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, 86 (2), 401-9

Liver X Receptor Agonist Regulation of Th17 Lymphocyte Function in Autoimmunity

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Liver X Receptor Agonist Regulation of Th17 Lymphocyte Function in Autoimmunity

Jihong Xu et al. J Leukoc Biol.

Abstract

CD4+ Th17 cells are believed to play an important role in the development of a variety of autoimmune diseases including EAE, an animal model of MS. Previously, we and others demonstrated that LXR agonists suppressed the activation of primary glial cells and blocked the development of EAE. The present studies demonstrated that the LXR agonist T0901317 suppressed IL-17A expression from splenocytes derived from Valpha2.3/Vbeta8.2 TCR transgenic mice and from MOG(35-55)-immunized C57BL/6 mice. Furthermore, in vitro treatment with IL-23 alone or in combination with MOG(35-55) induced IL-17A expression from splenocytes derived from MOG(35-55)-immunized mice, and T0901317 blocked this induction. In vitro treatment with the LXR agonist suppressed IL-23R expression by splenocytes. In addition, in vivo treatment with the LXR agonist suppressed IL-17A and IL-23R mRNA and protein expression in EAE mice. These studies suggest that LXR agonists suppress EAE, at least in part by suppressing IL-23 signaling. Recent studies indicate that the cytokines IL-21 and IL-22 are produced by Th17 cells and modulate immune responses. Our studies demonstrate that the LXR agonist T0901317 suppressed MOG(35-55)-induced expression of IL-21 and IL-22 mRNA in splenocytes derived from MOG(35-55)-immunized mice. Finally, we demonstrate that the LXR agonist T0901317 suppressed the development of EAE in an experimental paradigm involving treatment of established EAE. Collectively, these studies suggest that LXR agonists may be effective in the treatment of MS.

Figures

Figure 1.
Figure 1.
LXR agonist T0901317 inhibits cytokine production by splenocytes. Splenocytes from Vα2.3/Vβ8.2 TCR transgenic mice, were activated in vitro with 25 μg/ml MBPAc1–11 in the absence or presence of the indicated concentrations of T0901317 (μM). Supernatants were collected at 48 h, and IFN-γ (A) and IL-17A (B) concentrations were determined by ELISA. Splenocytes from C57BL/6 mice immunized with MOG35–55 11 days earlier were activated in vitro with 25 μg/ml MOG35–55 in the absence or presence of the indicated concentrations of T0901317 (T09; μM). Supernatants were collected at 48 h, and IFN-γ (C) and IL-17A (D) concentrations were determined by ELISA. Values represent the mean ± sem for a representative experiment run in triplicate. Three independent experiments were conducted. *, P < 0.05, and ***, P < 0.001, versus MBP-treated cultures (A and B) or MOG-treated cultures (C and D).
Figure 2.
Figure 2.
LXR agonist T0901317 inhibits IL-17 production by IL-23-activated splenocytes isolated from MOG-immunized mice. Splenocytes from C57BL/6 mice immunized with MOG35–55 11 days earlier were treated in vitro with different concentrations of IL-23 as indicated (A) or 2 ng/ml IL-23 and/or 2 μg/ml MOG35–55 in the absence or presence of the indicated concentrations of T0901317 (μM; B) or 20 ng/ml IL-23 in the absence or presence of the indicated concentrations of T0901317 (μM; C). Supernatants were collected at 48 h, and the IL-17A concentrations were determined by ELISA. Values represent the mean ± sem for a representative experiment run in triplicate. Four independent experiments were conducted. **, P < 0.01, and ***, P < 0.001, versus control (A) or ***, P < 0.001, versus IL-23 plus MOG35–55 (B) or ***, P < 0.001, versus IL-23 (C)-treated cultures.
Figure 3.
Figure 3.
LXR agonist T0901317 inhibits splenic T cell production of IL-17 and IFN-γ intracellular cytokine-staining analysis. Splenocytes from MOG35–55-immunized EAE mice were isolated by CD90 microbeads. Purified CD90+ T cells were stimulated with leukocyte activation cocktail [PMA, ionomycin (iono), and the protein transport inhibitor brefeldin A] in the absence or presence of the indicated concentrations of T0901317 (μM). IL-17A (A) or IFN-γ (B) protein levels were evaluated by intracellular cytokine staining and flow cytometry analysis. Results shown are representative of three independent experiments.
Figure 4.
Figure 4.
LXR agonist T0901317 inhibits IL-17A, IFN-γ, and IL-23R mRNA expression in splenocytes. Splenocytes from C57BL/6 mice immunized with MOG35–55 11 days earlier, were activated in vitro with 25 μg/ml MOG35–55 in the absence or presence of the indicated concentrations of T0901317 (μM). Total RNA was isolated. IL-17A (A), IFN-γ (B), and IL-23R (C) mRNA levels were determined by real-time quantitative RT-PCR. Results are expressed as fold changes relative to control, and all values were normalized against GAPDH. Values are mean ± sem from five independent experiments, and duplicate reactions were performed on each sample. *, P < 0.05, **, P < 0.01, ***, P < 0.001, versus MOG-treated cultures, and #, P < 0.05, versus control.
Figure 5.
Figure 5.
LXR agonist T0901317 inhibits IL-17A, IFN-γ, and IL-23R mRNA expression in EAE mice. C57BL/6 mice were treated (i.p.) with 25 mg/kg/day T0901317 (n=5) or vehicle (n=5), beginning 3 days before MOG35–55 immunization. Sham-operated mice (Control, n=3) were only injected with IFA. Spinal cords were harvested on Day 16 postimmunization, and total RNA was isolated. IL-17A (A), IFN-γ (B), and IL-23R (C) mRNA levels were determined by real-time quantitative RT-PCR. Results are expressed as fold changes relative to vehicle-treated EAE mice, and all values are normalized against GAPDH. Values are mean ± sem from five samples, and duplicate reactions were performed on each sample. ***, P < 0.001, versus vehicle-treated mice.
Figure 6.
Figure 6.
LXR agonist T0901317 suppresses IL-17A and IL-23R protein expression in EAE mice. C57BL/6 mice were injected (i.p.) with the vehicle daily (Lanes 1–5, n=5) or 25 mg/kg/day T0901317 (Lanes 6–10, n=5), beginning 3 days before MOG35–55 immunization. Sham mice (Control, Lanes 11–13, n=3) were only injected with IFA. Spinal cords were harvested on Day 16 postimmunization, and protein extracts were prepared. Protein expression for IL-17A and IL-23R (A) was evaluated by Western blot analysis as described in Materials and Methods. Protein levels were quantified by densitometry. Results are expressed in arbitrary units as the ratio of IL-17A or IL-23R relative to actin concentrations, and values represent the mean ± sem from five samples from vehicle or T0901317-treated EAE mice and three samples from sham-operated mice. **, P < 0.01, and ***, P < 0.001, versus vehicle-treated EAE mice (B).
Figure 7.
Figure 7.
LXR agonist T0901317 inhibits Th17 effector cytokine mRNA expression in splenocytes. Splenocytes from C57BL/6 mice immunized by MOG35–55, were activated in vitro with 25 μg/ml MOG35–55 in the absence or presence of the indicated concentrations of T0901317 (μM). Total RNA was isolated, and IL-21 (A) and IL-22 (B) mRNA levels were determined by real-time quantitative RT-PCR. Splenocytes from C57BL/6 mice immunized 11 days previously with MOG35–55 were isolated and activated in vitro with 20 ng/ml IL-23 in the absence or presence of the indicated concentrations of T0901317 (μM). Total RNA was isolated, and IL-17A (C) and IL-22 (D) mRNA levels were determined by real-time quantitative RT-PCR. Results are expressed as fold changes relative to control, and all values were normalized against GAPDH. Values are mean ± sem from at least three independent experiments, and duplicate reactions were performed on each sample. *, P < 0.05, **, P < 0.01, and ***, P < 0.001, versus MOG-treated cultures (A and B) or versus IL-23-treated cultures (C and D).
Figure 8.
Figure 8.
Postimmunization treatment with LXR agonist T0901317 suppresses the clinical severity of EAE. Mice were administered vehicle (•, n=8) or T0901317 (▴, n=9) daily via i.p. injection, as indicated. Mice were monitored daily for clinical disease. Mean clinical scores represent the average for all animals in a treatment group, and error bars represent se. Results are representative of two independent experiments. *, P < 0.05, **, P < 0.01, and ***, P < 0.001, for T0901317-treated versus vehicle-treated mice.

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