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.

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.

Characterizing memory T helper cells in patients with psoriasis, subclinical, or early psoriatic arthritis using a machine learning algorithm.

BACKGROUND: Psoriasis patients developing psoriatic arthritis (PsA) are thought to go through different phases. Understanding the underlying events in these phases is crucial to diagnose PsA early. Here, we have characterized the circulating memory T helper (Th) cells in psoriasis patients with or without arthralgia, psoriasis patients who developed PsA during follow-up (subclinical PsA), early PsA patients and healthy controls to elucidate their role in PsA development. METHODS: We used peripheral blood mononuclear cells of sex and age-matched psoriasis patients included in Rotterdam Joint Skin study (n=22), early PsA patients included in Dutch South West Early Psoriatic Arthritis Cohort (DEPAR) (n=23) and healthy controls (HC; n=17). We profiled memory Th cell subsets with flow cytometry and used the machine learning algorithm FlowSOM to interpret the data. RESULTS: Three of the 22 psoriasis patients developed PsA during 2-year follow-up. FlowSOM identified 12 clusters of memory Th cells, including Th1, Th2, Th17/22, and Th17.1 cells. All psoriasis and PsA patients had higher numbers of Th17/22 than healthy controls. Psoriasis patients without arthralgia had lower numbers of CCR6-CCR4+CXCR3+ memory Th cells and higher numbers of CCR6+CCR4-CXCR3-memory Th cells compared to HC. PsA patients had higher numbers of Th2 cells and CCR6+CCR4+CXCR3- cells, but lower numbers of CCR6+CCR4+CXCR3+ memory Th cells compared to HC. The number of CCR6+ Th17.1 cells negatively correlated with tender joint counts and the number of CCR6+ Th17 cells positively correlated with skin disease severity. CONCLUSIONS: Unsupervised clustering analysis revealed differences in circulating memory Th cells between psoriasis and PsA patients compared to HC; however, no specific subset was identified characterizing subclinical PsA patients.

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.

Interleukin-17A Drives IL-19 and IL-24 Expression in Skin Stromal Cells Regulating Keratinocyte Proliferation.

IL-17A has been shown to be up-regulated in psoriasis lesions and is central to psoriasis pathogenesis. IL-19, along with other IL-20 subfamily cytokines such as IL-20 and IL-24, is induced by IL-17A and contributes especially to epidermal hyperplasia in psoriasis. However, the regulation, cellular sources of IL-19 and whether targeting of IL-17A by biologics influence IL-19 expression is not completely understood. To investigate the regulation of IL-19 by IL-17A in psoriasis, the imiquimod-induced psoriasis mouse (IMQ) model was used. Enhanced expression of IL-17A in the IMQ model was achieved by anti-IL-10 antibody treatment. Assessments of skin inflammation macroscopically, by histology and flow cytometry, all confirmed increased psoriatic symptoms. Interestingly, depletion of IL-10 markedly upregulated IL-23/IL-17 pathway related cytokines followed by a significant increase in IL-19 and IL-24. The up-regulation of IL-19 and IL-24, but not IL-17A, coincided with increased keratinocyte proliferation. To investigate the cellular source and effects of biologics on IL-19, human skin fibroblasts from healthy controls and psoriasis patients were cultured alone or co-cultured with activated memory CD4+ T cells. Besides IL-1ß, IL-17A induced direct expression of IL-19 and IL-24 in skin fibroblasts and keratinocytes. Importantly, intrinsic higher expression of IL-19 in psoriatic skin fibroblasts was observed in comparison to healthy skin fibroblasts. Neutralization of IL-17A in the human skin fibroblast-T cell co-culture system significantly suppressed IL-19 and IL-24 expression. Together, our data show that IL-17A-induced IL-19 and IL-24 expression in skin stromal cells contribute to keratinocyte proliferation.

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.

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.