Abstract
Objective. Early recognition and treatment of juvenile idiopathic arthritis (JIA) can prevent joint damage and minimize side effects of medication. The balance between proinflammatory and antiinflammatory mechanisms is known to be important in JIA, and we therefore investigated T cell subsets including Th cells, autoaggressive Th17 cells, and regulatory T cells (Treg), including a novel Treg subset in peripheral blood (PB) and synovial fluid (SF) of patients with JIA.
Methods. Fifty children with JIA were enrolled in our study. Frequency, phenotype, and function of T lymphocytes in PB and SF were characterized using flow cytometry. Migration capabilities of PB and SF cells were compared.
Results. Synovial T cells showed different phenotype and function compared with PB T cells, with an increased proportion of memory T cells, expression of CCR4, CCR5, CXCR3, interleukin 23R, and an increased ratio of Th17 to Treg. Although Treg were increased in SF compared with the PB, we found a significant decrease in the numbers of peptidase inhibitor 16 (PI16)+ Treg in active joints compared with peripheral blood. Coexpression of CCR4 and CCR6 was reduced on PI16+ Treg in PB and SF of patients with JIA compared with healthy children, however the ability of these cells to migrate toward their ligands was unaffected.
Conclusion. This is a comprehensive characterization of novel PI16+ Treg and Th17 cells in matched blood and synovial fluid samples of patients with JIA. Despite an increased number of Treg within the inflamed joint, lower numbers of PI16+ Treg but high numbers of Th17 cells might contribute to the inability to control disease.
- JUVENILE IDIOPATHIC ARTHRITIS
- PEPTIDASE INHIBITOR 16
- Th CELLS
Juvenile idiopathic arthritis (JIA) is the most common rheumatologic disease of childhood, occurring in up to 1:500 children1. The effect on children with JIA can be significant, with pain, disability, deformity, growth failure, and osteoporosis2. The disease can affect not only the child’s education, social, and psychological functioning, but also his or her family3. As demonstrated, it is important to recognize and treat JIA early to prevent soft tissue deformities, irreversible joint damage, and medication side effects4.
T cells have been demonstrated to play key roles in the pathogenesis of JIA. Specifically, cells that produce interleukin 17 (IL-17; Th17) are believed to be the major inducers of severe autoimmune tissue inflammation and destruction4,5,6. By binding the chemokine CCL20 produced by activated monocytes, chemokine receptor CCR6-expressing Th17 cells migrate along a CCL20 gradient into sites of inflammation7,8, where they induce the production of proinflammatory cytokines and chemokines that attract neutrophils and other immune cells9. In the synovial fluid (SF) of patients with rheumatoid arthritis (RA), it is known that IL-17 is responsible for osteoclastogenesis10, and that it promotes cartilage and bone destruction and resorption11. IL-17 also promotes the generation, attraction, and expansion of further Th17 cells, which sustain the inflammatory response within the joint. However, little is known about T cells within the joint and peripheral blood (PB) in patients with JIA. Previous studies have shown that IL-17-producing cells are enriched in the synovial fluid of children with extended oligoarthritis compared with patients with persistent oligoarthritis in a reciprocal relationship with regulatory T cells (Treg)8. An inverse relationship between the increased synovial expression of the Th17 transcription factor RORC2 and the Treg transcription factor FOXP3 has also been demonstrated in the joints of children with JIA12.
As part of a search for surface surrogates of FOXP3 on human Treg, we have recently identified a novel surface molecule peptidase inhibitor 16 (PI16) as being over-expressed on expanded Treg compared with Th cells13. We found that PI16 is expressed by a T cell subset that expresses high levels of FOXP3. These cells have a memory phenotype (CD45RO+) and express high levels of the chemokines CCR4 and CCR6. In vitro assays show that PI16+ Treg cells are able to suppress Th cell function and also migrate to ligands CCL17 and CCL2014.
A detailed understanding of the mechanism(s) of inflammation in JIA may be crucial in helping to predict which children will develop severe or persistent disease. We have characterized T cell subsets including Th17 and Treg cells in PB and SF from patients to further understand the mechanisms involved, ultimately providing a target for the therapeutic intervention of JIA.
MATERIALS AND METHODS
Patients
When possible, paired PB and SF samples were obtained from children with JIA. The study cohort of 50 patients is summarized in Table 1. Thirty-one females and nineteen males, aged 6 months to 18 years, with mean age of onset of 9.0 ± 5.0 years, and mean disease duration of 3.4 ± 3.4 years, were included in the study. All patients with JIA fulfilled the revised International League of Associations for Rheumatology classification criteria for JIA1. Disease activity at the time of joint injection was calculated using Juvenile Arthritis Disease Activity Scores 10015. Our study was approved by the Women’s and Children’s Hospital Research Ethics Committee (REC2101/9/11) and written informed consent was obtained for all participants. Paired PB and SF samples were obtained at the time of therapeutic steroid joint injection and processed immediately. PB samples from healthy children used in our study were obtained from a parallel study which was approved by the Women’s and Children’s Hospital Research Ethics Committee (REC2264/3/13) and written informed consent was obtained for all participants.
Cell isolation and culture
A small aliquot of centrifuged SF was stored at −80°C for cytokine and chemokine analysis using the Cytometric Bead Array System (BD Biosciences, San Diego, CA, USA). PB mononuclear cells (PBMC) and SF mononuclear cells (SFMC) were isolated by standard density gradient centrifugation (Lymphoprep; Fresenius Kabi, Bad Homburg, Germany). Up to 5 × 106 PBMC and SFMC were stained with antibodies against surface molecules and analyzed by flow cytometry. For detection of intracellular cytokines, PBMC and SFMC samples were cultured in RPMI 1640 (HyClone Laboratories, South Logan, UT, USA) supplemented with 2 mM L-glutamine (SAFCBiosciences, Lenexa, KS, USA), penicillin/streptomycin (Sigma Aldrich, Steinheim, Germany), and 10% fetal calf serum (SAFCBiosciences). Cells were stimulated in 96 U-well culture plates (0.5–1 × 106/well) with Staphylococcus enterotoxin B (SEB; Sigma-Aldrich; 1 μg/ml) for 18 h and GolgiPlug (BD Biosciences, San Jose, CA, USA) was added after 2 h of stimulation. For detection of secreted cytokines, cells were stimulated as described but without the addition of GolgiPlug. Supernatants were collected and kept at −80°C until use.
Flow cytometry
For cell surface immunostaining, stimulated and unstimulated PBMC and SFMC (0.5–1 × 106/50 μl) were stained with monoclonal antibodies against CD4, CD8, CD25, CD27, CD45RA, CD62L, CD127, CD161, CCR2, CCR4, CCR5, CCR6, CXCR3 (BD Biosciences), FOXP3 (eBioscience, San Diego, CA, USA), IL-23R and transforming growth factor-βR (R&D Systems, Minneapolis, MN, USA), and PI16 (monoclonal antibody CRCBT-02-001 kindly provided by the Co-operative Research Centre for Biomarker Translation, Australia). For intracellular staining, stimulated cells were first stained with monoclonal antibodies against surface antigens and then fixed and permeabilized using Fix/Perm solution (BD Biosciences) in Eppendorf tubes. Cells were then washed with Perm/Wash buffer (BD Biosciences) and stained with monoclonal antibodies against intracellular antigens: IL-17A (eBioscience), IL-10, interferon-γ (IFN-γ), FOXP3, IL-22, and IL-27 (R&D Systems). Cell data were acquired on a FACSAria II flow cytometer and analyzed using FACSDiva software v 6.1.3 (BD Biosciences). For data analysis, 100,000 lymphocytes were acquired for each sample.
The cytokines IL-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-α, IFN-γ, and IL-17A were measured in SF and supernatants from stimulated PBMC and SFMC using the Human Th1/Th2/Th17 Cytometric Bead Array (CBA) Kit (provided by Homero Sepulveda, BD Biosciences, San Diego, CA, USA) and CBA Flex Kits according to the manufacturer’s instructions (BD Biosciences) and analyzed by flow cytometry. The chemokines CCL2, CCL3, CCL4, and CCL5 were measured in SF using the Human Chemokine Bead Array Kit according to the manufacturer’s instructions (BD Biosciences).
PI16 ELISA
Soluble PI16 was measured in blood plasma and SF using a PI16 ELISA kit (USCN Life Sciences Inc., Wuhan, China) according to the manufacturer’s instructions.
Chemotaxis assay
Recombinant human thymus and activation regulated chemokine (CCL17) and recombinant human macrophage inflammatory protein-3 alpha (CCL20) were purchased from Raybiotech (Norcross, GA, USA). Propidium iodide (Molecular Probes Inc.) was used to determine PBMC and SFMC cell viability. Chemotaxis assays were performed using Transwell plates with 5-μm pores (Corning, NY, USA). Bottom wells contained media alone, 100 ng/ml CCL17, 100 ng/ml CCL20, or 100ng/ml of both CCL17 and CCL20. PBMC and SFMC were isolated by standard-density gradient centrifugation. Upper wells were loaded with 1 × 106 cells per well and the cells were allowed to undergo chemotaxis for 2 h at 37°C. After chemotaxis, cells in the top (no migration) and bottom (migrated) wells were collected and stained with monoclonal antibodies against CD4, CD25, PI16, CCR4, and CCR6. The chemotactic index was calculated as previously shown by Nistala, et al8. The number of CD4+/CD25hi/PI16+ Treg cells that migrated in response to chemokine ligands was divided by the number of cells that migrated spontaneously to control medium.
Statistical analysis
The results are expressed as either mean ± SD or mean ± SEM. The data were analyzed using 2-tailed Student’s t test for paired and unpaired samples. P values < 0.05 were considered significant. Pearson correlations were used to measure the statistical relationship between samples.
RESULTS
Enrichment of memory T cells in SF compared with matching blood samples
To evaluate whether cells in the peripheral blood are indicative of those within active joints of patients with JIA, we measured the distribution of different T cell subtypes in SF compared with the PB in the same patients using flow cytometry. Although we saw no significant difference in the percentage of total CD4+ Th cells, when the CD4+ Th cells were further divided into subtypes (Figure 1A), we detected a clear enrichment of CD45RA- memory Th cells within SF (p < 0.0001). This included both CD27+ (mixed memory; p < 0.0001) and CD27– (effector memory, p < 0.0001) T cells. Equally, we observed an increase of memory Th1-like cells (CD45RA-/CD62L-) within SF (p < 0.0001). In contrast, the proportion of CD27+/CD45RA+-naive Th cells was significantly increased in PB compared with SF (p < 0.0001; Figure 1A).
Increased expression of chemokine and cytokine receptors on SF cells
To understand the recruitment of Th cells into the joints, we analyzed Th cells within SF and PB of patients with JIA and revealed a significant increased expression of the chemokine receptors CCR4, CCR5, and CXCR3 and the cytokine receptor IL23R within the SF (Figure 1B). We found an increase in the percentage of CXCR3+/CCR4- cells within the SF compared with PB (Figure 1B). Expression of CXCR3 on CCR4-negative cells indicated a Th1-like immune response. To analyze polarization of Th cells in SF compared with PB, we stimulated T cells overnight with SEB and analyzed intracellular cytokine production. We found a significant bias toward Th1 immune responses in SF, with Th cells producing more IFN-γ (19 ± 6.8%) and IL-10 (8.7 ± 6.2%) compared with PB (1.8 ± 1.8%, p < 0.001; and 0.4 ± 0.3%, p < 0.05, respectively). Further analysis of the supernatants of stimulated T cells revealed that PB T cells also secreted Th1 cytokines IL-2 [55.6 ng/ml (mean)] and IFN-γ (31 ng/ml) but in lower concentration than SF T cells (241.2 ng/ml and 90.3 ng/ml, respectively). IL-4 secretion was virtually undetected in the SF and PB. Proinflammatory cytokines IL-6 and TNF-α were secreted by both SF and PB Th cells.
Increased frequency of IL-17-producing cells in SF compared with matching blood samples
Analysis of stimulated CD4+ T cells from paired PB and SF samples revealed that SF contained a higher percentage of IL-17-producing cells [mean 3.4% (range 0.5–6.8)] than PB [mean 0.7% (range 0.2–1.1; p < 0.001)]. The concentration of IL-17 in the supernatant of stimulated SFMC was also higher compared with the supernatant of stimulated PBMC, and we found a strong correlation between the proportion of IL-17-producing T cells and concentration of IL-17 in the supernatants of stimulated cells (R = 0.95).
Phenotypic analysis of IL-17-producing cells in PB and SF
To test the hypothesis that IL-17-producing cells are responsible for tissue damage within active joints of patients with JIA, we characterized these cells in more detail (Figure 2). The analysis of IL-17-producing Th cells in SF confirmed that the majority of Th17 cells have a memory phenotype (CD45RA-/CD45RO+) and express CCR6 on their surface but do not express CD62L (Figure 2). Because published data describing the expression of CD161, CCR4, and CXCR3 on Th17 cells are inconsistent, we investigated the expression of these receptors and found that 69 ± 3.6% (mean ± SEM) of IL-17-producing cells expressed CD161, whereas only a few expressed CCR4 (5.9 ± 1.9%) and CXCR3 (4.7 ± 1.8%) on their cell surface (Figure 2B). Analysis of intracellular cytokine expression confirmed recently reported data that almost half of IL-17-producing cells also secreted IFN-γ (42 ± 4.9%) and 31.9 ± 5.5% produced IL-2216. It has been suggested that Th17 cells are controlled by IL-27 and IL-10. To investigate autocrine feedback mechanisms of IL-17-producing cells in patients with JIA, we analyzed the co-production of IL-17 and IL-10 or IL-27, respectively. Fifteen percent of IL-17-producing cells also produced IL-10 (15 ± 3.0%), whereas only 2.2 ± 1.5% co-produced IL-27 (Figure 2B).
In contrast, IL-17-producing cells in the PB had increased expression of CD62L (32.2% ± 7.2%, p < 0.01) and CXCR3 (17.5% ± 6.7%, p < 0.05), but comparable levels of CD161 (54.9% ± 3.1%; Figure 2C). Circulating IL-17 cells had reduced expression of CCR5 (14.1% ± 4.5%, p < 0.001), IFN-γ (4.1% ± 3.1%, p < 0.001), IL-22 (7.7% ± 7.5%, p < 0.001) and IL-10 (0.3% ± 0.1%, p < 0.01), compared with the SF cells (Figure 2C).
Treg and PI16
Figure 3 shows an increase in the proportion of Th17+ and CD4+/CD25hi/CD127low/FOXP3+ Treg within the SF compared with the PB of patients with JIA (Figure 3A and B, respectively). By analyzing the ratio between Th17 and Treg in SF and PB we demonstrated a difference in the relationship between Th17 and Treg with a ratio of 1:8 in blood and a ratio of 1:4 in active joints (Figure 3C). Figure 3B illustrates the significant increase of CD4+/CD25hi/CD127low/FOXP3+ regulatory T cells in SF (10.6 ± 0.8%) of patients with JIA compared with matched samples of PB (2.7 ± 0.2%; p < 0.0001).
As shown in Figures 4A, 4B, and 4C, there is a high variation of PI16 expression on CD4+ T cells. Total PI16+ expression was reduced (p = 0.003) in patients with oligoarthritis (Figure 4B), but there was no correlation between concentration of PI16 in plasma and SF. We found a significant decrease (p = 0.02) in the percentage of CD4+/CD25hi/CD127low Treg expressing PI16 in SF (15.8 ± 1.8%) compared with matched JIA blood samples (24.7 ± 3.1%; Figure 4D). We have recently shown that PI16+ Treg express high levels of FOXP3, CD45RO, and Th17-like chemokine receptors CCR4 and CCR614. PI16+ Treg were reduced in PB (p = 0.02) and SF (p = 0.05) of oligoarticular patients with JIA compared with the PB of age-matched healthy children (Figure 4F). In contrast, patients with juvenile psoriatic arthritis (JPsA), enthesitis-related arthritis (ERA), and systemic-onset JIA (SoJIA; other JIA) had variable levels of the PI16+ Treg cells within PB, but elevated levels within SF (Figure 4F). However, patient numbers in these cohorts are too low to draw any firm conclusions, and will require larger numbers to evaluate these findings further. CD4+/CD25hi/PI16+ Treg represented 1.2 ± 0.2% (mean ± SEM) of total CD4+ cells for healthy children compared with 0.48 ± 0.05% and 0.5 ± 0.11% in PB and SF in oligoarthritis patients with JIA, respectively. We saw no correlation between the percentage of circulating or synovial CD4+/CD25hi/PI16+ Treg and disease duration, arthritis medications, or antinuclear antibodies (ANA) status. Our original hypothesis was that PI16 may be shed at the site of inflammation; however, we saw no difference in the amount of soluble PI16 in plasma compared with that in SF of patients with JIA (Figure 4C).
Studies have suggested that Th17 cells express chemokine receptors CCR2, CCR4, and CCR6 but not CXCR3 and CCR517,18,19. We have confirmed that PI16+ Treg also coexpress chemokine receptors CCR4 and CCR6. As shown in Figure 5, the coexpression of these homing chemokines on PI16+ Treg was significantly lower in PB and SF of patients with JIA compared with healthy children (p = 0.02). CCR4+/CCR6+ coexpression was present on 68 ± 7.0% (mean ± SEM) PI16+ Treg in healthy children compared with 41.1 ± 6.7% and 43.1 ± 9.7% in blood and SF of patients with JIA, respectively (Figure 5).
Migration
The ability of CD4+/CD25hi/PI16+ Treg cells from PB and SF to migrate in response to the CCR4 and CCR6 ligands CCL17 and CCL20 was tested using 5 μm-pore Transwell plates. There was no significant difference in cell viability before migration of SFMC (92%) compared with PBMC (range 83–97%) as determined by propidium iodide staining. CD4+/CD25hi/PI16+ Treg cells migrated toward CCL17, CCL20, and CCL17 + CCL20 ligands (Figure 6). After 2 h chemotaxis, the chemotactic index of PI16+ Treg cells from healthy children was 11.4 ± 2.0, 12.4 ± 3.5 and 22.1 ± 2.3, in response to ligands CCL17, CCL20, and CCL17 + CCL20, respectively (Figure 6). A similar trend was observed in JIA patients with oligoarthritis with the chemotactic index of 14.4 ± 5.4, 7.2 ± 2.0, and 20.4 ± 5.1 in response to the chemokine ligands. In contrast, these PI16+ Treg cells from the SF did not migrate in response to ligands CCL17, CCL20, and/or CCL17 + CCL20, having a chemotactic index of only 4.4 ± 1.5 (p = 0.02), 3.2 ± 2.0 (p = 0.04), and 7.2 ± 2.6 (p = 0.002), respectively (Figure 6).
DISCUSSION
Inflammation in JIA persists as a consequence of continuing leukocyte recruitment and retention within synovial tissue and fluid20. The key immunological mechanisms causing joint inflammation are still largely uncharacterized. The identification of factors involved in inducing and regulating tissue damage in JIA may provide a tool to individualize treatment in the future. To investigate whether blood or SF reflect those mechanisms, we analyzed T cells, mainly Th17 and novel PI16 Treg cells in PB and SF of patients with JIA.
Our data show a significant enrichment of memory T cells in the SF compared with PB samples. Because CD27 is considered a reliable marker of T cell effector status21,22, co-staining of CD27 and CD45RA was used to determine activation and memory status of CD4+ T cells23. Our results are consistent with previous studies in patients with autoimmune disease, which have shown that memory T cells are recruited to the site of inflammation, where they become reactivated24,25. The subsequent enrichment of effector memory T cells within JIA SF is reflected in the decrease of CD27 expression on these cells. We found a balance between memory and naive CD4+ T cells in the blood of patients with JIA, similar to recently published data on RA and healthy controls25. In contrast, SF contained more Th1-like memory T cells (CD62L–/CD45RA–)26 than the matched blood samples, suggesting an enrichment of Th1 cells within SF. Although some studies have shown a Th1 bias in the SF of patients with RA and JIA27,28,29, other studies have refuted these findings30,31.
Th17 cells have been described as the master mediators of tissue damage in a variety of autoimmune diseases, with IFN-γ inhibiting Th17 cells and protecting tissues31. On the other hand, recent data demonstrate that Th1 and Th17 cells are independently capable of inducing disease in 2 established models of autoimmunity30. The enrichment of Th1 and Th17 cells within the inflamed joints of children with JIA as presented here indicates that both cell types may contribute to joint pathology.
Highly proinflammatory IL-17-secreting CD4+ T cells (Th17) have been shown to be the major mediators in the prolongation of inflammation and the induction and persistence of joint damage in adult patients with RA4,5,6 and more recently in JIA8,32,33. However, there is evidence that RA and JIA are different diseases34. We examined 50 children with JIA and showed that IL-17-producing cells were highly enriched within SF compared with the matched PB samples. The IL-17-producing cells were uniformly contained within the T helper memory subset (CD4+/CD45RA-) and expressed the chemokine receptor CCR6, which enables Th17 cells to migrate toward sites of inflammation. Th17 cells are also enriched in the PB in RA35. We found that blood IL-17-positive cells showed a phenotype similar to IL-17-producing cells in JIA SF. However, we did not see an increased proportion of IL-17-producing cells in PB, supporting one of the few studies on Th17 cells in JIA that suggests a direct effect of IL-17-producing cells at sites of inflammation8. Because the CCR6 ligand CCL20 has been shown to be upregulated in human Th17 cells and in the SF of patients with RA or JIA, it remains to be determined whether differences in the autocrine production of CCL20 contribute to differences in Th17 recruitment among these patients.
Conflicting data have been reported regarding the phenotype of IL-17-producing cells and we therefore investigated the expression of surface molecules and intracellular cytokines in cells from the synovial fluid of patients with JIA. A majority of IL-17-producing CD4+ cells expressed the C-type lectin-like receptor CD161, previously found on natural killer and CD8 T cells36. It has been shown that human IL-17-producing cells originate from a CD161+/CD4+ precursor cell37. Notably, CD161 is expressed on resting Th17 cells that can be activated by IL-23 and mediate destructive tissue inflammation38. Therefore, the increased expression of CD161 and IL-23R on IL-17-producing cells in the SF of patients with JIA may reflect the role of CD161 in supporting activation-induced T cell expansion and tissue destruction through additional costimulatory pathways.
In our study, the majority of IL-17-producing cells also expressed the chemokine receptor CCR5. It has been reported that CCR5 is expressed on activated T cells that show Th1 characteristics39,40, but also on memory T cells and Th17+ cells in healthy adults41. Interestingly, we found low levels of macrophage inflammatory protein (CCL3) but high levels of RANTES (CCL5) in the SF of our patients with JIA. Both are ligands that bind CCR5. This result contrasts with data from patients with RA in which high levels of both CCL3 and RANTES have been reported42,43,44,45, supporting the hypothesis that JIA and RA are clinically and mechanistically distinct arthritides.
Treg, like Th17 cells, are found in high numbers in the inflamed synovium of patients with RA46 and JIA47,48,49 compared with the circulating blood. Our data confirm this enrichment with increased CD4+/CD25hi/CD127low/FOXP3+Treg cells within the SF of patients with JIA. PI16 has been described as a novel marker for regulatory T cells13,14. PI16-positive Treg cells have suppressing activity in suppressor assays and show enhanced migration toward the inflammatory chemokines CCL17 and CCL20 compared with PI16-negative Treg in healthy adults. The role of PI16 on Treg is yet to be demonstrated, but our data suggest that PI16 identifies a distinct subset of functional memory Treg with the highest expression of the Treg transcription factor FOXP3, which can migrate to sites of inflammation and regulate the proinflammatory response at those sites14.
Although there is an enrichment of Treg in inflamed joints of patients with JIA, fewer of these Treg express PI16 compared with the Treg in the periphery, and the ratio between Treg and Th17 is reduced by half in the joint compared with the blood. That means that in contrast to blood, in the inflamed joint there are few Treg and even fewer PI16+ Treg to control the increased numbers of Th17 cells to keep the balance.
Oligoarticular JIA subjects had a reduced number of PI16+ Treg cells within the blood and SF when compared with healthy children and JIA patients with ERA, PsA, and SoJIA. However, the trend of an increase in the number of PI16+ Treg cells in patients with ERA, JPsA, and SoJIA has not been statistically analyzed because of the lack of sufficient patient numbers for each subtype. Further, we did not observe a correlation between the percentage of circulating or synovial PI16+ Treg cells and disease duration, medication, or ANA status in children with oligoarthritis.
Our original hypothesis was that PI16 might be shed within active joints or during the process of migrating toward joints. This hypothesis is not supported by the data, with similar levels of soluble PI16 observed within the plasma of healthy children and that of patients with JIA, as well as in the SF of patients with JIA. However, the degradation or loss of soluble PI16 from inflamed tissue has yet to be investigated.
We have shown that PI16+ Treg cells express the same chemokine receptors as Th17 cells (CCR4/CCR6), indicating similar homing characteristics. PB Treg from patients with JIA migrated toward inflammatory ligands CCL17 and CCL20, unlike those from SF of the same patients. We have shown that the failure of SFMC migration toward CCL17 and CCL20 is not due to poor viability but may be part of a generalized chemotactic defect. Nonetheless, we hypothesize that circulating PI16+ Treg and Th17 cells are capable of migrating to the same sites of inflammation, with the Treg controlling the Th17 immune response. A defect or reduced number of PI16+ Treg, on the other hand, might contribute to autoimmunity and consequently to tissue damage.
Our data show that IL-17-producing cells are enriched in the SF of patients with JIA compared with PB, supporting the hypothesis that IL-17-producing cells contribute to the tissue damage within active joints. In contrast to adult RA, our results show that PB does not reflect processes in JIA SF and therefore is less likely to be useful for disease diagnosis or prognosis. We hypothesize that in individuals who are able to bring inflammation under control, there is a balance between PI16-positive Treg and Th17 cells homing to the same sites of inflammation, with the Treg controlling the Th17 immune response. A reduced number of PI16-positive Treg within active joints of patients with JIA may contribute to their inability to regulate disease activity and consequently result in tissue damage. Therapeutic interventions to downregulate Th17 cells in the joint, or to increase the recruitment of functional (PI16+) Treg to the joint may provide a significant therapeutic benefit for patients with JIA.
Footnotes
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Supported by the Co-operative Research Centre (CRC) for Biomarker Translation, Australia.
- Accepted for publication June 4, 2012.
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