TGF-β is necessary for induction of IL-23R and Th17 differentiation by IL-6 and IL-23

https://doi.org/10.1016/j.bbrc.2009.05.140Get rights and content

Abstract

TGF-β and IL-6 induce Th17 differentiation, and IL-23 is required for expansion and maintenance of Th17 cells. Recently, it was shown that IL-6 up-regulates IL-23R mRNA in naive CD4+ T cells and therefore IL-6 and IL-23 synergistically promote Th17 differentiation. However, the molecular mechanism whereby IL-6 and IL-23 induce Th17 differentiation and the relevance to TGF-β remain unknown. Here, we found that IL-6 up-regulated IL-23R mRNA expression, and IL-6 and IL-23 synergistically augmented its protein expression. The combination induced Th17 differentiation, and TGF-β1 further enhanced it. IL-6 augmented endogenous TGF-β1 mRNA expression, whereas the amount of TGF-β produced was not enough to induce Th17 differentiation by IL-6 alone. However, unexpectedly, the up-regulation of IL-23R and induction of Th17 differentiation by IL-6 and IL-23 were almost completely inhibited by anti-TGF-β. These results suggest that the induction of IL-23R and Th17 differentiation by IL-6 and IL-23 is mediated through endogenously produced TGF-β.

Introduction

A novel T helper cell subset producing IL-17, called Th17, that mediates the inflammation associated with tissue-specific autoimmune diseases including experimental autoimmune encephalitis (EAE) and collagen-induced arthritis (CIA) has been elucidated [1], [2], [3]. Aggarwal et al. demonstrated that IL-23 elicits production of the proinflammatory cytokine IL-17 from CD4+ T cells of the effector and memory, but not naive, phenotype [4]. Langrish et al. also demonstrated that adoptive transfer of autoantigen-specific Th17 cells generated in vivo by IL-23 but not Th1 cells by IL-12, induces EAE as well as CIA [5]. Thereafter, Harrington et al. [6] and Park et al. [7] demonstrated that IL-23 can drive in vitro Th17 differentiation from naive CD4+ T cells when IFN-γ and IL-4 are neutralized by blocking Abs or genetic deficiency. Thus, IL-23 appeared to be required for Th17-mediated immunopathology. However, three new reports indicated that IL-23 is not essential for development of Th17 but that instead Th17 differentiation is driven by the combination of TGF-β and IL-6 [8], [9], [10]. TGF-β is an important cytokine that promotes the differentiation of anti-inflammatory Foxp3+ regulatory T (Treg) cells [11]. Bettelli et al. demonstrated that IL-6 is capable of inhibiting TGF-β-dependent Foxp3+ Treg cell induction and simultaneously TGF-β plus IL-6 promotes Th17 cell differentiation [9]. Mangan et al. also showed that in Th17-polarizing conditions induced by TGF-β, TGF-β induces IL-23R expression and confers IL-23 responsiveness and that IL-23 subsequently serves as a survival factor for committed Th17 [10]. More recently, the transcription factors RORγt and STAT3 were shown to be critically important for the development of Th17 cells [12], [13], [14], [15]. Taken together, these recent studies indicate that TGF-β and IL-6 plays a critical role in the differentiation of naive CD4+ T cells to Th17 cells, and that IL-23 is important for amplifying and/or stabilizing the Th17 phenotype.

Recently, it was further demonstrated that IL-6 functions to up-regulate IL-23R mRNA in naive CD4+ T cells and that IL-23 synergizes with IL-6 in promoting Th17 differentiation [15]. However, the molecular mechanism whereby IL-6 and IL-23 induce Th17 differentiation and the relevance to TGF-β remain unknown. In the present study, we found that IL-6 up-regulates IL-23R mRNA expression, whereas IL-23 is necessary to induce IL-23R at protein level in synergy with IL-6. Moreover, IL-6 augmented TGF-β mRNA expression in naive CD4+ T cells, but IL-6 alone was not enough to induce Th17 differentiation. Of note is that the induction of IL-23R and Th17 differentiation by IL-6 and IL-23 was highly dependent on TGF-β, which was produced endogenously by IL-6. Thus, the combination of IL-6 and IL-23 may play an important role for the induction of Th17 differentiation under certain circumstances where bioactive TGF-β production is limited.

Section snippets

Materials and methods

Cell culture and mice. Naive CD4+ T cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 50 μM 2-ME. BALB/c mice were purchased from Japan SLC (Hamamatsu, Japan). Mice transgenic for αβ TCR recognizing OVA323–339 (DO11.10, BALB/c background) were provided by Dr. T. Yoshimoto (Hyogo College of Medicine, Hyogo, Japan). STAT1-deficient mice of 129/Sv background and wild-type 129/Sv mice were purchased from Taconic Farms, Inc. (Germantown, NY). T-bet-deficient mice with BALB/c

IL-6 and IL-23 induces Th17 differentiation and TGF-β enhances it

It was previously demonstrated that IL-6 up-regulates IL-23R mRNA expression and therefore IL-6 and IL-23 synergistically induce Th17 differentiation [15]. To investigate the molecular mechanism whereby IL-6 and IL-23 induce Th17 differentiation, we first confirmed the effect of IL-6 and IL-23 on induction of Th17 differentiation. Naive CD4+ T cells (CD4+CD25CD44lowCD62L+) were purified by FACS sorting from spleen cells of BALB/c mice and activated with plate-coated anti-CD3/anti-CD28 in the

Discussion

It was previously shown that up-regulation of IL-23R expression by TGF-β was observed 6 days after the stimulation [10], whereas TGF-β alone failed to efficiently induce IL-23R up-regulation during the first 72 h after the stimulation (Fig. 2A). In contrast, IL-6 rapidly and efficiently up-regulated IL-23R at mRNA level and the combination of IL-6 and IL-23 further augmented the expression, especially, at protein level (Fig. 2). Moreover, IL-6 augmented TGF-β1 mRNA expression in naive CD4+ T

Acknowledgments

This study was supported in parts by Grant-in-Aid for Scientific Research, “High-Tech Research Center” Project, and “University-Industry Joint Research” Project from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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