Evaluation of the host cell response following Leishmania infantum infection

The Leishmaniases are a group of parasitic diseases caused by protozoa belonging to the Leishmania genus and transmitted by the bite of infected female phlebotomine sandflies, affecting both humans and other vertebrates, especially dogs. Mammalian leishmaniasis shows a worldwide distribution. Leishmania is endemic in at least 98 countries, with the highest number of cases in developing nations. There are three forms of the disease: cutaneous leishmaniasis (CL) is the most common form of leishmaniasis, mucocutaneous leishmaniasis (MCL) and visceral leishmaniasis (VL). The main species in Italy is Leishmania infantum responsible for both cutaneous and visceral form of the disease. The aim of this project was to investigate molecular response in macrophages infected with L. infantum, to better understand and characterize the interaction between Leishmania infantum and the host cells. The reference strain of L. infantum MHOM/TN/80/IPT1 was provided by the OIE Reference Laboratory National Reference Center for Leishmaniasis in Palermo. The in vitro infection model has been developed from a human monocytic cell lines U937 and THP-1 after differentiation with phorbol myristic acid and from murine primary macrophages. Cells were infected with a parasite-to-cell ratio of 10:1 showing about 30% and 60% of infected cells after 8 and 24 h, respectively, evaluated by cell counting and a quantitative PCR assay targeting the Leishmania kinetoplastid DNA. Using this infection protocol it was possible to analyze the molecular response in infected macrophages, revealing that Leishmania induced Akt phosphorylation and inhibited the caspase-3 cleavage, promoting host cell survival. Moreover, gene expression analysis of ER stress-related genes showed a mild but significant induction of the UPR markers. On the contrary, results showed that Leishmania inhibited the induction of UPR markers following treatment with a strong inducer of ER stress (tunicamycin and DTT). Additionally, to explore the gene expression modulation in host cells following L. infantum infection, experiments of RNA sequencing targeting total RNA of non-infected and infected U937 and Leishmania RNA (Splice Leader protocol) were performed at the Center for Infectious Disease Research in Seattle. Another mechanism triggered in response to ER stress involves the induction of microRNA by UPR. Among different miRNA found to be related to the UPR, we investigate the induction of mir-346 following the increase of the spliced XBP1, revealing a significant upregulation of mir-346 in both U937 and THP-1 derived macrophages infected with L. infantum reference strain and two canine clinical isolates. In conclusion, we suggested that L. infantum could promote host cells survival by inducing a mild ER stress response (subtle but significant increase in ER stress expression markers, delay/attenuation of the effects of ER stress inducers). Moreover, a significant up-regulation of the miR-346 has been detected, in both U937 and THP1- derived macrophages infected with L. infantum. However, to develop more effective/new therapeutic approaches targeting these molecular pathways, we need to deeply understand the intricated interactions between host molecular pathways and Leishmania parasites