Regulatory T cells (Treg cells) maintain immune homeostasis by limiting inflammatory responses. SOCS1 (suppressor of cytokine signaling 1), a negative regulator of cytokine signaling, is necessary for the suppressor functions of Treg cells in vivo, yet detailed mechanisms remain to be clarified. We found that Socs1−/− Treg cells produced high levels of IFN-γ and rapidly lost Foxp3 when transferred into Rag2−/− mice or cultured in vitro, even though the CNS2 (conserved noncoding DNA sequence 2) in the Foxp3 enhancer region was fully demethylated. Socs1−/− Treg cells showed hyperactivation of STAT1 and STAT3. Because Foxp3 expression was stable and STAT1 activation was at normal levels in Ifnγ−/−Socs1−/− Treg cells, the restriction of IFN-γ–STAT1 signaling by SOCS1 is suggested to be necessary for stable Foxp3 expression. However, Ifnγ−/−Socs1−/− Treg cells had hyperactivated STAT3 and higher IL-17A (IL-17) production compared with Ifnγ−/−Socs1+/+ Treg cells and could not suppress colitis induced by naive T cells in Rag2−/− mice. In vitro experiments suggested that cytokines produced by Socs1−/− Treg cells and Ifnγ−/−Socs1−/− Treg cells modulated antigen-presenting cells for preferential Th1 and Th17 induction, respectively. We propose that SOCS1 plays important roles in Treg cell integrity and function by maintaining Foxp3 expression and by suppressing IFN-γ and IL-17 production driven by STAT1 and STAT3, respectively.
A variety of pathologies of autoimmune diseases and allergic diseases are caused by the immune responses to self, environmental nonmicrobial antigens, and infectious agents. Regulatory T cells (Treg cells), which are characterized by expression of the Forkhead transcription factor, Foxp3, play an indispensable role in immunological tolerance, protecting the host from excessive immune responses (Hori et al., 2003; Sakaguchi, 2004; Sakaguchi et al., 2008; Belkaid and Tarbell, 2009). Foxp3 plays an essential role in the suppressive function of Treg cells (Wan and Flavell, 2007), and Foxp3 deficiency causes a multiorgan autoimmune disease as can be observed in the scurfy mouse and in patients with IPEX (immunodysregulation polyendocrinopathy enteropathy X-linked syndrome; Bennett and Ochs, 2001; Bennett et al., 2001; Brunkow et al., 2001). Foxp3 induction in natural Treg cells (nTreg cells) occurs in vivo during thymic differentiation, under the influence of relatively high avidity interactions of the TCR with self-antigens.
Although the suppression of autoimmunity by Treg cells is now well established, recently, nTreg cells have been shown to convert to effector/helper T cells (Komatsu et al., 2009). Although most Treg cells retain high Foxp3 expression after adoptive transfer to a nonpathogenic setting, 10–15% of Treg cells were found to lose Foxp3 expression after adoptive transfer into lymphopenic hosts. A recent study showed that half of Treg cells transferred into lymphopenic hosts did not die, but rather began producing IL-2 and IFN-γ (Komatsu et al., 2009). Additionally, multiple recent studies have demonstrated that in the inflammatory settings of autoimmunity, there is a loss of Foxp3 during inflammatory responses (Zhou et al., 2009; Murai et al., 2010). The adoptive transfer of Treg cells into Cd3ε−/− hosts, which retain B lymphocytes, resulted in the loss of Foxp3 expression and the generation of lapsed Treg cells that differentiated into follicular helper T cells in Peyer’s patches that promoted IgA class switching (Tsuji et al., 2009). Such exFoxp3 cells (Zhou et al., 2009) or lapsed Treg cells (Murai et al., 2010) develop an effector-memory phenotype, produce pathogenic cytokines, and can trigger the development of autoimmunity. Two possibilities were proposed regarding the developmental plasticity of Treg cells: (1) the lineage reprogramming of the conversion of committed Foxp3+ cells to Foxp3− cells or (2) the expansion of uncommitted Treg cells, which easily lose Foxp3 (Hori, 2010). In any case, the molecular basis for such Treg cell conversion and the signals that ensure the stability of Treg cells have not yet been clarified. On the contrary, recent work by Rubtsov et al. (2010) reported that highly purified Treg cells were very stable under physiological and inflammatory conditions. Such clarification is also necessary for the development of applications for transferring Treg cells to treat autoimmune diseases or to prevent rejections of transplantations.
SOCS1 (suppressor of cytokine signaling 1) is apparently defined as an important mechanism for the negative regulation of the cytokine–JAK–STAT pathway (Yoshimura et al., 2007), and uncontrolled IFN-γ signaling results from a deficiency of SOCS1. SOCS1 is highly expressed in Treg cells (Lu et al., 2009). It has been reported that SOCS1 expression is reduced in lupus-affected (NZB × NZW) F1 mice (Sharabi et al., 2009), and expression levels of SOCS1 are altered in patients with rheumatoid arthritis or systemic lupus erythematosus (Isomäki et al., 2007; Chan et al., 2010). Analyses of T cell–specific Socs1 conditional KO (cKO; LckCre-Socs1f/f) mice revealed that SOCS1-deficient effector T cells produce high levels of IFN-γ and low levels of IL-17A (IL-17; Tanaka et al., 2008). Although we have shown that the number of Foxp3-positive Treg cells in cKO mice is higher than that in WT mice (Lu et al., 2009), SOCS1-deficient Treg cells lost proper suppression functions in Treg cell–specific Socs1-cKO mice (Lu et al., 2010). However, detailed mechanisms for the impaired Treg cell function by SOCS1 deficiency remain to be clarified.
This study was undertaken to clarify the role of SOCS1 in the stability and suppressive function of Treg cells. We observed autoimmune phenotypes in T cell–specific Socs1-cKO mice, suggesting a defective Treg cell function in these mice. The defective suppression activity of SOCS1-deficient Treg cells was confirmed through the failure to suppress colitis in Rag2−/− mice by the cotransfer of naive T cells and Treg cells. In lymphopenic conditions, Socs1−/− Treg cells tended to lose Foxp3 and were easily converted into Th1-like IFN-γ–producing cells. Foxp3 levels were preserved in Ifnγ−/−Socs1−/− Treg cells, yet Ifnγ−/−Socs1−/− Treg cells could still not suppress colitis induced by the transfer of naive T cells. To our surprise, Ifnγ−/−Socs1−/− Treg cells produced extremely high levels of IL-17, thereby instructing Th17 cell differentiation of naive T cells in Rag2−/− mice. Il-17−/− naive T cells still caused colitis but were suppressed by the cotransfer of Ifnγ−/−Socs1−/− Treg cells, which suggests that Ifnγ−/−Socs1−/− Treg cells instruct naive T cells to become colitogenic by producing high levels of IL-17. In vitro experiments suggest that cytokines from Socs1−/− Treg cells play roles in the instruction of Th1 and Th17 through APCs. We propose that SOCS1 is a guardian of Treg cells because SOCS1 inhibits the conversion of Treg cells into Th1- and Th17-like IFN-γ– and IL-17–producing cells.
Autoimmune phenotypes caused by
LckCre- Socs1f/f mice and impaired suppression activity of SOCS1-deficient nTreg cells in vivo
As reported previously, T cell–specific Socs1-deleted (LckCre-Socs1f/f; cKO) mice survived >6 mo (Tanaka et al., 2008), yet all of these mice eventually developed dermatitis, splenomegaly, and lymphadenopathy with age (Fig. 1, A–C). Inflammatory cellular infiltrations in the skin, liver, and kidney were observed in these mice (Fig. 1 D). Serum Ig levels were much higher in cKO mice than in Socs1f/f (WT) mice, and dsDNA antibodies were detected as well (Fig. 1 E). These data suggest that cKO mice developed a systemic inflammatory and autoimmune disease.
The autoimmune phenotype of T cell–specific Socs1-cKO mice has been thought to be mostly caused by hyperactivation of effector T cells and the production of a huge amount of IFN-γ (Fig. 1 F). Because Ifnγ−/−LckCre-Socs1f/f mice did not develop dermatitis (not depicted), these phenotypes were dependent on IFN-γ. cKO mice and Socs1f/f littermates were crossed with knockin mice with Foxp3-IRES-GFP as reported previously (Wang et al., 2008), and the number of nTreg cells was increased in cKO-Foxp3GFP mice in the thymus (Fig. 1 G) and periphery (Zhan et al., 2009). Similar autoimmune phenotypes were observed in Treg cell–specific Socs1-deleted mice, Foxp3Cre-Socs1f/f mice (Lu et al., 2010), suggesting that SOCS1-deficient nTreg cells have a defective suppression activity.
The defective suppression activity of SOCS1-deficient Treg cells in vivo was confirmed by the failure to suppress colitis in Rag2−/− mice by the cotransfer of naive T cells (Ly5.1) and Treg cells (Ly5.2; Fig. 2 A and Fig. S1). Treg cells were purified by FACS sorting as CD3+CD4+CD25+GFP+ (>99% Foxp3 positive). 4 wk after transfer, the fraction of Foxp3-positive cells from Ly5.2-positive cKO-Foxp3GFP mice was significantly decreased compared with those from Ly5.2-positive WT-Foxp3GFP mice (Fig. 2 B). IFN-γ levels in the mesenteric LN in Rag2−/− mice cotransferred with naive T cells and Socs1−/− Treg cells were much higher than in Rag2−/− mice transferred with naive T cells and WT Treg cells (not depicted). These results indicate that the suppressive function of SOCS1-deficient Treg cells was impaired in lymphopenic conditions, concomitant with faster loss of Foxp3 expression, compared with that of WT Treg cells.
Loss of Foxp3 expression and conversion to Th1-like cells of SOCS1-deficient Treg cells in lymphopenic conditions
We then investigated the fate of Foxp3+ cells in lymphopenic conditions. Treg cells from WT-Foxp3GFP or cKO-Foxp3GFP mice were transferred into Rag2−/− mice, and 6 wk later, Foxp3 positivity was examined by flow cytometry. WT Treg cells lost Foxp3 expression in the single-transfer condition more significantly than in the cotransfer with naive T cells, yet they did not develop colitis, as shown previously (Komatsu et al., 2009). Surprisingly, SOCS1-deficient Treg cells transferred into Rag2−/− mice developed colitis (Fig. 2 C and Fig. S2). SOCS1-deficient Treg cells experienced a more profound loss than did WT Treg cells (63.1% Foxp3+ in WT Treg cells vs. 40.7% in SOCS1-deficient Treg cells; Fig. 2 D).
Not only Foxp3 but also other phenotypical markers of Treg cells, CD25 and CTLA4, which have been shown to be critical molecules in the suppression activity of Treg cells (Wing et al., 2008), were altered in transferred SOCS1-deficient Treg cells. Before transfer, SOCS1-deficient Treg cells expressed CD25 and CTLA4 at significantly higher levels than did WT Treg cells. After transfer, however, CD25 and CTLA4 were more profoundly down-regulated in Socs1−/− Treg cells than in WT Treg cells, in both the Foxp3-maintaining fraction (Foxp3+→+; Fig. 2 E) and the exFoxp3 fraction (Foxp3+→−; Fig. 2 E, top and bottom). In addition, transferred Socs1−/− Treg cells produced higher amounts of IFN-γ from both the Foxp3-maintaining and the Foxp3-losing fractions (Fig. 2 F, top and bottom). The Foxp3-maintaining fraction of SOCS1-deficient Foxp3+ Treg cells also produced IL-2 in addition to IFN-γ (Fig. 2 G, top and bottom). These results indicate that SOCS1-deficient Treg cells tend to lose Foxp3 expression and produce effector cytokines, thus suggesting that SOCS1 is necessary for preventing IFN-γ–producing exFoxp3 cell development.
To exclude the possibility that the higher number of Foxp3− cells in Foxp3+Socs1−/− Treg cell transfer was caused by an extraordinary expansion of contaminated Foxp3−Socs1−/− cells, activated/memory Socs1−/− or Socs1+/+ T cells (Ly5.2+CD44highCD62L−Foxp3− cells) were mixed to 3.3% in the cotransfer of Ly5.1 WT naive T cells and Treg cells (Fig. S3, top) or to 5% in the transfer of Ly5.1 WT Treg cells into Rag2−/− mice (Fig. S3, bottom). Any abnormal expansion of activated/memory Socs1−/− T cells was not observed, suggesting that the larger number of Foxp3− cells in Socs1−/− Treg cell transfer was not caused by rapid expansion of contaminated SOCS1-deficient Foxp3− T cells.
IL-10 from Treg cells has been shown to play an important role in the suppression of naive T cell–induced colitis in Rag2−/− mice (Maloy et al., 2003). Thus, we suspected that impaired IL-10 production from SOCS1-deficient Treg cells could be a mechanism for SOCS1-deficient Treg cell–mediated colitis. However, production of IL-10 was significantly higher in Socs1−/− Treg cells compared with WT Treg cells both before and after transfer, suggesting that IL-10 is not involved in the SOCS1-deficient Treg cell–mediated colitis (Fig. S4).
Rapid methylation of the CNS2 (conserved noncoding DNA sequence 2) region in the Foxp3 enhancer of committed SOCS1-deficient Treg cells
Like in Rag2−/− mice, Foxp3 levels were decreased more rapidly in Socs1−/− Treg cells than in WT Treg cells in vitro. CFSE dilution revealed that the loss of Foxp3 occurred mostly in divided Treg cells, suggesting that the loss of Foxp3 is associated with cell division or that cells that lost Foxp3 expression divide faster than Foxp3+ cells (Fig. 3 A). It has been reported that CD4+CD25+CD62Lhigh Treg cells maintained stable Foxp3 expression along with suppressive activity after in vitro expansion compared with CD4+CD25+CD62Llow Treg cells (Scalapino and Daikh, 2009). Thus, we compared Foxp3 expression levels in Socs1+/+ and Socs1−/− Treg cells between the CD62high and CD62Llow fraction after 3-d in vitro culture. The fraction of CD62Llow was much higher than that of CD62Lhigh in Socs1−/− Treg cells (Fig. 3 B). As reported, Foxp3 expression was decreased in the CD62Llow fraction in WT Treg cells, and Foxp3 was maintained in the CD62Lhigh fraction (Fig. 3 C, left). In Socs1−/− Treg cells, Foxp3 was also maintained in the CD62Lhigh fraction. However, the loss of Foxp3 expression in the CD62Llow fraction was significantly more drastic in Socs1−/− Treg cells than in WT Treg cells (Fig. 3 C, right).
Recently, it has been reported that the DNA methylation status of the CNS2 region in the Foxp3 enhancer is particularly important for maintenance of Foxp3 expression (Floess et al., 2007; Kim and Leonard, 2007; Zheng et al., 2010). To examine whether SOCS1-deficient Foxp3+ Treg cells contained uncommitted Treg cells or unstable inducible Treg cells (iTreg cells), we determined the methylation status of the CNS2 region in SOCS1-deficient Treg cells. As shown in Fig. 3 D, the CNS2 region of Treg cells from the LN of SOCS1-cKO mice were fully demethylated, similar to those of WT mice (Fig. 3 D). This indicates that >95% SOCS1-deficient Treg cells were committed and did not contain iTreg cells. After 3-d in vitro culture, the Foxp3-maintaining fraction (Foxp3+→+) retained full demethylation in the CNS2 region in both Socs1+/+ and Socs1−/− Treg cells. However, the CNS2 region in the exFoxp3 fraction (Foxp3+→−) was fully or partially methylated, and more methylation occurred in the SOCS1-defcient exFoxp3 cells than in the SOCS1-sufficient exFoxp3 cells (Fig. 3 D), which is consistent with a more rapid loss of Foxp3 in Socs1−/− Treg cells than in WT Treg cells.
In vivo, we also found hypermethylation of the CNS2 region in Foxp3− cells after transfer of Foxp3+ cells into Rag2−/− mice (Fig. 3 E). More intensive hypermethylation in the CNS2 region in Foxp3− cells was observed in Socs1−/− Treg cell transfer than in WT Treg cell transfer (Fig. 3 E; >90% methylated regions were 90% in Socs1−/− vs. 50% in Socs1+/+). Thus, the CNS2 region of the Foxp3 gene in exFoxp3 cells from SOCS1-deficient Treg cells was more easily hypermethylated not only in vitro but also in vivo than that from WT Treg cells (Fig. 3 E).
Accelerated conversion of SOCS1-deficient Treg cells into Th1- and Th17-like cells in vitro
It has been reported that Treg cells convert into Th1- or Th17-like cells in appropriate in vitro culture conditions (Yang et al., 2008). To investigate the mechanism of the loss of Foxp3 and enhanced IFN-γ production from Socs1−/− Treg cells in vivo, we performed an in vitro conversion assay. As shown in Fig. 4 A, when Foxp3+ cells were cultured in the presence of TCR stimulation and IL-2, much higher amounts of IFN-γ as well as of IL-17 were produced from Socs1−/− Treg cells than from WT Treg cells. IL-12 further up-regulated IFN-γ production, whereas IL-17 was up-regulated by IL-1 and IL-6 (Fig. 4 A). Interestingly, the production of IL-17 by IL-1 and IL-6 was dependent on TGF-β because anti–TGF-β antibody abrogated the effect of IL-1 and IL-6.
Because SOCS1 has been shown to be an essential negative regulator for the JAK–STAT pathway, the activation status of STAT1 and STAT3 were compared using immunoblotting and intracellular staining (Fig. 4 B). STAT5 was reported to be phosphorylated in SOCS1-deficient Treg cells at higher levels than in WT Treg cells, which is associated with the increase in the number of Treg cells (Lu et al., 2009). Much stronger tyrosine phosphorylation of both STAT1 and STAT3 were observed in freshly isolated SOCS1-deficient Treg cells than in WT Treg cells by both methods (Fig. 4 B). These data indicate that SOCS1 is necessary for the suppression of spontaneous activation of STAT1 and STAT3 in Treg cells.
Foxp3 was stabilized in
Ifnγ−/−Socs1−/− Treg cells
Because SOCS1 is a strong inhibitor for the IFN-γ–STAT1 pathway, we hypothesized that hyper-STAT1 activation led to abnormalities in SOCS1-deficient Treg cells. To address this hypothesis, we used Treg cells from Ifnγ−/−Socs1−/− mice.
Most of the Ifnγ−/−Socs1+/+ and Ifnγ−/−Socs1−/− Treg cells (CD4+CD25bright fraction) were CD62Lhigh and CD44high, suggesting that IFN-γ deficiency diminished CD62Llow Treg cell subsets (Fig. 5 A). Consistently, when Treg cells from both Ifnγ−/−Socs1+/+ and Ifnγ−/−Socs1−/− mice were transferred into Rag2−/− mice, colitis did not develop even 6 wk after transfer (Fig. 5 B). Foxp3 expression in Ifnγ−/−Socs1−/− Treg cells was as stable as in Ifnγ−/−Socs1+/+ Treg cells and WT Treg cells after transfer into Rag2−/− mice (Figs. 2 D and 5 C). After transfer, unlike in Socs1−/− Treg cells, CD25 and CTLA4 were maintained at significantly higher levels in Ifnγ−/−Socs1−/− Treg cells (Fig. 5 D, top and bottom). We examined the methylation status of the CNS2 region in both Ifnγ−/−Socs1+/+ and Ifnγ−/−Socs1−/− Treg cells in vitro. The CNS2 region of Treg cells from the LN of Ifnγ−/−Socs1+/+ and Ifnγ−/−Socs1−/− mice were fully demethylated (Fig. 5 E). After 3-d culture, the Foxp3-maintaining fraction (Foxp3+→+) retained full demethylation in the CNS2 region in both Ifnγ−/−Socs1+/+ and Ifnγ−/−Socs1−/− Treg cells. Methylation levels of the CNS2 region in the Ifnγ−/− exFoxp3 fraction (Foxp3+→−) were lower than those in the Ifnγ+/+ exFoxp3 fraction (Figs. 3 D and 5 E), suggesting that IFN-γ played a positive role in the methylation of the CNS2 region in Treg cells. The rate of methylation in the CNS2 region in Ifnγ−/−Socs1−/− exFoxp3 cells was lower than that in Ifnγ−/−Socs1+/+ exFoxp3 cells. These results were consistent with a stable Foxp3 expression in Ifnγ−/−Socs1+/+ and Ifnγ−/−Socs1−/− Treg cells after transfer into Rag2−/− mice.
Ifnγ−/−Socs1−/− Treg cells still lacked in vivo suppression activity because of stronger Th17 instruction
To test the suppression activity, Treg cells from Ifnγ−/−Socs1+/+ or Ifnγ−/−Socs1−/− mice were then transferred into Rag2−/− mice with naive T cells. 4 wk after transfer, Treg cells from Ifnγ−/−Socs1+/+ mice suppressed colitis elicited by naive T cells (Fig. 6 A and Fig. S7). The suppression activity of Ifnγ−/− Treg cells was lower than that of Ifnγ+/+ Treg cells (Figs. 2 A and 6 A), yet recipient mice did not develop severe colitis 4 wk after T cell transfer. To our surprise, Ifnγ−/−Socs1−/− Treg cells could not prevent colitis, which was confirmed by body weight loss and histological examination (Fig. 6 A and Fig. S5). Expression of Foxp3 in Ifnγ−/−Socs1+/+ Treg cells as well as in Ifnγ−/−Socs1−/− Treg cells was maintained in recipient Rag2−/− mice (Fig. 6 B), just as it was in the transfer of Treg cells alone (Fig. 2 B). Therefore, Foxp3 maintenance is not sufficient for the suppression of effector T cell functions in vivo.
To understand these unexpected results, we examined the production of inflammatory cytokines from Treg cells or naive/effector cells transferred into Rag2−/− mice. IL-10 levels were not significantly different between Ifnγ−/−Socs1+/+ and Ifnγ−/−Socs1−/− Treg cells (Fig. S4). Because Th17 has also been implicated in colitis (Chaudhry et al., 2009; Durant et al., 2010), we then compared IFN-γ and IL-17 production from naive/effector (Ly5.1+) T cells (Fig. 6 C, top) and Treg cells (Ly5.1−, Ly5.2+; Fig. 6 C, middle) in the LN of transferred recipient mice, after which percent positivity values of cytokine production were determined by flow cytometry (Fig. 6 C, bottom). As shown in Fig. 6 C, when naive T cells alone were transferred into Rag2−/− mice, a portion of these cells converted into effector cells that produced mainly IFN-γ, but with very low levels of IL-17 (Fig. 6 C, a). When naive T cells and WT Treg cells were cotransferred, differentiation of Th1-like cells from naive T cells was completely suppressed (Fig. 6 C, b). However, when naive T cells and Socs1−/− Treg cells were cotransferred, both IFN-γ– and IL-17–producing cells appeared from transferred naive T cells, which is consistent with the failure of suppression of colitis by Socs1−/− Treg cells (Fig. 6 C, c). Thus, SOCS1-deficient Treg cells promoted Th1 and Th17 cell differentiation from naive T cells, whereas WT Treg cells did not. In contrast, when naive T cells and Ifnγ−/−Socs1+/+ Treg cells were cotransferred, a significant fraction of IL-17–producing cells appeared from transferred naive T cells (Fig. 6 C, d). IL-17 was also produced from transferred Treg cells. These results could explain why Ifnγ−/− Treg cells did not prevent colitis very efficiently (Fig. 6 A). Much more intense IL-17 production was observed from both naive T cells and Treg cell fractions in recipient mice cotransferred with naive T cells and Ifnγ−/−Socs1−/− Treg cells (Fig. 6 C, e). These data suggest that the cytokine profile of Treg cells instructs the direction of differentiation of Th1 or Th17 from naive T cells in lymphopenic conditions.
Instruction of Th1 and Th17 cell differentiation by cytokines from Treg cells through APCs in vitro
To confirm these instructions by Treg cells, we first measured IL-17 from IFN-γ–deficient Treg cells in in vitro culture experiments. As expected, Ifnγ−/− Treg cells produced higher amounts of IL-17 than did WT Treg cells, and IL-17 production was further enhanced by SOCS1 deficiency (Fig. 7 A). These data suggest that IFN-γ usually suppressed IL-17 production from Treg cells, which skewed Th1 cell differentiation from naive T cells. Coincidently, hyper-STAT1 activation was not observed in Ifnγ−/−Socs1−/− Treg cells, although STAT3 activation was still high (Fig. 7 B, top and bottom). Thus, hyper STAT3 activation could account for high levels of IL-17 production from Ifnγ−/−Socs1−/− Treg cells.
The effect of culture supernatants of Treg cells on Th1/Th17 cell differentiation was then examined (Fig. 7 C). Culture supernatants of Treg cells were added to the co-culture of naive T cells only or in the presence or absence of APCs (Fig. 7 C). In the absence of APCs, culture supernatants from Socs1+/+ Treg cells, Socs1−/− Treg cells, or Ifnγ−/−Socs1−/− Treg cells did not promote Th1 or Th17 cell differentiation, suggesting that cytokines from Treg cells modify the APCs for accelerated Th1 or Th17 cell differentiation form naive T cells (Fig. 7 C, top). Supernatants from Socs1−/− Treg cells instructed naive T cells to differentiate into IL-17–producing effector cells when cultured with LPS-untreated APCs (Fig. 7 C, middle; and Fig. S6, right). This finding is consistent with our observations that Socs1−/− Treg cells produced IL-17 (Fig. 4 A) and induced more Th17 in vivo (Fig. 6 C). This instruction of naive T cells was augmented by Ifnγ−/−Socs1−/− Treg cells, which produced the largest amount of IL-17 (Fig. 7 A), consistent with the results shown in Fig. 6 C. When APCs were activated by LPS (Fig. 7 C, bottom; and Fig. S6, left), naive T cells, after the addition of supernatant of Socs1−/− Treg cells, were instructed to differentiate into IFN-γ–producing effector cells, which is again consistent with higher IFN-γ production from Socs1−/− Treg cells (Fig. 4 A), and induced more Th1 in vivo (Fig. 6 C). Ifnγ−/−Socs1−/− Treg cells did not induce Th1 in LPS-treated APCs, suggesting that IFN-γ production from Treg cells plays an important role in the Th1 instruction of naive T cells. Both IFN-γ and IL-17 antibodies abolished Th1 and Th17 instruction, respectively, confirming that IFN-γ and IL-17 from SOCS1-deficeint Treg cells play essential roles in Th1 and Th17 induction from naive T cells (Fig. S6).
To confirm the role of APCs, we examined the effect of anticytokine antibodies on the in vitro instruction experiments (Fig. S6). Th1 instruction was eliminated by IL-12 antibodies, suggesting that IL-12 from APCs promoted Th1 cell differentiation. Consistent with this notion, Socs1−/− Treg cells could not suppress colitis induced by Ifnγr1−/− naive T cells transferred into Rag2−/− mice (Fig. S7). This experiment suggested that IFN-γ from Socs1−/− Treg cells still induced colitogenic T cells from Ifnγr1−/− naive T cells. In contrast, anti–IL-6 antibody completely inhibited and anti–TGF-β antibody partially inhibited Th17 instruction. Anti–IL-23 antibody did not affect Th17 cell differentiation. These results suggest that IL-17 from cultured Treg cells stimulated IL-6 and TGF-β production from APCs, which promoted Th17 cell differentiation from naive T cells.
To confirm this possibility, we used IL-17–deficient naive T cells for the transfer. As shown in Fig. 7 D, Rag2−/− mice transferred with IL-17–deficient naive T cells developed colitis, which was less severe than in WT naive T cell transfer. However, Socs1−/− Treg cells caused extremely severe colitis (Fig. 7 D), which was triggered by the large levels of IFN-γ (not depicted). As we expected, Ifnγ−/−Socs1−/− Treg cells as well as Ifnγ−/−Socs1+/+ Treg cells suppressed colitis induced by IL-17–deficient naive T cell transfer (Fig. 7 D). These data further support our hypothesis that Ifnγ−/− Treg cells instruct Th17 cell differentiation of naive T cells, thereby suppressing colitis less efficiently, and these processes are accelerated when SOCS1 is deficient in Ifnγ−/− Treg cells.
In this study, we demonstrated that SOCS1 is necessary for Treg stability and suppressor functions: SOCS1 protects Treg cells from harmful effects of inflammatory cytokines, which promote the loss of Foxp3 expression and the conversion into Th1/Th17-like effector cells. Recent studies have shown that Treg cells rapidly lose Foxp3 expression upon the transfer into a lymphopenic host (Komatsu et al., 2009) or in inflammatory conditions (Zhou et al., 2009). Such exFoxp3 cells (Zhou et al., 2009) or lapsed Treg cells (Murai et al., 2010) develop an effector-memory phenotype, produce pathogenic cytokines, and can trigger the development of autoimmunity. Multiple studies have suggested that Treg cells isolated from inflammatory sites express reduced amounts of Foxp3, possibly increasing susceptibility to autoimmunity (Wan and Flavell, 2007; Tang et al., 2008).
Foxp3 expression is regulated by various factors, such as Smad2/3 (Takimoto et al., 2010), Runx1 (Kitoh et al., 2009; Rudra et al., 2009), STAT5 (Yao et al., 2007), c-Rel (Hori, 2010), and DNA methylation of the CNS2 region in the Foxp3 enhancer (Zheng et al., 2010). DNA methylation of CNS2 is particularly important because CNS2 is required for Foxp3 expression in the progeny of dividing Treg cells (Zheng et al., 2010). Surprisingly, we found that the CNS2 region of freshly isolated SOCS1-deficient Treg cells was fully demethylated, indicating that they are committed Foxp3+ Treg cells. In Rag2−/− mice as well as in in vitro culture, part of the committed Foxp3+ cells expanded and converted into Foxp3− cells (i.e., exFoxp3 cells). The CNS2 region in SOCS1-deficient exFoxp3 cells was more methylated than that in WT Treg cells. Also, because the CNS2 methylation status of Treg cells was altered after 3-d in vitro culture and there was a partially methylated CNS2 region, it is unlikely that all of the exFoxp3 cells were caused by the expansion of contaminated Foxp3− effector T cells. Accordingly, we concluded that SOCS1 contributes to Treg cell integrity by maintaining stable Foxp3 expression.
Our present study indicated that the excessive IFN-γ–STAT1 signals resulted in faster loss of Foxp3 expression in Treg cells in lymphopenic conditions as well as in an in vitro culture system. One possibility is that the IFN-γ–STAT1 pathway increases reprogramming from SOCS1-deficient Treg cells to effector cells. IFN-γ may promote the expansion or development of memory type CD62Llow cells because we observed that the CD62Llow fraction was at normal levels in IFN-γ/SOCS1 double-deficient Treg cells. STAT1 may antagonize STAT5, yet this is unlikely because we could not observe the reduction of STAT5 phosphorylation in our SOCS1-deficient Treg cells. STAT1 has also been shown to inhibit the TGF-β–Smad pathway (Tanaka et al., 2008). The phenotypes of Smad2/3-deficient Treg cells were similar to those observed in SOCS1-deficient Treg cells (Takimoto et al., 2010). Thus, the suppression of Smad signaling by STAT1 could be one of the mechanisms behind the reprogramming of Treg cells. The relationship between these factors and the reprogramming of Treg cells remain to be clarified.
Recently, Rubtsov et al. (2010) reported that highly purified Treg cells were very stable under physiological and inflammatory conditions. Lu et al. (2010) reported that Foxp3 expression of SOCS1-deficient Treg cells from Foxp3Cre-Socs1f/f mice was maintained in the presence of naive and effector T cells, although the suppression activity of Socs1−/− Treg cells was defective and IFN-γ was produced from Socs1−/− Treg cells with STAT1 hyperactivation. Thus, Foxp3 expression in Socs1−/− Treg cells from Foxp3Cre-Socs1f/f mice was more stable than that in Socs1−/− Treg cells from LckCre-Socs1f/f mice. Because SOCS1 of LckCre-Socs1f/f mice is deleted in the Treg cell progenitors, whereas SOCS1 of Foxp3Cre-Socs1f/f mice is deleted after Treg cell maturation, this discrepancy may be explained by differences in the developmental conditions of Treg cells. It is also possible that in LckCre-Socs1f/f mice, cytokines from Socs1−/− effector T cells could contribute to more drastic phenotypic changes in Socs1−/− Treg cells.
A recent study has demonstrated that during lethal Toxoplasma gondii infection, the exposure of WT Treg cells to high amounts of Th1 inflammatory mediators superimposes a Th1 effector program on Treg cells of not only the Foxp3-losing portion but also the Foxp3-maintaining portion (Oldenhove et al., 2009). Thus, the conversion of Treg cells to Th1 is one of the mechanisms for uncontrolled Th1 response in STAT1-hyperactivated Treg cells, and this process is usually protected by SOCS1. Recently, Lu et al. (2010) reported on the defective suppression activity of SOCS1-deficient Treg cells and IFN-γ production from these Treg cells by STAT1 hyperactivation. In addition, our data suggest the intriguing possibility that hyperactivation of STAT3 in Treg cells facilitates the conversion to Th17-like cells, which leads to an uncontrolled response in effector Th17 cells. STAT5, in contrast, enhances Foxp3 expression and the number of Treg cells (Lu et al., 2009).
Like STAT1, STAT3 has been reported to inhibit Foxp3 expression during iTreg/Th17 cell differentiation together with TGF-β (Takimoto et al., 2010). However, the role of STAT3 in Foxp3 stability in Treg cells seems to be more complicated. STAT3-deficient Treg cells have been shown to expand less efficiently than WT Treg cells in lymphopenic conditions (Durant et al., 2010) and lose their suppression activity, especially for Th17 (Chaudhry et al., 2009; Durant et al., 2010). Foxp3 was stable in Ifnγ−/−Socs1−/− Treg cells with hyperactivated STAT3. Thus, it is unlikely that the hyperactivation of STAT3 is linked to Foxp3 instability. Previously, we have shown that STAT3 activation is reduced in SOCS1-deficient naive T cells in response to IL-6 and TGF-β caused by SOCS3 induction (Tanaka et al., 2008). The mechanism of hyperactivation of STAT3 in SOCS1-deficient Treg cells remains to be investigated. Although Foxp3 expression of Ifnγ−/−Socs1−/− Treg cells is stabilized, these Ifnγ−/−Socs1−/− Treg cells were still defective in terms of the suppression of IL-17 production from both Treg cells and effector T cells in Rag2−/− mice. This indicates that the stable expression of Foxp3 is not a sufficient condition for the suppression of effector Th17 cells. This may be linked to a high conversion potential of Treg cells to Th17 cells. In a recent study using IL-17–RFP reporter mice with a Foxp3-GFP reporter, Yang et al. (2008) demonstrated the presence of a RFP+GFP+ transient phase upon T cell activation in vitro and in vivo. Thus, it is highly possible that both Th17 and Treg cell programs could occur simultaneously in one cell, which underlies the conversion of Foxp3-positive Treg cells to IL-17–producing cells. We observed that STAT3 was hyperactivated in Socs1−/− Treg cells as well as in Ifnγ−/−Socs1−/− Treg cells. Although we do not have direct proof demonstrating that STAT3 hyperactivation is responsible for the conversion of Treg cells to Th17, it is likely true because IL-17 production from nTreg cells was enhanced by IL-6 and IL-1 and because STAT3, in addition to TGF-β signals, has been shown to be an essential factor for the generation of Th17.
SOCS1-deficient Treg cells, which convert to Th1- and Th17-like cells, seem to instruct naive T cells to differentiate into the same cytokine-producing effector T cells. This can also explain why STAT1 or STAT3 hyperactivated Treg cells cannot control Th1 or Th17 effectors, respectively. Intriguingly, previous studies have indicated that STAT1–T-bet (Koch et al., 2009) and STAT3 (Chaudhry et al., 2009; Durant et al., 2010) in Treg cells are required for Th1 and Th17 suppression, respectively. Our current study, as well as a previous study (Lu et al., 2010), clearly indicates that both the lack of STAT1/3 and unrestrained STAT1/3 activation in Treg cells leads to a severe failure of immunological tolerance.
Anti–IFN-γ, –IL-17, –IL-12, and –IL-6 antibodies nearly completely inhibited Th1 and Th17 cell differentiation (Fig. S6). Thus, we clarified that IFN-γ from Th1-converted Treg cells and IL-17 from Th17-converted Treg cells preferentially induced effector Th1 and Th17 cell differentiation, respectively, by modulating APCs. It has been reported that Treg cells interact with DCs before naive T cells and down-regulate the expression of CD80/86 caused by high levels of CTLA4 on Treg cells (Onishi et al., 2008). Because SOCS1-deficient Treg cells lose CTLA4 expression, they may not be able to suppress the expression levels of co-stimulator ligands. It has been reported that DCs treated with IFN-γ preferentially induce Th1 (Hanada et al., 2003), whereas IL-17–treated DCs preferentially induce Th17 cell differentiation because of the higher production of IL-1β, IL-6, and IL-23 from IL-17–treated DCs (Sutton et al., 2009). IL-17 may not directly induce Th17 cell differentiation in the absence of APCs because IL-17 has been reported to actually reduce Th17 cell differentiation (Smith et al., 2008). These previous studies support our results, and IFN-γ and IL-17 from Treg cells are thought to instruct effector T cell differentiation into Th1 or Th17 by modulating APCs.
In conclusion, we propose the idea that SOCS1 is an important guardian of Treg cells. Our findings also raise the interesting possibility that up-regulation of SOCS1 in Treg cells at appropriate levels reinforces Treg cell functions because SOCS1 may protect Treg cells from harmful effects of inflammatory cytokines that accelerate the conversion of Treg cells into effectors. These findings may improve Treg cell therapy for autoimmune diseases and organ transplantations.
MATERIALS AND METHODS
LckCre-Socs1f/f (T cell–specific Socs1-cKO; Tanaka et al., 2008) and Socs1f/f (littermate control; WT) mice (sex and age matched) were used. cKO mice and Socs1f/f littermates crossed with knockin mice with Foxp3-IRES-GFP (WT-Foxp3GFP mice and cKO-Foxp3GFP mice) were provided by B. Malissen (Université de la Méditerranée, Marseille, France; Wang et al., 2008). Ifnγ−/−, Ifnγ−/−Socs1−/−, Il17−/−, and Ifnγr1−/− mice with the C57BL/6J background were described previously (Nakae et al., 2002; Hanada et al., 2003; Suzue et al., 2003). Mice were kept in conventional conditions in Keio University. All experiments using these mice were approved by and performed according to the guidelines of the Animal Ethics Committee of Keio University.
Tissue samples were obtained from the proximal and distal colon, skin, liver, and kidneys and then fixed in 10% neutral buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin (H&E). Assessments of the severity of colitis were performed using the histopathologic score as described previously (Inagaki-Ohara et al., 2004).
In vivo suppression assay and transfer colitis model in
Flow cytometry–purified 4 × 105 CD4+CD25−CD62L+CD44− naive T cells from WT mice were injected intravenously into Rag2−/− mice in combination with 2 × 105 CD3+CD4+CD25+Foxp3GFP cells from WT-Foxp3GFP or cKO-Foxp3GFP mice (8 wk old and sex matched) or 2 × 105 CD3+CD4+CD25bright Treg cells from Ifnγ−/−Socs1+/+ or Ifnγ−/−Socs1−/− mice (8 wk old and sex matched). Mice were observed daily and weighed weekly. 4 wk after cell transfer, the mice were sacrificed for experiments, and colon sections were stained with H&E. Tissues were graded semiquantitatively. Histological grades were assigned in a blinded manner.
Transfer of Treg cells into
Flow cytometry–purified 2 × 105 CD3+CD4+CD25+Foxp3GFP cells from WT-Foxp3GFP or cKO-Foxp3GFP mice (8 wk old and sex matched) or 2 × 105 CD3+CD4+CD25bright Treg cells from Ifnγ−/−Socs1+/+ or Ifnγ−/−Socs1−/− mice (8 wk old and sex matched) were injected intravenously into Rag2−/− mice. Mice were observed daily and weighed weekly. 6 wk after cell transfer, the mice were sacrificed for experiments.
Flow cytometry, cell sorting, and cytokine secretion assays.
Cell surface staining and flow cytometric analysis of CD3, CD4, CD25, CD62L, and CD44 (all from eBioscience) expression were performed as described previously (Fontenot et al., 2005). For the isolation of Treg cells, CD4+ T cells were positively selected with magnetic-activated cell sorting (Miltenyi Biotec), and CD3+CD4+CD25+Foxp3GFP cells or CD3+CD4+CD25bright cells were further purified using a FACSAria cell sorter (BD). The purity of the sorted populations was invariably >99%. Intracellular staining of Foxp3, IL-2, IFN-γ, IL-17A, and CTLA4 (all from eBioscience) was performed after fixation and permeabilization according to the manufacturer’s instructions. To measure T cell cytokine production, cells were stimulated with 50 ng/ml PMA and 250 ng/ml ionomycin in the presence of Golgi Plug (BD) for 4 h at 37°C before staining. For phospho-STAT1 and phospho-STAT3 staining, Phosflow Lyse/Perm buffer and Perm Buffer III (BD) were used according to the manufacturer’s instructions.
In vitro culture of Treg cells.
To examine effector T cell differentiation, 105 Treg cells/well (CD3+CD4+CD25+ Foxp3GFP cells or CD3+CD4+CD25bright cells) from WT-Foxp3GFP, cKO-Foxp3GFP, Ifnγ−/−Socs1+/+, or Ifnγ−/−Socs1−/− mice (8 wk old and sex matched) were stimulated with anti-CD3/anti-CD28 mAb–coated beads (Dynal) at a 1:1 cell/bead ratio for 72 h, with 10 ng/ml IL-2 in 96-well, flat-bottom plates.
In vitro effector T cell differentiation with supernatants of cultured Treg cells.
3 × 105 WT naive T cells/well were cultured for 5 d without, with LPS-untreated, or with 1 µg/ml LPS–stimulated APCs (7 × 105 T cell–depleted spleen cells/well) and supernatants from 2-d cultured 2 × 105 Socs1+/+, Socs1−/−, and Ifnγ−/−Socs1−/− Treg cells/well. Flow cytometric analysis of IFN-γ and IL-17A was performed on these cells. These were also observed with 10 µg/ml anti–IFN-γ, –IL-12, –IL-17, –IL-6, –TGF-β, or –IL-23 antibodies.
Supernatants from cultures of Treg cells with or without the indicated cytokines were harvested and pooled. Where indicated, 20 ng/ml IL-1α, 20 ng/ml IL-1β, 40 ng/ml IL-6, 50 ng/ml IL-12, 50 ng/ml IL-23, and 10 µg/ml anti–TGF-β mAb (1D11) were added. IFN-γ and IL-17A concentrations were measured using commercially available mouse ELISA Ready-S (eBioscience). All samples were run in triplicate.
Western blot analysis.
Cell lysates from Treg cells (2 × 105 cells) from WT-Foxp3GFP, cKO-Foxp3GFP, Ifnγ−/−Socs1+/+, and Ifnγ−/−Socs1−/− mice were resolved by SDS-PAGE subjected to Western blot analysis. The membranes were immunoblotted with various antibodies, and the bound antibodies were visualized with horseradish peroxidase–conjugated antibodies against rabbit or mouse IgG (Jackson ImmunoResearch Laboratories, Inc.), using Chemi-Lumi One Super Western blotting detection reagents (Nacalai Tesque). Antibodies against STAT1, phospho-STAT1, STAT3, phospho-STAT3 (all from Cell Signaling Technology), and tubulin (Sigma-Aldrich) were used to visualize the corresponding proteins.
Reverse transcription PCR analysis.
Total RNA was prepared using a nucleospin RNA XS (MACHEREY-NAGEL). RNA was reverse transcribed to cDNA with random primers (Applied Biosystems) and a high capacity cDNA reverse transcription kit (Applied Biosystems) in accordance with the manufacturer’s protocol. To determine the cellular expression level of each gene, quantitative real-time PCR analysis was performed using a C1000 Thermal Cycler (Bio-Rad Laboratories). The PCR mixture consisted of 5 µl of KAPA SYBR FAST qPCR kits (Kapa Biosystems), 15 pmol of forward and reverse primers, and the cDNA samples in a total volume of 10 µl. Relative RNA abundance was determined based on control-HPRT abundance.
Genomic DNA extracted from freshly isolated or 3-d cultured 2 × 105 Treg cells from male WT-Foxp3GFP, cKO-Foxp3GFP, Ifnγ−/−Socs1+/+, and Ifnγ−/−Socs1−/− mice (8 wk old and sex matched) was digested with BamHI. 2 × 105 Treg cells from male WT-Foxp3GFP and cKO-Foxp3GFP mice before transfer or 4 wk after transfer into Rag2−/− mice were also examined. H2O was added to 500 ng of the digested DNA to a volume of 19 µl, and 1.2 µl of 6N NaOH was added; the mixture was then incubated at 37°C for 15 min. Next, 120 µl of 3.6N Na bisulfite, 0.57 mM hydroquinone, and 0.3N NaOH were added. Samples were then treated with 15 cycles of 95°C for 30 s to 50°C for 15 min. The reaction was then desalted using the Wizard DNA cleanup system (Promega) and eluted with 50 µl TE (Tris-EDTA). 3 µl of 5N NaOH was added, and the samples were incubated for 5 min at room temperature. The products were then ethanol precipitated and dissolved with 50 µl of TE buffer. The CNS2 region was PCR amplified with the primer set (forward, 5′-TTTTGGGTTTTTTTGGTATTTAAGA-3′; reverse, 5′-TTAACCAAATTTTTCTACCATTAAC-3′) and T/A cloned into a pGEM-T-Easy vector (Promega). 10 inserted plasmids from each condition were purified and sequenced. 2 × 105 Treg cells before transfer and 4 wk after transfer into Rag2−/− mice were also analyzed.
For statistical analysis, we used the Student’s t test.
Online supplemental material.
Fig. S1 shows that the suppressive activity of SOCS1-deficient Treg cells is attenuated in Rag2−/− mice. Fig. S2 shows colitis caused by SOCS1-deficient Treg cells in Rag2−/− mice. Fig. S3 shows that the loss of Foxp3 expression in Treg cells is not caused by outgrowth of activated memory T cell contamination of injected donor cells in Rag2−/− mice. Fig. S4 shows expression levels of IL-10 of Treg cells before and after transfer in Rag2−/− mice. Fig. S5 shows that the suppressive activity of Ifnγ−/−Socs1−/− Treg cells is attenuated in Rag2−/− mice. Fig. S6 shows that anti–IFN-γ, –IL-17, –IL-12, and –IL-6 antibodies nearly completely inhibit Th1 and Th17 cell differentiation through APCs. Fig. S7 shows that Socs1−/− Treg cells cannot suppress colitis induced by the transfer of Ifnγr1−/− naive T cells into Rag2−/− mice.
We thank N. Shiino for providing technical assistance and N. Aizawa for manuscript preparation.
This work was supported by special grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Japan Society for the Promotion of Science, the Takeda Science Foundation, the Uehara Memorial Foundation, the Mitsubishi Pharma Research Foundation, the Kanae Foundation for the Promotion of Medical Science, the Mochida Memorial Foundation, and the Program for the Promotion of Fundamental Studies in Health Science of the National Institute of Biomedical Innovation (to A. Yoshimura).
The authors have no conflicting financial interests.