Suzana Passos Chaves

Modulation of P2X7 purinergic receptor in macrophages by Leishmania amazonensis and its role in parasite elimination.

The purinergic P2X(7) receptor is a membrane protein of leucocytes involved in the clearance of intracellular bacteria such as Chlamydia and Mycobacterium. In this work, we investigated the role and modulation of macrophage P2X(7)R in intracellular infection with the protozoan parasite Leishmania amazonensis. Upon infection, isolated murine macrophages displayed enhanced expression of P2X(7)R and were significantly more responsive to extracellular ATP (ATPe)-induced pore opening, as demonstrated by the increased uptake of Lucifer Yellow. This was extended to the in vivo situation, where cells from established cutaneous lesions were more sensitive to ATPe than cells from uninfected mice. ATP treatment of infected macrophages inhibited parasite growth, and this was prevented by pre-treatment with oxidized ATP, a selective antagonist of P2X(7)R. Parasite killing was unlikely due to induction of nitric oxide production or cytolysis of infected macrophage, as those functions were unaltered with parasite-effective ATPe concentrations. A direct drug effect is also unlike, as ATPe enhanced axenic parasite growth. We found that leishmanial infection rendered wild-type but not P2X(7)R-deficient macrophages more prone to ATP-induced apoptosis. These results show that macrophage infection with L. amazonensis leads to enhanced expression of functional P2X(7)R, that upon ligation with ATPe helps in the elimination of the parasites by an as yet unclear mechanism possibly involving host cell apoptosis.

Infection with Leishmania amazonensis upregulates purinergic receptor expression and induces host-cell susceptibility to UTP-mediated apoptosis

Nucleotides are released into the extracellular milieu from infected cells and cells at inflammatory sites. The extracellular nucleotides bind to specific purinergic (P2) receptors and thereby induce a variety of cellular responses including anti‐parasitic effects. Here we investigated whether extracellular nucleotides affect leishmanial infection in macrophages, and found that UTP reduces strongly the parasite load in peritoneal macrophages. Ultrastructural analysis of infected cells revealed that UTP induced morphological damage in the intracellular parasites. Uridine nucleotides also induced dose‐dependent apoptosis of macrophages and production of ROI and RNI only in infected macrophages. The intracellular calcium measurements of infected cells showed that the response to UTP, but not UDP, increased the sensitivity and amplitude of cytosolic Ca2+ changes. Infection of macrophages with Leishmania upregulated the expression of P2Y2 and P2Y4 receptor mRNA. The data suggest indirectly that Leishmania amazonensis infection induces modulation and heteromerization of P2Y receptors on macrophages. Thus UTP modulates the host response against L. amazonensis infection. UTP and UTP homologues should therefore be considered as novel components of therapeutic strategies against cutaneous leishmaniasis.

Serine proteases of Leishmania amazonensis as immunomodulatory and disease-aggravating components of the crude LaAg vaccine

We previously demonstrated that intradermal and intramuscular vaccination with Leishmania amazonensis promastigote antigens (LaAg) increases the susceptibility of BALB/c mice to cutaneous leishmaniasis. In this study, we investigated the role played by serine and cysteine proteases as disease-promoting components of LaAg. Mice were immunized by the intramuscular route with LaAg that was pre-treated with a pool of serine or cysteine protease inhibitors (SPi and CPi, respectively) prior to infection with L. amazonensis. Neutralization of either enzyme type reversed the disease-promoting effect of LaAg, as seen by the slower lesion development. However, the parasite burden was only effectively controlled in mice receiving SPi-treated LaAg. Protection was associated with diminished production of TGF-beta and particularly IL-10 in response to parasite antigens by the lesion-draining lymph node cells of vaccinated mice relative to control. In vitro, soluble proteases isolated from LaAg (LaSP-Sol) directly activated IL-4, IL-10 and TGF-beta production by immune cells. Like native LaAg, vaccination with LaSP-Sol primed mice to respond to parasite challenge with a strong Jones-Mote cutaneous hypersensitivity reaction, and increased susceptibility to infection. Furthermore, neutralization of serine but not cysteine proteases blocked the capacity of LaAg to sensitize mice for Jones-Mote reaction. Together, these results indicate that soluble serine proteases are key components of LaAg responsible for its disease-promoting immunity.

The C-terminal extension of Leishmania pifanoi amastigote-specific cysteine proteinase Lpcys2: A putative function in macrophage infection

Cysteine proteinases have been implicated in many aspects of protozoan parasite pathogenesis. These hydrolases are normally found as zymogens, and some classes in trypanosomatids possess a long C-terminal extension (CTE), for which no function has been assigned. In this paper we hypothesize that the CTE domain of Lpcys2, the abundant lysosomal cysteine proteinase of Leishmania pifanoi amastigotes, is involved in host cell infection. Confirming previous reports that this peptide is highly immunogenic in Trypanosoma cruzi, we detected antibodies against CTE in sera of leishmaniasis patients. We produced a polyclonal antibody specific to Lpcys2 CTE and determined that this antibody was capable of recognizing both L. pifanoi and Leishmania amazonensis cysteine proteinases. Using this antibody, we detected a predominant localization of Lpcys2 CTE in the lysosome and flagellar pocket of cultured axenic amastigotes of both parasite species; however, its location was shifted towards the surface of the parasites during macrophage infection. We examined the role of Lpcys2 CTE in macrophage infection and found a significant reduction in the percentage of infected cells when macrophages were infected with L. pifanoi and L. amazonensis in the presence of anti-CTE antibody. This study suggests a role for leishmanial cysteine proteinases CTE at early stages of infection.

Intranasal vaccination with extracellular serine proteases of Leishmania amazonensis confers protective immunity to BALB/c mice against infection

BackgroundPreviously, we demonstrated that unlike subcutaneous or intramuscular vaccination, intranasal vaccination of BALB/c mice with whole Leishmania amazonensis antigens leads to protection against cutaneous leishmaniasis. Here, the role of parasite serine proteases in the protective immunity was investigated.FindingsSerine Proteases were partially purified from both soluble (LaSP-Sol) and extracellular (LaSP-Ex) Leishmania amazonensis promastigote extracts by aprotinin-agarose chromatography. BALB/c mice were intranasally immunized with LaSP-Sol and LaSP-Ex prior to infection with L. amazonensis. LaSP-Ex but not LaSP-Sol vaccination led to significantly smaller lesions and parasite burdens as compared with non-vaccinated controls. Protection was accompanied by systemic Th1 polarization with increased IFN-γ and decreased IL-4 and IL-10 splenic production. Likewise, increased production of IFN-γ, IL-12 and IL-4 concomitant with decreased TGF-β and TNF-α was locally observed in the infected footpad.ConclusionThis study indicates that extracellular serine proteases of L. amazonensis are strong candidates for a more defined intranasal vaccine against cutaneous leishmaniasis.

Serine Proteases and Vaccines against Leishmaniasis: A Dual Role

Serine proteases are involved in several biochemical processes that are essential for the biology of pathogens, including Leishmania sp. Considering their importance, an interest in serine proteases for vaccine development against leishmaniasis has been raised. As targets, these enzymes have demonstrated a dual role in a vaccine against leishmaniasis, both protective and a counter-protective, depending on the conditions that they are evaluated. In this work, serine proteases or inhibitors of them that have been used as components of vaccines to Leishmania sp. are presented, aiming to disseminate the knowledge gained about these proteases and their potential in potential vaccine against leishmaniasis.

Efficacy of intranasal LaAg vaccine against Leishmania amazonensis infection in partially resistant C57Bl/6 mice

BackgroundWe have previously demonstrated that intranasal vaccination of highly susceptible BALB/c mice with whole Leishmania amazonensis antigens (LaAg) leads to protection against murine cutaneous leishmaniasis. Here, we evaluate the response of partially resistant C57BL/6 mice to vaccination as a more representative experimental model of human cutaneous leishmaniasis.MethodsC57BL/6 mice from different animal facilities were infected with L. amazonensis (Josefa strain) to establish the profile of infection. Intranasal vaccination was performed before the infection challenge with two doses of 10 μg of LaAg alone or associated with the adjuvant ADDAVAX® by instillation in the nostrils. The lesion progression was measured with a dial caliper and the parasite load by limited dilution assay in the acute and chronic phases of infection. Cytokines were quantified by ELISA in the homogenates of infected footpads.ResultsC57BL/6 mice from different animal facilities presented the same L. amazonensis infection profile, displaying a progressive acute phase followed by a controlled chronic phase. Parasites cultured in M199 and Schneider’s media were equally infective. Intranasal vaccination with LaAg led to milder acute and chronic phases of the disease. The mechanism of protection was associated with increased production of IFN-gamma in the infected tissue as measured in the acute phase. Association with the ADDAVAX® adjuvant did not improve the efficacy of intranasal LaAg vaccination. Rather, ADDAVAX® reduced vaccination efficacy.ConclusionThis study demonstrates that the efficacy of adjuvant-free intranasal vaccination with LaAg is extendable to the more resistant C57Bl/6 mouse model of infection with L. amazonensis, and is thus not exclusive to the susceptible BALB/c model. These results imply that mucosal immunomodulation by LaAg leads to peripheral protection irrespective of the genetic background of the host.

Periodate‐oxidized ATP modulates macrophage functions during infection with Leishmania amazonensis

Previously, we showed that treating macrophages with ATP impairs the intracellular growth of Leishmania amazonensis, and that the P2X7 purinergic receptor is overexpressed during leishmaniasis. In the present study, we directly evaluated the effect of periodate‐oxidized ATP (oATP) on parasite control in Leishmania‐infected macrophages. We found that oATP impaired the attachment/entrance of L. amazonensis promastigotes to C57BL/6 mouse macrophages in a P2X7 receptor‐independent manner, as macrophages from P2X7−/− mice were similarly affected. Although oATP directly inhibited the growth of axenic promastigotes in culture, promoted rapid ultrastructural alterations, and impaired Leishmania internalization by macrophages, it did not affect intracellular parasite multiplication. Upon infection, phagosomal acidification was diminished in oATP‐treated macrophages, accompanied by reduced endosomal proteolysis. Likewise, MHC class II molecules expression and ectoATPase activity was decreased by oATP added to macrophages at the time of parasite infection. These inhibitory effects were not due to a cytotoxic effect, as no additional release of lactate dehydrogenase was detected in culture supernatants. Moreover, the capacity of macrophages to produce nitric oxide and reactive oxygen species was not affected by the presence of oATP during infection. We conclude that oATP directly affects extracellular parasite integrity and macrophage functioning.

Intralesional uridine-5′-triphosphate (UTP) treatment induced resistance to Leishmania amazonensis infection by boosting Th1 immune responses and reactive oxygen species production

Leishmania amazonensis is the etiologic agent of cutaneous leishmaniasis, an immune-driven disease causing a range of clinical symptoms. Infections caused by L. amazonensis suppress the activation and function of immune cells, including macrophages, dendritic cells, and CD4+ T cells. In this study, we analyzed the course of infection as well as the leishmanicidal effect of intralesional UTP treatment in L. amazonensis-infected BALB/c mice. We found that UTP treatment reduced the parasitic load in both footpad and lymph node sites of infection. UTP also boosted Th1 immune responses, increasing CD4+ T cell recruitment and production of IFN-γ, IL-1β, IL-12, and TNF-α. In addition, the role of UTP during innate immune response against L. amazonensis was evaluated using the air pouch model. We observed that UTP augmented neutrophil chemoattraction and activated microbicidal mechanisms, including ROS production. In conclusion, our data suggested an important role for this physiological nucleotide in controlling L. amazonensis infection, and its possible use as a therapeutic agent for shifting immune responses to Th1 and increasing host resistance against L. amazonensis infection.

Effects of Bone Marrow Mesenchymal Stromal Cell Therapy in Experimental Cutaneous Leishmaniasis in BALB/c Mice Induced by Leishmania amazonensis

Cutaneous leishmaniasis remains both a public health and a therapeutic challenge. To date, no ideal therapy for cutaneous leishmaniasis has been identified, and no universally accepted therapeutic regimen and approved vaccines are available. Due to the mesenchymal stromal cell (MSC) immunomodulatory capacity, they have been applied in a wide variety of disorders, including infectious, inflammatory, and allergic diseases. We evaluated the potential effects of bone marrow MSC therapy in a murine model of cutaneous leishmaniasis. In vitro, coculture of infected macrophages with MSC increased parasite load on macrophages in comparison with controls (macrophages without MSCs). In vivo, BALB/c mice were infected with 2 × 106 Leishmania amazonensis (Josefa strain) promastigotes in the footpad. 7 and 37 days after infection, animals were treated with 1 × 105 MSCs, either intralesional (i.l.), i.e., in the same site of infection, or intravenously (i.v.), through the external jugular vein. Control animals received the same volume (50 µL) of phosphate-buffered saline by i.l. or i.v. routes. The lesion progression was assessed by its thickness measured by pachymetry. Forty-two days after infection, animals were euthanized and parasite burden in the footpad and in the draining lymph nodes was quantified by the limiting dilution assay (LDA), and spleen cells were phenotyped by flow cytometry. No significant difference was observed in lesion progression, regardless of the MSC route of administration. However, animals treated with i.v. MSCs presented a significant increase in parasite load in comparison with controls. On the other hand, no harmful effect due to MSCs i.l. administered was observed. The spleen cellular profile analysis showed an increase of IL-10 producing T CD4+ and TCD8+ cells in the spleen only in mice treated with i.v. MSC. The excessive production of IL-10 could be associated with the disease-aggravating effects of MSC therapy when intravenously administered. As a conclusion, in the current murine model of L. amazonensis-induced cutaneous disease, MSCs did not control the damage of cutaneous disease and, depending on the administration route, it could result in deleterious effects.