[Elucidating and characterizing the dynamic biological processes that account for the sustainability of Leishmania populations]. / Décrypter et caractériser les processus biologiques dynamiques qui rendent compte de la pérennité des populations de Leishmania.

To attempt resolving this issue accurately, it was necessary to anchor our experimental approaches in the observations and pioneering work of our predecessors, notably Alphonse Laveran, Louis Parrot, Edmond and Étienne Sergent. The latter, among other things, had identified as natural hosts of leishmaniasis, rodent populations with which hematophagous telmophagous sand fly populations cohabited closely.When human populations emerged in these natural ecosystems, after the sedentarization of Homo sapiens, more or less important disturbances would have led to a transition of sand fly hematophagy, from zoophilia, to zoo-anthropophilia and anthropophilia.The creation of infrastructures that allow the breeding and integration into experimental groups of both holobiont sand flies and holobiont laboratory rodents (rats, mice, hamsters, etc.) remains crucial. With such infrastructures, it becomes possible to grasp and characterize the multilateral dynamic processes - mostly clinically silent - that account for the biogenesis of tissue and/or cellular niches protecting populations of Leishmania developmental morphotypes, including those ensuring host-to-host transmission, albeit in small numbers.

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.

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.

Intracellular protozoan parasites: living probes of the host cell surface molecular repertoire.

Intracellular protozoans co-evolved with their mammalian host cells a range of strategies to cope with the composite and dynamic cell surface features they encounter during migration and infection. Therefore, these single-celled eukaryotic parasites represent a fascinating source of living probes for precisely capturing the dynamic coupling between the membrane and contractile cortex components of the cell surface. Such biomechanical changes drive a constant re-sculpting of the host cell surface, enabling rapid adjustments that contribute to cellular homeostasis. As emphasized in this review, through the design of specific molecular devices and stratagems to interfere with the biomechanics of the mammalian cell surface these parasitic microbes escape from dangerous or unfavourable microenvironments by breaching host cell membranes, directing the membrane repair machinery to wounded membrane areas, or minimizing membrane assault using discretion and speed when invading host cells for sustained residence.

Sensing of red blood cells with decreased membrane deformability by the human spleen.

The current paradigm in the pathogenesis of several hemolytic red blood cell disorders is that reduced cellular deformability is a key determinant of splenic sequestration of affected red cells. Three distinct features regulate cellular deformability: membrane deformability, surface area-to-volume ratio (cell sphericity), and cytoplasmic viscosity. By perfusing normal human spleens ex vivo, we had previously showed that red cells with increased sphericity are rapidly sequestered by the spleen. Here, we assessed the retention kinetics of red cells with decreased membrane deformability but without marked shape changes. A controlled decrease in membrane deformability (increased membrane rigidity) was induced by treating normal red cells with increasing concentrations of diamide. Following perfusion, diamide-treated red blood cells (RBCs) were rapidly retained in the spleen with a mean clearance half-time of 5.9 minutes (range, 4.0-13.0). Splenic clearance correlated positively with increased membrane rigidity (r = 0.93; P < .0001). To determine to what extent this increased retention was related to mechanical blockade in the spleen, diamide-treated red cells were filtered through microsphere layers that mimic the mechanical sensing of red cells by the spleen. Diamide-treated red cells were retained in the microsphilters (median, 7.5%; range, 0%-38.6%), although to a lesser extent compared with the spleen (median, 44.1%; range, 7.3%-64.0%; P < .0001). Taken together, these results have implications for understanding the sensitivity of the human spleen to sequester red cells with altered cellular deformability due to various cellular alterations and for explaining clinical heterogeneity of RBC membrane disorders.

Unveiling and Characterizing Early Bilateral Interactions between Biofilm and the Mouse Innate Immune System.

A very substantial progress has been made in our understanding of infectious diseases caused by invasive bacteria. Under their planktonic forms, bacteria transiently reside in the otherwise sterile mammal body tissues, as the physiological inflammation insures both their clearance and repair of any tissue damage. Yet, the bacteria prone to experience planktonic to biofilm developmental transition still need to be studied. Of note, sessile bacteria not only persist but also concur preventing the effectors and regulators of the physiological inflammation to operate. Thus, it is urgent to design biologically sound experimental approaches aimed to extract, at the earliest stage, immune signatures of mono-bacteria planktonic to biofilm developmental transition in vivo and ex vivo. Indeed, the transition is often the first event to which succeeds the "chronicization" process whereby classical bacteria-targeting therapies are no more efficacious. An in vivo model of micro-injection of Staphylococcus aureus planktonic or biofilm cells in the ear pinna dermis of laboratory transgenic mice with fluorescent immune cells is proposed. It allows visualizing, in real time, the range of the early interactions between the S. aureus and myeloid cell subsets- the resident macrophages and dendritic cells, the recruited neutrophil granulocytes/polymorphonuclear neutrophils, monocytes otherwise known to differentiate as macrophages or dendritic cells. One main objective is to extract contrasting immune signatures of the modulation of the physiological inflammation with respect to the two bacterial lifestyles.

Reduced erythrocyte deformability associated with hypoargininemia during Plasmodium falciparum malaria.

The mechanisms underlying reduced red blood cell (RBC) deformability during Plasmodium falciparum (Pf) malaria remain poorly understood. Here, we explore the possible involvement of the L-arginine and nitric oxide (NO) pathway on RBC deformability in Pf-infected patients and parasite cultures. RBC deformability was reduced during the acute attack (day0) and returned to normal values upon convalescence (day28). Day0 values correlated with plasma L-arginine levels (r = 0.69; p = 0.01) and weakly with parasitemia (r = -0.38; p = 0.006). In vitro, day0 patient's plasma incubated with ring-stage cultures at 41°C reduced RBC deformability, and this effect correlated strongly with plasma L-arginine levels (r = 0.89; p < 0.0001). Moreover, addition of exogenous L-arginine to the cultures increased deformability of both Pf-free and trophozoite-harboring RBCs. NO synthase activity, evidenced in Pf-infected RBCs, induced L-arginine-dependent NO production. These data show that hypoargininemia during P. falciparum malaria may altogether impair NO production and reduce RBC deformability, particularly at febrile temperature.

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?

Surface area loss and increased sphericity account for the splenic entrapment of subpopulations of Plasmodium falciparum ring-infected erythrocytes.

Ex vivo perfusion of human spleens revealed innate retention of numerous cultured Plasmodium falciparum ring-infected red blood cells (ring-iRBCs). Ring-iRBC retention was confirmed by a microsphiltration device, a microbead-based technology that mimics the mechanical filtering function of the human spleen. However, the cellular alterations underpinning this retention remain unclear. Here, we use ImageStream technology to analyze infected RBCs' morphology and cell dimensions before and after fractionation with microsphiltration. Compared to fresh normal RBCs, the mean cell membrane surface area loss of trophozoite-iRBCs, ring-iRBCs and uninfected co-cultured RBCs (uRBCs) was 14.2% (range: 8.3-21.9%), 9.6% (7.3-12.2%) and 3.7% (0-8.4), respectively. Microsphilters retained 100%, ∼50% and 4% of trophozoite-iRBCs, ring-iRBCs and uRBCs, respectively. Retained ring-iRBCs display reduced surface area values (estimated mean, range: 17%, 15-18%), similar to the previously shown threshold of surface-deficient RBCs retention in the human spleen (surface area loss: >18%). By contrast, ring-iRBCs that successfully traversed microsphilters had minimal surface area loss and normal sphericity, suggesting that these parameters are determinants of their retention. To confirm this hypothesis, fresh normal RBCs were exposed to lysophosphatidylcholine to induce a controlled loss of surface area. This resulted in a dose-dependent retention in microsphilters, with complete retention occurring for RBCs displaying >14% surface area loss. Taken together, these data demonstrate that surface area loss and resultant increased sphericity drive ring-iRBC retention in microsphilters, and contribute to splenic entrapment of a subpopulation of ring-iRBCs. These findings trigger more interest in malaria research fields, including modeling of infection kinetics, estimation of parasite load, and analysis of risk factors for severe clinical forms. The determination of the threshold of splenic retention of ring-iRBCs has significant implications for diagnosis (spleen functionality) and drug treatment (screening of adjuvant therapy targeting ring-iRBCs).

High content analysis of primary macrophages hosting proliferating Leishmania amastigotes: application to anti-leishmanial drug discovery.

BACKGROUND/OBJECTIVES: Human leishmaniases are parasitic diseases causing severe morbidity and mortality. No vaccine is available and numerous factors limit the use of current therapies. There is thus an urgent need for innovative initiatives to identify new chemotypes displaying selective activity against intracellular Leishmania amastigotes that develop and proliferate inside macrophages, thereby causing the pathology of leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: We have developed a biologically sound High Content Analysis assay, based on the use of homogeneous populations of primary mouse macrophages hosting Leishmania amazonensis amastigotes. In contrast to classical promastigote-based screens, our assay more closely mimics the environment where intracellular amastigotes are growing within acidic parasitophorous vacuoles of their host cells. This multi-parametric assay provides quantitative data that accurately monitors the parasitic load of amastigotes-hosting macrophage cultures for the discovery of leishmanicidal compounds, but also their potential toxic effect on host macrophages. We validated our approach by using a small set of compounds of leishmanicidal drugs and recently published chemical entities. Based on their intramacrophagic leishmanicidal activity and their toxicity against host cells, compounds were classified as irrelevant or relevant for entering the next step in the drug discovery pipeline. CONCLUSIONS/SIGNIFICANCE: Our assay represents a new screening platform that overcomes several limitations in anti-leishmanial drug discovery. First, the ability to detect toxicity on primary macrophages allows for discovery of compounds able to cross the membranes of macrophage, vacuole and amastigote, thereby accelerating the hit to lead development process for compounds selectively targeting intracellular parasites. Second, our assay allows discovery of anti-leishmanials that interfere with biological functions of the macrophage required for parasite development and growth, such as organelle trafficking/acidification or production of microbicidal effectors. These data thus validate a novel phenotypic screening assay using virulent Leishmania amastigotes growing inside primary macrophage to identify new chemical entities with bona fide drug potential.

Microsphiltration: a microsphere matrix to explore erythrocyte deformability.

The altered deformability of erythrocytes infected with Plasmodium falciparum is central in malaria -pathogenesis, as it influences the hemodynamic properties of the infected cell and its retention in the spleen. Exported parasite proteins, as well as the shape and volume of the parasite itself, influence the deformability of the infected erythrocyte. To explore changes in erythrocyte deformability, we have developed a new method, called microsphiltration, based on filtration of erythrocytes through a mixture of metal microspheres that mimic the geometry of inter-endothelial splenic slits. As P. falciparum develops in its host cell, the retention rates observed in microspheres correlate with the progressive decrease of erythrocyte deformability and with the retention rates in the spleen. The yields of microsphiltration separation allow for molecular analyses of subpopulations with distinct mechanical phenotypes.

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.

A switch in infected erythrocyte deformability at the maturation and blood circulation of Plasmodium falciparum transmission stages.

Achievement of malaria elimination requires development of novel strategies interfering with parasite transmission, including targeting the parasite sexual stages (gametocytes). The formation of Plasmodium falciparum gametocytes in the human host takes several days during which immature gametocyte-infected erythrocytes (GIEs) sequester in host tissues. Only mature stage GIEs circulate in the peripheral blood, available to uptake by the Anopheles vector. Mechanisms underlying GIE sequestration and release in circulation are virtually unknown. We show here that mature GIEs are more deformable than immature stages using ektacytometry and microsphiltration methods, and that a switch in cellular deformability in the transition from immature to mature gametocytes is accompanied by the deassociation of parasite-derived STEVOR proteins from the infected erythrocyte membrane. We hypothesize that mechanical retention contributes to sequestration of immature GIEs and that regained deformability of mature gametocytes is associated with their release in the bloodstream and ability to circulate. These processes are proposed to play a key role in P falciparum gametocyte development in the host and to represent novel and unconventional targets for interfering with parasite transmission.

Quantitative assessment of sensing and sequestration of spherocytic erythrocytes by the human spleen.

Splenic sequestration of RBCs with reduced surface area and cellular deformability has long been recognized as contributing to pathogenesis of several RBC disorders, including hereditary spherocytosis. However, the quantitative relationship between the extent of surface area loss and splenic entrapment remains to be defined. To address this issue, in the present study, we perfused ex vivo normal human spleens with RBCs displaying various degrees of surface area loss and monitored the kinetics of their splenic retention. Treatment with increasing concentrations of lysophosphatidylcholine resulted in a dose-dependent reduction of RBC surface area at constant volume, increased osmotic fragility, and decreased deformability. The degree of splenic retention of treated RBCs increased with increasing surface area loss. RBCs with a > 18% average surface area loss (> 27% reduced surface area-to-volume ratio) were rapidly and completely entrapped in the spleen. Surface-deficient RBCs appeared to undergo volume loss after repeated passages through the spleen and escape from splenic retention. The results of the present study for the first time define the critical extent of surface area loss leading to splenic entrapment and identify an adaptive volume regulation mechanism that allows spherocytic RBCs to prolong their life span in circulation. These results have significant implications for understanding the clinical heterogeneity of RBC membrane disorders.

Early systemic bacterial dissemination and a rapid innate immune response characterize genetic resistance to plague of SEG mice.

BACKGROUND: Although laboratory mice are usually highly susceptible to Yersinia pestis, we recently identified a mouse strain (SEG) that exhibited an exceptional capacity to resist bubonic plague and used it to identify immune mechanisms associated with resistance. METHODS: The kinetics of infection, circulating blood cells, granulopoiesis, lesions, and cellular populations in the spleen, and cytokine production in various tissues were compared in SEG and susceptible C57BL/6J mice after subcutaneous infection with the virulent Y. pestis CO92. RESULTS: Bacterial invasion occurred early (day 2) but was transient in SEG/Pas mice, whereas in C57BL/6J mice it was delayed but continuous until death. The bacterial load in all organs significantly correlated with the production of 5 cytokines (granulocyte colony-stimulating factor, keratinocyte-derived chemokine (KC), macrophage cationic peptide-1 (MCP-1), interleukin 1α, and interleukin 6) involved in monocyte and neutrophil recruitment. Indeed, higher proportions of these 2 cell types in blood and massive recruitment of F4/80(+)CD11b(-) macrophages in the spleen were observed in SEG/Pas mice at an early time point (day 2). Later times after infection (day 4) were characterized in C57BL/6J mice by destructive lesions of the spleen and impaired granulopoiesis. CONCLUSION: A fast and efficient Y. pestis dissemination in SEG mice may be critical for the triggering of an early and effective innate immune response necessary for surviving plague.

Critical role of the neutrophil-associated high-affinity receptor for IgE in the pathogenesis of experimental cerebral malaria.

The role of the IgE-FcεRI complex in malaria severity in Plasmodium falciparum-hosting patients is unknown. We demonstrate that mice genetically deficient for the high-affinity receptor for IgE (FcεRIα-KO) or for IgE (IgE-KO) are less susceptible to experimental cerebral malaria (ECM) after infection with Plasmodium berghei (PbANKA). Mast cells and basophils, which are the classical IgE-expressing effector cells, are not involved in disease as mast cell-deficient and basophil-depleted mice developed a disease similar to wild-type mice. However, we show the emergence of an FcεRI(+) neutrophil population, which is not observed in mice hosting a non-ECM-inducing PbNK65 parasite strain. Depletion of this FcεRI(+) neutrophil population prevents ECM, whereas transfer of this population into FcεRIα-KO mice restores ECM susceptibility. FcεRI(+) neutrophils preferentially home to the brain and induce elevated levels of proinflammatory cytokines. These data define a new pathogenic mechanism of ECM and implicate an FcεRI-expressing neutrophil subpopulation in malaria disease severity.

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.

The sensing of poorly deformable red blood cells by the human spleen can be mimicked in vitro.

Retention of poorly deformable red blood cells (RBCs) by the human spleen has been recognized as a critical determinant of pathogenesis in hereditary spherocytosis, malaria, and other RBC disorders. Using an ex vivo perfusion system, we had previously shown that retention of Plasmodium falciparum-infected RBCs (Pf-RBCs) occur in the splenic red pulp, upstream from the sinus wall. To experimentally replicate the mechanical sensing of RBCs by the splenic microcirculation, we designed a sorting device where a mixture of 5- to 25-µm-diameter microbeads mimics the geometry of narrow and short interendothelial splenic slits. Heated RBCs, Pf-RBCs, and RBCs from patients with hereditary spherocytosis were retained in the microbead layer, without hemolysis. The retention rates of Pf-RBCs were similar in microbeads and in isolated perfused human spleens. These in vitro results directly confirm the importance of the mechanical sensing of RBCs by the human spleen. In addition, rigid and deformable RBC subpopulations could be separated and characterized at the molecular level, and the device was used to deplete a stored RBC population from its subpopulation of rigid RBCs. This experimental approach may contribute to a better understanding of the role of the spleen in the pathogenesis of inherited and acquired RBC disorders.

Development of the malaria parasite in the skin of the mammalian host.

The first step of Plasmodium development in vertebrates is the transformation of the sporozoite, the parasite stage injected by the mosquito in the skin, into merozoites, the stage that invades erythrocytes and initiates the disease. The current view is that, in mammals, this stage conversion occurs only inside hepatocytes. Here, we document the transformation of sporozoites of rodent-infecting Plasmodium into merozoites in the skin of mice. After mosquito bite, ∼50% of the parasites remain in the skin, and at 24 h ∼10% are developing in the epidermis and the dermis, as well as in the immunoprivileged hair follicles where they can survive for weeks. The parasite developmental pathway in skin cells, although frequently abortive, leads to the generation of merozoites that are infective to erythrocytes and are released via merosomes, as typically observed in the liver. Therefore, during malaria in rodents, the skin is not just the route to the liver but is also the final destination for many inoculated parasites, where they can differentiate into merozoites and possibly persist.