Arginase I induction by modified lipoproteins in macrophages: a peroxisome proliferator-activated receptor-gamma/delta-mediated effect that links lipid metabolism and immunity - PubMed (original) (raw)

Alejandro Gallardo-Soler et al. Mol Endocrinol. 2008 Jun.

Abstract

Macrophages are phagocytic cells that play essential roles in innate immunity and lipid homeostasis. The uptake of modified lipoproteins is an important early event in the development of atherosclerosis. We analyzed the ability of modified low-density lipoprotein (LDL) (oxidized and acetylated) to alter the expression and activity of arginases (ArgI and ArgII) in macrophages. We show that ArgI expression is potently induced by both oxidized and acetylated LDL in macrophages. We further show that this effect is mediated by peroxisome proliferator-activated receptors (PPAR). ArgI expression is highly responsive to agonists for PPARgamma and PPARdelta but not PPARalpha. Moreover, the induction of ArgI by both PPAR agonists and IL-4 is blocked in macrophages from PPARgamma- and PPARdelta-deficient mice. Functionally, PPAR activity induces macrophage activation toward a more Th2 immune phenotype in a model of Leishmania major infection. We show that PPARgamma and -delta ligands promote intracellular amastigote growth in infected macrophages, and this effect is dependent on both PPAR expression and Arg activity. Collectively, our results strongly suggest that ArgI is a key marker of the alternative program triggered by PPAR in macrophages.

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Figures

Fig. 1.

Fig. 1.

Modified Lipoproteins Induce Arginase I in BDMD A, Analysis of arginase activity in cells treated with increasing concentrations of oxLDL, acLDL, and nLDL for 24 h; B, time course of arginase induction in BMDM treated with 30 μ

m

of each type of LDL; C, macrophages were pretreated overnight with 1 μ

m

GW9662 and then stimulated with 30 μ

m

modified LDL for 24 h; D, arginase I and II protein expression. Proteins were analyzed as described in Materials and Methods. β-Actin was used as loading control. IL-4, (2.5 ng/ml, 24 h) was used as positive control. c, Control; 9662+ox; GW9662 plus oxLDL. E, Arginase I and arginase II expression levels determined by TaqMan quantitative real-time PCR. The experimental conditions for D and E were identical to those in C. Data are expressed as the mean ±

sd

of triplicate measurements. F, Macrophages were loaded with LDL for 24 h and then used for RNA extraction. CD36, AP2, PPARγ, and ADRP gene expression were analyzed by real-time PCR SYBR Green assays and normalized to 36B4 gene expression.

Fig. 2.

Fig. 2.

PPARγ and -δ Agonists Induce Arginase I in BMDM A and B, Dose response of arginase induction with different PPARγ agonists. Cells were treated with agonists for 24 h and enzyme activity measured as described in Materials and Methods; C, arginase activity in cells treated with increasing concentrations of PPARδ agonist GW0742 for 24 h; D, arginase I and arginase II mRNA expression levels from cells treated with the indicated ligands for 24 h. The relative fold induction was calculated by using 18S mRNA as internal control and TaqMan real-time quantitative PCR; E, protein expression by Western blot. The concentrations of agonists used in D or E were 1 μ

m

PPARγ agonists rosiglitazone (Rosi), GW1929, or GW7845; 100 n

m

PPARδ agonist GW0742; 2 μ

m

of the PPARα agonists GW7647 and WY14643, and 0.5 μ

m

9-cRA. Lm Liver extract, used as positive control for arginase I. β-Actin was used as loading control. Data represent the mean values and

sd

of replicate cultures of four independent experiments. **, P < 0.01 by the Student’s t test at 25 n

m

, compared with the control value.

Fig. 3.

Fig. 3.

The Induction of Arginase by PPARγ and -δ Ligands Is Increased in the Presence of the RXR Ligand 9-cRA A, Arginase activity of cells treated with PPARγ agonists, added at the same dose as in Figs. 1 and 2 (white bars) or in combination with 100 n

m

9-cRA for 24 h; B, arginase activity in cells treated with 100 n

m

GW0742 with or without 100 n

m

9-cRA for 24 h; C, dose response of arginase induction by 24 h treatment with increasing concentrations of 9-cRA; E, mRNA relative induction of PPAR target genes CD36, AP2, and ADRP. BMDM were treated for 24 h with 1 μ

m

concentration of the indicated PPAR agonists, and relative gene expression was analyzed by real-time PCR SYBR Green assays, normalized to 36B4 expression, as previously described. **, P < 0.01 by the Student’s t test at 50 n

m

, compared with the control value.

Fig. 4.

Fig. 4.

Arginase I Induction by PPARγ and -δ Is Inhibited by PPAR Antagonists and Reduced in Macrophages from PPARγ- and -δ-Deficient Mice A, BMDM were pretreated overnight with the PPAR antagonists GW9662 or GW5393 and then triggered with the corresponding agonists for 24 h. After this time, arginase activity was measured in cell lysates. B, Immunoblots show arginase I protein inhibition by PPAR antagonists and increased amount of protein in the presence of PPAR-RXR heterodimer. IL-4 is shown as positive control. C, Arginase I relative expression in PPARγ WT and KO peritoneal macrophages with or without 100 n

m

GW7845 for 12 h or PPARδ WT and KO cultures treated with 100 n

m

0742 for 12 h. Finally, the relative induction of arginase I to IL-4 was analyzed in cells from PPARγ WT and KO pretreated overnight with 2.5ng/ml IL-4 and then with the PPARγ ligand for another 12 h. **, P < 0.01 by the Student’s t test for arginase I expression by PPARγ and -δ agonists in experiments with macrophages from PPARγ- and PPARδ-deficient mice vs. the WT mice; *, P < 0.05 for arginase I expression by IL-4 in cells from PPARγ WT cells compared with those from the knockout.

Fig. 5.

Fig. 5.

PPARγ/δ-Dependent Arginase I Induction Promotes the Growth of L. major Inside Macrophages A, Micrographs of _L. major_-infected BMDM. Cells were treated with PPAR agonists (1 μ

m

7845 or 100 n

m

  1. for 2 h or alternatively pretreated with 1 μ

m

concentration of each antagonist for 24 h and PPAR agonist during 2 h and finally infected with stationary phase L. major promastigotes for 24 h. After this time, cultures were stained as described in Materials and Methods. Original magnification, ×100. B, Enumeration of intracellular amastigotes in infected BMDM. Data are the results of counting the parasites from micrographs of A, where the errors bars represent results obtained from three independent macrophage infections performed in triplicate. White bars are control infected cultures or cells pretreated 2 h with PPAR agonists, infected, and counted 24 h after infection. PPAR antagonists were added 24 h before the agonists, and 100 μ

m

Nor-NOHA (black bars) was added at the same time as the agonists. **, P < 0.01 by the Student’s t test of intracellular parasite counts of infected cells treated with PPAR ligands alone compared with those pretreated with PPAR antagonists of nor-NOHA.

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