Activation Pathways of Murine Macrophages by Lipophosphoglycan from Strains of Leishmania major (FV1 and LV39)

Abstract

Lipophosphoglycan (LPG) is an important Leishmania virulence factor. It is the most abundant surface glycoconjugate in promastigotes, playing an important role in the interaction with phagocytic cells. While LPG is known to modulate the macrophage immune response during infection, the activation mechanisms triggered by this glycoconjugate have not been fully elucidated. This work investigated the role that LPGs purified from two strains of Leishmania major (FV1 and LV39) play in macrophage activation, considering the differences in their biochemical structures. Bone marrow-derived macrophages from BALB/c mice were stimulated with 10 μg/mL purified LPG from the LV39 and FV1 strains. We then measured the production of nitric oxide (NO) and cytokines, the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and the activation of MAPK pathways. LPG from the LV39 strain, which has longer poly-galactosylated side chains, induced a more pro-inflammatory profile than that from the FV1 strain. This included higher production of NO, TNF-α, and PGE2, and increased expression of COX-2 and iNOS. Additionally, the phosphorylation of ERK-1/2 and JNK was elevated in macrophages exposed to LPG from the LV39 strain. No difference in IL-10 production was observed in cells stimulated by both LPG. Thus, intraspecific structural differences in LPG contribute to distinct innate immune responses in macrophages.

Keywords: Leishmania major; Lipophosphoglycan; cytokines; inflammatory mediators; leishmaniasis; macrophage.

Figure 1

Purified LPG from L. major strain LV39 induces higher nitric oxide and PGE2 production via iNOS and COX-2. BMDM primed with IFN-γ (100 ng/mL) were stimulated for 24 h with 10 μg/mL of LPG purified from L. major FV1 or LV39. LPS (300 ng/mL) was used as a positive control. (A) Inos and COX-2 Western blot analysis from cell lysate 24 h after LPG stimulation. Actin was used as a loading control. (B) Nitric oxide (NO) production was measured in culture supernatants by the Griess reaction. (D) PGE₂ and (F) LTB₄ were dosed in culture supernatants by ELISA. (G) PGE₂/LTB₄ ratio. Bars represent means ± SD of quadruplicates. Representative densitometry ratios of iNOS bands (C) and COX-2 bands (E) by actin bands (C,E) were obtained by ImageJ/Fiji. The statistical analysis was performed using the ANOVA test followed by the Newman–Keuls post-test using multiple comparisons between the experimental groups (****p < 0.0001) and (####p < 0.0001) compared to the control group. NS: nonstimulated; FV1 and LV39: L. major LPGs. LPS: lipopolysaccharide.

Figure 2

Signaling pathways triggered by purified LPG from L. major. BMDM were stimulated with 10 μg/mL of purified LPG from the FV1 or LV39 strains for 5, 15, 30, and 60 min. LPS (300 ng/mL) was used as a positive control. The bands indicate phosphorylation of ERK1/2 (A) and JNK (B) in cell lysates evaluated by Western blotting. Representative densitometry ratios of p-ERK (C) and p-JNK (D) by total-ERK expression were obtained by ImageJ/Fiji. The statistical analysis was performed using the ANOVA test followed by the Newman–Keuls post-test using multiple comparisons between the experimental groups (*p < 0.05). NS: nonstimulated; FV1 and LV39: L. major LPGs. LPS: lipopolysaccharide.

Figure 3

Impact of purified L. major LPG cytokine production. BMDM primed with IFN-γ (100 ng/mL) were stimulated for 24 h with 10 μg/mL of LPG purified from L. major FV1 or LV39. LPS (300 ng/mL) was used as a positive control. The production of (A) TNF-α and (B) IL-10 was evaluated from culture supernatants. Bars represent means ± SD performed in quadruplicates. The analysis was performed using the ANOVA test followed by the Newman–Keuls post-test using multiple comparisons between the experimental groups (*p < 0.05; **p < 0.01) and (##p < 0.01; ###p < 0.001) when compared to the NS control group). Ns: nonstimulated; FV1 and LV39: L. major LPGs. LPS: lipopolysaccharide.

Figure 4

Inhibition of ERK, COX-2, and PKC pathways and induction of PPARy negatively impact the capacity of nitric oxide production by L. major LPG LV39. BMDM were primed with IFN-γ (100 ng/mL) for 24 h. Afterward, they were pretreated for 1 h with 10 μM rosiglitazone (PPAR-γ agonist), 20 μM BIS (PKC inhibitor), 50 μM PD98059 (ERK1/2 inhibitor), or 1 μM NS398 (COX-2 inhibitor). Then, the stimulus with purified LPG LV39 was added for 24 h. (A) Nitric oxide (NO) production was measured in culture supernatants by the Griess reaction. (B) Cell viability was assessed by Alamar Blue. (C) Bands were obtained by Western blotting from culture lysates. (D) Representative densitometry quantification iNOS and actin expression were obtained by ImageJ/Fiji. Bars represent means ± SD of experiments performed in quadruplicates. The analysis was performed by an ANOVA test followed by the Newman–Keuls post-test used for multiple comparisons between experimental groups (***p < 0.001; ****p < 0.0001, and ####p < 0, 0001 when compared to the NS control group). NS: nonstimulated; FV1 and LV39: L. major LPGs. LPS: lipopolysaccharide.

Figure 5

Inhibition of ERK, COX-2, and PKC pathways and induction of PPAR-γ alter the ability to produce IL-6 and MCP-1 by macrophages stimulated with L. major LPG LV39. BMDM primed with IFN-γ (100 ng/mL) were pretreated with rosiglitazone (PPAR-γ agonist), BIS (PKC inhibitor), PD98059 (ERK1/2 inhibitor), and NS398 (COX-2 inhibitor) for 1 h. Then, cells were stimulated for 24 h with 10 μg/mL of LPG purified from L. major FV1 or LV39. LPS (300 ng/mL) was used as a positive control. (A) IL-6, (B) MCP-1, TNF-α (C), and IL-10 (D) dosage was performed with culture supernatants by Luminex. Bars represent means ± SD of experiments performed in quadruplicates. The analysis was performed using the ANOVA test followed by the Newman–Keuls post-test used for multiple comparisons between the experimental groups (***p < 0.001) and (##p < 0.01; ###p < 0.001 ####p < 0.0001) compared to the NS control group). NS: nonstimulated; FV1 and LV39: L. major LPGs. LPS: lipopolysaccharide.