Human Dendritic Cell Maturation Is Modulated by Leishmania mexicana through Akt Signaling Pathway

Abstract

Dendritic cells (DC) along with macrophages are the main host cells of the intracellular parasite Leishmania. DC traverse a process of maturation, passing through an immature state with phagocytic ability to a mature one where they can modulate the immune response through the secretion of cytokines. Several studies have demonstrated that Leishmania inhibits DC maturation. Nevertheless, when cells are subjected to a second stimulus such as LPS/IFN-γ, they manage to mature. In the maturation process of DC, several signaling pathways have been implicated, importantly MAPK. On the other hand, Akt is a signaling pathway deeply involved in cell survival. Some Leishmania species have shown to activate MAPK and Akt in different cells. The aim of this work was to investigate the role of ERK and Akt in the maturation of monocyte-derived DC (moDC) infected with L. mexicana. moDC were infected with L. mexicana metacyclic promastigotes, and the phosphorylation of ERK and Akt, the expression of MHCII and CD86 and IL-12 transcript, and secretion were determined in the presence or absence of an Akt inhibitor. We showed that L. mexicana induces a sustained Akt and ERK phosphorylation, while the Akt inhibitor inhibits it. Moreover, the infection of moDC downregulates CD86 expression but not MHCII, and the Akt inhibitor reestablishes CD86 expression and 12p40 production. Thus, L. mexicana can modulate DC maturation though Akt signaling.

Keywords: Akt; CD86; ERK; IL-12; Leishmania mexicana; MHC-II; dendritic cells; maturation.

Figure 1

moDC were infected with L. mexicana metacyclic promastigotes, which was prevented when Akt was inhibited. moDC were adhered overnight to poly-L-lysine-coated eight-well glass chamber slides. On the next day, cells were pre-treated with Akt and Erk inhibitors and then infected with L. mexicana as described previously. Afterwards, cells were fixed with methanol and stained with Giemsa stain (A), photomicrographs at 400×. Parasite loads are expressed as the average number of intracellular amastigotes per 100 infected cells (B). Also, percentages of infected cells per condition were determined (C). Data are expressed as the mean ± SEM of 4 independent experiments. (** p ≤ 0.001; **** p < 0.00001).

Figure 2

The infection of moDC with L. mexicana metacyclic promastigotes induced Akt phosphorylation, while a specific inhibitor for Akt (iAkt) prevented it. moDC were incubated in medium alone or with a specific inhibitor for Akt (iAkt) for 2 h and infected with L. mexicana metacyclic promastigotes. Cells were lysed, and the total extract was resolved in 12% SDS-PAGE and electro-transferred, and the membranes were incubated with anti-phospho-Akt and anti-actin. (A) Akt phosphorylation kinetics during 24 h. Representative blot of three independent experiments (B). The induced Akt phosphorylation in infected moDC was prevented with the Akt inhibitor. Data are expressed as the mean ± SEM of four independent experiments. (** p ≤ 0.001; *** p < 0.00001).

Figure 3

The infection of moDC with L. mexicana metacyclic promastigotes induced the phosphorylation of ERK, which diminished when Akt was inhibited. moDC were incubated in medium alone or with a specific inhibitor for Akt (iAkt) for 2 h and infected with L. mexicana metacyclic promastigotes. Cells were lysed, and the total extract was resolved in 12% SDS-PAGE and electro-transferred, and the membranes were incubated with the antibodies anti-phospho-ERK and anti-actin. (A) ERK phosphorylation kinetics during 24 h, with a peak between 12–24 h. Representative blot of five independent experiments. (B) The phosphorylation of ERK induced by the infection of moDC with L. mexicana was prevented when Akt was inhibited with a specific inhibitor. Data are expressed as the mean ± SEM of five independent experiments. (* p < 0.05; ** p ≤ 0.01; *** p < 0.0001).

Figure 4

The infection of moDC with L. mexicana metacyclic promastigotes diminished the expression of the costimulatory molecule CD86, which was reestablished when Akt was inhibited. moDC were treated with specific inhibitors for ERK and Akt to determine the role of both kinases in the expression of the costimulatory molecule CD86 as a feature of moDC maturation. Cells were then infected with L. mexicana metacyclic promastigotes for 24 h and stained with FITC-anti-CD86 to analyze the expression of this molecule by flow cytometry. Data are expressed as media ± SEM of three independent experiments. (* p < 0.05; ** p ≤ 0.01).

Figure 5

The infection of moDC with L. mexicana metacyclic promastigotes and the inhibition of Akt or ERK did not affect the presence of HLA-DR. moDC were treated with specific inhibitors for ERK and Akt to determine the role of both kinases in the expression of HLA-DR as a feature of moDC maturation. Cells were then infected with L. mexicana metacyclic promastigotes for 24 h and stained with FITC-anti-HLA-DR to analyze the expression of this molecule by flow cytometry. Data are expressed as media ± SEM of three independent experiments.

Figure 6

The infection of moDC with L. mexicana metacyclic promastigotes diminished IL-12 p40 Scheme 12. by moDC. Afterwards, they were infected with L. mexicana metacyclic promastigotes, total RNA was isolated from cells and retrotranscribed, and the cDNA obtained from each sample was assayed for PCR amplification of a 674 bp sequence of human IL-12 p40 gene and a 138 bp sequence of human GAPDH gene (A). Densitometric values obtained from three independent experiments are indicated (B). Also, culture supernatants were recovered for IL-12 p40 determination by ELISA (C). LPS (100 ng/mL) was used as a positive control for the production of IL-12p40. Data are expressed as media ± SEM of three independent experiments. (* p < 0.05; ** p ≤ 0.01; *** p < 0.0001; **** p < 0.00001).