Dynamic imaging reveals surface exposure of virulent Leishmania amastigotes during pyroptosis of infected macrophages.

Leishmania spp. are obligate intracellular parasites that infect phagocytes, notably macrophages. No information is available on how Leishmania parasites respond to pyroptosis of their host cell, which is known to limit microbial infection. Here, we analyzed the pyroptotic process and the fate of intracellular amastigotes at the single-cell level using high-content real-time imaging. Bone marrow-derived macrophages were infected with virulent Leishmania amazonensis amastigotes and sequentially treated with lipopolysaccharide and ATP to induce pyroptosis. Real-time monitoring identified distinct pyroptotic phases, including rapid decay of the parasitophorous vacuole (PV), progressive cell death and translocation of the luminal PV membrane to the cell surface in 40% of macrophages, resulting in the extracellular exposure of amastigotes that remained anchored to PV membranes. Electron microscopy analyses revealed an exclusive polarized orientation of parasites, with the anterior pole exposed toward the extracellular milieu, and the parasite posterior pole attached to the PV membrane. Exposed parasites retained their full infectivity towards naïve macrophages suggesting that host cell pyroptosis may contribute to parasite dissemination.

Use of Optical Imaging Technology in the Validation of a New, Rapid, Cost-Effective Drug Screen as Part of a Tiered In Vivo Screening Paradigm for Development of Drugs To Treat Cutaneous Leishmaniasis.

In any drug discovery and development effort, a reduction in the time of the lead optimization cycle is critical to decrease the time to license and reduce costs. In addition, ethical guidelines call for the more ethical use of animals to minimize the number of animals used and decrease their suffering. Therefore, any effort to develop drugs to treat cutaneous leishmaniasis requires multiple tiers of in vivo testing that start with higher-throughput efficacy assessments and progress to lower-throughput models with the most clinical relevance. Here, we describe the validation of a high-throughput, first-tier, noninvasive model of lesion suppression that uses an in vivo optical imaging technology for the initial screening of compounds. A strong correlation between luciferase activity and the parasite load at up to 18 days postinfection was found. This correlation allows the direct assessment of the effects of drug treatment on parasite burden. We demonstrate that there is a strong correlation between drug efficacy measured on day 18 postinfection and the suppression of lesion size by day 60 postinfection, which allows us to reach an accurate conclusion on drug efficacy in only 18 days. Compounds demonstrating a significant reduction in the bioluminescence signal compared to that in control animals can be tested in lower-throughput, more definitive tests of lesion cure in BALB/c mice and Golden Syrian hamsters (GSH) using Old World and New World parasites.

Cross reactivity of S. aureus to murine cytokine assays: A source of discrepancy.

Staphylococcus aureus is one of the versatile Gram positive bacteria causing a range of diseases. Upon challenge, host immune cells recognize S. aureus and mount diverse immune responses including production of pro-inflammatory cytokines such as IL-1ß and TNF-α. These cytokines are important mediators of inflammation which can be detected via various immunological methods such as enzyme linked immunosorbent assay (ELISA) and immunoblotting. In the current study, we found that a number of clinical isolates as well as laboratory strains of S. aureus exhibited cross reactivity with ELISA antibodies for murine IL-1ß and TNF-α assays. This cross reactivity generates exaggerated false positive signals which can be a source of discrepancy for the understanding of real immune responses against S. aureus infection by host immune cells.

A combined luciferase imaging and reverse transcription polymerase chain reaction assay for the study of Leishmania amastigote burden and correlated mouse tissue transcript fluctuations.

Laboratory mice display features of bona fide hosts for parasites such as Leishmania major and Leishmania donovani. Characterizing the amastigote population size fluctuations and the mouse transcript abundance accounting for these fluctuations demands the capacity to record in real time and integrate quantitative multiparametric datasets from the host tissues where these processes occur. To this end, two technologies, luciferase-expressing Leishmania imaging and a very sensitive quantitative analysis of both Leishmania and mouse transcripts, were combined. After the inoculation of either L. major or L. donovani, the amastigote population size fluctuations - increase, plateau and reduction - were monitored by bioluminescence. It allowed a limited number of mice to be selected for further analysis of both mouse and amastigote transcripts using the real-time quantitative polymerase chain reaction assay we set up. The illustrative examples displayed in the present analysis highlight a correlation between the transcriptional signatures displayed by mouse tissues with the amastigote burden fluctuations. We argue that these two combined technologies will have the potential to provide further insights on complex phenotypes driven by Leishmania developmental programs in the tissues of the mammal hosts.

Going ballistic: Leishmania nuclear subversion of host cell plasticity.

Intracellular parasites have evolved intricate strategies to subvert host cell functions for their own survival. These strategies are particularly damaging to the host if the infection involves immune cells, as illustrated by protozoan parasites of the genus Leishmania that thrive inside mononuclear phagocytic cells, causing devastating immunopathologies. While the impact of Leishmania infection on host cell phenotype and functions has been well documented, the regulatory mechanisms underlying host cell subversion were only recently investigated. Here we summarize the current knowledge on how Leishmania infection affects host nuclear activities and propose thought-provoking new concepts on the reciprocal relationship between epigenetic and transcriptional regulation in host cell phenotypic plasticity, its potential subversion by the intracellular parasite, and its relevance for host-directed therapy.

Early curative applications of the aminoglycoside WR279396 on an experimental Leishmania major-loaded cutaneous site do not impair the acquisition of immunity.

Topical therapy is an attractive approach for the treatment of Leishmania major cutaneous leishmaniasis (CL). WR279396, an expanded-spectrum aminoglycoside ointment, is now in phase 3 trials. Because the application of a cream is easier than the injection of pentavalent antimony, many patients with CL will likely be treated with WR279396 soon after the onset of a lesion. However, this new therapeutic approach may impair the acquisition of immunity. We evaluated the impact of early topical therapy on acquired immunity in an optimized mouse model of L. major-induced CL. The efficacy of the WR279396 ointment in this model has been established previously. Acquired immunity was defined as the absence of lesions upon reinoculation of the same parasite isolate at a different skin site. Bioluminescence-based follow-up of luciferase-expressing L. major loads was also performed. In this model, the control of L. major loads at the initial inoculation site and the acquisition of immunity are simultaneous (day 22 postinoculation). The clinical and parasitological efficacies of WR279396 applied as early as day 11 postinoculation, i.e., during the L. major multiplication phase, did not impair the acquisition of immunity to a second L. major challenge. This is reassuring from the perspective of the wide deployment of WR279396-based therapy in foci where L. major is endemic.

Targeting Macrophage Histone H3 Modification as a Leishmania Strategy to Dampen the NF-κB/NLRP3-Mediated Inflammatory Response.

Aberrant macrophage activation during intracellular infection generates immunopathologies that can cause severe human morbidity. A better understanding of immune subversion strategies and macrophage phenotypic and functional responses is necessary to design host-directed intervention strategies. Here, we uncover a fine-tuned transcriptional response that is induced in primary and lesional macrophages infected by the parasite Leishmania amazonensis and dampens NF-κB and NLRP3 inflammasome activation. Subversion is amastigote-specific and characterized by a decreased expression of activating and increased expression of de-activating components of these pro-inflammatory pathways, thus revealing a regulatory dichotomy that abrogates the anti-microbial response. Changes in transcript abundance correlate with histone H3K9/14 hypoacetylation and H3K4 hypo-trimethylation in infected primary and lesional macrophages at promoters of NF-κB-related, pro-inflammatory genes. Our results reveal a Leishmania immune subversion strategy targeting host cell epigenetic regulation to establish conditions beneficial for parasite survival and open avenues for host-directed, anti-microbial drug discovery.

Leishmania amazonensis Subverts the Transcription Factor Landscape in Dendritic Cells to Avoid Inflammasome Activation and Stall Maturation.

Leishmania parasites are the causative agents of human leishmaniases. They infect professional phagocytes of their mammalian hosts, including dendritic cells (DCs) that are essential for the initiation of adaptive immune responses. These immune functions strictly depend on the DC's capacity to differentiate from immature, antigen-capturing cells to mature, antigen-presenting cells-a process accompanied by profound changes in cellular phenotype and expression profile. Only little is known on how intracellular Leishmania affects this important process and DC transcriptional regulation. Here, we investigate these important open questions analyzing phenotypic, cytokine profile and transcriptomic changes in murine, immature bone marrow-derived DCs (iBMDCs) infected with antibody-opsonized and non-opsonized Leishmania amazonensis (L.am) amastigotes. DCs infected by non-opsonized amastigotes remained phenotypically immature whereas those infected by opsonized parasites displayed a semi-mature phenotype. The low frequency of infected DCs in culture led us to use DsRed2-transgenic parasites allowing for the enrichment of infected BMDCs by FACS. Sorted infected DCs were then subjected to transcriptomic analyses using Affymetrix GeneChip technology. Independent of parasite opsonization, Leishmania infection induced expression of genes related to key DC processes involved in MHC Class I-restricted antigen presentation and alternative NF-κB activation. DCs infected by non-opsonized parasites maintained an immature phenotype and showed a small but significant down-regulation of gene expression related to pro-inflammatory TLR signaling, the canonical NF-kB pathway and the NLRP3 inflammasome. This transcriptomic profile was further enhanced in DCs infected with opsonized parasites that displayed a semi-mature phenotype despite absence of inflammasome activation. This paradoxical DC phenotype represents a Leishmania-specific signature, which to our knowledge has not been observed with other opsonized infectious agents. In conclusion, systems-analyses of our transcriptomics data uncovered important and previously unappreciated changes in the DC transcription factor landscape, thus revealing a novel Leishmania immune subversion strategy directly acting on transcriptional control of gene expression. Our data raise important questions on the dynamic and reciprocal interplay between trans-acting and epigenetic regulators in establishing permissive conditions for intracellular Leishmania infection and polarization of the immune response.

A flavivirus protein M-derived peptide directly permeabilizes mitochondrial membranes, triggers cell death and reduces human tumor growth in nude mice.

Dengue viruses belong to the Flavivirus family and are responsible for hemorrhagic fever in Human. Dengue virus infection triggers apoptosis especially through the expression of the small membrane (M) protein. Using isolated mitochondria, we found that synthetic peptides containing the C-terminus part of the M ectodomain caused apoptosis-related mitochondrial membrane permeabilization (MMP) events. These events include matrix swelling and the dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)). Protein M Flavivirus sequence alignments and helical wheel projections reveal a conserved distribution of charged residues. Moreover, when combined to the cell penetrating HIV-1 Tat peptide transduction domain (Tat-PTD), this sequence triggers a caspase-dependent cell death associated with DeltaPsi(m) loss and cytochrome c release. Mutational approaches coupled to functional screening on isolated mitochondria resulted in the selection of a protein M derived sequence containing nine residues with potent MMP-inducing properties on isolated mitochondria. A chimeric peptide composed of a Tat-PTD linked to the 9-mer entity triggers MMP and cell death. Finally, local administration of this chimeric peptide induces growth inhibition of xenograft prostate PC3 tumors in immuno-compromised mice, and significantly enhances animal survival. Together, these findings support the notion of using viral genomes as valuable sources to discover mitochondria-targeted sequences that may lead to the development of new anticancer compounds.

Imaging visceral leishmaniasis in real time with golden hamster model: Monitoring the parasite burden and hamster transcripts to further characterize the immunological responses of the host.

Characterizing the clinical, immunological and parasitological features associated with visceral leishmaniasis is complex. It involves recording in real time and integrating quantitative multi-parametric data sets from parasite infected host tissues. Although several models have been used, hamsters are considered the bona fide experimental model for Leishmania donovani studies. To study visceral leishmaniasis in hamsters we generated virulent transgenic L. donovani that stably express a reporter luciferase protein. Two complementary methodologies were combined to follow the infectious process: in vivo imaging using luciferase-expressing Leishmania and real time RT-PCR to quantify both Leishmania and host transcripts. This approach allows us: i) to assess the clinical outcome of visceral leishmaniasis by individual monitoring of hamster weight, ii) to follow the parasite load in several organs by real time analysis of the bioluminescence in vivo and through real time quantitative PCR analysis of amastigote parasite transcript abundance ex vivo, iii) to evaluate the immunological responses triggered by the infection by quantifying hamster transcripts on the same samples and iv) to limit the number of hamsters selected for further analysis. The overall data highlight a correlation between the transcriptional cytokine signatures of hamster affected tissues and the amastigote burden fluctuations, thus providing new insights into the immunopathological process driven by L. donovani in the tissues of mammalian hosts. Finally, they suggest organ-specific immune responses.

New insights into experimental visceral leishmaniasis: Real-time in vivo imaging of Leishmania donovani virulence.

Visceral leishmaniasis is an insidious neglected disease with worldwide distribution. It is caused by parasites from the Leishmania donovani complex, which are able to be transmitted by different species of phlebotomine sand flies and to infect numerous mammal hosts. Despite the high number of people infected or at risk, and the remarkable quantity of studies focusing on this disease, a proper experimental model to efficiently decipher the infectious process of visceral leishmaniasis taking into account the nuances of parasite's virulence and the duration of the infection is still lacking. Therefore, using golden Syrian hamsters and BALB/c mice, state-of-the-art genetic manipulation applied on a fully virulent L. donovani strain and in vivo imaging approaches, we describe herein three benefits for experimental visceral leishmaniasis: (i) the development of a double transfected bioluminescent (firefly luciferase) and fluorescent (E2-crimson) virulent strain of L. donovani (Ld1S_luci_E2-crimson), favoring a wide range of both in vivo and in vitro investigations, (ii) the establishment of a non-invasive mouse model to evaluate the infectious process during visceral leishmaniasis and the parasite's virulence in real time, allowing longitudinal studies with the same animals, and (iii) the elaboration of a suitable method to reinstate (and verify anew) the virulence in a population of attenuated parasites, by recovering persistent parasites from chronic infected mice. Consequently, these results open up new perspectives on the study of visceral leishmaniasis, especially in the fields of therapeutics and vaccinology, since the model described herein renders now possible long-lasting follow up studies, with easy and accurate day-by-day verifications of the infection status along with a reduced number of laboratory animals. TRIAL REGISTRATION: ClinicalTrials.gov 2013-0047.

The hepatitis B virus X protein abrogates Bcl-2-mediated protection against Fas apoptosis in the liver.

The role of the hepatitis B virus protein HBx in liver cell proliferation and apoptosis remains controversial. Using a transgenic mouse model, we have recently shown that HBx stimulates the apoptotic turnover of hepatocytes, independently of p53. In this paper, we tested whether the proapoptotic function of HBx can interfere with Bcl-2 during hepatic apoptosis in vivo. HBx transgenic mice were crossed with PK-hBcl-2 mice that are protected against Fas killing by constitutive overexpression of Bcl-2 in hepatocytes. In a lethal challenge with Fas antibodies, HBx expressed at low levels restored sensitivity to Fas-mediated apoptosis and fulminant hepatic failure in mice overexpressing Bcl-2. Furthermore, cytochrome c release from mitochondria and caspase 3 activation were restored to normal levels in HBx/Bcl-2 mice during transduction of the Fas signal. Thus, the proapoptotic activity of HBx overcomes or bypasses the inhibitory effect of Bcl-2 against Fas cytotoxicity. This effect was not apparently mediated through downregulation of the PK-hBcl-2 transgene or via delocalization of the Bcl-2 protein, and a direct interaction of HBx with Bcl-2, Bcl-X(L) or Bax could not be evidenced in yeast two-hybrid assays. We further show that apoptosis induced by ectopic expression of HBx is associated with mitochondrial membrane alterations and caspase 3 activation. Our data indicate that the dominant function of HBx upon Bcl-2-regulated control of apoptosis might play an important role in the pathogenesis of chronic hepatitis B.

Reduction of antibiotic use in the community reduces the rate of colonization with penicillin G-nonsusceptible Streptococcus pneumoniae.

BACKGROUND: There is a lack of evidence documenting the impact of optimized antibiotic use on the rates of colonization with penicillin G-nonsusceptible Streptococcus pneumoniae (PNSP) in children. This study evaluates the effect of community-based intervention strategies on the prevalence of pnsp colonization. METHODS: A controlled, population-based pharmacoepidemiological trial was conducted from January through May 2000. Three French geographic areas were selected on the basis of demographic similarities. Two intervention strategies were implemented: (1) reduced antibiotic use, which was achieved by not prescribing antibiotics for presumed viral respiratory tract infections (the prescription-reduction group); and (2) better adaptation of dose and duration (the dose/duration group). A control group received no intervention. The target population was children aged 3-6 years who were attending kindergarten. Oropharyngeal pneumococcus colonization and antibiotic use were monitored throughout the 5-month study. RESULTS: The prescription-reduction, dose/duration, and control groups included 601, 483, and 405 children, respectively. The interventions induced significantly larger decreases in antibiotic use in the prescription-reduction group (-18.8%) and dose/duration group (-17.1%) than in the control group (-3.8%), and the rates of PNSP colonization were initially similar for the 3 groups (52.5%, 55.1%, and 50.0%, respectively). At the end of the 5-month study, the rates of PNSP colonization were 34.5% for the prescription-reduction group (P=.05) and 44.3% for the dose/duration group (P=.8), compared with 46.2% for the control group. CONCLUSIONS: Intensive educational strategies aimed at optimizing antibiotic use can significantly reduce the rate of PNSP colonization in areas with high resistance rates.

Lack of control of T cell apoptosis under HAART. Influence of therapy regimen in vivo and in vitro.

BACKGROUND: Increased and premature T cell apoptosis is recognized as a feature of HIV infection, and its normalization during highly active antiretroviral therapy (HAART) is thought to contribute to quantitative CD4 T cell restoration. DESIGN: Cross-sectional study of spontaneous, CD3- and CD95-mediated apoptosis in lymphocytes from 53 HIV-infected individuals taking HAART. METHODS: Overnight stimulation of peripheral blood mononuclear cells (PBMC) with coated anti-CD3 or anti-CD95 monoclonal antibodies or incubation overnight in medium. Apoptosis in CD4 and CD8 T cells was measured by flow cytometry. For in vitro assay of antiretroviral drugs, normal PBMC were prestimulated with anti-CD3 monoclonal antibodies and apoptosis was induced by ligation of CD95. The expression of active caspase-8 and caspase-3 was examined by flow cytometry. RESULTS: We report for the first time that important levels of T cell apoptosis may persist under HAART, in spite of a rise in CD4 T cells from baseline and a sustained suppression of plasmatic viral load. Spontaneous CD3- or CD95-induced apoptosis levels were inversely correlated with the in vivo number of CD4 T cells and the CD4/CD8 ratio, but not with the viral load or duration of antiretroviral therapy. Regimens including lamivudine are associated with persistent T cell apoptosis, particularly following CD95 ligation. Lamivudine was also found to stimulate in vitro CD95-induced apoptosis and caspase activation in pre-activated T lymphocytes from healthy donors. CONCLUSION: The immunomodulatory effect of lamivudine may be one of the contributing factor to increased levels of T cell apoptosis under HAART. The data suggest that there is a requirement for physiological apoptosis during HAART.

Multiparametric flow cytometric analysis of biochemical and functional events associated with apoptosis and oncosis using the 7-aminoactinomycin D assay.

Apoptosis and primary necrosis are the two modes of cell death induced by a lethal injury. The majority of structural and biochemical events occurring during cell death can be analysed by flow cytometry. The 7-aminoactinomycin D (7-AAD) assay can be used to detect the loss of membrane integrity during apoptosis of murine thymocytes and human peripheral lymphocytes. We describe here new applications of the 7-AAD assay. It can be applied to a variety of cell lines of different origins, including adherent cell lines, and it allows the co-detection of lipidic antigens such as phosphatidylserine (PS) residues, and biochemical processes linked to apoptosis, such as the loss of mitochondrial transmembrane potential, cardiolipin peroxidation, the expression of the 7A6 mitochondrial antigen and DNA fragmentation. Thus, this assay is a noninvasive method particularly adapted to the analysis of biochemical events associated with cell death. Finally, we show that this assay is not specific for apoptosis since it detects oncosis, the early stage of primary necrosis.

A combined luciferase-expressing Leishmania imaging/RT-qPCR assay provides new insights into the sequential bilateral processes deployed in the ear pinna of C57BL/6 mice.

Leishmania/L. major was identified as the etiological agent of human localized cutaneous leishmaniasis. L. major metacyclic promastigotes/MP - the infectious form transmitted by sand flies - were enriched from axenically-derived cultures and inoculated into the dermis of mice (10(3) or 10(4) luciferase-expressing L. major MP inoculated into the C57BL/6 mouse ear pinna). Quantitative readout assays were then combined with imaging of this L. major-hosting skin site and established i) that a specific period of time - depending upon the L. major load used for the inoculation - is required for the L. major-hosting ear pinna to be continuously populated by a balanced population of functional regulatory and effector T lymphocytes, and that ii) this balance coincides with persisting low numbers of amastigotes in more or less rapidly healing skin. This approach also established that, whatever the MP inoculum load delivered to the primary site, the immune processes that reduce the L. major amastigote population also account for concomitant immunity, namely remodelling of the secondary site - where 10(4) MP were delivered - as a clinically silent niche hosting a small L. major population.

Reprogramming neutral lipid metabolism in mouse dendritic leucocytes hosting live Leishmania amazonensis amastigotes.

BACKGROUND: After loading with live Leishmania (L) amazonensis amastigotes, mouse myeloid dendritic leucocytes/DLs are known to undergo reprogramming of their immune functions. In the study reported here, we investigated whether the presence of live L. amazonensis amastigotes in mouse bone marrow-derived DLs is able to trigger re-programming of DL lipid, and particularly neutral lipid metabolism. METHODOLOGY/PRINCIPAL FINDINGS: Affymetrix-based transcriptional profiles were determined in C57BL/6 and DBA/2 mouse bone marrow-derived DLs that had been sorted from cultures exposed or not to live L. amazonensis amastigotes. This showed that live amastigote-hosting DLs exhibited a coordinated increase in: (i) long-chain fatty acids (LCFA) and cholesterol uptake/transport, (ii) LCFA and cholesterol (re)-esterification to triacyl-sn-glycerol (TAG) and cholesteryl esters (CE), respectively. As these neutral lipids are known to make up the lipid body (LB) core, oleic acid was added to DL cultures and LB accumulation was compared in live amastigote-hosting versus amastigote-free DLs by epi-fluorescence and transmission electron microscopy. This showed that LBs were both significantly larger and more numerous in live amastigote-hosting mouse dendritic leucocytes. Moreover, many of the larger LB showed intimate contact with the membrane of the parasitophorous vacuoles hosting the live L. amazonensis amastigotes. CONCLUSIONS/SIGNIFICANCE: As leucocyte LBs are known to be more than simple neutral lipid repositories, we set about addressing two related questions. Could LBs provide lipids to live amastigotes hosted within the DL parasitophorous vacuole and also deliver? Could LBs impact either directly or indirectly on the persistence of L. amazonensis amastigotes in rodent skin?

Distinct transcriptional signatures of bone marrow-derived C57BL/6 and DBA/2 dendritic leucocytes hosting live Leishmania amazonensis amastigotes.

BACKGROUND/OBJECTIVES: The inoculation of a low number (10(4)) of L. amazonensis metacyclic promastigotes into the dermis of C57BL/6 and DBA/2 mouse ear pinna results in distinct outcome as assessed by the parasite load values and ear pinna macroscopic features monitored from days 4 to 22-phase 1 and from days 22 to 80/100-phase 2. While in C57BL/6 mice, the amastigote population size was increasing progressively, in DBA/2 mice, it was rapidly controlled. This latter rapid control did not prevent intracellular amastigotes to persist in the ear pinna and in the ear-draining lymph node/ear-DLN. The objectives of the present analysis was to compare the dendritic leukocytes-dependant immune processes that could account for the distinct outcome during the phase 1, namely, when phagocytic dendritic leucocytes of C57BL/6 and DBA/2 mice have been subverted as live amastigotes-hosting cells. METHODOLOGY/PRINCIPAL FINDINGS: Being aware of the very low frequency of the tissues' dendritic leucocytes/DLs, bone marrow-derived C57BL/6 and DBA/2 DLs were first generated and exposed or not to live DsRed2 expressing L. amazonensis amastigotes. Once sorted from the four bone marrow cultures, the DLs were compared by Affymetrix-based transcriptomic analyses and flow cytometry. C57BL/6 and DBA/2 DLs cells hosting live L. amazonensis amastigotes do display distinct transcriptional signatures and markers that could contribute to the distinct features observed in C57BL/6 versus DBA/2 ear pinna and in the ear pinna-DLNs during the first phase post L. amazonensis inoculation. CONCLUSIONS/SIGNIFICANCE: The distinct features captured in vitro from homogenous populations of C57BL/6 and DBA/2 DLs hosting live amastigotes do offer solid resources for further comparing, in vivo, in biologically sound conditions, functions that range from leukocyte mobilization within the ear pinna, the distinct emigration from the ear pinna to the DLN of live amastigotes-hosting DLs, and their unique signalling functions to either naive or primed T lymphocytes.

Sorting of Leishmania-bearing dendritic cells reveals subtle parasite-induced modulation of host-cell gene expression.

Once in the mouse skin, Leishmania (L) amazonensis amastigotes are hosted by professional mononuclear phagocytes such as dendritic cells (DCs). When monitored after parasite inoculation, the frequency of amastigote-hosting DCs is very low (<1%) in both the skin and skin-draining lymph nodes. Therefore, we designed and validated an efficient procedure to purify live amastigotes-hosting DCs with the objective to facilitate quantitative and qualitative analysis of such rare cells. To this end, a L. amazonensis transgenic parasite expressing DsRed2 fluorescent protein was generated and added to mouse bone marrow-derived DC cultures. Then, a high speed sorting procedure, performed in BSL-2 containment, was setup to pick out only DCs hosting live amastigotes. This study reveals, for the first time, a unique transcript pattern from sorted live amastigotes-hosting DCs that would have been undetectable in unsorted samples. It was indeed possible to highlight a significant and coordinated up-regulation of L-arginine transporter and arginase2 transcripts in Leishmania-hosting DCs compared to un-parasitized DCs. These results indicate that arginine catabolism for polyamine generation is dominating over L-arginine catabolism for NO generation. In conclusion, this approach provides a powerful method for further characterisation, of amastigote-hosting DCs in the skin and the skin-draining lymph nodes.

Imaging Leishmania development in their host cells.

The interactions between the Leishmania parasite and its mammalian host cells are strongly regulated dynamic processes that take place at the molecular, cellular and organ level. Several different interaction models have been developed to take advantage of the development of imaging technologies. Here, we first review how conventional imaging techniques have been applied to fixed Leishmania-loaded tissue and cell samples. Then, we show how transgenic Leishmania expressing fluorescent or bioluminescent reporters allowed characterization of their tissue and cell host niches. Most notably, the use of whole-body imaging or intravital microscopy techniques has allowed accurate real-time monitoring of parasites in their environment. Finally, we discuss how innovative imaging technologies will allow further refinement of our understanding of the interplay between Leishmania and its hosts.