Rheumatoid arthritis (RA) is a chronic inflammatory disorder characterized by autoimmunity, infiltration of the joint synovium by activated inflammatory cells, and progressive destruction of cartilage and bone. Although the exact cause of RA is not clear, T cells seem to play a crucial role in the initiation and perpetuation of the chronic inflammation in RA.
The Th1 cell subset has long been considered to play a predominant role in inflammatory arthritis, because T cell clones from RA synovium were found to produce large amounts of interferon-γ (IFNγ) (1). Recently, interleukin-17 (IL-17)–producing Th17 cells have been identified, and this newly discovered T cell population appears to play a critical role in the development of various forms of autoimmune arthritis in experimental animals, such as those with glucose-6-phosphate isomerase–induced arthritis (2) and collagen-induced arthritis (CIA) (3). Conversely, IFNγ has antiinflammatory effects on the development of experimental arthritis (4, 5). IL-17 is spontaneously produced by RA synovium (6), and the percentage of IL-17–positive CD4+ T cells was increased in the peripheral blood mononuclear cells of patients with RA compared with healthy control subjects (7). It is therefore necessary to determine if autoimmune arthritis is a Th1- or a Th17-associated disorder.
The lineage commitment of each Th cell subset from naive CD4+ T cells is dependent on the expression of specific transcription factors induced under the particular cytokine environment. Differentiation of Th1 cells is dependent on the expression of the transcription factor T-bet, which is induced by IFNγ/STAT-1 signaling pathways and directly activates the production of IFNγ (8, 9). Similarly, Th17 cell differentiation in mice is dependent on the transcription factor retinoic acid receptor–related orphan nuclear receptor γt (RORγt) induced by transforming growth factor β (TGFβ) and IL-6 (10). Previous studies showed that these transcription factors negatively regulate the differentiation of other T cell subsets by direct co-interaction and/or indirect effects of cytokines produced from each T cell subset (11, 12). How the predominant differentiation of CD4+ T cells affects the development of autoimmune arthritis remains unclear, however.
In the present study, CIA was induced in C57BL/6 (B6) mice and T-bet–transgenic (Tg) mice under the control of the CD2 promoter. The results showed that CIA was significantly suppressed in T-bet–Tg mice as compared with B6 mice. IL-17 production was not detected in type II collagen (CII)–reactive T cells from T-bet–Tg mice, and a significant reduction in IL-17 production by CII-reactive CD4+ T cells from T-bet–Tg mice was observed even when they were cocultured with splenic dendritic cells (DCs) from B6 mice. IFNγ production was also reduced in T-bet–Tg mice as compared with B6 mice, and levels of IFNγ in CII-reactive CD4+ T cells from T-bet–Tg mice were not different from those in B6 mice. Inhibition of Th17 cell differentiation and predominant differentiation of Th1 cells under an in vitro condition favoring Th17 cell differentiation was observed in T-bet–Tg mice, and surprisingly, this inhibition was also observed in T-bet–Tg/IFNγ−/− mice. These results indicate suppression of Th17 cell differentiation by overexpression of T-bet, but not IFNγ. Our findings support the notion that the suppression of autoimmune arthritis in T-bet–Tg mice might be due to the direct inhibition of Th17 cell differentiation by T-bet overexpression in T cells.
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Recent studies showed that IL-17 plays a crucial role in the development of CIA (3) and other types of experimental arthritis (2). In contrast, it has been reported that IFNγ can suppress IL-17 production in vitro (16) and has antiinflammatory effects on the development of experimental arthritis (4, 5). T-bet is a transcription factor known to induce the differentiation of naive CD4+ T cells to Th1 cells (8). Although the absence of T-bet can result in severe IL-17–mediated experimental autoimmune myocarditis via dysregulation of IFNγ (17), several studies have shown that T-bet is essential for the development of several models of autoimmunity, such as experimental autoimmune encephalitis (18, 19), colitis (20), and diabetes mellitus (21). Nevertheless, the effect of T-bet expression on Th17 cell differentiation and function during arthritis remains unclear.
T-bet–Tg mice overexpress T-bet and mainly produce IFNγ in their T cells (14). Previous studies in T-bet–Tg mice suggested that overexpression of T-bet and a predominant Th1 response affect the pathogenesis of various diseases (14, 22, 23). To examine whether T-bet overexpression on T cells affects the regulation of autoimmune arthritis, we induced CIA in T-bet–Tg mice and found marked suppression of CIA in T-bet–Tg mice.
To determine the reason for the low incidence of CIA in T-bet–Tg mice, we measured CII-reactive cytokine production and expression in vitro. IL-17 production from CII-reactive CD4+ T cells and Il17a expression were reduced in T-bet–Tg mice as compared with B6 mice. Although a predominant Th1 cell response was reported by Ishizuka et al (14), CII-specific IFNγ production was reduced in T-bet–Tg mice, and no significant difference was observed in Ifng expression between B6 mice and T-bet–Tg mice. Furthermore, Il12a expression was significantly higher in T-bet–Tg mice than in B6 mice, suggesting that overexpression of T-bet on T cells seems to affect innate immune cells, because the main producers of IL-12 are DCs and macrophages, not CD4+ T cells.
In criss-cross coculture experiments with CD4+ T cells and splenic DCs from B6 mice and T-bet–Tg mice, CII-reactive IL-17 production was also reduced even when CD4+ T cells from T-bet–Tg mice were cocultured with DCs from B6 mice, although there was no significant difference in IL-17 production by CD4+ T cells from B6 mice cocultured with DCs from either B6 mice or T-bet–Tg mice. In contrast, no difference in IFNγ production was observed under all coculture conditions examined. Moreover, suppression of RORγt expression and high expression of T-bet on CD4+ T cells were observed even when CD4+ T cells from T-bet–Tg mice were cocultured with DCs from B6 mice. These findings indicate that T-bet overexpression on CD4+ T cells might suppress CII-reactive IL-17 production resulting from suppression of RORγt expression in an IFNγ-independent manner, and that overexpression of T-bet has no direct effect on DC function.
CII-specific IgG levels correlate well with the development of arthritis (15). We observed significant suppression of CII-specific IgG production in the T-bet–Tg mice as compared with the B6 mice. A previous study showed that IL-17 is required for anti-CII antibody production (3). Therefore, the suppression of anti-CII antibody formation might be due to lower CII-reactive IL-17 production in T-bet–Tg mice.
To evaluate the low cytokine response to CII in T-bet–Tg mice, we analyzed lymphocytes obtained after immunization from draining lymph nodes and spleen. The percentage and absolute number of T cells tended to be lower in both the draining lymph nodes and spleen of T-bet–Tg mice compared with B6 mice. Moreover, significantly lower numbers of total thymocytes and an abnormal proportion of T precursor cells were observed in T-bet–Tg mice. The latter phenomenon could be due to T-bet transgene expression on double-negative thymic cells in T-bet–Tg mice. Because previous observations showed that T-bet interferes with GATA-3 function (11) and that GATA-3 was required for the development of early thymic T cells (24), one of the reasons for abnormal T cell development in the thymus might be the dysfunction of GATA-3 by overexpression of T-bet. These results suggest that overexpression of T-bet in thymic T cells affects T cell development, is responsible for the low number of T cells in spleen and lymph nodes, and is related to the low cytokine production against CII in T-bet–Tg mice.
To assess the effect of T-bet on CD4+ T cell differentiation in T-bet–Tg mice, we performed in vitro induction of Th17 cells. Analysis of T-bet–Tg mice showed a reduction in IL-17–producing CD4+ T cells and an increase in IFNγ-producing CD4+ T cells in spite of the condition favoring Th17 differentiation, which indicates suppression of Th17 cell differentiation and predominance of Th1 cell differentiation in vitro in T-bet–Tg mice. These results did not contradict the previous findings that the phenotype of polarized Th1 cells was not affected by Th cell–polarizing conditions (25). It is possible that suppression of CII-reactive IL-17 production in T-bet–Tg mice was not associated with IFNγ. For this reason, we generated T-bet–Tg/IFNγ−/− mice and performed in vitro induction of Th17 cells in these mice. Surprisingly, in T-bet–Tg/IFNγ−/− mice, the levels of IL-17–producing CD4+ T cells were also markedly reduced under Th17 cell differentiation–favoring conditions, indicating an IFNγ-independent suppressive pathway against Th17 cell differentiation. Although previous studies showed that suppression of Th17 cell differentiation was mediated through IFNγ signal transduction (16), our findings allow us to propose a new hypothesis: Th17 cell differentiation is regulated by a pathway that is distinct from the IFNγ signaling pathway. Therefore, we suggest that T-bet expression either directly or indirectly suppresses Th17 cell differentiation via an IFNγ-independent mechanism.
Tbx21 expression was significantly higher in T-bet–Tg mice as compared with B6 mice, and FACS analysis of CII-reactive CD4+ T cells revealed a significantly higher percentage of T-bet+ cells among the CD4+ T cell subset in T-bet–Tg mice. While there was no significant difference in the percentage of RORγt+ cells among the CD4+ T cell subset in T-bet–Tg mice as compared with B6 mice, Rorc expression was down-regulated on CII-reactive CD4+ T cells in T-bet–Tg mice. In the case of CD4+ T cells under conditions favoring Th17 cell differentiation, RORγt expression on CD4+ T cells from T-bet–Tg mice was lower than that on cells from B6 mice. Interestingly, most of the RORγt+ cells also expressed T-bet in T-bet–Tg mice, and the proportion of IL-17–producing RORγt+ T cells in the CD4+ cell subset was lower in T-bet–Tg mice than in B6 mice. These findings support the notion that overexpression of T-bet not only suppresses RORγt expression on CD4+ T cells, but also inhibits the production of IL-17 from RORγt+ T cells.
Previous studies showed that RORγt expression is positively regulated by several transcription factors, such as runt-related transcription factor 1 (RUNX-1), interferon regulatory factor 4, and STAT-3 (26–28). Lazarevic et al (29) recently reported that T-bet prevented RUNX-1–mediated activation of the gene encoding RORγt, followed by the suppression of Th17 cell differentiation. In addition to direct promotion of RORγt expression, RUNX-1 also acts as a coactivator, together with RORγt, and induces the expression of Il17a and Il17f (26); therefore, T-bet inhibits IL-17 production by RORγt+ cells induced by RUNX-1 (29). Although further studies will be required to identify the effect of T-bet overexpression on the function of RUNX-1, it might be associated with the suppression of Th17 cell differentiation that was observed in the T-bet–Tg mice.
In conclusion, our results demonstrated that overexpression of T-bet in T cells suppressed the development of autoimmune arthritis. The regulatory mechanism of CIA might involve dysfunction of CII-reactive Th17 cell differentiation by overexpression of T-bet via IFNγ-independent pathways. These findings should enhance our understanding of the pathogenesis of autoimmune arthritis and help in the development of new therapies for RA.
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- MATERIALS 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. Sumida 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. Sugihara, Hayashi, Yoh, Takahashi, Matsumoto, Sumida.
Acquisition of data. Kondo, Yao, Tahara.
Analysis and interpretation of data. Kondo, Iizuka, Wakamatsu, Tsuboi, Matsumoto.