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. 2017 Jun 14;19(1):137.
doi: 10.1186/s13075-017-1349-2.

Leucine-rich alpha 2 glycoprotein promotes Th17 differentiation and collagen-induced arthritis in mice through enhancement of TGF-β-Smad2 signaling in naïve helper T cells (V体育平台登录)

Hayato Urushima  1 , Minoru Fujimoto  2   3 , Takashi Mishima  1 , Tomoharu Ohkawara  1 , Hiromi Honda  1 , Hyun Lee  1 , Hirohisa Kawahata  4 , Satoshi Serada  1   5 , Tetsuji Naka  1   5
Affiliations

Leucine-rich alpha 2 glycoprotein promotes Th17 differentiation and collagen-induced arthritis in mice through enhancement of TGF-β-Smad2 signaling in naïve helper T cells

Hayato Urushima et al. Arthritis Res Ther. .

Abstract

Background: Leucine-rich alpha 2 glycoprotein (LRG) has been identified as a serum protein elevated in patients with active rheumatoid arthritis (RA). Although the function of LRG is ill-defined, LRG binds with transforming growth factor (TGF)-β and enhances Smad2 phosphorylation VSports手机版. Considering that the imbalance between T helper 17 (Th17) cells and regulatory T cells (Treg) plays important roles in the pathogenesis of RA, LRG may affect arthritic pathology by enhancing the TGF-β-Smad2 pathway that is pivotal for both Treg and Th17 differentiation. The purpose of this study was to explore the contribution of LRG to the pathogenesis of arthritis, with a focus on the role of LRG in T cell differentiation. .

Methods: The differentiation of CD4 T cells and the development of collagen-induced arthritis (CIA) were examined in wild-type mice and LRG knockout (KO) mice. To examine the influence of LRG on T cell differentiation, naïve CD4 T cells were isolated from LRG KO mice and cultured under Treg- or Th17-polarization condition in the absence or presence of recombinant LRG. V体育安卓版.

Results: In the CIA model, LRG deficiency led to ameliorated arthritis and reduced Th17 differentiation with no influence on Treg differentiation. By addition of recombinant LRG, the expression of IL-6 receptor (IL-6R) was enhanced through TGF-β-Smad2 signaling. In LRG KO mice, the IL-6R expression and IL-6-STAT3 signaling was attenuated in naïve CD4 T cells, compared to wild-type mice. V体育ios版.

Conclusions: Our findings suggest that LRG upregulates IL-6R expression in naïve CD4 T cells by the enhancement of TGF-β-smad2 pathway and promote Th17 differentiation and arthritis development. VSports最新版本.

Keywords: IL-6 receptor; Leucine rich alpha2 glycoprotein; Smad2; TGF-β; Th17 V体育平台登录. .

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Figures

Fig. 1
Fig. 1
Leucine-rich alpha 2 glycoprotein (LRG) knockout (KO) mice are protected against collagen-induced arthritis (CIA). Male wild-type (WT) mice and LRG KO mice were subjected to CIA. a The serum levels of LRG in WT mice (n = 6–8) on indicated days after arthritis induction were determined by ELISA. Values are mean ± SD; **p < 0.01, Dunnett’s test. b Representative immunohistochemical analysis of LRG in wrist and ankle joints from non-treated WT mice or mice with CIA on day 35 after arthritis induction. c Representative macroscopic joint symptoms in WT mice or LRG KO mice on day 35 after arthritis induction. d, e The symptoms of arthritis were scored (0–4 per limb) until day 70. The arthritis score (d) and maximum arthritis score (e) are shown for WT mice (n = 12) and LRG KO mice (n = 11). Values are mean ± SD: *p < 0.05, Mann-Whitney U test. f Representative histological appearance of joints from a WT mouse and LRG KO mouse on day 35 after arthritis induction. Joints were stained with hematoxylin and eosin (left and middle panel) or safranin O (right panel)
Fig. 2
Fig. 2
Leucine-rich alpha 2 glycoprotein (LRG) deficiency results in impaired differentiation of T helper (Th)-17 cells after induction of arthritis. a Representative macroscopic images (left), the weight (middle) and the cell number (right) of inguinal lymph nodes in wild-type (WT) mice (n = 16) and LRG knockout (LRG KO) mice (n = 16) with or without arthritis induction (day 27): *p < 0.05. b Inguinal lymph node cells on day 27 were isolated from WT mice (n = 8) and LRG KO mice (n = 8) and stimulated by chicken type-2 collagen for 3 days. The percentages of IL-17+ cells gated on CD4+ cells were determined by flow cytometry (left). The levels of IL-17 in cell culture supernatants were analyzed by ELISA (right): *p < 0.05. c Percentages of CD4 + Foxp3 + cells (Treg) and CD4 + IFN-γ + cells (Th1) of the same population as b were determined by flow cytometry. d The serum levels of cytokines and anti-collagen type-2 antibodies in WT mice (n = 8) and LRG KO mice (n = 8) on day 27 after induction of collagen-induced arthritis (CIA) were analyzed by ELISA: *p < 0.05. All statistical analyses were performed using Student’s t test. NT non-treated, n.s. not significant, IFN interferon
Fig. 3
Fig. 3
Recombinant leucine-rich alpha 2 glycoprotein (LRG) enhances transforming growth factor beta (TGF-β)-induced Smad2 phosphorylation in naïve CD4 T cells and promotes T regulatory cell differentiation. a Naïve CD4 T cells were isolated from LRG knockout (LRG KO) mice and were stimulated by TGF-β (0.5 or 2 ng/mL) in the presence or absence of recombinant LRG (LRG + (1 μg/mL) or LRG -) for 30 minutes. Representative data from three independent experiments are shown (upper panel). The levels of phosphorylated Smad2 relative to total Smad2 were quantified using imageJ and values (mean ± SD; n =3) are shown (bottom panel): *p < 0.05, **p < 0.01, one-way analysis of variance followed by the Bonferroni test. NT non-treated. b Naïve CD4 T cells were isolated from wild-type (WT) or LRG KO mice (n = 3, respectively). The expression of endoglin was determined by western blotting. MS-1, mouse endothelial cell, was used as a positive control (PC) for the expression of endoglin. c Naïve CD4 T cells were obtained from LRG KO mice and stimulated by TGF-β (0, 0.5 and 2.5 ng/mL) in the absence or presence of recombinant LRG (LRG + (1 μg/mL) or LRG -) for 30 minutes. The phosphorylation of Smad1 and Smad2 were examined by western blotting. L929, mouse fibroblast, was used as a PC. GAPDH glyceraldehyde-3-phosphate dehydrogenase
Fig. 4
Fig. 4
Recombinant leucine-rich alpha 2 glycoprotein (LRG) enhances differentiation of T helper 17 (Th17) cells under the Th17 polarization condition. a Naïve CD4 T cells (n = 3) were treated for 3 days with anti-CD3/CD28 beads and transforming growth factor beta (TGF-β) (0, 0.125 or 0.5 ng/mL (Treg polarization condition)) in the presence or absence of LRG (LRG + (1 μg/mL) or LRG -). The percentage of CD4+Foxp3+ T regulatory (Treg) cells was determined by flow cytometry (right): *p < 0.05. Representative data are shown (left panel). b, c, d Naïve CD4 T cells were isolated from the spleen and lymph nodes of LRG knockout mice (LRG KO) (n = 3) and cultured for 3 days with anti-CD3/CD28 beads in the presence or absence of recombinant LRG (LRG + (1 μg/mL) or LRG -) under a non-polarization condition without cytokines or the Th17 polarization condition with TGF-β (2 ng/mL) and IL-6 (100 ng/mL). b The expression of IL-17 (left) and RORγt (right) mRNA was analyzed by quantitative PCR: *p < 0.05. c The percentage of IL-17+ cells in the CD4+ population under the Th17 polarization condition was determined by flow cytometry. Values are mean ± SD, n = 3 (left panel). Representative data are shown (right panel). d The induction of Treg cells under a non-polarization condition or under the Th17 polarization condition was examined by flow cytometry. All statistical analyses were performed using Student’s t test. NT; non-treated
Fig. 5
Fig. 5
Leucine-rich alpha 2 glycoprotein (LRG) contributes to the IL-6-STAT3 signaling in naïve CD4 T cells by upregulating IL-6 receptor expression. a Naïve CD4 T cells were isolated from wild-type (WT) mice and stimulated by IL-6 (100 ng/mL) or IL-6 with transforming growth factor beta (TGF-β) (2 ng/mL) in the absence or presence of recombinant LRG (1 μg/mL) for 30 minutes. Phosphorylation of Smad2 and p38 was determined by western blotting. Relative band intensities of pSmad2 and p-p38, as normalized by Smad2 and p38, respectively, are depicted at the bottom of the bands. b Naïve CD4 T cells were isolated from WT or LRG knockout (LRG KO) mice and stimulated by IL-6 (100 ng/mL) in the absence or presence of TGF-β (2 ng/mL) for 30 minutes. STAT3 phosphorylation was determined by western blotting. Relative band intensities of pSTAT3 normalized by STAT3 are depicted at the bottom of the bands. c Splenocytes were isolated from WT and LRG KO mice (n = 3 for each group) and stimulated by IL-6 (100 ng/mL) for 30 minutes. After intracellular staining, phosphorylated STAT3 in CD4 + CD62 + CD25 - cells was determined by flow cytometry. Representative data are shown in the left panel and mean fluorescence intensity of phosphorylated STAT3 (mean ± SD) is shown in the right panel: *p < 0.05. GAPDH glyceraldehyde-3-phosphate dehydrogenase
Fig. 6
Fig. 6
Leucine-rich alpha 2 glycoprotein (LRG) upregulates the expression of IL-6 receptor (IL-6R) in naïve CD4 T cells presumably through the enhancement of transforming growth factor beta (TGF-β) signaling. a, b Naïve CD4 T cells were isolated from the spleen and lymph nodes of wild-type (WT) or LRG knockout (LRG KO) mice (n = 8 in each group) and the expression of IL-6R and gp130 were examined. a Representative data from immunocytochemical analysis of IL-6R. b The expression of IL-6R or gp130 in naïve CD4 T cells was analyzed by flow cytometry and representative data are shown in the left panel. Mean values of mean fluorescence intensity (MFI) ± SD are shown in the right panel: **p < 0.01. c, d Naïve CD4 T cells obtained from LRG KO mice (n = 3) were stimulated by TGF-β (1 ng/mL) (c) or TGF-β (0.2 or 1 ng/mL) with IL-6 (100 ng/mL) (d). After 24 h of culture, IL-6R expression was analyzed by flow cytometry. Values are mean ± SD: *p < 0.05. e The serum levels of soluble IL-6 receptor alpha (IL-6Ra) of WT or LRG KO mice on day 27 of CIA were determined by ELISA. f Naïve CD4 T cells derived from WT and LRG KO mice were stimulated by IL-6 (20 or 50 ng/ mL) in the presence or absence of soluble IL-6R (20 ng/mL) for 30 minutes. STAT3 phosphorylation was determined by western blotting. Relative band intensities of pSTAT3 normalized by STAT3 are depicted at the bottom of the bands. NT non-treated
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