Background: The dominant, cell surface lipophosphoglycan (LPG) ofLeishmaniais 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, twoLeishmaniaspecies from the New World that express structural variations of side chains of LPG repeat units were examined. In this context, the procyclic form ofL. braziliensisLPG (strain M2903), is devoid of side chains, while theL. infantumLPG (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 withL. braziliensisLPG, had a higher TNF-α, IL-1β, IL-6 and NO production than those stimulated with that ofL. infantum. Furthermore, the LPGs from the two species resulted in differential kinetics of signaling via MAPK activation.L. infantumLPG exhibited a gradual activation profile, whereasL. braziliensisLPG showed a sharp but transient activation.L. braziliensisLPG was able to activate NF-kB.Conclusion: These data suggest that two biochemically distinct LPGs were able to differentially modulate macrophage functions.

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. Leishmania infantum (syn. L. chagasi) and L. braziliensis are the causative agents of VL and CL, respectively, in the New World. A vital part of the parasite's life cycle involves the circumvention of the host immune system and the infection of macrophages. This work focused on an important class of surface glycoconjugates, the glycoinositolphospholipids (GIPLs), and their role in the interaction with murine macrophages. GIPLs are expressed on every stage of the parasite life cycle and are the most abundant molecules on its surface. Here we show that these molecules modulate many macrophage functions such as cytokine production, release of nitric oxide and differentially activate MAPK. Although the GIPLs of both New World species are capable of modulating the same mechanisms, they do so to different degrees requiring an examination of their glycan composition. We show that L. infantum synthesize mannose rich GIPLs whereas L. braziliensis express galactose rich GIPLs.

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(β1,4)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. Leishmania amazonensis, a member of the Leishmania mexicana complex, is the causative agent of localized cutaneous leishmaniasis (LCL) and anergic diffuse cutaneous leishmaniasis (ADCL) [1,2]. It is widely distributed throughout the Amazon basin, where it infects a wide range of terrestrial rodents and, less frequently, marsupials. Its main vector is Lutzomyia flaviscutellata (Diptera: Psychodidae) widely distributed in South America and a recent study has predicted its expansion towards South of Brazil [3]. Moreover, Lutzomyia migonei (França, 1920) can also harbor the infection of this species [4,5]. Although its transmission to man is very uncommon, L. amazonensis triggers an incurable and disseminated form of cutaneous leishmaniasis [2,6]. However, most of the mechanisms involved in L. amazonensis pathogenesis are still unknown, especially those related to surface molecules. Glycoconjugates have been extensively characterized as important for the establishment of infection as they protect the parasite from the early action of the host immune system and therefore acting as invasive/evasive strategies. Consequently, we here present the role of lipophosphoglycan (LPG) of L. amazonensis in the interaction with vertebrate and invertebrate hosts.

Parasites are responsible for the most neglected tropical diseases, affecting over a billion people worldwide (WHO, 2015) and accounting for billions of cases a year and responsible for several millions of deaths. Research on extracellular vesicles (EVs) has increased in recent years and demonstrated that EVs shed by pathogenic parasites interact with host cells playing an important role in the parasite's survival, such as facilitation of infection, immunomodulation, parasite adaptation to the host environment and the transfer of drug resistance factors. Thus, EVs released by parasites mediate parasite‐parasite and parasite‐host intercellular communication. In addition, they are being explored as biomarkers of asymptomatic infections and disease prognosis after drug treatment. However, most current protocols used for the isolation, size determination, quantification and characterization of molecular cargo of EVs lack greater rigor, standardization, and adequate quality controls to certify the enrichment or purity of the ensuing bioproducts. We are now initiating major guidelines based on the evolution of collective knowledge in recent years. The main points covered in this position paper are methods for the isolation and molecular characterization of EVs obtained from parasite‐infected cell cultures, experimental animals, and patients. The guideline also includes a discussion of suggested protocols and functional assays in host cells

Leishmania infection causes considerable human morbidity and may develop into a deadly visceral form in endemic regions. The parasite infects macrophages where they can replicate intracellularly. Furthermore, they modulate host immune responses by using virulence factors (lipophosphoglycan, glycoprotein-63, and others) that promote survival inside the cells. Extracellular vesicles (EVs) released by parasites are important for cell-cell communication in the proinflammatory milieu modulating the establishment of infection. However, information on the ability of EVs from different Leishmania species to modulate inflammatory responses is scarce, especially from those species causing different clinical manifestations (visceral vs. cutaneous). The purpose of this study was to compare macrophage activation using EVs from three Leishmania species from New World including L. infantum, L. braziliensis, and L. amazonensis. EVs were released from promastigote forms, purified by ultracentrifugation and quantitated by Nanoparticle Tracking Analysis (NTA) prior to murine macrophage exposure. NTA analysis did not show any differences in the EV sizes among the strains. EVs from L. braziliensis and L. infantum failed to induce a pro-inflammatory response. EVs from both L. infantum WT and LPG-deficient mutant (LPG-KO) did not show any differences in their interaction with macrophages, suggesting that LPG solely was not determinant for activation. On the other hand, EVs from L. amazonensis were immunomodulatory inducing NO, TNF-α, IL-6, and IL-10 via TLR4 and TLR2. To determine whether such activation was related to NF-κB p65 translocation, THP-1 macrophage cells were exposed to EVs. In the same way, only EVs from L. amazonensis exhibited a highly percentage of cells positive for NF-κB. Our results suggest an important role of EVs in determining the pattern of immune response depending on the parasite species. For L. infantum, LPG was not determinant for the activation.