Lutzomyia longipalpis Saliva or Salivary Protein LJM19 Protects against Leishmania braziliensis and the Saliva of Its Vector, Lutzomyia intermedia

Leishmaniasis, caused by parasitic protozoa Leishmania, is transmitted by bites of female sand flies that, during blood-feeding, inject humans with parasites and saliva. Sand fly saliva has been investigated as a potential vaccine candidate. It was previously shown that immunization with Lutzomyia longipalpis saliva or salivary proteins protects against cutaneous and visceral leishmaniasis. In the present study, we evaluated if immunization with Lu. longipalpis saliva or DNA plasmid coding for a specific sand fly salivary protein (LJM19) can protect hamsters against L. braziliensis plus another sand fly saliva. Immunization with saliva or LJM19 DNA plasmid induced a mononuclear cell infiltrate which can be a marker of protection. The immune response induced by immunization with these insect molecules was able to protect animals against L. braziliensis infection as shown by the significant reduction in lesion size, parasite load in the ear and draining lymph node. These data show the important role of immune response against sand fly saliva components, suggesting the possibility to develop vaccines using a single component of saliva against Leishmania transmitted by different vectors

Lutzomyia longipalpis Saliva Triggers Lipid Body Formation and Prostaglandin E2 Production in Murine Macrophages

After the injection of saliva into the host's skin by sand flies, a transient erythematous reaction is observed, which is related to an influx of inflammatory cells and the release of various molecules that actively facilitate the blood meal. It is important to understand the specific mechanisms by which sand fly saliva manipulates the host's inflammatory responses. Herein, we report that saliva from Lutzomyia (L.) longipalpis, a widespread Leishmania vector, induces early production of eicosanoids. Intense formation of intracellular organelles called lipid bodies (LBs) was noted within those cells that migrated to the site of saliva injection. In vitro and ex vivo, sand fly saliva was able to induce LB formation and PGE2 release by macrophages. Interestingly, PGE2 production induced by L. longipalpis saliva was dependent on intracellular mechanisms involving phosphorylation of signaling proteins such as PKC-α and ERK-1/2 and subsequent activation of cyclooxygenase-2. Thus, this study provides new insights into the pharmacological properties of sand fly saliva and opens new opportunities for intervening with the induction of the host's inflammatory pathways by L. longipalpis bites

Discovery of Markers of Exposure Specific to Bites of Lutzomyia longipalpis, the Vector of Leishmania infantum chagasi in Latin America

Leishmania parasites are transmitted by the bite of an infected vector sand fly that injects salivary molecules into the host skin during feeding. Certain salivary molecules can produce antibodies and can be used as an indicator of exposure to a vector sand fly and potentially the disease it transmits. Here we identified potential markers of specific exposure to the sand fly Lutzomyia longipalpis, the vector of visceral leishmaniasis in Latin America. Initially, we determined which of the salivary proteins produce antibodies in humans, dogs, and foxes from areas endemic for the disease. To identify potential specific markers of vector exposure, we produced nine different recombinant salivary proteins from Lu. longipalpis and tested for their recognition by individuals exposed to another human-biting sand fly, Lu. intermedia, that transmits cutaneous leishmaniasis and commonly occurs in the same endemic areas as Lu. longipalpis. Two of the nine salivary proteins were recognized only by humans exposed to Lu. longipalpis, suggesting they are immunogenic proteins and may be useful in epidemiological studies. The identification of specific salivary proteins as potential markers of exposure to vector sand flies will increase our understanding of vector–human interaction, bring new insights to vector control, and in some instances act as an indicator for risk of acquiring disease

Enhanced Leishmania braziliensis Infection Following Pre-Exposure to Sandfly Saliva

Parasites of the genus Leishmania cause a variety of diseases known as leishmaniasis, that are transmitted by bites of female sand flies that, during blood-feeding, inject humans with parasites and saliva. It was shown that, in mice, immunity to sand-fly saliva is able to protect against the development of leishmaniasis. We have investigated, in the present study, whether this finding extends the sand fly species Lutzomyia intermedia, which is responsible for transmission of Leishmania braziliensis, a parasite species able to cause destructive skin lesions that can be fatal if left untreated. We observed that mice injected with sand fly saliva develop a specific immune response against salivary proteins. Most importantly, however, this immune response was unable to protect mice against a challenge infection with L. braziliensis, indicating that exposure to this sand fly saliva is harmful to the host. Indeed, subjects with cutaneous leishmaniasis have a higher immune response against L. intermedia saliva. These findings indicate that the anti-saliva immune response to sand fly saliva plays an important role in the outcome of leishmaniasis caused by L. braziliensis, in both mice and humans, and emphasize possible hurdles in the development of vaccines based on sand fly saliva

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

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

Immunity to Lutzomyia intermedia Saliva Modulates the Inflammatory Environment Induced by Leishmania braziliensis

Transmission of Leishmania parasites occurs during blood feeding, when infected female sand flies inject humans with parasites and saliva. Chemokines and cytokines are secreted proteins that regulate the initial immune responses and have the potential of attracting and activating cells. Herein, we studied the expression of such molecules and the cellular recruitment induced by salivary proteins of the Lutzomyia intermedia sand fly. Of note, Lutzomyia intermedia is the main vector of Leishmania braziliensis, a parasite species that causes cutaneous leishmaniasis, a disease associated with the development of destructive skin lesions that can be fatal if left untreated. We observed that L. intermedia salivary proteins induce a potent cellular recruitment and modify the expression profile of chemokines and cytokines in mice. More importantly, in mice previously immunized with L. intermedia saliva, the alteration in the initial inflammatory response was even more pronounced, in terms of the number of cells recruited and in terms of gene expression pattern. These findings indicate that an existing immunity to L. intermedia sand fly induces an important modulation in the initial immune response that may, in turn, promote parasite multiplication, leading to the development of cutaneous leishmaniasis

Specific Humoral Immunity versus Polyclonal B Cell Activation in Trypanosoma cruzi Infection of Susceptible and Resistant Mice

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects 10–12 million people in Latin America. Patent parasitemia develops during acute disease. During this phase, polyclonal B cell activation has been reported to generate high levels of serum antibody with low parasite specificity, and delayed protective humoral immunity, which is necessary to prevent the host from succumbing to infection. In this manuscript, data show that relatively resistant mice have improved parasite-specific humoral immunity and decreased polyclonal B cell activation compared to susceptible mice. Parasite-specific humoral immunity was associated with differential expansion of B cell subsets and T cells in the spleen, as well as with increased Th1 and decreased Th2 cytokine production. These data suggest that host susceptibility/genetic biases impact the development of humoral responses to infection. Th2 cytokines are generally associated with improved antibody responses. In the context of T. cruzi infection of susceptible mice, Th2 cytokines were associated with increased total antibody production concomitant with delayed pathogen-specific humoral immunity. This study highlights the need to consider the effect of host biases when investigating humoral immunity to any pathogen that has reported polyclonal B cell activation during infection