Glycoinositolphospholipids from Leishmania braziliensis and L. infantum: modulation of innate immune system and variations in carbohydrate structure

Abstract

The essential role of the lipophosphoglycan (LPG) of Leishmania in innate immune response has been extensively reported. However, information about the role of the LPG-related glycoinositolphospholipids (GIPLs) is limited, especially with respect to the New World species of Leishmania. GIPLs are low molecular weight molecules covering the parasite surface and are similar to LPG in sharing a common lipid backbone and a glycan motif containing up to 7 sugars. Critical aspects of their structure and functions are still obscure in the interaction with the vertebrate host. In this study, we evaluated the role of those molecules in two medically important South American species Leishmania infantum and L. braziliensis, causative agents of visceral (VL) and cutaneous Leishmaniasis (CL), respectively. GIPLs derived from both species did not induce NO or TNF-α production by non-primed murine macrophages. Additionally, primed macrophages from mice (BALB/c, C57BL/6, TLR2-/- and TLR4-/-) exposed to GIPLs from both species, with exception to TNF-α, did not produce any of the cytokines analyzed (IL1-β, IL-2, IL-4, IL-5, IL-10, IL-12p40, IFN-γ) or p38 activation. GIPLs induced the production of TNF-α and NO by C57BL/6 mice, primarily via TLR4. Pre incubation of macrophages with GIPLs reduced significantly the amount of NO and IL-12 in the presence of IFN-γ or lipopolysaccharide (LPS), which was more pronounced with L. braziliensis GIPLs. This inhibition was reversed after PI-specific phospholipase C treatment. A structural analysis of the GIPLs showed that L. infantum has manose rich GIPLs, suggestive of type I and Hybrid GIPLs while L. braziliensis has galactose rich GIPLs, suggestive of Type II GIPLs. In conclusion, there are major differences in the structure and composition of GIPLs from L. braziliensis and L. infantum. Also, GIPLs are important inhibitory molecules during the interaction with macrophages.

Figure 1. Types of GIPLs.

For information on M2, M3, iM2, GIPL-A, isoM3 and isoM4, see introduction. Fatty acid chains vary in different GIPL species: The predominant type fatty acid in Type-1 and Hybrid GIPLs is C_18∶0, in type-2 GIPLs the predominant lipids are C_18∶0, C_22∶0 C_24∶0 and C_26∶0. “R” in Type 1 GIPLs represent a protein linked to the GIPL structure by a ethanolamine phosphate residue (e.g. gp63 surface metalloprotease) , .

Figure 2. Nitrite production by BALB/c primed macrophages after stimulation with different concentrations of GIPLs.

C, negative control; IFN-γ, gamma-interferon; LPS, lipopolysaccharide; Gb, L. braziliensis GIPLs; Gi, L. infantum GIPLs. Cells were primed with IFN-γ (3 IU/ml) for 18 h prior to the addition of the GIPLs or LPS (positive control). Non primed cells and primed cells without the addition of a new stimulus were also used as controls. ANOVA test was performed and P<0.05 was considered significant. Results are the representation of three experiments in triplicate.

Figure 3. Nitrite and TNF-α production by primed macrophages after stimulation with GIPLs and parasites.

C, negative control; Gb, L. braziliensis GIPLs; Gi, L. infantum GIPLs; Lb, L. braziliensis live promastigotes and Li, L. infantum live promastigotes. Cells were pre-incubated with IFN-γ (3 IU/ml) for 18 h then 25 µg/mL of GIPLs or 100 ng/mL of LPS was added. Supernatants were collected 48 hours later, in (A) NO concentrations were measured by Griess reaction and in (B) TNF-α concentrations determined by flow cytometry. ANOVA test was performed and P<0.05 was considered significant.

Figure 4. Modulation of nitrite, TNF-α and IL-12 production by Leishmania GIPLs in BALB/c macrophages.

Cells were incubated with GIPLs (25 µg/ml) from L. braziliensis (Gb) and L. infantum (Gi) for 15 min prior to stimulation with IFN-γ (100 IU/ml) (A) or LPS (100 ng/mL) (B). Nitrite content was measured by Griess reaction; TNF-α and IL-12 concentrations were measured by ELISA. P<0.05 was considered significant. Results are the representation of three experiments.

Figure 5. Modulation of nitrite production by macrophages stimulated with intact and PI-PLC treated GIPLs.

Mouse peritoneal macrophages were incubated with GIPLs (25 µg/ml) from L. braziliensis (Gb), L. infantum (Gi), PI-PLC treated L. braziliensis GIPLs (Gb PI-PLC) and L. infantum PI-PLC treated GIPLs (Gi PI-PLC) for 15 min prior to stimulation with IFN- γ (100 IU/ml). Nitrite content was measured by Griess reaction on the supernatants after 24 h. Student “t” test was performed and P<0.05 was considered significant. Results are the mean of two experiments.

Figure 6. Activation of MAPKs (ERK and p38) by Leishmania GIPLs in BALB/c peritoneal macrophages.

Mouse peritoneal macrophages were stimulated for 30 min with 25 µg/mL of GIPLs. Dually phosphorylated MAPKs were detected by western blot. C, negative control; Gb, L. braziliensis GIPLs and Gi, L. infantum GIPLs. Also cells were incubated with GIPLs prior to stimulation with LPS; total ERK content as a normalizing protein.

Figure 7. Thin layer chromatography (TLC) of Leishmania glycoinositolphospholipids (GIPLs).

(A) Purified intact GIPLs: Lane 1, L. braziliensis GIPLs (Gb); lane 2, L. infantum GIPLs (Gi) and lane 3, L. donovani GIPLs (Gd). The assignments for L. donovani structures are: isoM2 as Manα1-3Manα1-4GlcN-PI; isoM3 as Manα1-6(Manα1-3)Manα1-4GlcN-PI and isoM4 as Manα1-2Manα1-6(Manα1-3)Manα1-4GlcN-PI . LPG, lipophosphoglycan; GPI, glicosyl phosphatidylinositol. (B) Deaminated GIPLs. Lane 1, L. braziliensis untreated GIPLs (Gb); lane 2, deaminated L. braziliensis GIPLs (Gb deam.); lane 3, L. infantum untreated GIPLs (Gi) and Lane 4, deaminated L. infantum GIPLs (Gi deam.).

Figure 8. Fluorophore-assisted carbohydrate electrophoresis (FACE) of Leishmania GIPLs.

Lane 1, oligoglucose ladder represented by G2-G7; lane 2, L. braziliensis GIPLs (Gb) and lane 3, L. infantum GIPLs (Gi) and lane 4, L. donovani GIPLs (Gd).

Figure 9. Monosaccharide profile of Leishmania glycoinositolphospholipids (GIPLs).

(A) Fluorophore-assisted carbohydrate electrophoresis (FACE). Lane 1, standards represented by galactose, glucose and mannose (100 µg/ml); lane 2, L. braziliensis GIPLs (Gb); lane 3, L. infantum GIPLs (Gi) and Lane 4, L. donovani GIPLs (Gd). (B) High performance liquid chromatography (HPLC). Gal, galactose; Glc, glucose and Man, mannose.