Transcriptional inhibition of interleukin-12 promoter activity in Leishmania spp.-infected macrophages

Abstract

To establish and persist within a host, Leishmania spp. parasites delay the onset of cell-mediated immunity by suppressing interleukin-12 (IL-12) production from host macrophages. Although it is established that Leishmania spp.-infected macrophages have impaired IL-12 production, the mechanisms that account for this suppression remain to be completely elucidated. Using a luciferase reporter assay assessing IL-12 transcription, we report here that Leishmania major, Leishmania donovani, and Leishmania chagasi inhibit IL-12 transcription in response to interferon-gamma, lipopolysaccharide, and CD40 ligand and that Leishmania spp. lipophosphoglycan, phosphoglycans, and major surface protein are not necessary for inhibition. In addition, all the Leishmania spp. strains and life-cycle stages tested inhibited IL-12 promoter activity. Our data further reveal that autocrine-acting host factors play no role in the inhibitory response and that phagocytosis signaling is necessary for inhibition of IL-12.

Figure 1

Leishmania infection inhibits IL-12p40 transcription in murine macrophage cell line. RAW264.7 cells stably transfected with human IL-12p40-LUC construct were infected with L. major strain V1 and subsequently treated with 3 μg/ml CD40L ± 100 U/ml IFN-γ or 1 μg/ml LPS ± 100 U/ml IFN-γ. Samples were run in triplicate and mean relative luciferase units ± standard deviation is reported. * Infected values were significantly different from uninfected, stimulated controls (P < 0.05). † Uninfected, stimulated values were different from uninfected, untreated controls (P < 0.05). One representative of 3 independent experiments is presented.

Figure 2

Leishmania-induced IL-12p40 inhibition is independent of species and strain. RAW264.7 cells stably transfected with human IL-12p40-LUC construct were infected with different L. donovani (A) or L. major (B) substrains and treated with 100 U/ml IFN-γ plus 1 μg/ml LPS. Samples were run in triplicate and mean relative luciferase units ± standard deviation is reported. * Infected values were significantly different from uninfected, stimulated controls (P < 0.05). † Uninfected, stimulated values were different from uninfected, untreated controls (P < 0.05). In each case 1 representative of 2 independent experiments is shown.

Figure 3

Inhibition of IL-12p40Luc by different Leishmania spp. life-cycle stages. RAW264.7 cells stably transfected with human IL-12p40-LUC construct were infected with L. major strain V1 life-cycle stages for 16 hr followed by 8 hr of LPS (1 μg/ml) stimulation. Life-cycle stages (met = metacyclic; log = log-phase promastigotes; am = amastigotes). Samples were run in triplicate and mean relative luciferase units ± standard deviation is reported. * Infected values were significantly different from uninfected, stimulated controls (P < 0.05). † Uninfected, stimulated values were different from uninfected, untreated controls (P < 0.05). One representative of 2 independent experiments is shown.

Figure 4

Inhibition of IL-12p40Luc by Leishmania spp. products. RAW264p7 cells stably transfected with human IL-12p40-LUC construct were infected with L. major strain V1 or treated with parasite products for 16 hr followed by 8 hr of LPS (1 μg/ml) stimulation. (A) Parasite products (Live = live metacyclic infection; Lysate = freeze–thaw lysates; HK = heat-killed metacyclics). (B) Secreted products (V1 = live metacyclic infection; Neat, Dil, Conc = cultured parasite supernantants either diluted or concentrated). Samples were run in triplicate and mean relative luciferase units ± standard deviation is reported. * Infected values were significantly different from uninfected, stimulated controls (P < 0.05). † Untreated, stimulated values were different from untreated, media controls (P < 0.05). In each case 1 of 3 independent experiments is shown.

Figure 5

Inhibition of IL-12p40Luc by Leishmania is not dependent on LPG, PG, or GP63. RAW264.7 cells stably transfected with human IL-12p40-LUC construct were infected with Leishmania for 16 hr followed by 8 hr of LPS (1 μg/ml) stimulation. (A) Leishmania donovani wild-type, LPG-deficient, and PG-deficient mutants. (B) Leishmania mexicana wild-type and GP63-deficient mutant. Samples were run in triplicate and mean relative luciferase units ± standard deviation is reported. * Infected values were significantly different from uninfected, stimulated controls (P < 0.05). † Uninfected, stimulated values were different from uninfected, untreated controls (P < 0.05). One representative of 2 independent experiments is shown.

Figure 6

Inhibition of IL-12p40Luc by Leishmania does not involve autocrine MP factors; 1 × 10⁶ RAW264.7 cells stably transfected with human IL-12p40-LUC construct were plated in lower wells and 1 × 10⁶ were plated in upper wells of a transwell system. Cells were infected with L. major at a 5:1 ratio for 2 hr followed by IFN-γ (100 U/ml) stimulation for 16 hr and LPS (1 μg/ml) stimulation for 8 hr. Luciferase values were detected from cells in lower chambers. Samples were run in triplicate and mean relative luciferase units ± standard deviation is reported. * Infected values were significantly different from uninfected, stimulated controls (P < 0.05). † Uninfected, stimulated values were different from uninfected, untreated controls (P < 0.05). One representative of 3 independent experiments is shown.

Figure 7

Inhibition of IL-12p40Luc by Leishmania requires phagocytosis. RAW264.7 cells stably transfected with human IL-12p40-LUC construct were untreated (A, C) or pretreated with 1 μM cytochalasin D (B, D), then infected with L. major strain V1 and subsequently treated with 1 μg/ml LPS ± 100 U/ml IFN-γ. Samples were run in triplicate, treated with IFN-γ + LPS, and assessed for luciferase production (A, B) or endogenous levels of IL-12p40 by quantitative RT-PCR (C, D). Mean percentage expression ± standard deviation of is reported. * Infected values were significantly different from uninfected, stimulated controls (P < 0.05). One representative of 2 independent experiments is presented.