Ghrelin Inhibits the Differentiation of T Helper 17 Cells through mTOR/STAT3 Signaling Pathway

<div><p>Enhanced activity of interleukin 17 (IL-17) producing T helper 17 (Th17) cells plays an important role in autoimmune and inflammatory diseases. Significant loss of body weight and appetite is associated with chronic inflammation and immune activation, suggesting the cross talk between immune and neuroendocrine systems. Ghrelin has been shown to regulate the organism immune function. However, the effects of ghrelin on the differentiation of Th17 cells remain elusive. In the present study, we observed the enhanced differentiation of Th17 cells in spleens of growth hormone secretagogue receptor 1a (GHSR1a)^-/- mice. Treatment of ghrelin repressed Th17 cell differentiation in a time- and concentration-dependent manner. Phosphorylation of mammalian target of rapamycin (mTOR) and signal transducer and activator of transcription 3 (STAT3) was increased in the spleens of GHSR1a^-/- mice. Activation of mTOR signaling by injection of Cre-expressiong adenovirus into tuberous sclerosis complex 1 (TSC1) ^loxp/loxp mice increased the differentiation of Th17 cells in spleen, which was associated with an increment in the phosphorylation of STAT3. Activation of mTOR signaling by leucine or overexpression of p70 ribosome protein subunit 6 kinase 1 (S6K1) activated mTOR signaling in isolated T cells, while reversed the ghrelin-induced inhibition of iTh17 cell differentiation. In conclusion, mTOR mediates the inhibitory effect of ghrelin on the differentiation of Th17 cells by interacting with STAT3.</p></div

Ghrelin inhibited the differentiation of Th17 cells <i>in vitro</i>.

<p>(A&B) Total T cells were isolated from mouse spleens and induced to differentiate into Th17 cells with TGF-β (5 ng/ml) and IL-6 (20 ng/ml), then treated with ghrelin with final concentration (A) and time (B) as indicated. The mRNA level of RORγt was analyzed with RT-PCR. (C) Differentiated Th17 cells were treated with ghrelin (10^–8 M). The mRNA level of IL-17A was analyzed with RT-PCR. (D)The concentration of IL-17A in the supernatant was examined with ELISA. (E&F) Cells were stimulated with PMA, Ionomycin and Brefeldin A for 4–6 hours. The percentage of IL-17A⁺ cells in splenic total T cells (E) and CD4⁺ T cells (F) was analyzed with flow cytometry. Shown is the representative of three independent experiments. *<i>P</i><0.05 versus control; ^#<i>P</i><0.05 versus ghrelin-treated alone.</p

Overexpression of S6K1 rescued the inhibitory effect of ghrelin on Th17 cells.

<p>Total T cells were isolated from mouse spleens and induced differentiation to Th17 cells. Adenovirus was used to infect activated T cells to overexpress S6K1, the critical downstream molecule in mTOR signaling pathway. (A) The phosphorylation of S6 was analyzed with Western Blot. Relative protein signal intensity was quantified. (B&C) Differentiated Th17 cells were treated with adenovirus expressing S6K1 (Ad-S6K1) or GFP (Ad-GFP) with final titer indicated. The mRNA level of RORγt (B) and IL-17A (C) were analyzed with RT-PCR. (D) The concentration of IL-17A in the supernatant was examined with ELISA. (E&F) The percentage of IL-17A⁺ cells in splenic total T cells (E) and CD4⁺ T cells (F) was analyzed with flow cytometry. Shown is the representative of three independent experiments. *<i>P</i><0.05 versus control; ^#<i>P</i><0.05 versus ghrelin-treated alone.</p

STAT3 was positively regulated by mTOR.

<p>(A) Total T cells were isolated from the spleen of TSC1^loxp/loxp mice injected with Ad-GFP and Ad-Cre. The phosphorylation of STAT3 was analyzed with Western Blot. (B) Differentiated Th17 cell was stimulated with ghrelin and/or leucine. The phosphorylation of STAT3 was analyzed with Western Blot. (C) Adenovirus was used to infect differentiated Th17 cells to overexpress S6K1. The phosphorylation of STAT3 was analyzed with Western Blot. (D) Differentiated Th17 cells were stimulated with ghrelin and/or colivelin. The phosphorylation of S6 was analyzed with Western Blot. Relative protein signal intensity was quantified.</p

STAT3 signaling pathway was involved in the inhibitory effect of ghrelin on Th17 cells.

<p>(A) Total T cells were isolated from spleens of GHSR1a^WT and GHSR1a^-/- mice. The phosphorylation of STAT3 was analyzed with Western Blot. (B) Total T cells were isolated from mouse spleens and induced differentiation to Th17 cells, then treated with ghrelin (10^–8 M). The phosphorylation of STAT3 was analyzed with Western Blot. (C) Differentiated Th17 cells were pre-treated with or without Colivelin (100 pM), then treated with or without ghrelin (10^-8M). The phosphorylation of STAT3 was analyzed with Western Blot. Relative protein signal intensity was quantified. (D&E) The mRNA level of RORγt (D) and IL-17A (E) was analyzed with RT-PCR. (F) The concentration of IL-17A in the supernatant was examined with ELISA. (G&H) The percentage of IL-17A⁺ cells in splenic total T cells (G) and CD4⁺ T cells (H) was analyzed with flow cytometry. Shown is the representative of three independent experiments. *<i>P</i><0.05 versus control; ^#<i>P</i><0.05 versus ghrelin-treated alone.</p

Enhanced differentiation of Th17 cells in GHSR1a^-/- mice.

<p>8 to 10-wk-old GHSR1a^WT (n = 5) and GHSR1a^-/- (n = 6) male mice were fed standard chow. (A) Total T cells were isolated from the spleen of GHSR1a^WT and GHSR1a^-/- mice. The mRNA level of GHSR1a was evaluated to verify the deficiency of GHSR1a. (B&C) The mRNA levels of RORγt and IL-17A in splenic total T cells and CD4⁺ T cells were analyzed with RT-PCR. (D) The concentration of IL-17A in serum was examined with ELISA. (E&F) The percentage of IL-17A⁺ cells in splenic total T cells (E) and CD4⁺ T cells (F) was analyzed with flow cytometry. Shown is the representative of three independent experiments. *<i>P</i><0.05 versus wild type control.</p

mTOR signaling pathway mediated the inhibitory effect of ghrelin on Th17 cells.

<p>(A) Total T cells were isolated from spleens of GHSR1a ^WT and GHSR1a^-/- mice. The phosphorylation of S6 was analyzed with Western Blot. Relative protein signal intensity was quantified. (B) 8 to 10-wk-old TSC1^loxp/loxp mice were injected intravenously through caudal vein with GFP or Cre virus for 4 weeks. The location of Ad-GFP in spleen was observed using immunoluorescence microscopy. (C) Total T cells were isolated from the spleens of mice injected with Ad-GFP or Ad-Cre. The expression of TSC1 and the phosphorylation of S6 were analyzed with Western Blot. Relative protein signal intensity was quantified. (D) Total T cells were isolated from the spleens of mice injected with Ad-GFP and Ad-Cre. The mRNA levels of RORγt and IL-17A were analyzed with RT-PCR. (E) The concentration of IL-17A in mice serum was examined with ELISA. *<i>P</i><0.05 versus Ad-GFP injected group.</p

Leucine rescued the inhibitory effect of ghrelin on Th17 cells.

<p>Total T cells were isolated from mouse spleens and induced differentiation to Th17 cells. (A) Differentiated Th17 cells were treated with ghrelin (10^–8 M) and leucine (1 mM). The phosphorylation of S6 was analyzed with Western Blot. Relative protein signal intensity was quantified. (B&C) Differentiated Th17 cells were treated with ghrelin (10^–8 M) and leucine with final concentration as indicated. The mRNA levels of RORγt (B) and IL-17A (C) were analyzed with RT-PCR. (D) Differentiated Th17 cells were treated with ghrelin (10^–8 M) and leucine (1 mM). The concentration of IL-17A in the supernatant was examined with ELISA. (E&F) The percentage of IL-17A⁺ cells in splenic total T cells (E) and CD4⁺ T cells (F) was analyzed with flow cytometry. Shown is the representative of three independent experiments. *<i>P</i><0.05 versus control; ^#<i>P</i><0.05 versus ghrelin-treated alone.</p

List and sequences of primers used in RT-PCR experiments.

<p>List and sequences of primers used in RT-PCR experiments.</p