Rheumatoid arthritis (RA) is characterized by autoimmunity, synovial hyperplasia, progressive destruction of cartilage and bone, neoangiogenesis, and infiltration of joint synovium by activated inflammatory leukocytes, such as Th1 lymphocytes and monocytes. However, in the synovium of RA patients, it is difficult to detect the Th1 cytokine interferon-γ (IFNγ), whereas relatively high expression levels of interleukin-17A (IL-17A) can be detected (1). Since IL-17A is a T cell factor produced by the novel T helper cell subset Th17, the question arises whether RA is a Th1-associated or Th17-associated disorder. Identification and subsequent modulation of the pathogenic T helper cell subset is a major goal in the treatment of RA.
Classically, T helper cells have been divided into 2 different subsets, namely, Th1 and Th2. Th1 cells are associated with the elimination of intracellular pathogens. In contrast, Th2 cells are characterized by the production of IL-4, IL-5, and IL-13 and are involved in the eradication of parasitic worms and the development of allergic responses. Differentiation of Th1 cells from naive CD4+ T cells is dependent on the IL-12/STAT-4 and IFNγ/STAT-1/T-bet signaling pathways. Th2 cell differentiation is dependent on IL-4 signaling, via the activation of STAT-6, resulting in the expression of the transcription factor GATA-3. GATA-3 further stabilizes the differentiation of Th2 cells by autoactivating its own expression and by increasing the accessibility of the Th2 cytokine cluster (2, 3).
Because Th1 and Th2 cells appear to arise in a mutually exclusive manner, various mechanisms of counter-regulation exist. It has been reported that GATA-3 negatively regulates the differentiation of Th1 cells by down-regulating STAT-4 and IL-12 receptor β2 (4). Furthermore, differentiation of Th1 and Th2 cells is regulated by the interchromosomal association of Th1 and Th2 loci (5). However, T-bet interferes with the binding of GATA-3 to its target DNA through a physical interaction with GATA-3 (6). The function of GATA-3 as a principal Th1/Th2 switch was shown in studies of conditional gene targeting, in which deletion of GATA-3 was sufficient to induce differentiation of Th1 cells in the absence of IL-12 and IFNγ. Moreover, GATA-3 deficiency in Th2 cells resulted in the reduced maintenance and diminished responsiveness of Th2 cells (7, 8). In addition to its regulatory role during differentiation of Th1 cells, GATA-3 acts as a negative regulator of Treg cells by directly inhibiting the expression of the Treg cell–associated factor FoxP3 (9).
Furthermore, GATA-3 is required for embryogenesis, which has been shown in studies of targeted GATA-3 gene disruption in which embryonic lethality was observed at day 11 after gestation (10). Moreover, GATA-3 plays essential roles throughout the phases of T cell development, including effects on the early stages of differentiation of the CD4 and CD8 double-negative T cells, selection of T cell receptor β (TCRβ), and selection of CD4+ T cells (11–15).
In the past few years, a new T helper cell subset has been identified. This subset produces IL-17 and is therefore referred to as Th17 (16, 17). IL-17 is a proinflammatory cytokine produced by activated CD4+ T cells and has been implicated in a range of autoimmune diseases, including RA, multiple sclerosis, and psoriasis (1, 18, 19). In mouse models of arthritis, IL-17 expression has been implicated in the development of inflammation and bone destruction (20–24).
Induction of the differentiation of murine Th17 cells is critically dependent on the functions of transforming growth factor β (TGFβ), IL-6, and the transcription factors retinoic acid–related orphan receptor α (RORα), RORγt, and interferon regulatory factor 4. Additionally, the cytokines IL-21 and IL-23 have been reported to promote IL-17 expression. JAK-STAT signaling, via the phosphorylation of STAT-3, is required to induce IL-17 expression. STAT-3 phosphorylation is, in turn, negatively regulated by suppressor of cytokine signaling 3 (SOCS-3) (for review, see ref. 25).
As mentioned above, various mechanisms, acting through GATA-3, exist to regulate the differentiation of Th1 and Th2 cells. However, it is still unclear whether GATA-3 influences the differentiation and functions of Th17 cells in chronic inflammatory diseases. In this study, we investigated the effects of GATA-3 expression on the function of IL-17–producing cells in experimental arthritis. For this purpose, arthritis was induced in CD2 T cell–specific GATA-3 (CD2–GATA-3)–transgenic mice, an experimental model in which expression of GATA-3 is controlled by the human CD2 promoter and the locus-control region, resulting in enforced GATA-3 expression throughout T cell development (26).
Our results show that enforced GATA-3 expression during the progression of methylated bovine serum albumin (mBSA)–induced arthritis in mice leads to a striking suppression of severe joint inflammation and bone erosion. Interestingly, we found that this effect was associated with a remarkable reduction in the fraction of IL-17–producing CD4+ T cells, but not IFNγ-producing CD4+ T cells, isolated from the spleens and draining lymph nodes. Furthermore, we observed an enhanced capacity of Th2 cytokine expression and a reduced expression of Th17-associated genes in the transgenic mice. In addition, IL-17 expression was even more reduced in the peripheral CD4+ T cells isolated from the inflamed knee joints of transgenic mice. Taken together, these findings show that enforced GATA-3 expression in T cells results in a reduction in severe joint-destructive inflammation in mice, and this occurs in conjunction with reduced Th17 cell differentiation, but no reduced Th1 cell differentiation, both systemically and locally in the inflamed knee joints.
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The transcription factor GATA-3 is a master regulator of differentiation of Th2 cells and expression of Th2 cytokines. A lot of knowledge has been gained with regard to the role of GATA-3 in the regulation of Th1 and Th2 cell differentiation. However, the effect of GATA-3 expression on Th17 cell differentiation and function during arthritis is unclear.
In this report, we demonstrate that enforced T cell–specific expression of GATA-3 in an experimental arthritis model suppresses severe joint inflammation and bone erosion, and this is associated with reduced Th17 cell differentiation. Wild-type mice developed severe arthritis, in which joint inflammation was associated with bone erosions and massive infiltration of inflammatory cells, whereas only mild joint inflammation was observed in CD2–GATA-3–transgenic mice. This suppressed joint inflammation was associated with reduced IL-17 expression by CD4+ T cells obtained from the spleen, draining lymph nodes, and synovium.
Surprisingly, enforced GATA-3 expression had no suppressive effect on IFNγ; instead, GATA-3 overexpression moderately enhanced the expression of IFNγ by CD4+ T cells. This was also reflected in the enhanced gene expression of Th1-associated factors such as T-bet, STAT-1, and STAT-4 in sorted splenic effector T cells. However, the coexpression profile of a large fraction of CD4+ T cells, which coexpressed both IL-4 and IFNγ at the single-cell level (Figure 4A), has to be taken into account, which makes it hard to judge whether these cells are true Th1 cells. In addition, involvement of the strong Th1-skewing potential of CFA may explain the inability of GATA-3 to suppress the expression of IFNγ, which is indeed the case when CD2–GATA-3–transgenic CD4+ T cells are cultured in vitro (26). However, the fact that expression of IFNγ is elevated in CD2–GATA-3–transgenic mice, while IL-17 expression is suppressed, indicates that the induction of arthritis in mBSA-immunized mice is mediated by Th17 cells, rather than by Th1 cells. A similar observation in support of the critical role of IL-17–producing CD4+ T cells was made in models of autoimmune collagen-induced arthritis (20, 42).
The role of IL-17+IFNγ+ double-positive cells in the pathology of arthritis is still unclear. These cells have been found in studies of collagen-induced arthritis (ref. 20 and Lubberts E, et al: unpublished observations), as well as in studies of patients with early RA (Colin EM, et al: unpublished observations). In the present study, we could hardly detect expression of both IL-17 and IFNγ in single CD4+ T cells of CD2–GATA-3–transgenic mice, whereas substantial fractions of these double-positive cells were found in wild-type mice. This might indicate a pathogenic potential of IL-17+ IFNγ+CD4+ T cells in arthritis.
The finding of a large fraction, ∼17%, of wild-type joint-infiltrating CD4+ T cells that expressed IL-17 indicates the local function of IL-17 expression in joint inflammation. In particular, in these joint-infiltrating CD4+ T cells, enforced GATA-3 expression significantly suppressed IL-17 expression. However, the results of this study do not reveal whether the reduced IL-17A–producing T cells in the inflamed synovium could be attributed to local regulation of IL-17 expression or to less migration of these IL-17–producing cells to the site of joint inflammation or to both.
Moreover, enforced GATA-3 expression greatly enhanced the expression of IL-4 in these cells. It has been described that IL-4 has a suppressive potential on destructive arthritis, and that IL-4 can inhibit the differentiation of Th17 cells (43). However, treatment of mBSA-immunized mice with high doses, of ∼1.2 mg, of neutralizing anti–IL-4 mAb per mouse did not increase joint inflammation or IL-17 production in CD2–GATA-3–transgenic mice (results not shown). Although we cannot exclude the possibility that not all of the IL-4–expressing population is neutralized by this antibody treatment, these data suggest that the protection against severe arthritis and prevented expansion of Th17 cells may not be fully dependent on IL-4.
The reduced expression of IL-17 by CD2–GATA-3–transgenic CD4+ T cells could possibly be the result of an effect on the expression of Th17 cell–associated factors. Analyses of gene expression indicated a suppressed gene expression of RORγt in sorted effector CD4+ T cells, while expression of STAT-3 was enhanced. However, gene expression of SOCS-3, a negative regulator of STAT-3 phosphorylation, was enhanced as well. In addition, enforced GATA-3 expression resulted in reduced gene expression of TNFα, but not of IL-21 and IL-22.
It has been described that GATA-3 is negatively regulated by TGFβ, which is required for differentiation of Th17 and Treg cells (44). This negative regulation in Treg cells is possibly required to inhibit GATA-3 expression, as revealed in its capacity to directly inhibit FoxP3 expression (9). A speculative hypothesis could be that a similar mechanism is present in Th17 cells, whereby GATA-3 is capable of directly or indirectly inhibiting the differentiation of Th17 cells by repressing the expression of RORγt. Future experiments are required to unravel the direct or indirect effects of GATA-3 on RORγt expression.
Enforced GATA-3 expression was not sufficient to provide complete suppression of joint inflammation nor could it provide complete blockade of IL-17 expression. In particular, at day 1 after arthritis induction, no differences between CD2–GATA-3–transgenic and wild-type mice were observed in terms of joint inflammation and IL-17 expression. This might suggest that GATA-3 is not capable of inhibiting the induction of Th17 cells, but that GATA-3 is more likely to be involved in the regulation of the survival or pathogenic function of Th17 cells.
Taken together, the results in this report demonstrate the suppressive capacity of enforced GATA-3 expression on severe joint inflammation and bone erosion during murine arthritis, and show that this effect is associated with inhibited Th17 cell differentiation. These findings indicate that selective modulation of specific transcription factors (for example, up-regulation of GATA-3 or down-regulation of RORγt) might lead to new therapeutic applications, which may improve on the currently available therapies and eventually even help us reach the goal of preventing the development of chronic inflammatory diseases such as RA.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
Dr. Lubberts 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 design. Van Hamburg, Boon, Hendriks, Lubberts.
Acquisition of data. Van Hamburg, Mus, de Bruijn, Vogel, Boon, Cornelissen, Asmawidjaja.
Analysis and interpretation of data. Van Hamburg, Mus, de Bruijn, Cornelissen, Hendriks, Lubberts.
Manuscript preparation. Van Hamburg, Boon, Hendriks, Lubberts.
Statistical analysis. Van Hamburg.