Lipophosphoglycans from <i>Leishmania amazonensis</i> Strains Display Immunomodulatory Properties via TLR4 and Do Not Affect Sand Fly Infection

<div><p>The immunomodulatory properties of lipophosphoglycans (LPG) from New World species of <i>Leishmania</i> have been assessed in <i>Leishmania infantum</i> and <i>Leishmania braziliensis</i>, the causative agents of visceral and cutaneous leishmaniasis, respectively. This glycoconjugate is highly polymorphic among species with variation in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO₄ backbone of repeat units. Here, the immunomodulatory activity of LPGs from <i>Leishmania amazonensis</i>, the causative agent of diffuse cutaneous leishmaniasis, was evaluated in two strains from Brazil. One strain (PH8) was originally isolated from the sand fly and the other (Josefa) was isolated from a human case. The ability of purified LPGs from both strains was investigated during <i>in vitro</i> interaction with peritoneal murine macrophages and CHO cells and <i>in vivo</i> infection with <i>Lutzomyia migonei</i>. In peritoneal murine macrophages, the LPGs from both strains activated TLR4. Both LPGs equally activate MAPKs and the NF-κB inhibitor p-IκBα, but were not able to translocate NF-κB. <i>In vivo</i> experiments with sand flies showed that both stains were able to sustain infection in <i>L</i>. <i>migonei</i>. A preliminary biochemical analysis indicates intraspecies variation in the LPG sugar moieties. However, they did not result in different activation profiles of the innate immune system. Also those polymorphisms did not affect infectivity to the sand fly.</p></div

Procedures for extraction, purification, preliminary characterization of <i>L</i>. <i>amazonensis</i> LPG, interaction with vertebrate cells and <i>L</i>. <i>migonei</i>.

<p>Late log phase cells were harvested and washed with PBS. For studies with vector, <i>L</i>. <i>migonei</i> midguts were dissected on days 2 and 4 post feeding containing <i>L</i>. <i>amazonensis</i> from each strain. Parasite cell pellets were subject to extraction with organic solvents as described elsewhere. For purification, the solvent E extract was dried under N₂ evaporation and applied into a phenyl-Sepharose column. The purified LPG was used for biological and immunological assays.</p

LPGs purified of <i>L</i>. <i>amazonensis</i> do not induce translocation of NF-κB through TLRs.

<p>CHO cells expressing TLR2 (TLR2+), TLR4 (TLR4+), or neither (TLR2-/TLR4-) were either untreated (purple line) or treated (green line) with LPGs from both strains of <i>L</i>. <i>amazonensis</i>. Legend: PH8 and Josefa LPGs (0.2 and 0.02 μg), Controls: LPS (TLR4 control) and <i>S</i>. <i>aureus</i> (S.a.) (TLR2 control). CD25 expression was measured by flow cytometry 18 h after stimulation. Results shown as percentage of CD25 expression on stimulated cells minus percentage of CD25 expression on non-stimulated cells.</p

Activation of p38/JNK (A) and p-IκBα (B) in peritoneal murine macrophages (C57BL/6, TLR2 -/- and TLR4 -/-) by <i>L</i>. <i>amazonensis</i> LPGs (PH8 and Josefa).

<p>Macrophages were stimulated for 5, 15, 30, 45 and 60 min with 10 μg/mL of LPG from <i>L</i>. <i>amazonensis</i> PH8 and Josefa strains. Dually phosphorylated MAPKs (p38 and JNK) and p-IκBα were detected by Western blot analysis. C- = negative control; C+ = <i>E</i>. <i>coli</i> extract, positive control (100 ng/mL, 45 min). Total p38 content was used as the normalizing protein.</p