A recently described subset of CD4+ T helper cells, referred to as Th17 cells, has been suggested to be pathogenic in several murine models of chronic inflammatory disorders, such as experimental autoimmune encephalomyelitis (1), collagen-induced arthritis (2), and inflammatory bowel disorders (3–8). Th17 cells have also been described in humans, and these cells have been found to be at least partially different from murine Th17 cells (9, 10).
Of particular note, human Th17 cells express molecules that are distinct from those expressed by Th1 cells, such as interleukin-23 receptor (IL-23R) and retinoic acid receptor–related orphan receptor C (RORC). However, there are also other molecules that are shared with Th1 cells, such as IL-12Rβ2 and T-box protein 21 (10). Moreover, in IL-17A–producing human cells, at least part of the cells were found to also produce interferon-γ (IFNγ) (referred to as Th17/Th1 cells), and both Th17 and Th17/Th1 cells exhibit plasticity toward Th1 cells after culturing in the presence of IL-12 (10). Furthermore, human Th17 cells originate in response to stimulation from IL-1β and IL-23, without any critical need for transforming growth factor β (11). More recently, we and other investigators (12, 13) have shown that virtually all human memory Th17 cells are contained within the CD161+ cell fraction of circulating and tissue-infiltrating CD4+ T cells, and that they originate from CD161+ precursors present in umbilical cord blood and newborn thymus (12, 14).
Juvenile idiopathic arthritis (JIA), the most common form of persistent arthritis in children, is a broad term describing a clinically heterogeneous group of arthritides of unknown cause, the onset of which typically begins before age 16 years (15). Several immunologic abnormalities have been characterized both in patients with rheumatoid arthritis (RA) (16) and in children with JIA (17, 18). Many of these abnormalities are common to both diseases, whereas others are related to a specific disease subtype. The synovial membrane shows pronounced infiltration of mononuclear cells, including T cells, B cells, macrophages, dendritic cells, and plasma cells (19, 20). T cell infiltrates predominantly consist of Th1-skewed cells, which were thought to have a central role in disease pathogenesis (21). After the association between Th17 cells and several human autoimmune diseases, including RA, was observed, some studies found increased levels of IL-17A, as well as the presence of Th17 cells and the expression of their transcription factor, RORC, in the synovial fluid (SF) of children with JIA (22–26).
In this study, we assessed the phenotypic and functional features of SF CD4+ T cells from the affected joints of children with oligoarticular-onset JIA, and compared these features to those of circulating CD4+ T cells from the same patients and to those of peripheral blood mononuclear cells (PBMCs) from healthy children. We found an accumulation of CD4+CD161+ T cells producing both IL-17A and IFNγ, which probably originated from the shift of Th17 cells to the production of IFNγ, in the SF of children with JIA, and this finding correlated with parameters of disease activity, suggesting that these cells play a major role in the inflammatory processes of JIA.
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The results of this study demonstrate an accumulation of CD4+ T cells that have the capacity to produce IFNγ (Th1 cells) or both IFNγ and IL-17A (Th17/Th1 cells), but not IL-17A alone (Th17 cells), in the SF of children with oligoarticular-onset JIA, when compared to cells from the PB. These findings are partially in agreement with the results reported in other investigations (22–24), in which increased levels of IL-17A, IL-17A–producing cells, and RORC have been demonstrated in the SF of children with JIA. In addition, we also observed an enrichment for CD4+CD161+ T cells in the JIA SF, the majority of which exhibited either the Th17/Th1 phenotype or the nonclassic Th1 (RORC+IFNγ+IL-17−) phenotype.
In order to explain the minimal presence of Th17 cells in the SF of children with JIA, as well as the prevalence of Th17/Th1 cells and, even more, of Th1 cells (part of which expressed CD161), we hypothesized that a shifting of Th17 cells to the production of IFNγ could occur in the SF microenvironment. Accordingly, while the majority of purified Th17 cells from the PB of both healthy children and children with JIA maintained their cytokine profile after in vitro culture, virtually all of the cells present in the JIA SF spontaneously shifted, under the same experimental conditions, to the Th1 profile.
Of note, we found significantly increased levels of IL-12 in the SF of JIA patients when compared to the plasma levels of IL-12 in the same patients. Moreover, Th17 cells from the PB of healthy children were induced to shift to Th1 cells when cultured in vitro in the presence of JIA SF, to an extent similar to that induced by culturing of the cells in the presence of recombinant IL-12. More importantly, this effect was completely reversed by a neutralizing anti–IL-12 mAb, strongly suggesting that the shift was related mainly to the activity of IL-12 present in the SF. Accordingly, a trend toward a positive correlation, although not statistically significant (P < 0.1), between SF levels of IL-12 and the proportions of CD161+ Th17/Th1 cells or CD161+ Th1 cells was observed. In contrast, culturing CD161+ Th1 cells in the presence of IL-1β plus IL-23, both of which are cytokines involved in the differentiation of Th17 cells (12), never induced the cells to shift to the production of IL-17A (results not shown). These findings are in agreement with our previous results obtained by assessing the shifting ability of human Th17 and Th1 clones that had been generated from the intestinal mucosa of patients with Crohn's disease (10).
Finally, and most importantly, spectratyping analysis demonstrated that both Th17 cells and nonclassic Th1 cells in the SF exhibited a similar TCRBV repertoire, despite their different cytokine profiles. This finding was strengthened by our assessment, at the clonotype level, of 3 TCRBV families that were found to be expanded. These findings, together with the demonstration that CD4+CD161+ T cells that produce both IL-17A and IFNγ can only originate from Th17 cells, but not from Th1 cells, strongly suggest that CD161+ Th17/Th1 cells, as well as CD161+ Th1 cells, present in the SF of children with JIA are derived from CD161+ Th17 cells. The late plasticity of Th17 cells to Th1 cells was recently observed in a mouse study, in which the effect was found to be related to the activity of IL-12, or the prolonged exposure to IL-23, on Th17 cells (36). Furthermore, similar results were recently reported by Nistala et al (37), who showed Th17 plasticity to Th1 driven by the inflammatory environment in human autoimmune arthritis. Very recently, the instability of the Th17 phenotype has been definitively demonstrated, at a genetic level, in mice (38).
Another interesting finding emerging from this study was the demonstration that the proportions of total CD4+CD161+ T cells, as well as that of CD4+CD161+ T cells producing both IL-17A and IFNγ (Th17/Th1 cells), present in the SF of affected joints correlated with the ESR and serum levels of CRP in the same patients. This suggests that CD4+CD161+ T cells and CD4+CD161+ Th17/Th1 cells may play a role in the activity of the disease. Moreover, the demonstration that CD4+CD25+FoxP3+ T cells are significantly enriched in the CD161− cell fraction, in comparison with that in the CD161+ cell fraction, is consistent with this interpretation.
At present, the reason that CD4+CD161+ T cells show increased expression in the SF of JIA patients is unclear. One possibility is that naive CD4+CD161+CCR6+ T cells that behave as precursors of Th17 cells (12) are recruited in the joints at early stages of the disease, wherein they differentiate into Th17, Th17/Th1, and, finally, Th1 cells, in response to environmental cytokines. Another possibility is that the memory-type subpopulation of CD4+CD161+CCR6+ Th17 cells are recruited to the inflamed joints, wherein they differentiate into Th17/Th1 cells and then into Th1 cells, in response to IL-12. Consistent with both of these hypotheses, one study showed that CCL20, the natural ligand of CCR6, is produced by SF monocytic cells and accumulates in the SF of JIA patients (39).
The respective role of Th17, Th17/Th1, and Th1 cells in the pathogenesis of chronic inflammatory disorders is still debated. It has indeed been shown that Th17 cells can promote pancreatic inflammation, but they only induce type 1 diabetes mellitus (DM) efficiently in lymphopenic mice after their conversion into Th1 cells (40). Accordingly, highly purified Th17 cells from BDC2.5NOD mice shift into Th1-like cells in NOD/SCID recipient mice. More importantly, the development of type 1 DM was prevented by treatment with anti-IFNγ–specific antibodies, but not with anti–IL-17A–specific antibodies (41). Moreover, in the murine model of the autoimmune disorder known as proteoglycan-induced arthritis, Th1 cells, but not Th17 cells, are pathogenic (42).
The results of this study strongly support the concept that among CD4+ T cells, the subset of CD161+ T cells are the most important in maintaining the inflammatory process, and that within this cell subset, a shifting may occur from Th17 to Th17/Th1 cells, and even to nonclassic Th1 cells, which is probably related to the activity of IL-12, even if other, still-unidentified cytokines present in the inflammatory microenvironment might be involved. Thus, it is reasonable to suggest that this family of CD161+ T cells is different from classic CD161− Th1 cells, probably because of its ability to produce other proinflammatory components that still need to be identified.
The debate on the respective role of Th17, Th17/Th1, and Th1 cells in the pathogenesis of JIA is particularly important for the development of possible biologic therapeutic strategies. In this view, it has recently been suggested that the neutralization of IL-17 could be a potential novel goal for the treatment of immune-mediated arthritides, by showing that a humanized anti–IL-17 mAb added to oral disease-modifying antirheumatic drugs improved the signs and symptoms of RA in patients (43). In contrast, ustekinumab, a humanized mAb that inhibits receptor-binding of IL-12 and IL-23, reduced the signs and symptoms of psoriatic arthritis (44), suggesting that both Th1 and Th17 cells cooperate in disease pathogenesis. Our results support this latter observation and identify CD161 as a link between Th17, Th17/Th1, and part of Th1 cells. This finding provides the opportunity to interfere, at the same time, with all of the cell populations, whose presence in the SF from affected joints seems to be related to biologic parameters of disease activity, thus prompting us to hypothesize that targeting of CD161 may represent a possible approach for the treatment of JIA.
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- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Romagnani had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Cosmi, Cimaz, L. Maggi, Santarlasci, Liotta, E. Maggi, Romagnani, Annunziato.
Acquisition of data. L. Maggi, Santarlasci, Capone, Borriello, Frosali, Querci, Simonini, Barra, Piccinni.
Analysis and interpretation of data. Cosmi, Liotta, De Palma, E. Maggi, Romagnani, Annunziato.