3D-Hepatocyte Culture Applied to Parasitology: Immune Activation of Canine Hepatic Spheroids Exposed to Leishmania infantum.

The application of innovative three-dimensional (3D) spheroids cell culture strategy to Parasitology offers the opportunity to closely explore host-parasite interactions. Here we present a first report on the application of 3D hepatic spheroids to unravel the immune response of canine hepatocytes exposed to Leishmania infantum. The liver, usually considered a major metabolic organ, also performs several important immunological functions and constitutes a target organ for L. infantum infection, the etiological agent of canine leishmaniasis (CanL), and a parasitic disease of major veterinary and public health concern. 3D hepatic spheroids were able to sense and immunologically react to L. infantum parasites, generating an innate immune response by increasing nitric oxide (NO) production and enhancing toll-like receptor (TLR) 2 and interleukin-10 gene expression. The immune response orchestrated by canine hepatocytes also lead to the impairment of several cytochrome P450 (CYP450) with possible implications for liver natural xenobiotic metabolization capacity. The application of meglumine antimoniate (MgA) increased the inflammatory response of 3D hepatic spheroids by inducing the expression of Nucleotide oligomerization domain (NOD) -like receptors 1 and NOD2 and TLR2, TLR4, and TLR9 and enhancing gene expression of tumour necrosis factor α. It is therefore suggested that hepatocytes are key effector cells and can activate and orchestrate the immune response to L. infantum parasites.

Central Asian Rodents as Model Animals for Leishmania major and Leishmania donovani Research.

The clinical manifestation of leishmaniases depends on parasite species, host genetic background, and immune response. Manifestations of human leishmaniases are highly variable, ranging from self-healing skin lesions to fatal visceral disease. The scope of standard model hosts is insufficient to mimic well the wide disease spectrum, which compels the introduction of new model animals for leishmaniasis research. In this article, we study the susceptibility of three Asian rodent species (Cricetulus griseus, Lagurus lagurus, and Phodopus sungorus) to Leishmania major and L. donovani. The external manifestation of the disease, distribution, as well as load of parasites and infectiousness to natural sand fly vectors, were compared with standard models, BALB/c mice and Mesocricetus auratus. No significant differences were found in disease outcomes in animals inoculated with sand fly- or culture-derived parasites. All Asian rodent species were highly susceptible to L. major. Phodopus sungorus showed the non-healing phenotype with the progressive growth of ulcerative lesions and massive parasite loads. Lagurus lagurus and C. griseus represented the healing phenotype, the latter with high infectiousness to vectors, mimicking best the character of natural reservoir hosts. Both, L. lagurus and C. griseus were also highly susceptible to L. donovani, having wider parasite distribution and higher parasite loads and infectiousness than standard model animals.

Meglumine Antimoniate and Miltefosine Combined With Allopurinol Sustain Pro-inflammatory Immune Environments During Canine Leishmaniosis Treatment.

Canine leishmaniosis (CanL) caused by Leishmania infantum is a zoonotic disease of global concern. Antileishmanial drug therapies commonly used to treat sick dogs improve their clinical condition, although when discontinued relapses can occur. Thus, the current study aims to evaluate the effect of CanL treatments in peripheral blood, lymph node, and bone marrow cytokine profile associated with clinical recovery. Two groups of six dogs diagnosed with CanL were treated with miltefosine combined with allopurinol and meglumine antimoniate combined with allopurinol (MT+A and MG+A), respectively. At diagnosis and after treatment, during a 3-month follow-up, clinical signs, hematological and biochemical parameters, urinalysis results and antileishmanial antibody titers were registered. Furthermore, peripheral blood, popliteal lymph node, and bone marrow samples were collected to assess the gene expression of IL-2, IL-4, IL-5, IL-10, IL-12, TNF-α, TGF-ß, and IFN-γ by qPCR. In parallel, were also evaluated samples obtained from five healthy dogs. Both treatment protocols promoted the remission of clinical signs as well as normalization of hematological and biochemical parameters and urinalysis values. Antileishmanial antibodies returned to non-significant titers in all dogs. Sick dogs showed a generalized upregulation of IFN-γ and downregulation of IL-2, IL-4, and TGF-ß, while gene expression of IL-12, TNF-α, IL-5, and IL-10 varied between groups and according to evaluated tissue. A trend to the normalization of cytokine gene expression was induced by both miltefosine and meglumine antimoniate combined therapies. However, IFN-γ gene expression was still up-regulated in the three evaluated tissues. Furthermore, the effect of treatment in the gene expression of cytokines that were not significantly changed by infection, indicates that miltefosine and meglumine antimoniate combined therapy directly affects cytokine generation. Both combined therapies are effective in CanL treatment, leading to sustained pro-inflammatory immune environments that can compromise parasite survival and favor dogs' clinical cure. In the current study, anti-inflammatory and regulatory cytokines do not seem to play a prominent role in CanL or during clinical recovery.

Intracellular and extracellular effector activity of mouse neutrophils in response to cutaneous and visceral Leishmania parasites.

Neutrophils are short-lived phagocytic cells equipped with several receptors for pathogen recognition and phagocytosis and have intracellular and extracellular effector mechanisms that can inactivate pathogens. Leishmaniases are diseases caused by different species of Leishmania that mainly afflicts poorer populations of tropical and subtropical regions and immunocompromised individuals. Thus, the present study aims to investigate the effector response of murine neutrophils to species of Leishmania causing American cutaneous leishmaniasis and zoonotic visceral leishmaniasis by evaluating pattern recognition receptors (PRR) and intracellular and extracellular effector microbicide activity. When exposed to Leishmania parasites, mouse neutrophils produced superoxide, released enzymes in the extracellular space and generated neutrophil extracellular traps, although PRR gene expression is negatively regulated. L. infantum, L. guyanensis, and L. shawi inhibited enzymatic activity, whereas L. amazonensis reduced the emission of extracellular structures. These findings indicate that although neutrophils trigger several microbicide mechanisms, Leishmania parasites can manipulate extracellular effector mechanisms. The present study also provides evidence that neutrophils can internalize parasites by coiling phagocytosis.

Canine neutrophils activate effector mechanisms in response to Leishmania infantum.

Canine leishmaniosis caused by L. infantum is a severe zoonotic disease. Although macrophages are the definitive host cells, neutrophils are the first cells to encounter the parasite soon after its inoculation in the dermis by the phlebotomine vector. To study the interaction of dog neutrophils and L. infantum promastigotes, blood neutrophils were isolated from healthy donors and the infection was established in vitro. In the majority of the dogs, L. infantum was efficiently phagocytized by neutrophils, and oxidative (superoxide production) and non-oxidative (neutrophil elastase exocytosis) intracellular effector mechanisms were activated, but the release of neutrophil extracellular traps was minimized. Furthermore, promastigotes and culture supernatants induced neutrophil migration, but the prior contact with Leishmania inhibits chemotaxis, which might contribute to neutrophil retention at the inoculation site. Neutrophil-parasite interaction resulted in a decrease in parasite viability, although some intracellular promastigotes survive and maintain their proliferative capacity. These findings indicate that dog neutrophils are competent effector cells able to control the initial L. infantum infection. However, some parasites evade intracellular effector mechanisms and can be transferred to the definitive host cell, the macrophage, contributing to the development of canine leishmaniosis.

Hemisynthetic trifluralin analogues incorporated in liposomes for the treatment of leishmanial infections.

Leishmaniasis, a vector-borne parasitic disease caused by Leishmania protozoa, is one of the most neglected tropical diseases in terms of drug discovery and development. Current treatment is based on a limited number of chemotherapeutic agents all of which present either/or resistance issues, severe toxicities and adverse reactions associated with extended treatment regimens, and high cost of therapy. Dinitroanilines are a new class of drugs with proven in vitro antileishmanial activity. In previous work a liposomal formulation of one dinitroaniline (TFL) was found to be active against Leishmania parasites in a murine model of visceral leishmaniasis (VL) and in the treatment of experimental canine leishmaniasis. In this study we have investigated the use of dinitroaniline analogues (TFL-A) associated to liposomes, as means to further improve TFL antileishmanial activity. The potential of the liposomal formulations was assessed in vitro against Leishmania infantum promastigotes and intracellular amastigotes and in vivo in a murine model of zoonotic VL. Free and liposomal TFL-A were active in vitro against Leishmania parasites, and they also exhibited reduced cytotoxicity and haemolytic activity. Treatment of infected mice with liposomal TFL-A reduced the amastigote loads in the spleen up to 97%, compared with the loads for untreated controls. These findings illustrate that chemical synthesis of new molecules associated with the use of Nano Drug Delivery Systems that naturally target the diseased organs could be a promising strategy for effective management of VL.