Caratterizzazione in-vitro e in-vivo della risposta immunitaria mediata da macrofagi in infezioni da Babesia divergens

Human and bovine Babesiosis is a zoonosis caused by parasites of the genus Babesia spp. The infection is mainly transmitted by ticks of the genus Ixodes, and by other routes such as blood transfusion from asymptomatic donors. The cycle of asexual reproduction of B.divergens takes place inside the erythrocytes and outside, where the stage of free merozoite is capable of infecting new erythrocytes. Despite its interest as a pathogen for humans, there is very little current information about the process of recognition of the pathogen by the innate immune system and the activation of the consequent antiparasitic response. In order to achieve a better understanding of the immunity response necessary for the control of babesiosis, the role of macrophages in the activation of the innate response seems to be of prime importance. The purpose of the present work was a characterization of the immune response against the parasite mediated from macrophages. For this reason, we performed a series of in-vitro experiments in which human macrophages isolated from peripheral blood, were differentiated in GM-CSF and M-CSF subpopulation, and infected with free merozoite (FM). In order to evaluate the immune response activated from macrophages against the parasite, the level of proinflammatory cytokines was evaluated by Bioassay to determinate the IFN antiviral activity, ELISA and qPCR analysis. An animal model was established to identify the course of the infection in mice; thirteen C57BL/6J mice were infected at day 0 with B.divergens in vitro cultures at 30% of parasitaemia. The infected mice were evaluated for 21 days, the evolution of the animals after the inoculation was compared to control animals by haematological analysis and histological assays of spleen, liver and kidneys. The detection of the parasite was performed by Giemsa stain and qPCR. The results of in-vitro experiment demonstrate the detection of B.divergens in the form of FM by macrophages. Therefore,macrophages can contribute to the induction of an antiparasitic response mediated by release of pro-inflammatory cytokines. 24h post infection was detected a predominant release of IL-6 by FM GM-CSF subpopulation with a concentration of 21.499,94±4.102 [mean±SD] pg/ml (Pvalue<0,01), and a higher release of TNF&#945; with a concentration of 5.367,92±1.025 [mean±SD] pg/ml by FR GM-CSF (Pvalue <0.05).The expression of IFN type I was predominant at 6h post infection in FM GM-CSF and FM M-CSF correspond to the control. The gene induction of IL-12 was principal mediated by FM GM-CSF with a first pick at 9h post infection and a second one at 52h post infection respect to the control. In vivo experiment showed that mice developed an asymptomatic infection; none of the infected animal showed clinical signs of disease during the infection thanks to the correct functioning of the immune system and what was thought to be a key role played by spleen macrophages in response to infection. In order to support this thesis, in the future, the research team proposes to carry out a second in vivo experimentation in which a depletion of the spleen macrophages will be performed on C57BL/6J mice using clodronate liposomes at the different evolution steps pathology.