Two biochemically distinct lipophosphoglycans from Leishmania braziliensis and Leishmania infantum trigger different innate immune responses in murine macrophages

BACKGROUND: The dominant, cell surface lipophosphoglycan (LPG) of Leishmania is a multifunctional molecule involved in the interaction with vertebrate and invertebrate hosts. Although the role of LPG on infection has been extensively studied, it is not known if LPG interspecies variations contribute to the different immunopathologies of leishmaniases. To investigate the issue of interspecies polymorphisms, two Leishmania species from the New World that express structural variations of side chains of LPG repeat units were examined. In this context, the procyclic form of L. braziliensis LPG (strain M2903), is devoid of side chains, while the L. infantum LPG (strain BH46) has up to three glucoses residues in the repeat units. METHODS: Mice peritoneal macrophages from Balb/c, C57BL/6 and knock-out (TLR2 -/-, TLR4 -/-) were primed with IFN-γ and stimulated with purified LPG from both species. Nitric oxide and cytokine production, MAPKs (ERK, p38 and JNK) and NF-kB activation were evaluated. RESULTS: Macrophages stimulated with L. braziliensis LPG, had a higher TNF-α, IL-1β, IL-6 and NO production than those stimulated with that of L. infantum. Furthermore, the LPGs from the two species resulted in differential kinetics of signaling via MAPK activation. L. infantum LPG exhibited a gradual activation profile, whereas L. braziliensis LPG showed a sharp but transient activation. L. braziliensis LPG was able to activate NF-kB. CONCLUSION: These data suggest that two biochemically distinct LPGs were able to differentially modulate macrophage functions

Glycoinositolphospholipids from Leishmania braziliensis and L. infantum: Modulation of Innate Immune System and Variations in Carbohydrate Structure

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

Fluorophore-assisted carbohydrate electrophoresis (FACE) of <i>Leishmania</i> GIPLs.

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

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

<p>Cells were incubated with GIPLs (25 µg/ml) from <i>L. braziliensis</i> (Gb) <i>and L. infantum</i> (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.</p

Monosaccharide profile of <i>Leishmania</i> glycoinositolphospholipids (GIPLs).

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