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, 58 (3), 875-87

Interleukin-17-producing T Cells Are Enriched in the Joints of Children With Arthritis, but Have a Reciprocal Relationship to Regulatory T Cell Numbers

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Interleukin-17-producing T Cells Are Enriched in the Joints of Children With Arthritis, but Have a Reciprocal Relationship to Regulatory T Cell Numbers

Kiran Nistala et al. Arthritis Rheum.

Abstract

Objective: To identify interleukin-17 (IL-17)-producing T cells from patients with juvenile idiopathic arthritis (JIA), and investigate their cytokine production, migratory capacity, and relationship to Treg cells at sites of inflammation, as well as to test the hypothesis that IL-17+ T cell numbers correlate with clinical phenotype in childhood arthritis.

Methods: Flow cytometry was used to analyze the phenotype, cytokine production, and chemokine receptor expression of IL-17-producing T cells in peripheral blood and synovial fluid mononuclear cells from 36 children with JIA, in parallel with analysis of forkhead box P3 (FoxP3)-positive Treg cells. Migration of IL-17+ T cells toward CCL20 was assessed by a Transwell assay. Synovial tissue was analyzed by immunohistochemistry for IL-17 and IL-22.

Results: IL-17+ T cells were enriched in the joints of children with JIA as compared with the blood of JIA patients (P = 0.0001) and controls (P = 0.018) and were demonstrated in synovial tissue. IL-17+ T cell numbers were higher in patients with extended oligoarthritis, the more severe subtype of JIA, as compared with patients with persistent oligoarthritis, the milder subtype (P = 0.046). Within the joint, there was an inverse relationship between IL-17+ T cells and FoxP3+ Treg cells (r = 0.61, P = 0.016). IL-17+,CD4+ T cells were uniformly CCR6+ and migrated toward CCL20, but synovial IL-17+ T cells had variable CCR4 expression. A proportion of IL-17+ synovial T cells produced IL-22 and interferon-gamma.

Conclusion: This study is the first to define the frequency and characteristics of "Th17" cells in JIA. We suggest that these highly proinflammatory cells contribute to joint pathology, as indicated by relationships with clinical phenotypes, and that the balance between IL-17+ T cells and Treg cells may be critical to outcome.

Figures

Figure 1
Figure 1
Enrichment of interleukin-17 (IL-17)–positive, CD4+ T cells in synovial fluid from patients with juvenile idiopathic arthritis (JIA) and correlation with clinical subtype. A, Representative dot plots of paired samples of peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) from a patient with JIA. Cells were stained for surface expression of CD3 and CD4 and then for intracellular expression of IL-17 and were analyzed by flow cytometry, gated on live lymphocytes and CD3+ cells. Numbers in each compartment are the percentage of cells. B, Numbers of IL-17+ cells as a percentage of CD4+ T cells in SFMCs from JIA patients (n = 28), PBMCs from JIA patients (n = 22), and PBMCs from healthy controls (n = 9). Bars show the median. C, Numbers of IL-17+ cells as a percentage of CD4+ T cells in PBMCs (PB) and SFMCs (SF) from patients with persistent (n = 9 PBMC samples; n = 12 SFMC samples) and extended (n = 6 PBMC samples; n = 13 SFMC samples) oligoarticular JIA (O-JIA) and in PBMCs from healthy controls (n = 9). Bars show the median.
Figure 2
Figure 2
Characterization of interleukin-17 (IL-17)–positive T cells in patients with juvenile idiopathic arthritis (JIA). Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) from JIA patients were analyzed by flow cytometry. A and B, Representative dot plots of paired PBMCs and SFMCs stained for CD3, IL-17, and T cell receptor γ/δ (TCRγ/δ) (A) as well as for CD3, IL-17, and CD8 (B), both gated on live lymphocytes and CD3+ cells. C, Representative dot plot of JIA SFMCs stained for CD3, CD4, CD45RO, and IL-17 gated on live lymphocytes, CD3+ cells, and CD4+ cells. Numbers in each compartment are the percentage of cells.
Figure 3
Figure 3
Inverse relationship between the number of synovial forkhead box P3 (FoxP3)–positive regulatory T cells and interleukin-17 (IL-17)–positive, CD4+ T cells. A, Representative dot plots of paired samples of peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) from a patient with juvenile idiopathic arthritis (JIA) and of samples of PBMCs from a healthy control subject. Cells were stained for surface expression of CD3, CD4, and CD25 and then for intracellular expression of FoxP3 and were analyzed by flow cytometry, gated on live lymphocytes and CD3+,CD4+ cells. Numbers in each compartment are the percentage of cells. B, Numbers of FoxP3+ cells as a percentage of CD4+ T cells in PBMCs and SFMCs from patients with persistent (n = 8) and extended (n = 5) oligoarticular JIA (O-JIA) and in PBMCs from 6 healthy controls. Bars show the median. C, Comparison of the numbers of IL-17+ and FoxP3+ cells as percentages of CD4+ T cells in SFMCs from 15 JIA patients. Linear regression was performed on log-transformed data.
Figure 4
Figure 4
Divergent patterns of chemokine receptor CCR4 expression of interleukin-17 (IL-17)–positive, CD4+ T cells in peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) from patients with juvenile idiopathic arthritis (JIA). A, Representative dot plots of paired PBMCs and SFMCs from JIA patients, showing CCR6 and CCR4 expression on IL-17+,CD4+ T cells. Cells were analyzed by flow cytometry, gated on live lymphocytes and CD4+ cells. Numbers in each compartment are the percentage of cells. Results are representative of 1 experiment of 5 experiments performed. B, Percentage of all CD4+ T cells and IL-17+,CD4+ T cells expressing CCR6 and CCR4 in the peripheral blood and synovial fluid of 5 patients with JIA. Values are the mean and SEM.
Figure 5
Figure 5
Dysregulated cytokine expression by interleukin-17 (IL-17)–positive, CD4+ T cells in the synovial fluid of juvenile idiopathic arthritis (JIA) patients. A, Representative dot plots of paired samples of peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) from patients with JIA. Cells were stained for intracellular cytokines IL-17, interferon-γ (IFNγ), IL-22, and IL-4 and analyzed by flow cytometry, gated on live lymphocytes and CD3+,CD4+ cells. Numbers in each compartment are the percentage of cells. B, Immunohistologic localization of IL-17 and IL-22 in JIA synovial tissue. Frozen sections of synovial biopsy tissue were stained for CD4, IL-17, and IL-22 by standard immunohistochemical techniques. Tissue sections show typical hypertrophied synovium and dense inflammatory infiltrate. Positive cells expressing the marker of interest are stained brown; arrow shows typical positive cells. Bar = 20 μm.
Figure 6
Figure 6
Preferential migration of interleukin-17 (IL-17)–positive, CD4+ T cells to the CCR6 ligand CCL20 as compared with CD4+ T cells. Purified CD4+ T cells from patients with juvenile idiopathic arthritis (JIA) and healthy controls were migrated to 10 ng/ml, 100 ng/ml, or 1,000 ng/ml of CCL20 or RPMI 1640–0.5% bovine serum albumin (BSA). A, Cells from healthy controls analyzed by intracellular cytokine staining for IL-17 and interferon-γ (IFNγ) before (left) and after migration to control medium (RPMI 1640–0.5% BSA) (middle) or 1,000 ng/ml of CCL20 (right). Cells were analyzed by flow cytometry, gated on live CD3+,CD4+ cells. Numbers in each compartment are the percentage of cells. Results are representative of 1 experiment of 5 experiments performed. B, Response of IL-17+,CD4+ T cells and all CD4+ T cells to a titration of the CCL20 gradient. Data are expressed as the chemotactic index (ratio of cells migrating to CCL20 divided by cells migrating to control medium). Values are the mean ± SEM of 5 experiments.

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