Astilbin promotes the induction of regulatory NK1.1 CD4+ NKG2D+ T cells through the PI3K, STAT3, and MAPK signaling pathways

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Highlights

Abstract

Astilbin is a potential agent for autoimmune and inflammatory diseases and has a protective effect in mice with DSS-induced colitis. NK1.1 CD4+ NKG2D+ T cells are a subpopulation of regulatory T cells that produce TGF-β1 and IL-10. Whether astilbin directly promotes the induction of NK1.1 CD4+ NKG2D+ T cells and whether these astilbin-stimulated T cells exert an immune-regulatory role remain unclear. Here, we show that astilbin efficiently induces the production of NK1.1 CD4+ NKG2D+ T cells with high expressions of TGF-β1, IL-10, CCR6, and CCR9 in a dose-dependent manner ex vivo. These regulatory T cells also substantially inhibit the activities of CD8+ T cells and macrophages. Intraperitoneal injection of astilbin ameliorates the severity of colitis with an increase in the frequency of NK1.1 CD4+ NKG2D+ T cells in the colon tissue of DSS-treated mice. Moreover, adoptive transfer of NK1.1 CD4+ NKG2D+ T cells induced by astilbin remarkably protects against the onset of DSS-induced colitis. Finally, the PI3K, STAT3, and MAPK signaling pathways are involved in the induction of NK1.1 CD4+ NKG2D+ T cells by astilbin. Taken together, our study elucidates a new immune-regulatory mechanism of astilbin by inducing the regulatory NK1.1 CD4+ NKG2D+ T cells and indicates a potential clinical use of astilbin for patients with inflammatory bowel diseases.

Introduction

Astilbin is a natural compound isolated from Rhizoma smilacis glabrae with anti-inflammatory and anti-oxidative effects that acts as an immunosuppressive factor in several autoimmune experimental models, such as concanavalin A-induced liver injury [1], delayed-type hypersensitivity [2], contact dermatitis [3], and collagen-induced arthritis [4]. Astilbin also exerts its regulatory properties by regulating lymphocyte behaviors. Studies indicated that astilbin is able to modulate the cytokine profiles of immune cells [5]. Astilbin is shown to induce IL-10 expression but inhibit TNF-α and IFN-γ expression in lymph node cells in contact hypersensitivity model [6]. Meanwhile, astilbin suppresses IL-17 production in Th17 cells and ameliorates experimental autoimmune myasthenia gravis [7]. Our group also reported that astilbin retards DSS-induced murine colitis by upregulating CD103+ dendritic cells (DCs) with high TGF-β1 and IL-10 production [8]. Furthermore, astilbin is proved to regulate T cells adhesion [1], [9] and migration [2] in different contexts. It is also indicated, by several studies, that astilbin might induce regulatory T cells in protecting autoimmune diseases [7].

As an activating receptor, NKG2D broadly expresses on NK, NKT, γδT, and CD8+ T cells but hardly shows on CD4+ T cells [10], [11]. Under tumor and inflammation, NKG2D can be induced on CD4+ T cells and is involved with pathological processes. In our research, CD4+ NKG2D+ T cells are further divided into two major subsets based on their NK1.1 expression [12]. NK1.1+ CD4+ NKG2D+ T cells generally exert pro-inflammatory properties by producing IFN-γ and TNF-α and participate in multiple autoimmune diseases and infections, including Wegener’s granulomatosis [13], Crohn’s disease [14], rheumatoid arthritis [15], multiple sclerosis [16], alopecia areata [17], and cytomegalovirus infections [18]. On the contrary, NK1.1 CD4+ NKG2D+ T cells mediate anti-inflammatory effects by producing TGF-β1 and IL-10 and could be induced in patients with tumors [19], [20], [21], [22], [23], autoimmune thyroid disorders [24], and juvenile-onset of systemic lupus erythematosus [25].

NK1.1 CD4+ NKG2D+ T cells have been induced in pCD86-RAE-1 transgenic mice [22], mice with DSS-induced colitis [12], and tumor-bearing mice [23]. This T cell subset is distinct from the conventional regulatory T cells due to its negative expression of Foxp3 and CD25 [12]. The activation of PI3K p85 is initiated by TCR/NKG2D co-ligation in NK1.1 CD4+ NKG2D+ T cells, followed by the activation of JNK/AP-1, STAT3, and NF-κB, and then contribute to TGF-β1 transcription [26]. Considering the immune-regulatory role of astilbin, its effects on regulatory NK1.1CD4+ NKG2D+ T cells and its molecular mechanisms were investigated.

Section snippets

Mice and cells

C57BL/6 mice (female, 6 weeks old, 16–20 g) were obtained from the Comparative Medicine Center of Yangzhou University (Yangzhou, China). Care and use of mice were performed according to the leading protocol approved by the Animal Experiment Ethics Committee of Yangzhou University (Approval ID: SYXK [Su] 2017-0044). A human T- cell acute lymphoblastic leukemia Jurkat cell line was purchased from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI 1640 medium

Astilbin promotes the induction of NK1.1 CD4+ NKG2D+ T cells ex vivo

Effects of astilbin on NKG2D expression in CD4+ T cells were examined. After 48 h of astilbin treatment, NKG2D was remarkably induced in the CD4+ T cells in a dose-dependent manner (Fig. 1A and 1B). Meanwhile, the transcription of NKG2D on CD4+ T cells was similarly upregulated by the astilbin treatment in a dose-dependent manner (Fig. 1C). In addition, the expression of NKG2D in a human T-cell acute lymphoblastic leukemia cell line (Jurkat cells) was slightly promoted by astilbin at 40 μg/ml

Discussion

This study elucidated a novel mechanism by which astilbin induces the regulatory NK1.1 CD4+ NKG2D+ T cells to attenuate DSS-induced colitis in mice. Astilbin promoted the freshly isolated NK1.1 CD4+ T or Jurkat cells to express NKG2D ex vivo. NK1.1 CD4+ NKG2D+ T cells induced by astilbin produced TGF-β1 and IL-10 and exhibited a potential migratory capacity towards peripheral tissues via CCR6 and CCR9 expressions. Furthermore, these NK1.1 CD4+ NKG2D+ T cells suppressed the activity of CD8+

Authors contributions

WG, BD and YZ designed and financed the study. SH and KW performed the experiments and drafted the manuscript. JW, YX, ZL and GL analyzed the data. WX, YD and XJ edited the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No. 81671547, No. 81873866, No. 81873867), Natural Science Foundation of Jiangsu Province, China (Grant No. BK20161339, Grant No. BK20160479); the “Six peaks” Talent Project of Jiangsu Province, and the Postgraduate Research & Practice Innovation Program of Jiangsu Province, China (KYCX18_2383).

Declaration of Competing Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.

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