Leishmania infantum Defective in Lipophosphoglycan Biosynthesis Interferes With Activation of Human Neutrophils.

Visceral leishmaniasis (VL) is often associated with hematologic manifestations that may interfere with neutrophil response. Lipophosphoglycan (LPG) is a major molecule on the surface of Leishmania promastigotes, which has been associated with several aspects of the parasite-vector-host interplay. Here, we investigated how LPG from Leishmania (L.) infantum, the principal etiological agent of VL in the New World, influences the initial establishment of infection during interaction with human neutrophils in an experimental setting in vitro. Human neutrophils obtained from peripheral blood samples were infected with either the wild-type L. infantum (WT) strain or LPG-deficient mutant (∆lpg1). In this setting, ∆lpg1 parasites displayed reduced viability compared to WT L. infantum; such finding was reverted in the complemented ∆lpg1+LPG1 parasites at 3- and 6-h post-infection. Confocal microscopy experiments indicated that this decreased survival was related to enhanced lysosomal fusion. In fact, LPG-deficient L. infantum parasites more frequently died inside neutrophil acidic compartments, a phenomenon that was reverted when host cells were treated with Wortmannin. We also observed an increase in the secretion of the neutrophil collagenase matrix metalloproteinase-8 (MMP-8) by cells infected with ∆lpg1 L. infantum compared to those that were infected with WT parasites. Furthermore, collagen I matrix degradation was found to be significantly increased in ∆lpg1 parasite-infected cells but not in WT-infected controls. Flow cytometry analysis revealed a substantial boost in production of reactive oxygen species (ROS) during infection with either WT or ∆lpg1 L. infantum. In addition, killing of ∆lpg1 parasites was shown to be more dependent on the ROS production than that of WT L. infantum. Notably, inhibition of the oxidative stress with Apocynin potentially fueled ∆lpg1 L. infantum fitness as it increased the intracellular parasite viability. Thus, our observations demonstrate that LPG may be a critical molecule fostering parasite survival in human neutrophils through a mechanism that involves cellular activation and generation of free radicals.

In Vitro Differentiation of Human Dendritic Cells and their Markers in Leishmania Infection.

Leishmaniasis comprises a collection of clinical manifestations associated with the infection of obligate intracellular protozoans, Leishmania. The life cycle of Leishmania parasites consists of two alternating life stages (amastigotes and promastigotes), during which parasites reside within either arthropod vectors or vertebrate hosts, respectively. Notably, the complex interactions between Leishmania parasites and several cells of the immune system largely influence the outcome of infection. Importantly, although macrophages are known to be the main host niche for Leishmania replication, parasites are also phagocytosed by other innate immune cells, such as neutrophils and dendritic cells (DCs). DCs play a major role in bridging the innate and adaptive branches of immunity and thus orchestrate immune responses against a wide range of pathogens. The mechanisms by which Leishmania and DCs interact remain unclear and involve aspects of pathogen capture, the dynamics of DC maturation and activation, DC migration to draining lymph node (dLNs), and antigen presentation to T cells. Although a large body of studies support the notion that DCs play a dual role in modulating immune responses against Leishmania, the participation of these cells in susceptibility or resistance to Leishmania remains poorly understood. After infection, DCs undergo a maturation process associated with the upregulation of surface major histocompatibility complex (MHC) II, in addition to costimulatory molecules (namely, CD40, CD80, and CD86). Understanding the role of DCs in infection outcome is crucial to developing therapeutic and prophylactic strategies to modulate the immune response against Leishmania. This paper describes a method for the characterization of Leishmania-DC interaction. This detailed protocol provides guidance throughout the steps of DC differentiation, the characterization of cell surface molecules, and infection protocols, allowing scientists to investigate DC response to Leishmania infection and gain insight into the roles played by these cells in the course of infection.

Screening organic repellent compounds against Lutzomyia longipalpis (Diptera: Psychodidae) present in plant essential oils: Bioassay plus an in silico approach.

In the Americas, Lutzomyia longipalpis is the most relevant sand fly species for the transmission of visceral leishmaniasis. For its vector control in Brazil, insecticide spraying has not shown persistent reduction in disease prevalence while some sand fly populations are reported resistant to the insecticides used in spraying. The usage of repellents and personal protection behavior can reduce vector borne diseases prevalence. Therefore, the search for new repellent compounds is needed to use together with insecticide spraying, especially from natural sources to overcome the resistance developed by some sand fly populations to the compounds commercially used. In silico strategies have been applied together with repellency bioassays successfully identifying new bioactive compounds from natural sources. Thus, the present study aimed to screen repellent potential of neem (Azadirachta indica), citronella (Cymbopogon winterianus), bushy matgrass (Lippia alba) and 'alecrim do mato' (Lippia thymoides) essential oils against L. longipalpis and to identify potential repellent compounds by chemical analysis and in silico approach. Plant essential oils were extracted from leaves and repellency bioassays were performed on volunteers using colony reared L. longipalpis. Aside from neem oil, all other tested essential oil has shown a reduced number of sand fly bites using higher concentrations. Chemical composition from oils was assessed and its compounds were screened on a pharmacophore model using odorant binding protein 1 (OBP1). All essential oils were majorly composed of either oxygenated monoterpenes, except for the oil extracted from neem which was composed of sesquiterpene hydrocarbons. Molecular docking was performed with the compounds that best superimposed in the OBP1 pharmacophore model, identifying those binding to OBP4, which is associated with insect repellency behavior. Citronellol, Citronellol acetate, Citronellal and Geranyl acetate showed similar interactions with OBP4 binding site as DEET. Thus, it is suggested that these compounds are able to bind to L. longipalpis OBP4 generating repellent behavior in sand flies.

Investigating the Phagocytosis of Leishmania using Confocal Microscopy.

Phagocytosis is an orchestrated process that involves distinct steps: recognition, binding, and internalization. Professional phagocytes take up Leishmania parasites by phagocytosis, consisting of recognizing ligands on parasite surfaces by multiple host cell receptors. Binding of Leishmania to macrophage membranes occurs through complement receptor type 1 (CR1) and complement receptor type 3 (CR3) and Pattern Recognition Receptors. Lipophosphoglycan (LPG) and 63 kDa glycoprotein (gp63) are the main ligands involved in macrophage-Leishmania interactions. Following the initial recognition of parasite ligands by host cell receptors, parasites become internalized, survive, and multiply within parasitophorous vacuoles. The maturation process of Leishmania-induced vacuoles involves the acquisition of molecules from intracellular vesicles, including monomeric G protein Rab 5 and Rab 7, lysosomal associated membrane protein 1 (LAMP-1), lysosomal associated membrane protein 2 (LAMP-2), and microtubule-associated protein 1A/1B-light chain 3 (LC3). Here, we describe methods to evaluate the early events occurring during Leishmania interaction with the host cells using confocal microscopy, including (i) binding (ii) internalization, and (iii) phagosome maturation. By adding to the body of knowledge surrounding these determinants of infection outcome, we hope to improve the understanding of the pathogenesis of Leishmania infection and support the eventual search for novel chemotherapeutic targets.

Cell Migration and Cell Adhesion Assays to Investigate Leishmania-Host Cell Interaction.

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-resolving localized cutaneous lesions to a highly fatal visceral form of the disease. An estimated 12 million people worldwide are currently infected, and another 350 million face risk of infection. It is known that host cells infected by Leishmania parasites, such as macrophages or dendritic cells, can migrate to different host tissues, yet how migration contributes to parasite dissemination and homing remains poorly understood. Therefore, assessing these parasites' ability to modulate host cell response, adhesion, and migration will shed light on mechanisms involved in disease dissemination and visceralization. Cellular migration is a complex process in which cells undergo polarization and protrusion, allowing them to migrate. This process, regulated by actin and tubulin-based microtubule dynamics, involves different factors, including the modulation of cellular adhesion to the substrate. Cellular adhesion and migration processes have been investigated using several models. Here, we describe a method to characterize the migratory aspects of host cells during Leishmania infection. This detailed protocol presents the differentiation and infection of dendritic cells, the analysis of host cell motility and migration, and the formation of adhesion complexes and actin dynamics. This in vitro protocol aims to further elucidate mechanisms involved in Leishmania dissemination within vertebrate host tissues and can also be modified and applied to other cell migration studies.

Leishmania-Induced Dendritic Cell Migration and Its Potential Contribution to Parasite Dissemination.

Leishmania, an intracellular parasite species, causes lesions on the skin and in the mucosa and internal organs. The dissemination of infected host cells containing Leishmania is crucial to parasite survival and the establishment of infection. Migratory phenomena and the mechanisms underlying the dissemination of Leishmania-infected human dendritic cells (hDCs) remain poorly understood. The present study aimed to investigate differences among factors involved in hDC migration by comparing infection with visceral leishmaniasis (VL) induced by Leishmaniainfantum with diverse clinical forms of tegumentary leishmaniasis (TL) induced by Leishmaniabraziliensis or Leishmania amazonensis. Following the infection of hDCs by isolates obtained from patients with different clinical forms of Leishmania, the formation of adhesion complexes, actin polymerization, and CCR7 expression were evaluated. We observed increased hDC migration following infection with isolates of L. infantum (VL), as well as disseminated (DL) and diffuse (DCL) forms of cutaneous leishmaniasis (CL) caused by L. braziliensis and L. amazonensis, respectively. Increased expression of proteins involved in adhesion complex formation and actin polymerization, as well as higher CCR7 expression, were seen in hDCs infected with L. infantum, DL and DCL isolates. Together, our results suggest that hDCs play an important role in the dissemination of Leishmania parasites in the vertebrate host.

Unbalanced production of LTB4/PGE2 driven by diabetes increases susceptibility to cutaneous leishmaniasis.

Poorly controlled diabetes mellitus leads to several comorbidities, including susceptibility to infections. Hyperglycemia increases phagocyte responsiveness, however immune cells from people with diabetes show inadequate antimicrobial functions. We and others have shown that aberrant production of leukotriene B4 (LTB4) is detrimental to host defense in models of bacterial infection. Here, we will unveil the consequences of high glucose in the outcome of Leishmania braziliensis skin infection in people with diabetes and determine the role of LTB4 in human phagocytes. We show that diabetes leads to higher systemic levels of LTB4, IL-6 and TNF-α in cutaneous leishmaniasis. Only LTB4 correlated with blood glucose levels and healing time in diabetes comorbidity. Skin lesions of people with leishmaniasis and diabetes exhibit increased neutrophil and amastigote numbers. Monocyte-derived macrophages from these individuals showed higher L. braziliensis loads, reduced production of Reactive Oxygen Species and unbalanced LTB4/PGE2 ratio. Our data reveal a systemic inflammation driven by diabetes comorbidity in opposition to a local reduced capacity to resolve L. braziliensis infection and a worse disease outcome.

Corrigendum: Immunization of Experimental Dogs With Salivary Proteins From Lutzomyia longipalpis, Using DNA and Recombinant Canarypox Virus Induces Immune Responses Consistent With Protection Against Leishmania infantum.

[This corrects the article DOI: 10.3389/fimmu.2018.02558.].

Lutzomyia longipalpis Saliva Induces Heme Oxygenase-1 Expression at Bite Sites.

Sand flies bite mammalian hosts to obtain a blood meal, driving changes in the host inflammatory response that support the establishment of Leishmania infection. This effect is partially attributed to components of sand fly saliva, which are able to recruit and activate leukocytes. Our group has shown that heme oxygenase-1 (HO-1) favors Leishmania survival in infected cells by reducing inflammatory responses. Here, we show that exposure to sand fly bites is associated with induction of HO-1 in vivo. Histopathological analyses of skin specimens from human volunteers experimentally exposed to sand fly bites revealed that HO-1 and Nrf2 are produced at bite sites in the skin. These results were recapitulated in mice ears injected with a salivary gland sonicate (SGS) or exposed to sand fly bites, indicating that vector saliva may be a key factor in triggering HO-1 expression. Resident skin macrophages were the main source HO-1 at 24-48 h after bites. Additionally, assays in vivo after bites and in vitro after stimulation with saliva both demonstrated that HO-1 production by macrophages was Nrf2-dependent. Collectively, our data demonstrates that vector saliva induces early HO-1 production at the bite sites, representing a major event associated with establishment of naturally-transmitted Leishmania infections.

Immunization of Experimental Dogs With Salivary Proteins From Lutzomyia longipalpis, Using DNA and Recombinant Canarypox Virus Induces Immune Responses Consistent With Protection Against Leishmania infantum.

Metacyclic Leishmania promastigotes are transmitted by sand flies that inject parasites and saliva into the host's skin. Previous studies have demonstrated that DNA plasmids encoding Lutzomyia longipalpis salivary proteins LJM17 and LJL143, when used to immunize dogs, resulted in a systemic and local Th1 cell-mediated immunity that interfered in parasite survival in vitro. Here we evaluated the ability of these same salivary antigens to induce anti-Leishmania immunity and to confer protection by immunizing dogs using a novel vaccination strategy more suitable for use in the field. The strategy consisted of a single dose of plasmid followed by two doses of recombinant Canarypoxvirus (rCanarypoxvirus) expressing L. longipalpis salivary proteins (LJM17 or LJL143). Thirty days after the final immunization, dogs were intradermally challenged with 107Leishmania infantum promastigotes in the presence of L. longipalpis saliva. We followed the experimentally infected dogs for 10 months to characterize clinical, parasitological, and immunological parameters. Upon vaccination, all immunized dogs presented strong and specific humoral responses with increased serum concentrations of IFN-γ, TNF, IL-7, and IL-15. The serum of dogs immunized with LJM17 also exhibited high levels of IL-2, IL-6, and IL-18. L. infantum infection was established in all experimental groups as evidenced by the presence of anti-Leishmania IgG, and by parasite detection in the spleen and skin. Dogs immunized with LJM17-based vaccines presented higher circulating levels of IFN-γ, IL-2, IL-6, IL-7, IL-15, IL-18, TNF, CXCL10, and GM-CSF post-infection when compared with controls. Results demonstrated that relevant Leishmania-specific immune responses were induced following vaccination of dogs with L. longipalpis salivary antigen LJM17 administered in a single priming dose of plasmid DNA, followed by two booster doses of recombinant Canarypox vector. Importantly, a significant increase in pro-inflammatory cytokines and chemokines known to be relevant for protection against leishmaniasis was evidenced after challenging LJM17-vaccinated dogs as compared to controls. Although similar results were observed following immunization with LJL143, the pro-inflammatory response observed after immunization was attenuated following infection. Collectively, these data suggest that the LJM17-based vaccine induced an immune profile consistent with the expected protective immunity against canine leishmaniosis. These results clearly support the need for further evaluation of the LJM17 antigen, using a heterologous prime-boost vaccination strategy against canine visceral leishmaniosis (CVL).

Biomarkers for Zoonotic Visceral Leishmaniasis in Latin America.

In Latin America, zoonotic visceral leishmaniasis (ZVL) arising from infection by L. infantum is primarily transmitted by Lutzomyia longipalpis sand flies. Dogs, which are chronic reservoirs of L. infantum, are considered a significant risk factor for acquisition of ZVL due to their close proximity to humans. In addition, as a vector-borne disease the intensity of exposure to vector sand flies can also enhance the risk of developing ZVL. Traditionally, IFN-γ and IL-10 are considered as the two main cytokines which determine the outcome of visceral leishmaniasis. However, more recently, the literature has demonstrated that different mediators, such as lipid mediators (PGE-2, PGF-2 alfa, LTB-4, resolvins) and other important inflammatory and anti-inflammatory cytokines are also involved in the pathogenicity of ZVL. Analysis of a greater number of mediators allows for a more complete view of disease immunopathogenesis. Additionally, our knowledge has expanded to encompass different biomarkers associated to disease severity and healing after specific treatments. These parameters can also be used to better define new potential targets for vaccines and chemotherapy for ZVL. Here, we will provide an overview of ZVL biomarkers identified for both humans and dogs and discuss their merits and shortcomings. We will also discuss biomarkers of vector exposure as an additional tool in our arsenal to combat ZVL.

Autophagic Induction Greatly Enhances Leishmania major Intracellular Survival Compared to Leishmania amazonensis in CBA/j-Infected Macrophages.

CBA mouse macrophages control Leishmania major infection yet are permissive to Leishmania amazonensis. Few studies have been conducted to assess the role played by autophagy in Leishmania infection. Therefore, we assessed whether the autophagic response of infected macrophages may account for the differential behavior of these two parasite strains. After 24 h of infection, the LC3-II/Act ratio increased in both L. amazonensis- and L. major-infected macrophages compared to uninfected controls, but less than in chloroquine-treated cells. This suggests that L. amazonensis and L. major activate autophagy in infected macrophages, without altering the autophagic flux. Furthermore, L. major-infected cells exhibited higher percentages of DQ-BSA-labeled parasitophorous vacuoles (50%) than those infected by L. amazonensis (25%). However, L. major- and L. amazonensis-induced parasitophorous vacuoles accumulated LysoTracker similarly, indicating that the acidity in both compartment was equivalent. At as early as 30 min, endogenous LC3 was recruited to both L. amazonensis- and L. major-induced parasitophorous vacuoles, while after 24 h a greater percentage of LC3 positive vacuoles was observed in L. amazonensis-infected cells (42.36%) compared to those infected by L. major (18.10%). Noteworthy, principal component analysis (PCA) and an hierarchical cluster analysis completely discriminated L. major-infected macrophages from L. amazonensis-infected cells accordingly to infection intensity and autophagic features of parasite-induced vacuoles. Then, we evaluated whether the modulation of autophagy exerted an influence on parasite infection in macrophages. No significant changes were observed in both infection rate or parasite load in macrophages treated with the autophagic inhibitors wortmannin, chloroquine or VPS34-IN1, as well as with the autophagic inducers rapamycin or physiological starvation, in comparison to untreated control cells. Interestingly, both autophagic inducers enhanced intracellular L. amazonensis and L. major viability, while the pharmacological inhibition of autophagy exerted no effects on intracellular parasite viability. We also demonstrated that autophagy induction reduced NO production by L. amazonensis- and L. major-infected macrophages but not alters arginase activity. These findings provide evidence that although L. amazonensis-induced parasitophorous vacuoles recruit LC3 more markedly, L. amazonensis and L. major similarly activate the autophagic pathway in CBA macrophages. Interestingly, the exogenous induction of autophagy favors L. major intracellular viability to a greater extent than L. amazonensis related to a reduction in the levels of NO.

Lutzomyia longipalpis Saliva Drives Interleukin-17-Induced Neutrophil Recruitment Favoring Leishmania infantum Infection.

During bloodfeeding, the presence of sand fly saliva in the hemorrhagic pool where Leishmania is also inoculated modulates the development of host immune mechanisms creating a favorable environment for disease progression. To date, information obtained through experimental models suggests that sand fly saliva induces cellular recruitment and modulates production of eicosanoids. However, the effect of sand fly saliva in the different steps of the inflammatory response triggered by Leishmania remains undefined. Here we further investigate if interaction of Lutzomyia longipalpis salivary gland sonicate (SGS) with different host cells present during the initial inflammatory events regulate Leishmania infantum infectivity. Initially, we observed that incubation of human peripheral blood mononuclear cells (PBMC) with Lu. longipalpis SGS in the presence of L. infantum significantly increased IL-10 but did not alter expression of IFN-γ and TNF-α by CD4+ T cells induced by the parasite alone. Interestingly, incubation of PBMC with Lu. longipalpis SGS alone or in the presence of L. infantum resulted in increased IL-17 production. The presence of IL-17 is related to neutrophil recruitment and plays an important role at the site of infection. Here, we also observed increased migration of neutrophil using an in vitro chemotactic assay following incubation with supernatants from PBMC stimulated with L. infantum and Lu. longipalpis SGS. Neutrophil migration was abrogated following neutralization of IL-17 with specific antibodies. Moreover, culture of human neutrophils with L. infantum in the presence of Lu. longipalpis SGS promoted neutrophil apoptosis resulting in increased parasite viability. Neutrophils operate as the first line of defense in the early stages of infection and later interact with different cells, such as macrophages. The crosstalk between neutrophils and macrophages is critical to determine the type of specific immune response that will develop. Here, we observed that co-culture of human macrophages with autologous neutrophils previously infected in the presence of Lu. longipalpis SGS resulted in a higher infection rate, accompanied by increased production of TGF-ß and PGE2. Our results provide new insight into the contribution of Lu. longipalpis SGS to L. infantum-induced regulation of important inflammatory events, creating a favorable environment for parasite survival inside different host cells.

Coinfection With Trypanosoma brucei Confers Protection Against Cutaneous Leishmaniasis.

Infection with certain bacteria, parasites, and viruses alters the host immune system to Leishmania major influencing disease outcome. Here, we determined the outcome of a chronic infection with Trypanosoma brucei brucei on cutaneous leishmaniasis (CL) caused by L. major. C57BL/6 mice infected with T. b. brucei were given a sub-curative treatment with diminazene aceturate then coinfected with L. major by vector bites. Our results revealed that infection with T. b. brucei controls CL pathology. Compared to controls, coinfected mice showed a significant decrease in lesion size (P < 0.05) up to 6 weeks post-infection and a significant decrease in parasite burden (P < 0.0001) at 3 weeks post-infection. Protection against L. major resulted from a non-specific activation of T cells by trypanosomes. This induced a strong immune response characterized by IFN-γ production at the site of bites and systemically, creating a hostile inflammatory environment for L. major parasites and conferring protection from CL.

Immunization with LJM11 salivary protein protects against infection with Leishmania braziliensis in the presence of Lutzomyia longipalpis saliva.

Leishmania is transmitted in the presence of sand fly saliva. Protective immunity generated by saliva has encouraged identification of a vector salivary-based vaccine. Previous studies have shown that immunization with LJM11, a salivary protein from Lutzomyia longipalpis, is able to induce a Th1 immune response and protect mice against bites of Leishmania major-infected Lutzomyia longipalpis. Here, we further investigate if immunization with LJM11 recombinant protein is able to confer cross-protection against infection with Leishmania braziliensis associated with salivary gland sonicate (SGS) from Lutzomyia intermedia or Lu. longipalpis. Mice immunized with LJM11 protein exhibited an increased production of anti-LJM11 IgG, IgG1 and IgG2a and a DTH response characterized by an inflammatory infiltrate with the presence of CD4+ IFN-γ+ T cells. LJM11-immunized mice were intradermally infected in the ear with L. braziliensis in the presence of Lu. longipalpis or Lu. intermedia SGS. A significant reduction of parasite numbers in the ear and lymph node in the group challenged with L. braziliensis plus Lu. longipalpis SGS was observed, but not when the challenge was performed with L. braziliensis plus Lu. intermedia SGS. A higher specific production of IFN-γ and absence of IL-10 by lymph node cells were only observed in LJM11 immunized mice after infection. After two weeks, a similar frequency of CD4+ IFN-γ+ T cells was detected in LJM11 and BSA groups challenged with L. braziliensis plus Lu. longipalpis SGS, suggesting that early events possibly triggered by immunization are essential for protection against Leishmania infection. Our findings support the specificity of saliva-mediated immune responses and reinforce the importance of identifying cross-protective salivary antigens.

Heme Drives Oxidative Stress-Associated Cell Death in Human Neutrophils Infected with Leishmania infantum.

Free heme is an inflammatory molecule capable of inducing migration and activation of neutrophils. Here, we examine the heme-driven oxidative stress-associated cell death mechanisms in human neutrophils infected with Leishmania infantum, an etiologic agent of visceral leishmaniasis (VL). We first performed exploratory analyses in a population of well characterized treatment-naïve VL patients as well as uninfected controls, who were part of previously reported studies. We noted a positive correlation between serum concentrations of heme with heme oxygenase-1 (HO-1) and lactate deydrogenase, as well as, a negative correlation between heme values and peripheral blood neutrophils counts. Moreover, in vitro infection with L. infantum in the presence of heme enhanced parasite burden in neutrophils, while increasing the production of reactive oxygen species and release of neutrophilic enzymes. Additional experiments demonstrated that treatment of infected neutrophils with ferrous iron (Fe+2), a key component of the heme molecule, resulted in increased parasite survival without affecting neutrophil activation status. Furthermore, stimulation of infected neutrophils with heme triggered substantial increases in HO-1 mRNA expression as well as in superoxide dismutase-1 enzymatic activity. Heme, but not Fe+2, induced oxidative stress-associated cell death. These findings indicate that heme promotes intracellular L. infantum survival via activation of neutrophil function and oxidative stress. This study opens new perspectives for the understanding of immunopathogenic mechanisms involving neutrophils in VL.

Prostaglandin E2/leukotriene B4 balance induced by Lutzomyia longipalpis saliva favors Leishmania infantum infection.

BACKGROUND: Eicosanoids and sand fly saliva have a critical role in the Leishmania infection. Here, we evaluated the effect of Lutzomyia longipalpis salivary gland sonicate (SGS) on neutrophil and monocyte recruitment and activation of eicosanoid production in a murine model of inflammation. METHODS: C57BL/6 mice were inoculated intraperitonealy with Lutzomyia longipalpis SGS or Leishmania infantum or both, followed by analyses of cell recruitment, parasite load and eicosanoid production. RESULTS: Intraperitoneal injection of Lutzomyia longipalpis SGS together with Leishmania infantum induced an early increased parasite viability in monocytes and neutrophils. L. longipalpis SGS increased prostaglandin E2 (PGE2), but reduced leukotriene B4 (LTB4) production ex vivo in peritoneal leukocytes. In addition, the pharmacological inhibition of cyclooxygenase 2 (COX-2) with NS-398 decreased parasite viability inside macrophages during Leishmania infection in the presence of L. longipalpis SGS arguing that PGE2 production is associated with diminished parasite killing. CONCLUSIONS: These findings indicate that L. longipalpis SGS is a critical factor driving immune evasion of Leishmania through modulation of PGE2/LTB4 axis, which may represent an important mechanism on establishment of the infection.

The immunobiology of Leishmania braziliensis infection.

Leishmaniases are a group of diseases caused by protozoa of the genus Leishmania that affect millions of people worldwide. These diseases are caused by distinct Leishmania species, of which L. braziliensis, a New World representative of the Leishmania genus, has been the least studied. Although leishmaniasis caused by L. braziliensis induces a range of clinical manifestations ranging from mild localized lesions to severe mucosal involvement, few studies have focused on elucidating the immune mechanisms behind this pathology. In this review, we focus on the immunobiology of L. braziliensis infection, emphasizing the innate and adaptive immune responses and taking into consideration both studies performed in endemic areas and experimental models of infection. Additionally, we address recent findings regarding the role of sand fly saliva in disease immunopathogenesis and vaccine development.

Heme oxygenase-1 promotes the persistence of Leishmania chagasi infection.

Visceral leishmaniasis (VL) remains a major public health problem worldwide. This disease is highly associated with chronic inflammation and a lack of the cellular immune responses against Leishmania. It is important to identify major factors driving the successful establishment of the Leishmania infection to develop better tools for the disease control. Heme oxygenase-1 (HO-1) is a key enzyme triggered by cellular stress, and its role in VL has not been investigated. In this study, we evaluated the role of HO-1 in the infection by Leishmania infantum chagasi, the causative agent of VL cases in Brazil. We found that L. chagasi infection or lipophosphoglycan isolated from promastigotes triggered HO-1 production by murine macrophages. Interestingly, cobalt protoporphyrin IX, an HO-1 inductor, increased the parasite burden in both mouse and human-derived macrophages. Upon L. chagasi infection, macrophages from Hmox1 knockout mice presented significantly lower parasite loads when compared with those from wild-type mice. Furthermore, upregulation of HO-1 by cobalt protoporphyrin IX diminished the production of TNF-α and reactive oxygen species by infected murine macrophages and increased Cu/Zn superoxide dismutase expression in human monocytes. Finally, patients with VL presented higher systemic concentrations of HO-1 than healthy individuals, and this increase of HO-1 was reduced after antileishmanial treatment, suggesting that HO-1 is associated with disease susceptibility. Our data argue that HO-1 has a critical role in the L. chagasi infection and is strongly associated with the inflammatory imbalance during VL. Manipulation of HO-1 pathways during VL could serve as an adjunctive therapeutic approach.

Human mucosal leishmaniasis: neutrophils infiltrate areas of tissue damage that express high levels of Th17-related cytokines.

Mucosal leishmaniasis (ML) is characterised by severe tissue destruction. Herein, we evaluated the involvement of the IL-17-type response in the inflammatory infiltrate of biopsy specimens from 17 ML patients. IL-17 and IL-17-inducing cytokines (IL-1ß, IL-23, IL-6 and TGF-ß) were detected by immunohistochemistry in ML patients. IL-17(+) cells exhibited CD4(+), CD8(+) or CD14(+) phenotypes, and numerous IL-17(+) cells co-expressed the CC chemokine receptor 6 (CCR6). Neutrophils, a hallmark of Th17-mediated inflammation, were regularly detected in necrotic and perinecrotic areas and stained positive for neutrophil elastase, myeloperoxidase and MMP-9. Taken together, these observations demonstrate the existence of Th17 cells in ML lesions associated with neutrophils in areas of tissue injury and suggest that IL-17 is involved in ML pathogenesis.