CD8α− DCs from GM-CSF treated mice that produce significantly lower levels of pro-inflammatory cytokines IL-12 and IL-1β compared to the DCs from control mice can induce and/or expand Foxp3+ Tregs and suppress autoimmune diseases 
. While pro-inflammatory cytokines have been generally associated with the induction of inflammation and autoimmunity, some studies have shown that they may provide positive signals for the induction of Tregs 
. Several studies have implicated IL-12 and other Th1 cytokines in autoimmune diseases 
. Recent studies have shown IL-1β as an important mediator of Th17 response in humans and is often associated with autoimmunity 
. In contrast, IL-1β has also been shown to provide co-stimulation and/or act as a growth factor for Foxp3+ Tregs 
. Therefore, in the current study we evaluated the effects of IL-12 and IL-1β on Foxp3 expression in T cells that were activated in the presence or absence of CD8α− DCs. Our observations showed that these cytokines have contrasting effects on Foxp3 expression in activated T cells. While the presence of IL-12 in the culture abrogated Foxp3 expression in T cells, IL-1β in the presence of reduced levels of IL-12 promoted Foxp3 expression. These results showed that IL-12 and IL-1β have opposing effects on Tregs. Our results also show that these secreted factors do not significantly modulate DC properties but have a direct effect on T cells.
IL-12 is recognized for its ability to promote Th1 type of pathogenic T cell response in many autoimmune conditions 
and can overcome immune tolerance by suppressing Foxp3+ Tregs. In a recent study, Brahmachari et al
. have shown that the p40 subunit of IL-12 can down-regulate Foxp3 expression via the production of nitric oxide 
. Our observation that IL-12 abrogates Foxp3 expression in T cells during activation further confirms the pro-inflammatory and the potential pathogenic effects of this cytokine in autoimmunity.
IL-1β, on the other hand, has pleiotropic effects and can alter cell signaling, migration and cytokine production, and influence T cell differentiation differently under different conditions 
. IL-1β has been shown to break peripheral tolerance by facilitating the expansion of effector T cells and is implicated in autoimmune diseases such as rheumatoid arthritis 
. Lately, IL-1β has been shown to be critical for the generation of IL-17 secreting T helper cells in humans. IL-23 when combined with IL-1β has been shown to induce and maintain pathogenic Th17 cells 
. Our current finding of a distinct role for IL-1β in promoting Foxp3 expression in T cells is in apparent contradiction to the above findings, but is in agreement with recent observations of Brinster et al
. who showed that IL-1β expands Foxp3+ Tregs in the presence of anti-CD3 Ab and splenic DCs 
. In another study, treatment of murine myeloid DCs with rapamycin, an immunosuppressive agent, resulted in the production of IL-1β by phenotypically immature DCs, which were refractive to inflammatory stimuli and thus were unable to activate T cell responses 
. Moreover, differentiation of DCs from monocytes was impaired when they were pre-treated with low doses of IL-1β, and these DCs were unable to effectively stimulate an immune response 
. However, our observations failed to show a significant direct modulatory effect of IL-1β on DCs We noted expression of IL-1 receptor transcripts in both DCs and T cells (). Recent studies have shown increased expression of IL-1 receptor on Tregs as compared to conventional T cells as well as increased IL-1β signaling in Tregs 
. Therefore, the higher levels of expression of IL-1 receptors on T cells may be determined by the lineage characteristics of T cells and this may have contributed to the direct effects of IL-1β on T cells noted in this study.
Although nTregs constitutively express Foxp3 during resting stage, expression level of this transcription factor is down-regulated upon in vitro
. This could explain why only less than 50% of CD4+CD25+ T cells expressed Foxp3 after activation in our control cultures. However, our observations indicate that IL-1β, when added to the T cell activation cultures, helps maintain Foxp3 expression in a significantly higher proportion of nTregs upon activation and proliferation (). This suggested that IL-1β may be promoting the expansion of naturally existing CD25+Foxp3+ T cells and/or inducing Foxp3 expression in CD25+Foxp3− effector T cells. The ability of IL-1β to promote higher levels of TGF-β1 production by T cells upon activation suggested that new Foxp3+ T cells could be induced in these cultures. T cells activated in the presence of IL-1β also produced significantly higher levels of IL-2 compared to control T cells. This indicated that these cytokines (i.e. TGF-β1 and IL-2) may have critical roles in inducing and/or maintaining Foxp3 expression in T cells that were activated in the presence of IL-1β.
TGF-β is a pleiotropic cytokine that can facilitate either regulatory or inflammatory responses depending on the levels of other cytokines present in the microenvironment 
. While TGF-β in combination with IL-2 is responsible for the survival of naïve T cells and helps maintain peripheral tolerance, it is also responsible for the differentiation of pathogenic Th17 cells in association with pro-inflammatory cytokines such as IL-6 
. Recent studies have conclusively demonstrated a critical role for IL-2 not only in the homeostasis and functioning of nTregs, but also in the generation of adaptive Tregs 
. Our observation that neutralization of IL-2 or TGF-β1 can reverse IL-1β-dependent increase in Foxp3+ T cell frequencies in the culture supports the notion that IL-1β promotes TGF-β1 and IL-2 dependent Foxp3 expression in T cells.
The nTregs are not known to produce significant amounts of IL-2 upon activation 
. Therefore, effector T cells are considered the primary source of IL-2 when total CD4+ T cells are activated using anti-CD3 and anti-CD28 Abs. Importantly, although most nTregs constitutively express high levels of CD25 on the surface, a significant proportion of CD4+CD25+ T cells enriched from normal mice could be Foxp3− activated effector T cells. This would explain why both CD4+CD25+ and CD4+CD25− T populations produced considerable amounts of IL-2 upon activation in our study. Our observation that only CD4+CD25+ T cells, but not CD4+CD25− cells produced TGF-β1 indicated that enriched CD4+CD25+ T cell preparation contains both Tregs and activated effector T cells, and this population, but not nTregs or CD4+CD25− cells, can produce significant amounts both TGF-β1 and IL-2 during activation leading to induction and/or maintenance of Foxp3 expression in them; the exogenous IL-1β enhances this effect.
Studies using purified GFP+Foxp3+ cells from Foxp3-GFP knock-in mice showed little or no proliferation when activated using anti-CD3 and anti-CD28 Abs with or without IL-1β. Although a considerable number of Foxp3+ T cells express TGF-β1 upon activation in the presence of IL-1β, it appears that IL-1β by itself does not promote Foxp3+ T cell proliferation. The inability of Foxp3+ cells to proliferate was most likely due to their failure to produce sufficient amounts of IL-2. In fact earlier studies have demonstrated that Foxp3+ nTregs require high amounts of exogenous IL-2 for their proliferation 
. In this regard, secretion of much higher amounts of TGF-β1 by CD4+CD25+ T cells, upon activation in the presence of IL-1β, suggested that it likely acted as an autocrine and helped sustain Foxp3 expression, while IL-2 secreted by Foxp3−CD25+ T cells facilitated proliferation of Foxp3+ cells. In fact, a recent study has shown that IL-1β-induced IL-2 production by Foxp3− T cells is essential for IL-1β-mediated expansion of Foxp3 T cells 
Very little is known about how IL-1β regulates IL-2 and TGF-β production. While studies have reported a negative correlation between IL-1β and TGF-β 
, Tao Lu et al
. showed that TGF-β and IL-1β cross activate each other in a dose-dependent manner 
. Earlier studies have also shown the induction of TGF-β by IL-1β in different human and animal tissues 
. Moreover, IL-1β has been shown to increase TGF-β1 in articular chondrocytes through the activation of AP-4 and AP-1 binding to the TGF-β1 gene promoter 
. Therefore, it appears that IL-1β and TGF-β regulate the expression of each other in different immune cells, and that this regulation is dependent upon their absolute and relative concentrations as well as on the presence of other cytokines such as IL-2. Our observation that TGF-β secretion and Foxp3 expression by Tregs is dependent on the concentration of IL-1β as well as presence of IL-2 in the culture supports this notion. While we have no direct evidence on the regulation of IL-2 production by IL-1β, the increased IL-2 production by effector T cells in the presence of IL-1β is likely the result of this cytokine's ability to enhance T cell proliferation upon activation.
Overall, our observations show that IL-1β can 1) enhance the proliferation of effector T cells upon activation, 2) increase the production of IL-2 by these proliferating cells, 3) enhance the activation and proliferation of Foxp3+ cells in the presence of IL-2 from effector T cells, 4) increase the production of TGF-β1 by Foxp3+ cells and 5) promote IL-2 and TGF-β1 dependent Foxp3 expression in both Tregs and effector T cells. The observation that IL-1β-exposed CD4+CD25+ cells can suppress effector T cell response in vitro and autoimmune thyroiditis in CBA/j mice suggest that the increase in the frequency of Foxp3+ Tregs in IL-1β-treated cultures may account for the enhanced suppression of autoimmune response when compared to controls. It must be noted that the control Tregs could also suppress autoimmune disease but to a lesser extent.
In conclusion, our study demonstrates a new role for IL-1β in promoting and enhancing Treg function. Considering that this cytokine is also produced during inflammatory responses, it is reasonable to assume that IL-1β has an immunoregulatory role. Based on our observations, we speculate that one of the functions of IL-1β produced during an inflammation may be to promote the suppressor function of Tregs to avoid autoimmune and lymphoproliferative responses.