Rabiaa M. Sghaier

MicroRNA Expression Profile in Human Macrophages in Response to Leishmania major Infection

Background Leishmania (L.) are intracellular protozoan parasites able to survive and replicate in the hostile phagolysosomal environment of infected macrophages. They cause leishmaniasis, a heterogeneous group of worldwide-distributed affections, representing a paradigm of neglected diseases that are mainly embedded in impoverished populations. To establish successful infection and ensure their own survival, Leishmania have developed sophisticated strategies to subvert the host macrophage responses. Despite a wealth of gained crucial information, these strategies still remain poorly understood. MicroRNAs (miRNAs), an evolutionarily conserved class of endogenous 22-nucleotide non-coding RNAs, are described to participate in the regulation of almost every cellular process investigated so far. They regulate the expression of target genes both at the levels of mRNA stability and translation; changes in their expression have a profound effect on their target transcripts. Methodology/Principal Findings We report in this study a comprehensive analysis of miRNA expression profiles in L. major-infected human primary macrophages of three healthy donors assessed at different time-points post-infection (three to 24 h). We show that expression of 64 out of 365 analyzed miRNAs was consistently deregulated upon infection with the same trends in all donors. Among these, several are known to be induced by TLR-dependent responses. GO enrichment analysis of experimentally validated miRNA-targeted genes revealed that several pathways and molecular functions were disturbed upon parasite infection. Finally, following parasite infection, miR-210 abundance was enhanced in HIF-1α-dependent manner, though it did not contribute to inhibiting anti-apoptotic pathways through pro-apoptotic caspase-3 regulation. Conclusions/Significance Our data suggest that alteration in miRNA levels likely plays an important role in regulating macrophage functions following L. major infection. These results could contribute to better understanding of the dynamics of gene expression in host cells during leishmaniasis.

An in silico immunological approach for prediction of CD8+ T cell epitopes of Leishmania major proteins in susceptible BALB/c and resistant C57BL/6 murine models of infection

It is well established that MHC class II restricted-CD4 T cells are dominant during the development of immunity against Leishmania (L) in the C57BL/6-resistant mouse strain. However and in agreement with a number of previous observations indicating that specific CD8 T cells are primed during natural infection or vaccination in humans, a great deal of evidence obtained recently with the susceptible BALB/c murine model of infection by Leishmania major indicates that CD8 T cells participate in both pathogenesis and immunity to cutaneous leishmaniasis. Our goal herein was to identify in silico all parasitic peptides present in the whole L. major predicted proteome, using several public computational systems for the prediction of peptide binding to all MHC (histocompatibility complex-2) molecules in BALB/c and C57BL/6 mice (Syfpeithi, Rankpep, PRED(BALB/c) and Bimas). Peptides that were predicted to bind to different H2 molecules were then analysed for their homology with any of the murine proteins annotated so far, using the BLAST algorithm. Sets of selected peptides for each H2 molecule were defined by different prediction systems and compared to each other. Surprisingly, the results showed that a higher number of L. major peptides were predicted to bind H2 BALB/c molecules and very few or none to bind H2 C57BL/6 molecules. Our finding illustrates how a hybrid immuno-computational approach may be useful for biologists to target an in silico set of selected proteins to define potential candidate antigens for experimental vaccination with greater accuracy as well as a reduced number of T cell antigens.

Genetic micro-heterogeneity of Leishmania major in emerging foci of zoonotic cutaneous leishmaniasis in Tunisia.

Tunisia is endemic for zoonotic cutaneous leishmaniasis (ZCL), a parasitic disease caused by Leishmania (L.) major. ZCL displays a wide clinical polymorphism, with severe forms present more frequently in emerging foci where naive populations are dominant. In this study, we applied the multi-locus microsatellite typing (MLMT) using ten highly informative and discriminative markers to investigate the genetic structure of 35 Tunisian Leishmania (L.) major isolates collected from patients living in five different foci of Central Tunisia (two old and three emerging foci). Phylogenetic reconstructions based on genetic distances showed that nine of the ten tested loci were homogeneous in all isolates with homozygous alleles, whereas one locus (71AT) had a 58/64-bp bi-allelic profile with an allele linked to emerging foci. Promastigote-stage parasites with the 58-bp allele tend to be more resistant to in vitro complement lysis. These results, which stress the geographical dependence of the genetic micro-heterogeneity, may improve our understanding of the ZCL epidemiology and clinical outcome.

Comparative genomics of Tunisian Leishmania major isolates causing human cutaneous leishmaniasis with contrasting clinical severity.

Zoonotic cutaneous leishmaniasis caused by Leishmania (L.) major parasites affects urban and suburban areas in the center and south of Tunisia where the disease is endemo-epidemic. Several cases were reported in human patients for which infection due to L. major induced lesions with a broad range of severity. However, very little is known about the mechanisms underlying this diversity. Our hypothesis is that parasite genomic variability could, in addition to the host immunological background, contribute to the intra-species clinical variability observed in patients and explain the lesion size differences observed in the experimental model. Based on several epidemiological, in vivo and in vitro experiments, we focused on two clinical isolates showing contrasted severity in patients and BALB/c experimental mice model. We used DNA-seq as a high-throughput technology to facilitate the identification of genetic variants with discriminating potential between both isolates. Our results demonstrate that various levels of heterogeneity could be found between both L. major isolates in terms of chromosome or gene copy number variation (CNV), and that the intra-species divergence could surprisingly be related to single nucleotide polymorphisms (SNPs) and Insertion/Deletion (InDels) events. Interestingly, we particularly focused here on genes affected by both types of variants and correlated them with the observed gene CNV. Whether these differences are sufficient to explain the severity in patients is obviously still open to debate, but we do believe that additional layers of -omic information is needed to complement the genomic screen in order to draw a more complete map of severity determinants.

Comparative analysis of macrophage transcriptome of four mice strains after L. Major infection

Leishmaniasis is a parasitic disease caused by a protozoan parasite of the genus Leishmania (L.), using the macrophage as the main host cell where it can survive and replicate. The infection outcome depends on the balance between the ability of the host to activate the macrophage microbocidal mechanisms to kill the parasite, and the pathogen’s ability to suppress the host immune response and survive in the harsh environment of phagolysosomes. This ability to circumvent the host immune response may be the result of pressure exerted by the parasite on macrophage gene expression in a way that promotes their survival and multiplication. In this study, we compared the expression kinetics of 82 Bone-Marrow-Derived Macropahge (BMDM) gene transcripts, from susceptible (BALB/c and PWK) and resistant (C57BL/6 and MBT) mice strains, following in vitro infection with L. major parasites, the causative agent of human zoonotic cutaneous leishmaniasis. These genes belong to several functional families e.g., IFN? pathway, TLRs, chemokines, nitric oxid production pathway or involved in various metabolic pathways. Our results showed a clear contrast between the different profiles according to the infection stage (early vs. middle or late) in the four mice strains: regardless to targeted transcripts, the BMDM gene expression profile reflects, at early stages (3h post-infection), the genetic mice background and their susceptibility to Leishmania infection; whereas there is no such correlation at later stages. Indeed, and independently of the gene function, macrophages of susceptible BALB/c and PWK mice showed a general inhibited expression profile while the resistant C57BL/6 and MBT mice expression profile levels are higher compared to non-infected BMDM. Strikingly, this observation was not seen at later times of infection (24h and 72h post-infection) and the expression level depends on each transcript; a direct relationship between the mouse background and the level of expression of a given gene being less obvious. Whether, this early strain-specific effect of parasites on BMDM is due to a general phenomenon that may be encountered with any phagocytosed particle or specific to L. major will be discussed. This study provides a strong argument that susceptibility versus resistance to Leishmania infection of mice with different genetic backgrounds, would be partly due to the innate immunity. These results indicate that these differences in activation of innate immunity illustrate, at least in part, the differences in clinical expression of experimental leishmaniasis in the four mice strains.

Do scars caused by past history of Leishmania major Ainfection may harbor persistent parasites

Based on the knowledge that a cured infection protects the individual from re-infection, the development of a vaccine to prevent leishmaniasis has been a goal for nearly a century. Indeed, it is generally believed that after healing of leishmaniasis, sterile cure is never achieved and that few residual living parasites will remain sequestered within some host cells that offer them a safe shelter and hence maintain anti-parasite immune memory. This statement is mainly supported by data from experimental leishmaniasis in mice of susceptible or resistant phenotype, in which, live parasites could be recovered from lesions even after healing, and in which disease reactivation can be obtained by immune manipulation even after apparent complete cure. Whether maintenance of a long-term immune effector memory in humans will also require persistence of live parasites is presently unknown but is very important in the perspective of a vaccine development. Our aim herein was to address the issue of Leishmania major parasite persistence vs. sterile healing in zoonotic cutaneous leishmaniasis (ZCL) by analyzing biopsies of scars from healed volunteers. Skin-punch scars’ biopsies (n=59, range of scar age: 1– 5 years) have been obtained from volunteers (18–55 years old) living in two ZCL endemic foci, who had a confirmed past history, are clinically cured of ZCL and who gave their written consent. The whole protocol was approved by the local IRB. The specimens were taken under sterile conditions and local anaesthesia. Each specimen was divided into three parts: (i) the first sample was processed for quantitative real time PCR, (ii) the second was cultured in vitro in enriched medium and (iii) the third was inoculated into the footpad of susceptible BALB/c mice which were kept under observation for five months. For in vitro isolation and after microscopic observation for at least 8 weeks, all cultures were found negative. For in vivo isolation, biopsy-inoculated mice were killed five months later, and skin fragments, draining lymph nodes and spleen were inoculated into culture medium, observed carefully for at least 8 weeks before being also designated as negative. PCR results were found negative for the majority of biopsies. These results indicate that any potential persistent living parasites after cure are unlikely to be sequestered in ZCL scars. This issue is of importance in strategies for control of leishmaniasis and requires further discussion.

Evaluation of anti-proliferative and anti-inflammatory activities of Pelagia noctiluca venom in Lipopolysaccharide/Interferon-γ stimulated RAW264.7 macrophages

Components of Pelagia noctiluca (P. noctiluca) venom were evaluated for their anticancer and nitric Oxide (NO) inhibition activities. Three fractions, out of four, obtained by gel filtration on Sephadex G75 of P. noctiluca venom revealed an important selective anti-proliferative activity on several cell lines such as human bladder carcinoma (RT112), human glioblastoma (U87), and human myelogenous leukemia (K562) but not on mitogen-stimulated peripheral blood mononuclear cells. Interestingly, P. noctiluca components showed an important dose-dependent anti-inflammatory activity, through inhibition of NO production via transcriptional regulation of Inducible NO Synthase (iNOS), in IFN-γ/LPS stimulated RAW 264.7 macrophages. These data strongly suggest that P. noctiluca venom could be used as a natural inhibitor of cancer cell lines and a potent anti-inflammatory agent for the treatment of anti-inflammatory diseases.