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. 2009 Feb;77(2):685-93.
doi: 10.1128/IAI.00718-08. Epub 2008 Dec 1.

Phosphoinositide 3-kinase-dependent Inhibition of Dendritic Cell interleukin-12 Production by Giardia Lamblia

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Free PMC article

Phosphoinositide 3-kinase-dependent Inhibition of Dendritic Cell interleukin-12 Production by Giardia Lamblia

Joel Désiré Kamda et al. Infect Immun. .
Free PMC article

Abstract

Dendritic cell interactions with pathogenic microbes initiate and direct the development of subsequent adaptive responses. The protozoan pathogen Giardia lamblia infects the mammalian small intestine, leading to nutrient malabsorption and diarrhea but rarely causing inflammation. In order to begin to understand how the innate immune system responds to this parasite and shapes the eventual adaptive response, we examined the interaction between parasites and murine bone marrow-derived dendritic cells (DCs). DCs incubated with live parasites or parasite extracts displayed enhanced levels of CD40. The expression of CD80 and CD86 also increased, but less than was seen with lipopolysaccharide-activated DCs. Small amounts of interleukin-6 (IL-6) and tumor necrosis factor alpha were secreted by these DCs, whereas no IL-10 or IL-12 could be detected. Coincubation of DCs with parasite extracts along with known Toll-like receptor (TLR) ligands resulted in enhanced secretion of IL-10 and reduced secretion of IL-12. The levels of major histocompatibility complex class II, CD80, and CD86 were also reduced compared to DCs stimulated with TLR ligands alone. Finally, studies with an extracellular signal-regulated kinase 1/2 pathway inhibitor, a phosphoinositide 3-kinase (PI3K) inhibitor, and anti-IL-10 receptor antibody revealed that the PI3K pathway is the dominant mechanism of inhibition in DCs incubated with both lipopolysaccharide and Giardia. These data suggest that this parasite actively interferes with host innate immunity, resulting in an immune response able to control the infection but devoid of strong inflammatory signals.

Figures

FIG. 1.
FIG. 1.
Response of mouse bone marrow DCs to G. lamblia. One million DCs were incubated overnight with medium alone (filled curves), 1 mg of Giardia extract/ml (dashed lines), or 10 ng of LPS/ml (solid lines). CD11c+ cells were analyzed for CD80 (A), CD86 (B), and CD40 (C) expression by flow cytometry. Overlap in the LPS and Giardia stimulated cells for CD40 expression obscures the Giardia trace. Culture supernatants were analyzed for IL-12p70 and IL-10 (D). For analysis of IL-6 (E) and TNF-α (F), DCs were stimulated with 1 mg of Giardia extract/ml or 10 ng of LPS/ml in the presence or absence of 100 μg of polymyxin B/ml. IL-6 and TNF-α production were also measured in response to titrated amounts of Giardia extract (G) or to live Giardia trophozoites (H and I) at a 10:1 parasite/DC ratio. This ratio was equivalent to a total protein concentration of 1 mg/ml, as was used for parasite extracts. Medium alone and 10 ng of LPS/ml were also used as controls. N.D., none detected. Flow cytometry data are representative of four independent experiments. Cytokine data are presented as means ± the standard error of the mean (SEM) of duplicate cultures and are representative of three or more independent experiments each.
FIG. 2.
FIG. 2.
Inhibition of DC responses to LPS by Giardia extracts. DCs were cultured with the indicated amounts of Giardia extract, followed by addition of 10 ng of LPS/ml. IL-6 (A), TNF-α (B), IL-10 (C), and IL-12p70 (D) were assayed in the supernatant by ELISA in duplicates. Cytokine data are presented as means ± the SEM of duplicate cultures. These data represent two independent experiments, and the effect of 1 mg of extract/ml was tested in at least four additional experiments with similar results. *, P < 0.05.
FIG. 3.
FIG. 3.
Inhibition of DC responses to multiple TLR agonists by Giardia extracts. DCs were incubated with or without 1 mg of Giardia extract/ml, followed by the addition of medium alone, 10 ng of LPS/ml (TLR4), 0.5 μg of PAM3CSK4/ml (TLR2), 25 μg of poly(I:C)/ml (TLR3), or 5 μg of CpG/ml containing oligodeoxynucleotide (TLR9). IL-6 (A), TNF-α (B), IL-10 (C), and IL-12p70 (D) were assayed in the overnight supernatant in duplicates. Cytokine data are presented as means ± the SEM of duplicate cultures, and the data shown are representative of six or more independent experiments. *, P < 0.05 comparing cultures with or without Giardia extract.
FIG. 4.
FIG. 4.
Inhibition of DC surface molecule expression after treatment with Giardia extract and LPS. DCs from BALB/c bone marrow were cultured overnight with TLR agonists with (gray lines) or without (black lines) Giardia extracts as in Fig. 3. The cells were then stained with fluorochrome-conjugated antibodies to CD80, CD86, CD40, and MHC-II (Ad) and analyzed by flow cytometry. Only CD11c+ cells were analyzed. The data are representative of three independent experiments.
FIG. 5.
FIG. 5.
Effect of ERK activation on DC cytokine production. (A) DCs were incubated with 10 ng of LPS/ml, 1 mg of Giardia extract/ml, or both. Protein extracts were made 20 and 40 min after DC stimulation and Western blotted for phosphorylated ERK (p-Erk) or total Erk protein. DCs were pretreated with DMSO or the MEK1/2 inhibitor U0126 (10 μM) for 30 min prior to stimulation with 10 ng of LPS/ml, with or without 1 mg of Giardia extract/ml. (B and C) The levels of IL-10 (B) and IL-12p70 (C) were measured in supernatants of overnight cultures. Error bars represent the SEM of duplicate cultures. Western blot data are representative of three independent experiments. Cytokine data are presented as means ± the SEM of duplicate cultures and are representative of two independent experiments each.
FIG. 6.
FIG. 6.
Effect of IL-10 signaling on DC cytokine production. DCs were cultured with medium alone, with 10 ng of LPS/ml, with 1 mg of Giardia extract/ml, or with both LPS and Giardia extract in the presence of 10 μg/ml of either anti-IL-10R antibody or isotype control immunoglobulin G/ml. The levels of IL-10 and IL-12p70 were measured in supernatants of overnight cultures. Cytokine data are presented as means ± the SEM of duplicate cultures and are representative of four independent experiments.
FIG. 7.
FIG. 7.
Effect of PI3K signaling on DC cytokine production. (A to D) DCs were pretreated for 30 min with various concentrations of wortmannin prior to addition of 10 ng of LPS/ml with or without 1 mg of Giardia extract/ml. DCs pretreated with wortmannin were incubated with medium alone, with 10 ng of LPS/ml, with 1 mg of Giardia extract/ml, or both (C and D). The levels of IL-10 (A and C) and IL-12p70 (B and D) were measured in the supernatants of overnight cultures. Cytokine data are presented as means ± the SEM of duplicate cultures and are representative of four independent experiments. *, P < 0.05.

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