Luís Carlos Crocco Afonso

Immune response induced by New World Leishmania species in C57BL/6 mice

Abstract In the present study, C57BL/6 mice were inoculated with metacyclic Leishmania amazonensis or L. braziliensis promastigotes. While these animals were capable of controlling the infection by L. braziliensis, they developed chronic lesions with elevated numbers of parasites when infected by L. amazonensis. The differences in parasite control were associated with a decreased production of IFN-γ and TNF by lymph node cells from L. amazonensis-infected mice. Furthermore, these animals presented decreased spleen cell proliferation and activation of germinal centers. In addition, we compared the ability of these parasites to hydrolyze extracellular ATP and AMP. While the ATPase activity of both parasite species was similar, L. amazonensis promastigotes presented higher AMP hydrolytic activity. This increased activity may lead to an increased production of adenosine, which has been shown to present anti-inflammatory activity and may thus be involved in the establishment of the immunosuppression observed in mice infected by L. amazonensis.

Intramuscular immunization with p36(LACK) DNA vaccine induces IFN-γ production but does not protect BALB/c mice against Leishmania chagasi intravenous challenge

Acute visceral leishmaniasis is a progressive disease caused by Leishmania chagasi in South America. The acquisition of immunity following infection suggests that vaccination is a feasible approach to protect against this disease. Since Leishmania homologue of receptors for activated C kinase (LACK) antigen is of particular interest as a vaccine candidate because of the prominent role it plays in the pathogenesis of experimental Leishmania major infection, we evaluated the potential of a p36(LACK) DNA vaccine in protecting BALB/c mice challenged with L. chagasi. In this study, mice received intramuscular (i.m.) or subcutaneous (s.c.) doses of LACK DNA vaccine. We evaluated the production of vaccine-induced cytokines and whether this immunization was able to reduce parasite load in liver and spleen. We detected a significant production of interferon gamma by splenocytes from i.m. vaccinated mice in response to L. chagasi antigen and to rLACK protein. However, we did not observe a reduction in parasite load neither in liver nor in the spleen of vaccinated animals. The lack of protection observed may be explained by a significant production of IL-10 induced by the vaccine.

Immunochemotherapy in American Cutaneous Leishmaniasis: Immunological Aspects before and after Treatment

In this study, we evaluated the immune response of patients suffering from cutaneous leishmaniasis treated with two distinct protocols. One group was treated with conventional chemotherapy using pentavalent antimonium salts and the other with immunochemotherapy where a vaccine against cutaneous leishmaniasis was combined with the antimonium salt. Our results show that, although no differences were observed in the necessary time for complete healing of the lesions between the two treatments, peripheral blood mononuclear cells from patients treated by chemotherapy showed smaller lymphoproliferative responses at the end of the treatment than those from patients in the immunochemotherapy group. Furthermore, IFN-gamma production was also different between the two groups. While cells from patients in the chemotherapy group produced more IFN-gamma at the end of treatment, a significant decrease in this cytokine production was associated with healing in the immunochemotherapy group. In addition, IL-10 production was also less intense in this latter group. Finally, an increase in CD8+ -IFN-gamma producing cells was detected in the chemotherapy group. Together these results point to an alternative treatment protocol where healing can be induced with a decreased production of a potentially toxic cytokine.

Germ-free mice produce high levels of interferon-gamma in response to infection with Leishmania major but fail to heal lesions

In order to investigate the importance of the host microbiota on differentiation of T cell subsets in response to infection, Swiss/NIH germ-free mice and conventional (microbiota-bearing) mice were infected with Leishmania major, and lesion development, parasite loads, and cytokine production were assessed. Germ-free mice failed to heal lesions and presented a higher number of parasites at the site of infection than their conventional counterparts. In addition, histopathological analysis indicated a higher density of parasitized macrophages in lesions from germ-free mice than in conventional mice. The initial production of interleukin (IL)-12 and interferon-gamma (IFN-gamma) in germ-free mice was comparable to the conventional controls. Also, germ-free mice produced elevated levels of IFN-gamma and lower levels of IL-4 throughout the course of infection, suggesting the development of a Th1 response. Macrophages from germ-free mice exposed to IFN-gamma and infected with amastigotes in vitro were not as efficient at killing parasites as macrophages from conventional animals. These observations indicate that the microbiota is not essential for the development of Th1 immune responses, but seems to be important for macrophage activation.

The influence of ecto-nucleotidases on Leishmania amazonensis infection and immune response in C57B/6 mice.

Previous results from our laboratory and from the literature have implicated the expression of ecto-nucleotidases in the establishment of Leishmania infection. In the present study we evaluated the correlation between ecto-nucleotidasic activity and the infectivity of L. amazonensis promastigotes that were kept in culture for short or extended numbers of passages, a condition that is known to decrease parasite infectivity. We also analyzed the immune response associated with the infection by these parasites. As expected, we found that long-term cultured parasites induce the development of smaller lesions than the short-term cultured counterparts. Interestingly, long-term cultured parasites presented reduced ecto-nucleotidasic activity. In addition, cells recovered from animals infected with long-term cultured parasites produced higher amounts of IFN-gamma and have smaller parasite load, after 8weeks of infection. Furthermore, after 1week of infection, there is increased expression of the chemokine CCL2 mRNA in animals infected with short-term cultured parasites. Finally, infection of peritoneal macrophages by these parasites also shows marked differences. Thus, while short-term cultured parasites are able to infect a greater proportion of macrophages, cells infected by long-term cultured parasites express higher amounts of CXCL10 mRNA, which may activate these cells to kill the parasites. We suggest that the enzymes involved in metabolism of extracellular nucleotides may have an important role in infection by L. amazonensis, by acting directly in its adhesion to target cells and by modulating host cell chemokine production.

IgG isotype profile is correlated with cardiomegaly in Beagle dogs infected with distinct Trypanosoma cruzi strains.

A systematic study following infection by various strains of the protozoan parasite, Trypanosoma cruzi, and the simultaneous monitoring of the humoral immune response together with the elicited cellular response, could add greatly to our understanding of differences between strains of this important human pathogen. In that sense, acute and chronic infections with distinct T. cruzi strains (Y, Berenice-78 and ABC) in Beagle dogs were studied through a longitudinal evaluation of immunoglobulin G (IgG), IgG1 and IgG2 isotypes (by ELISA and flow cytometry (FC)), as well as measurements of peripheral blood mononuclear cell (PBMC) proliferation over a 100-week period, and their correlation with cardiomegaly. Our results show that infected animals presenting cardiomegaly showed lower or absent levels of IgG1 during the chronic phase of the infection, when compared to those that did not show an increase in heart weight. In that manner, our results suggest that IgG1 could be used as a marker for cardiac pathogenicity in Chagas disease.

Ecto-nucleotidase activities of promastigotes from leishmania (Viannia) braziliensis relates to parasite infectivity and disease clinical outcome.

Background Leishmania (Viannia) braziliensis has been associated with a broad range of clinical manifestations ranging from a simple cutaneous ulcer to destructive mucosal lesions. Factors leading to this diversity of clinical presentations are not clear, but parasite factors have lately been recognized as important in determining disease progression. Given the fact that the activity of ecto-nucleotidases correlates with parasitism and the development of infection, we evaluated the activity of these enzymes in promastigotes from 23 L. braziliensis isolates as a possible parasite-related factor that could influence the clinical outcome of the disease. Methodology/Principal Findings Our results show that the isolates differ in their ability to hydrolyze adenine nucleotides. Furthermore, we observed a positive correlation between the time for peak of lesion development in C57BL/6J mice and enzymatic activity and clinical manifestation of the isolate. In addition, we found that L. (V.) braziliensis isolates obtained from mucosal lesions hydrolyze higher amounts of adenine nucleotides than isolates obtained from skin lesions. One isolate with high (PPS6m) and another with low (SSF) ecto-nucleotidase activity were chosen for further studies. Mice inoculated with PPS6m show delayed lesion development and present larger parasite loads than animals inoculated with the SSF isolate. In addition, PPS6m modulates the host immune response by inhibiting dendritic cell activation and NO production by activated J774 macrophages. Finally, we observed that the amastigote forms from PPS6m and SSF isolates present low enzymatic activity that does not interfere with NO production and parasite survival in macrophages. Conclusions/Significance Our data suggest that ecto-nucleotidases present on the promastigote forms of the parasite may interfere with the establishment of the immune response with consequent impaired ability to control parasite dissemination and this may be an important factor in determining the clinical outcome of leishmaniasis.

Leishmania amazonensis impairs DC function by inhibiting CD40 expression via A2B adenosine receptor activation.

Dendritic cells (DCs) play an essential role in the modulation of immune responses and several studies have evaluated the interactions between Leishmania parasites and DCs. While extracellular ATP exhibits proinflammatory properties, adenosine is an important anti‐inflammatory mediator. Here we investigated the effects of Leishmania infection on DC responses and the participation of purinergic signalling in this process. Bone marrow‐derived dendritic cells (BMDCs) from C57BL/6J mice infected with Leishmania amazonensis, Leishmania braziliensis or Leishmania major metacyclic promastigotes showed decreased major histocompatibility complex (MHC) class II and CD86 expression and increased ectonucleotidase expression as compared with uninfected cells. In addition, L. amazonensis‐infected DCs, which had lower CD40 expression, exhibited a decreased ability to induce T‐cell proliferation. The presence of MRS1754, a highly selective A2B adenosine receptor antagonist at the time of infection increased MHC class II, CD86 and CD40 expression in L. amazonensis‐infected DCs and restored the ability of the infected DCs to induce T‐cell proliferation. Similar results were obtained through the inhibition of extracellular ATP hydrolysis using suramin. In conclusion, we propose that A2B receptor activation may be used by L. amazonensis to inhibit DC function and evade the immune response.

Histopathology of Leishmania major infection: revisiting L. major histopathology in the ear dermis infection model

We describe the relationship between lesion outcome and histopathological hallmarks in susceptible (BALB/c) and resistant (C57BL/6 and IL-4-deficient BALB/c) mouse strains over the course of a 12-week-infection with Leishmania major in the ear. The infiltration of mononuclear cells and polymorphonuclear cells occurred within 6 h and mononuclear cells predominated one week post-infection. Permissive intracellular growth of the pathogen was associated with non-healing lesions. In contrast, tissue damage and clearance of the parasite was observed in healing lesions and was associated with inducible nitric oxide synthase expression. The identification of the structural components of tissue reaction to the parasite in this study furthers our understanding of subjacent immune effector mechanisms.

Leishmania metacyclogenesis is promoted in the absence of purines.

Leishmania parasites, the causative agent of leishmaniasis, are transmitted through the bite of an infected sand fly. Leishmania parasites present two basic forms known as promastigote and amastigote which, respectively, parasitizes the vector and the mammalian hosts. Infection of the vertebrate host is dependent on the development, in the vector, of metacyclic promastigotes, however, little is known about the factors that trigger metacyclogenesis in Leishmania parasites. It has been generally stated that “stressful conditions” will lead to development of metacyclic forms, and with the exception of a few studies no detailed analysis of the molecular nature of the stress factor has been performed. Here we show that presence/absence of nucleosides, especially adenosine, controls metacyclogenesis both in vitro and in vivo. We found that addition of an adenosine-receptor antagonist to in vitro cultures of Leishmania amazonensis significantly increases metacyclogenesis, an effect that can be reversed by the presence of specific purine nucleosides or nucleobases. Furthermore, our results show that proliferation and metacyclogenesis are independently regulated and that addition of adenosine to culture medium is sufficient to recover proliferative characteristics for purified metacyclic promastigotes. More importantly, we show that metacyclogenesis was inhibited in sand flies infected with Leishmania infantum chagasi that were fed a mixture of sucrose and adenosine. Our results fill a gap in the life cycle of Leishmania parasites by demonstrating how metacyclogenesis, a key point in the propagation of the parasite to the mammalian host, can be controlled by the presence of specific purines.