Simvastatin promotes Th2-type responses through the induction of the chitinase family member Ym1 in dendritic cells

Abstract

Statins, best known for their lipid-lowering actions, also possess immunomodulatory properties. Recent studies have shown a Th2-biasing effect of statins, although the underlying mechanism has not been identified. In this study, we investigated whether simvastatin can exercise a Th2-promoting effect through modulation of function of dendritic cells (DCs) without direct interaction with CD4+ T cells. Exposure of DCs to simvastatin induced the differentiation of a distinct subset of DCs characterized by a high expression of B220. These simvastatin-conditioned DCs up-regulated GATA-3 expression and down-regulated T-bet expression in cocultured CD4+ T cells in the absence of additional simvastatin added to the coculture. The Th2-biased transcription factor profile induced by simvastatin-treated DCs also was accompanied by increased Th2 (IL-4, IL-5, and IL-13) and decreased Th1 (IFN-gamma) cytokine secretion from the T cells. The Th2-promoting effect of simvastatin was found to depend on the chitinase family member Ym1, known to be a lectin. Anti-Ym1 antibody abolished the Th2-promoting effect of simvastatin-treated DCs. Also, simvastatin was unable to augment Ym1 expression in DCs developed from STAT6-/- or IL-4R alpha-/- mice. Thus, modulation of Ym1 production by DCs identifies a previously undescribed mechanism of Th2 polarization by statin.

Fig. 1.

Phenotype and morphology of simvastatin-treated DCs. (A) Bone marrow progenitors were grown in the presence of granulocyte/macrophage colony-stimulating factor (10 ng/ml) for 6 days, and CD11c⁺ DCs were purified. CD11c⁺ DCs were cultured further in the presence or absence of simvastatin (1 μM) for 2 days. Phenotypic analysis then was performed by flow cytometry. Thin lines indicate staining with isotype controls. (B) Light and scanning electron microscopy of DCs cultured in the presence or absence of simvastatin. (C) Expression of CD11b, Gr1, CD19, and DEC-205 on DCs cultured in the presence or absence of simvastatin. (D) Effect of different doses of simvastatin on the expression of B220. The numbers denote percentages of cells that are specifically stained. The experiments were repeated at least four times with similar results.

Fig. 2.

DCs treated with simvastatin polarize naïve CD4⁺ T cells toward the Th2 phenotype in vitro. (A) DCs were treated with simvastatin (1 μM) or left untreated for 2 days. Cells were then washed and cultured with naïve DO11.10 CD4⁺ T cells and OVA peptide (5 μg/ml) for 5 days. Nuclear extracts were analyzed by immunoblotting with antibodies against GATA-3, T-bet, or CREB-1. Each blot was scanned by densitometry, and the ratio of GATA-3 or T-bet to CREB-1 was calculated and presented. ∗, P < 0.001; ∗∗, P < 0.001; ∗∗∗, P < 0.01; ∗∗∗∗, P < 0.01, relative to an untreated group. (B) Cytokines in culture supernatants as determined by ELISA. Data are the mean ± SEM of individual wells and are representative of three independent experiments. (C) Similar proliferation of DO11.10 TCR transgenic T cells induced by simvastatin-treated or control DCs. The experiment was repeated two times with similar results.

Fig. 3.

(A) Effect of simvastatin on the expression of costimulatory molecules on DCs. Thin lines indicate staining with isotype controls. (B) Addition of IFN-γ did not reverse Th2 polarization induced by simvastatin-treated DCs. Although IFN-γ did not inhibit GATA-3 expression, it augmented T-bet expression in simvastatin-treated DCs. (C) Addition of IL-12 significantly reduced both GATA-3 expression and Th2 cytokine production in a dose-dependent fashion. Culture supernatants in B and C were assayed for cytokine production by ELISA.

Fig. 4.

Increased STAT6-dependent Ym1 expression in DCs treated with simvastatin. Cell lysates (A) and culture supernatants (B) of control and simvastatin-treated DCs were subjected to immunoblotting with anti-Ym1 antibody. The experiment was repeated three times with similar results, and shown is a representative experiment. Ym1 secretion was undetectable when DCs were isolated from Stat6^−/− mice (B) or IL4Rα^−/− mice (C). (D) Addition of neutralizing antibodies to IL-4 (Upper) or IL-13 (Lower) to DC culture did not change the level of Ym1 induction by simvastatin. (E) Ym1 expression in DCs that were pretreated with simvastatin for 2 days, washed to remove statin, and then cultured for another 3 days.

Fig. 5.

Anti-Ym1 antibody reverses the ability of DCs treated with simvastatin to drive Th2 polarization. Bone marrow DCs were treated with simvastatin for 2 days and then cocultured with CD4⁺ T cells from DO11.10 mice in the presence of different doses of anti-Ym1 or control antibody (rabbit IgG) for 5 days. GATA-3 levels in nuclear extracts (A) was assessed and culture supernatants were analyzed by ELISA for IL-5 and IL-13 (B). ∗, P < 0.005; ∗∗, P < 0.005; ∗∗∗, P < 0.005; ∗∗∗∗, P < 0.005. Results are representative of three independent experiments. (C and D) Similar results were obtained in the case of splenic DCs. ∗, P < 0.001; ∗∗, P < 0.001; ∗∗∗, P < 0.005; ∗∗∗∗, P < 0.05.