Leishmania Lipophosphoglycan Triggers Caspase-11 and the Non-canonical Activation of the NLRP3 Inflammasome.

Activation of the NLRP3 inflammasome by Leishmania parasites is critical for the outcome of leishmaniasis, a disease that affects millions of people worldwide. We investigate the mechanisms involved in NLRP3 activation and demonstrate that caspase-11 (CASP11) is activated in response to infection by Leishmania species and triggers the non-canonical activation of NLRP3. This process accounts for host resistance to infection in macrophages and in vivo. We identify the parasite membrane glycoconjugate lipophosphoglycan (LPG) as the molecule involved in CASP11 activation. Cytosolic delivery of LPG in macrophages triggers CASP11 activation, and infections performed with Lpg1-/- parasites reduce CASP11/NLRP3 activation. Unlike bacterial LPS, purified LPG does not activate mouse CASP11 (or human Casp4) in vitro, suggesting the participation of additional molecules for LPG-mediated CASP11 activation. Our data identify a parasite molecule involved in CASP11 activation, thereby establishing the mechanisms underlying inflammasome activation in response to Leishmania species.

Intraspecies susceptibility of Leishmania (Viannia) braziliensis to antileishmanial drugs: Antimony resistance in human isolates from atypical lesions.

Leishmania (Viannia) braziliensis is the most common etiological agent of cutaneous and mucocutaneous leishmaniasis (MCL) in Latin America. An interesting aspect of the disease outcome caused by this species is the appearance of non-ulcerated atypical cutaneous leishmaniasis. Atypical (AT) lesions are often associated with therapeutic failure when treated with antimony(Sb)-based drugs. Refractory cases are not necessarily due to intrinsic parasite drug resistance. The status of in vitro drug susceptibility from L. braziliensis field isolates is less assessed than patient treatment outcome. In this work, L. braziliensis isolated from typical CL (6), MCL (1) and AT (3) lesions and vector (1) were tested for their susceptibility to amphotericin B (AmB), miltefosine (MIL), glucantime (GLU) and non-comercial meglumine antimoniate (MA). Overall, intracellular amastigotes of all isolates were sensitive to the tested antileishmanial drugs except AT lesions-derived strains 316, 330 and 340 that presented in vitro resistance against SbV-based drugs. Although susceptible to miltefosine - based on phenotypic screening - intramacrophagic quiescent amastigotes could restore infection. L. braziliensis promastigotes isolated from AT lesions also displayed 29% reduced capacity to infect human monocyte-derived macrophages when compared with parasites obtained from patients with typical lesions, MCL or from sand-fly. These data indicate differences in drug susceptibility and infectiveness among L. braziliensis isolated from patients exhibiting different types of lesions and highlight the importance of its characterization for drug response prediction outcome in clinical practice.

Lipophosphoglycans from Leishmania amazonensis Strains Display Immunomodulatory Properties via TLR4 and Do Not Affect Sand Fly Infection.

The immunomodulatory properties of lipophosphoglycans (LPG) from New World species of Leishmania have been assessed in Leishmania infantum and Leishmania braziliensis, 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(ß1,4)Man(α1)-PO4 backbone of repeat units. Here, the immunomodulatory activity of LPGs from Leishmania amazonensis, 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 in vitro interaction with peritoneal murine macrophages and CHO cells and in vivo infection with Lutzomyia migonei. 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. In vivo experiments with sand flies showed that both stains were able to sustain infection in L. migonei. 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.