Fernanda O. Novais

Neutrophils and Macrophages Cooperate in Host Resistance against Leishmania braziliensis Infection

Neutrophils play an active role in the control of infections caused by intracellular pathogens such as Leishmania. In the present study, we investigated the effect of neutrophil depletion at the time of Leishmania braziliensis infection of BALB/c mice and how neutrophils interact with the infected macrophage to promote parasite elimination. The in vivo depletion of neutrophils led to a significant increase in parasite load and enhanced the Th1-Th2 immune response in this experimental model of infection. BALB/c mice coinoculated with both parasites and live neutrophils displayed lower parasite burdens at the site of infection and in the draining lymph nodes. In vitro, we observed that live neutrophils significantly reduced the parasite load in L. braziliensis-infected murine macrophages, an effect not observed with Leishmania major. L. braziliensis elimination was dependent on the interaction between neutrophils and macrophages and was associated with TNF-α as well as superoxide production. Furthermore, cooperation between neutrophils and macrophages toward parasite elimination was also observed in experiments performed with L. braziliensis-infected human cells and, importantly, with two other New World Leishmania species. These results indicate that neutrophils play an important and previously unappreciated role in L. braziliensis infection, favoring the induction of a protective immune response.

Toward a Novel Experimental Model of Infection To Study American Cutaneous Leishmaniasis Caused by Leishmania braziliensis

ABSTRACT Leishmania spp. cause a broad spectrum of diseases collectively known as leishmaniasis. Leishmania braziliensis is the main etiological agent of American cutaneous leishmaniasis (ACL) and mucocutaneous leishmaniasis. In the present study, we have developed an experimental model of infection that closely resembles ACL caused by L. braziliensis. In order to do so, BALB/c mice were infected in the ear dermis with 105 parasites and distinct aspects of the infection were evaluated. Following inoculation, parasite expansion in the ear dermis was accompanied by the development of an ulcerated dermal lesion which healed spontaneously, as seen by the presence of a scar. Histological analysis of infected ears showed the presence of a mixed inflammatory infiltrate consisting of both mononuclear and polymorphonuclear cells. In draining lymph nodes, parasite replication was detected throughout the infection. In vitro restimulation of draining lymph node cells followed by intracellular staining showed an up-regulation in the production of gamma interferon (IFN-γ) and in the frequency of IFN-γ-secreting CD4+ and CD8+ T cells. Reverse transcription-PCR of ears and draining lymph node cells showed the expression of CC chemokines. The dermal model of infection with L. braziliensis herein is able to reproduce aspects of the natural infection, such as the presence of an ulcerated lesion, parasite dissemination to lymphoid areas, and the development of a Th1-type immune response. These results indicate that this model shall be useful to address questions related to the concomitant immunity to reinfection and parasite persistence leading to mucocutaneous leishmaniasis.

Cutaneous leishmaniasis: immune responses in protection and pathogenesis

Cutaneous leishmaniasis is a major public health problem and causes a range of diseases from self-healing infections to chronic disfiguring disease. Currently, there is no vaccine for leishmaniasis, and drug therapy is often ineffective. Since the discovery of CD4+ T helper 1 (TH1) cells and TH2 cells 30 years ago, studies of cutaneous leishmaniasis in mice have answered basic immunological questions concerning the development and maintenance of CD4+ T cell subsets. However, new strategies for controlling the human disease have not been forthcoming. Nevertheless, advances in our knowledge of the cells that participate in protection against Leishmania infection and the cells that mediate increased pathology have highlighted new approaches for vaccine development and immunotherapy. In this Review, we discuss the early events associated with infection, the CD4+ T cells that mediate protective immunity and the pathological role that CD8+ T cells can have in cutaneous leishmaniasis.

Enhanced Leishmania braziliensis Infection Following Pre-Exposure to Sandfly Saliva

Background Sand fly saliva has an array of pharmacological and immunomodulatory components, and immunity to saliva protects against Leishmania infection. In the present study, we have studied the immune response against Lutzomyia intermedia saliva, the main vector of Leishmania braziliensis in Brazil, and the effects of saliva pre-exposure on L. braziliensis infection employing an intradermal experimental model. Methodology/principal findings BALB/c mice immunized with L. intermedia salivary gland sonicate (SGS) developed a saliva-specific antibody response and a cellular immune response with presence of both IFN-γ and IL-4. The inflammatory infiltrate observed in SGS-immunized mice was comprised of numerous polymorphonuclear and few mononuclear cells. Mice challenged with live L. braziliensis in the presence of saliva were not protected although lesion development was delayed. The inoculation site and draining lymph node showed continuous parasite replication and low IFN-γ to IL-4 ratio, indicating that pre-exposure to L. intermedia saliva leads to modulation of the immune response. Furthermore, in an endemic area of cutaneous leishmaniasis, patients with active lesions displayed higher levels of anti-L. intermedia saliva antibodies when compared to individuals with a positive skin test result for Leishmania. Conclusion These results show that pre-exposure to sand fly saliva plays an important role in the outcome of cutaneous leishmaniasis, in both mice and humans. They emphasize possible hurdles in the development of vaccines based on sand fly saliva and the need to identify and select the individual salivary candidates instead of using whole salivary mixture that may favor a non-protective response.

Cytotoxic T cells mediate pathology and metastasis in cutaneous leishmaniasis.

Disease progression in response to infection can be strongly influenced by both pathogen burden and infection-induced immunopathology. While current therapeutics focus on augmenting protective immune responses, identifying therapeutics that reduce infection-induced immunopathology are clearly warranted. Despite the apparent protective role for murine CD8+ T cells following infection with the intracellular parasite Leishmania, CD8+ T cells have been paradoxically linked to immunopathological responses in human cutaneous leishmaniasis. Transcriptome analysis of lesions from Leishmania braziliensis patients revealed that genes associated with the cytolytic pathway are highly expressed and CD8+ T cells from lesions exhibited a cytolytic phenotype. To determine if CD8+ T cells play a causal role in disease, we turned to a murine model. These studies revealed that disease progression and metastasis in L. braziliensis infected mice was independent of parasite burden and was instead directly associated with the presence of CD8+ T cells. In mice with severe pathology, we visualized CD8+ T cell degranulation and lysis of L. braziliensis infected cells. Finally, in contrast to wild-type CD8+ T cells, perforin-deficient cells failed to induce disease. Thus, we show for the first time that cytolytic CD8+ T cells mediate immunopathology and drive the development of metastatic lesions in cutaneous leishmaniasis.

Vaccination with the Leishmania major ribosomal proteins plus CpG oligodeoxynucleotides induces protection against experimental cutaneous leishmaniasis in mice

In the present work we analyze the antigenicity of Leishmania major ribosomal proteins (LRP) in infected BALB/c mice. We show that BALB/c mice vaccinated with LRP in the presence of CpG oligodeoxynucleotides (CpG-ODN) were protected against the development of dermal pathology and showed a reduction in the parasite load after challenge with L. major. This protection was associated with the induction of an IL-12 dependent specific-IFN-gamma response mediated mainly by CD4(+) T cell, albeit a minor contribution of CD8(+) T cells cannot be ruled out. Induction of Th1 responses against LRP also resulted in a reversion of the Th2 responses associated with susceptibility. A marked reduction of IgG1 antibody titer against parasite antigens besides an impaired IL-4 and IL-10 cytokine production by parasite specific T cells was observed. In addition, we show that the administration of the LRP plus CpG-ODN preparation also conferred protection in the naturally resistant C57BL/6 mice. In this strain protection was associated with a LRP specific IFN-gamma production in lymph nodes draining the challenge site. We believe that these evolutionary conserved proteins, combined with adjuvants that favor Th1 responses, may be relevant components of a pan-Leishmania vaccine.

Human Classical Monocytes Control the Intracellular Stage of Leishmania braziliensis by Reactive Oxygen Species

Leishmania braziliensis are intracellular parasites that cause unique clinical forms of cutaneous leishmaniasis. Previous studies with other leishmania species demonstrated that reactive oxygen species (ROS) control promastigotes, the infective stage of the parasite, but not the amastigote form that exists in the mammalian host. Here we show that ROS inhibits growth of L. braziliensis amastigotes in resting monocytes, and that classical monocytes are primarily responsible for this control. ROS, but not nitric oxide, also contributed to killing of L. braziliensis by IFN-γ activated monocytes. Furthermore, by gene expression profiling of human lesions we found greater expression of genes associated with ROS, but not nitric oxide, compared to normal skin. This study shows that ROS are important for control of L. braziliensis both at the initial stages of infection, as well as at later time points, and highlights that monocyte subsets may play different roles during leishmaniasis.

Immunity to Lutzomyia intermedia saliva modulates the inflammatory environment induced by Leishmania braziliensis.

Background During blood feeding, sand flies inject Leishmania parasites in the presence of saliva. The types and functions of cells present at the first host-parasite contact are critical to the outcome on infection and sand fly saliva has been shown to play an important role in this setting. Herein, we investigated the in vivo chemotactic effects of Lutzomyia intermedia saliva, the vector of Leishmania braziliensis, combined or not with the parasite. Methods and Findings We tested the initial response induced by Lutzomyia intermedia salivary gland sonicate (SGS) in BALB/c mice employing the air pouch model of inflammation. L. intermedia SGS induced a rapid influx of macrophages and neutrophils. In mice that were pre-sensitized with L. intermedia saliva, injection of SGS was associated with increased neutrophil recruitment and a significant up-regulation of CXCL1, CCL2, CCL4 and TNF-α expression. Surprisingly, in mice that were pre-exposed to SGS, a combination of SGS and L. braziliensis induced a significant migration of neutrophils and an important modulation in cytokine and chemokine expression as shown by decreased CXCL10 expression and increased IL-10 expression. Conclusion These results confirm that sand fly saliva modulates the initial host response. More importantly, pre-exposure to L. intermedia saliva significantly modifies the hosts response to L. braziliensis, in terms of cellular recruitment and expression of cytokines and chemokines. This particular immune modulation may, in turn, favor parasite multiplication.

CD8+ T cells in cutaneous leishmaniasis: The good, the bad and the ugly

CD8+ T lymphocytes are components of the adaptive immune response and play an important role in protection against many viral and bacterial infections. However, their role in parasitic infections is less well understood. In leishmaniasis, a disease caused by intracellular protozoan parasites of the genus Leishmania, CD8+ T cells have been shown to be protective. However, increasing evidence indicates that CD8+ T cells may also exacerbate disease. In this review, we will describe the situations where CD8+ T cells are either good or bad for the outcome of the infection and attempt to reconcile the dual role played by CD8+ T cells in cutaneous leishmaniasis.

DETC Induces Leishmania Parasite Killing in Human In Vitro and Murine In Vivo Models: A Promising Therapeutic Alternative in Leishmaniasis

Background Chemotherapy remains the primary tool for treatment and control of human leishmaniasis. However, currently available drugs present serious problems regarding side-effects, variable efficacy, and cost. Affordable and less toxic drugs are urgently needed for leishmaniasis. Methodology/Principal Findings We demonstrate, by microscopy and viability assays, that superoxide dismutase inhibitor diethyldithiocarbamate (DETC) dose-dependently induces parasite killing (p<0.001) and is able to “sterilize” Leishmania amazonensis infection at 2 mM in human macrophages in vitro. We also show that DETC-induced superoxide production (p<0.001) and parasite destruction (p<0.05) were reverted by the addition of the antioxidant N-acetylcysteine, indicating that DETC-induced killing occurs through oxidative damage. Furthermore, ultrastructural analysis by electron microscopy demonstrates a rapid and highly selective destruction of amastigotes in the phagosome upon DETC treatment, without any apparent damage to the host cell, including its mitochondria. In addition, DETC significantly induced parasite killing in Leishmania promastigotes in axenic culture. In murine macrophages infected with Leishmania braziliensis, DETC significantly induced in vitro superoxide production (p = 0.0049) and parasite killing (p = 0.0043). In vivo treatment with DETC in BALB/C mice infected with Leishmania braziliensis caused a significant decrease in lesion size (p<0.0001), paralleled by a 100-fold decrease (p = 0.0087) in parasite burden. Conclusions/Significance Due to its strong leishmanicidal effect in human macrophages in vitro, its in vivo effectiveness in a murine model, and its previously demonstrated in vivo safety profile in HIV treatment, DETC treatment might be considered as a valuable therapeutic option in human leishmaniasis, including HIV/Leishmania co-infection.