All-Trans Retinoic Acid Promotes TGF-β-Induced Tregs via Histone Modification but Not DNA Demethylation on Foxp3 Gene Locus

et al. (2011) All-Trans Retinoic Acid Promotes TGF-b-Induced Tregs via Histone Modification but Not DNA Demethylation on Foxp3 Gene Locus. PLoS ONE 6(9): e24590. doi:10.1371/journal.pone.0024590 All-Trans Retinoic Acid Promotes TGF-b-Induced Tregs via Histone Modification but Not DNA Demethylation on Foxp3 Gene Locus Ling Lu Jilin Ma Zhiyuan Li Qin Lan Maogen Chen Ya Liu Zanxian Xia Julie Wang Yuanping Han Wei Shi Valerie Quesniaux Bernhard Ryffel David Brand Bin Li Zhongmin Liu Song Guo Zheng Niels Olsen Saraiva Camara, Universidade de Sao Paulo, Brazil Background: It has been documented all-trans retinoic acid (atRA) promotes the development of TGF-b-induced CD4+Foxp3+ regulatory T cells (iTreg) that play a vital role in the prevention of autoimmune responses, however, molecular mechanisms involved remain elusive. Our objective, therefore, was to determine how atRA promotes the differentiation of iTregs. Methodology/Principal Findings: Addition of atRA to nave CD4+CD252 cells stimulated with anti-CD3/CD28 antibodies in the presence of TGF-b not only increased Foxp3+ iTreg differentiation, but maintained Foxp3 expression through apoptosis inhibition. atRA/TGF-b-treated CD4+ cells developed complete anergy and displayed increased suppressive activity. Infusion of atRA/TGF-b-treated CD4+ cells resulted in the greater effects on suppressing symptoms and protecting the survival of chronic GVHD mice with typical lupus-like syndromes than did CD4+ cells treated with TGF-b alone. atRA did not significantly affect the phosphorylation levels of Smad2/3 and still promoted iTreg differentiation in CD4+ cells isolated from Smad3 KO and Smad2 conditional KO mice. Conversely, atRA markedly increased ERK1/2 activation, and blockade of ERK1/2 signaling completely abolished the enhanced effects of atRA on Foxp3 expression. Moreover, atRA significantly increased histone methylation and acetylation within the promoter and conserved non-coding DNA sequence (CNS) elements at the Foxp3 gene locus and the recruitment of phosphor-RNA polymerase II, while DNA methylation in the CNS3 was not significantly altered. Conclusions/Significance: We have identified the cellular and molecular mechanism(s) by which atRA promotes the development and maintenance of iTregs. These results will help to enhance the quantity and quality of development of iTregs and may provide novel insights into clinical cell therapy for patients with autoimmune diseases and those needing organ transplantation. - Funding: This work was supported by National Institutes of Health grants R01 AR059103, R01 AR051558, and R01 HL068597; ACR Within Our Reach; the Arthritis Foundation; an Outstanding Youth Scientist Award from the National Natural Science Foundation of China (30728007); the National Natural Science Foundation of China (30772150, 81001307, and 81100270); and Zhejiang Province National Natural Science Foundation of China (Y2090918). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. All-trans-retinoic acid (atRA), a Vitamin A derivative, has profound effects on embryonal morphogenesis, vision, reproduction, cell differentiation, growth, and immune homeostasis [1]. Deficiency of vitamin A leads to exacerbation of experimental colitis [2]. In the immune system, atRA plays important roles in regulating the functions of many different cell types [3]. Vitamin A and its derivatives are capable of ameliorating several models of autoimmunity, including inflammatory bowel disease, rheumatoid arthritis, type I diabetes, and experimental encephalomyelitis [45]. In addition to the inhibitory effect of atRA on T effector cell differentiation and function, atRA has also been shown to be capable of promoting murine CD4+Foxp3+ Tregs induced by TGF-b from conventional CD4+Foxp32 cells, either directly by enhancing TGF-b-driven Smad3 signaling in naive cells and/or indirectly by relieving the production of pro-inflammatory cytokines from murine memory effector cells [68]. Such approaches show great promise as these T cells have been shown effective in combating several immune-mediated disorders [9]. CD4+CD25+ T regulatory (Treg) cells play a critical role in establishing and maintaining self-tolerance. Therefore, enhancing the number and/or function of Tregs represents a potential treatment for patients with autoimmune disorders or those who undergo transplant rejection. atRA can strongly increase TGF-binduced Foxp3 expression and Treg conversion in vitro [6]. Under these conditions, atRA may enhance TGF-b signaling by increasing the expression and phosphorylation of Smad3. On the other hand, it has also been reported that expression of RAR can be increased through TGF-b signaling [10]. Therefore, atRA and TGF-b may cooperatively augment their mutual signaling to further enhance Foxp3 expression. However, the exact roles of atRA in these signaling pathways are less well understood. We recently reported that while the Smad pathway plays a less important role in the differentiation of Foxp3+ iTregs induced by TGF-b, ERK and JNK kinases which mainly use non-Smad pathways, may play a more significant role in this process [11]. Herein, we further demonstrate that adding atRA to cultures containing TGF-b not only increases Foxp3 expression and maintenance, but also enhances the suppressive activities of these Tregs in vitro and in vivo. Studies of the underlying mechanism responsible for these observations indicate that atRA upregulates ERK rather than Smad2/3 activation of the TGF-b down-stream signaling pathway. Additionally, Foxp3 induced by a combination of atRA and TGF-b displayed increased Foxp3 binding ability on chromatin compared to that induced by TGF-b alone. We further found that atRA enhances histone methylation and acetylation in Foxp3 promoter and its conserved non-coding DNA sequence elements (CNS2), rather than DNA CpG demethylation of CNS3 in the Foxp3 locus. Thus, atRA improves both the quantity and quality of Foxp3+ iTregs, findings which will be important in the development of superior cell therapies to treat autoimmune diseases and prevent organ transplantation rejection. atRA directly up-regulates Foxp3 and sustains its expression by CD4+ cells treated with TGF-b In agreement with previous reports [6], addition of atRA to cultures containing TGF-b significantly enhanced the proportions of CD4+CD25+Foxp3+ cells induced from naive CD4+ CD252Foxp32 (or GFP2 cells using WT or Foxp3 GFP knockin mice). This effect may reflect either direct Foxp3+ cell induction or a secondary effect through suppression of CD4+Foxp32 cell expansion [8]. In either case, total Foxp3 protein levels and Foxp3+ cell numbers increased significantly in CD4+ cells treated with the combination of atRA and TGF-b than those treated with TG (...truncated)