Hypoxia-Inducible Factor-1 Alpha Stabilization in Human Macrophages during Leishmania major Infection Is Impaired by Parasite Virulence.

Hypoxia-inducible factor-1 alpha (HIF-1α) is one of the master regulators of immune and metabolic cellular functions. HIF-1α, a transcriptional factor whose activity is closely related to oxygen levels, is a target for understanding infectious disease control. Several studies have demonstrated that HIF-1α plays an important role during the infectious process, while its role in relation to parasite virulence has not been addressed. In this work, we studied the expression levels of HIF-1α and related angiogenic vascular endothelial growth factor A (VEGF-A) in human macrophages infected with promastigotes of hypo- or hyper-virulent Leishmania major human isolates. L. major parasites readily subverted host macrophage functions for their survival and induced local oxygen consumption at the site of infection. In contrast to hypo-virulent parasites that induce high HIF-1α expression levels, hyper-virulent L. major reduced HIF-1α expression in macrophages under normoxic or hypoxic conditions, and consequently impeded the expression of VEGF-A mRNA. HIF-1α may play a key role during control of disease chronicity, severity, or outcome.

Treatment with synthetic lipophilic tyrosyl ester controls Leishmania major infection by reducing parasite load in BALB/c mice.

Synthesized lipophilic tyrosyl ester derivatives with increasing lipophilicity were effective against Leishmania (L.) major and Leishmania infantum species in vitro. These findings prompted us to test in vivo leishmanicidal properties of these molecules and their potential effect on the modulation of immune responses. The experimental BALB/c model of cutaneous leishmaniasis was used in this study. Mice were infected with L. major parasites and treated with three in vitro active tyrosyl esters derivatives. Among these tested tyrosylcaprate (TyC) compounds, only TyC10 exhibited an in vivo anti-leishmanial activity, when injected sub-cutaneously (s.c.). TyC10 treatment of L. major-infected BALB/c mice resulted in a decrease of lesion development and parasite load. TyC10 s.c. treatment of non-infected mice induced an imbalance in interferon γ/interleukin 4 (IFN-γ/IL-4) ratio cytokines towards a Th1 response. Our results indicate that TyC10 s.c. treatment improves lesions' healing and parasite clearance and may act on the cytokine balance towards a Th1 protective response by decreasing IL-4 and increasing IFN-γ transcripts. TyC10 is worthy of further investigation to uncover its mechanism of action that could lead to consider this molecule as a potential drug candidate.

Intra-Specific Diversity of Leishmania major Isolates: A Key Determinant of Tunisian Zoonotic Cutaneous Leishmaniasis Clinical Polymorphism.

The clinical expression of zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania (L.) major parasites has a broad spectrum ranging from asymptomatic infection to self-limited cutaneous sores or severe disease. In concert with the host immune responses, the vector variability and the number of bites, genetic variation between L. major isolates might impact on the clinical output of the disease. We investigated herein the intra-specific variability of L. major field isolates independently of host or vector factors and then tried to correlate parasite variability to ZCL severity in corresponding patients. Several assays were applied, i.e., in vivo pathogenicity of promastigotes in a BALB/c mice model, resistance/sensibility to complement lysis, in vitro growth kinetics, and expression of different lectins on the promastigote surface. Combining all these parameters allowed us to conclude that the resistance to complement lysis and PNA/Jacalin lectins binding to parasite surfaces are important markers of parasite virulence. These factors correlate significantly with clinic polymorphism of ZCL and modestly with genetic micro-heterogeneity, a characteristic we previously revealed with a MLMT profile.

Healed Lesions of Human Cutaneous Leishmaniasis Caused By Leishmania major Do Not Shelter Persistent Residual Parasites.

In human cutaneous leishmaniasis (HCL) caused by Leishmania (L.) major, the cutaneous lesions heal spontaneously and induce a Th1-type immunity that confers solid protection against reinfection. The same holds true for the experimental leishmaniasis induced by L. major in C57BL/6 mice where residual parasites persist after spontaneous clinical cure and induce sustainable memory immune responses and resistance to reinfection. Whether residual parasites also persist in scars of cured HCL caused by L. major is still unknown. Cutaneous scars from 53 volunteers with healed HCL caused by L. major were biopsied and the tissue sample homogenates were analyzed for residual parasites by four methods: i) microscope detection of amastigotes, ii) parasite culture by inoculation on biphasic medium, iii) inoculation of tissue exctracts to the footpad of BALB/c mice, an inbred strain highly susceptible to L. major, and iv) amplification of parasite kDNA by a highly sensitive real-time PCR (RT-PCR). Our results show that the scars of healed lesions of HCL caused by L. major do not contain detectable residual parasites, suggesting that this form likely induces a sterile cure at least within the scars. This feature contrasts with other Leishmania species causing chronic, diffuse, or recidivating forms of leishmaniasis where parasites do persist in healed lesions. The possibility that alternative mechanisms to parasite persistence are needed to boost and maintain long-term immunity to L. major, should be taken into consideration in vaccine development against L. major infection.

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.

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.

Genotype profile of Leishmania major strains isolated from tunisian rodent reservoir hosts revealed by multilocus microsatellite typing.

Zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania (L.) major parasites represents a major health problem with a large spectrum of clinical manifestations. Psammomys (P.) obesus and Meriones (M.) shawi represent the most important host reservoirs of these parasites in Tunisia. We already reported that infection prevalence is different between these two rodent species. We aimed in this work to evaluate the importance of genetic diversity in L. major parasites isolated from different proven and suspected reservoirs for ZCL. Using the multilocus microsatellites typing (MLMT), we analyzed the genetic diversity among strains isolated from (i) P. obesus (n = 31), (ii) M. shawi (n = 8) and (iii) Mustela nivalis (n = 1), captured in Sidi Bouzid, an endemic region for ZCL located in the Center of Tunisia. Studied strains present a new homogeneous genotype profile so far as all tested markers and showed no polymorphism regardless of the parasite host-reservoir origin. This lack of genetic diversity among these L. major isolates is the first genetic information on strains isolated from Leishmania reservoirs hosts in Tunisia. This result indicates that rodent hosts are unlikely to exert a selective pressure on parasites and stresses on the similarity of geographic and ecological features in this study area. Overall, these results increase our knowledge among rodent reservoir hosts and L. major parasites interaction.

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.