an acute, macrophage-mediated joint inflammation. Methods Peptidoglycan (PG) or streptococcal cell wall fragments (SCW) were intra-articularly injected into the knee joint of naïve wt or IL-23p19 deficient mice. Joint swelling was assessed by measuring joint thickness using a caliper. In addition, synovial expression of different cytokines was measured by specific ELISA and/or Q-PCR. Moreover, synovial explants of wt and IL-23p19 deficient mice were stimulated with SCW and cytokine levels were measured by ELISA. Results TLR2/NOD2-mediated streptococcal cell wall (SCW) and peptidoglycan (PG) induced acute joint inflammation in IL-23p19-deficient mice resulted in a profound reduction of local joint inflammation compared to control mice in both these models. Synovial IL-23p19 transcript was detected at 1.5 and 4 hours after arthritis induction and was further increased 1 day after PG injection with a peak at day 2. Interestingly, IL-23p19-/- mice showed a significant reduction in synovial TNF-a, but not IL-6 levels 4 hours after TLR2/NOD2-mediated arthritis induction in the knee joint. In line with this, reduced TNF-a levels were detected in the culture supernatant of SCW-stimulated synovial explants from IL-23p19-/- mice compared to control mice. Conclusions These data show a critical role for IL-23 in the development of a TLR2/NOD2-mediated acute joint inflammation and reveal a novel IL-23/TNFalpha axis in this process. Disclosure of Interest None Declared

SAT0043 Impaired B cell immunity in IL-27R knock-out mice in collagen induced arthritis

Background Collagen induced arthritis (CIA), a mouse model for rheumatoid arthritis, is both B cell and T helper (Th) 17 cell mediated. Interleukin 27 receptor (IL27R) signaling upregulates IL12 receptor expression on naïve T cells and inhibits Th17 differentiation. In addition, IL27R expression is upregulated on activated B cells. However, the role of IL27R signaling on B cells is not fully understood. We therefore aim to investigate the role of IL27R signaling in B cell immunity and the pathogenesis of CIA. Objectives To investigate the role of IL27R signalling on B cells in CIA. Methods For CIA, mice were immunized with complete Freund’s adjuvant and chicken collagen type II (CFA/CII) and boosted 21 days later. For AIA, mice were immunized with CFA and methylated bovine serum albumin (mBSA) and 7 days later a stimulus with mBSA was given directly in the knee joint. For B cell activation experiments, mice were immunized with DNP-Ficoll and sacrificed 7 days later. Results IL27R knockout mice showed a decreased CIA arthritis score compared to wild type controls. Analysis of B cells in CIA by immunohistochemistry and flow cytometry showed severely decreased germinal center formation in IL27R knockout mice. In addition, plasma cell formation and auto-antibody formation were decreased compared to controls, suggesting an intrinsic defect in B cell differentiation upon activation. To exclude that the phenotype of IL27R knockout mice in CIA was caused by a defect in Th17 pathogenicity, we made use of the Th17 mediated antigen induced arthritis (AIA) model, which is B cell independent. IL27R knockout animals developed comparable arthritis to controls. Based on these data we concluded that T cells respond normally in these mice. We therefore further analyzed B cell responses using the T cell independent DNP-Ficoll immunization model. IL27R knockout mice showed lower IgM and IgG3 plasma cell counts in spleen and lower DNP specific IgM and IgG3 antibody levels in serum. These data further support impaired B cell differentiation after activation. Conclusions Here we show that IL27R knockout mice are protected against full blown CIA. Th17 cells in these mice are functionally normal. However, terminal B cell differentiation appears to be impaired, suggesting a critical role for IL27R signaling in B cell immunity. Disclosure of Interest None Declared