Geneviève Milon

Retention of Plasmodium falciparum ring-infected erythrocytes in the slow, open microcirculation of the human spleen

The current paradigm in Plasmodium falciparum malaria pathogenesis states that young, ring-infected erythrocytes (rings) circulate in peripheral blood and that mature stages are sequestered in the vasculature, avoiding clearance by the spleen. Through ex vivo perfusion of human spleens, we examined the interaction of this unique blood-filtering organ with P falciparum-infected erythrocytes. As predicted, mature stages were retained. However, more than 50% of rings were also retained and accumulated upstream from endothelial sinus wall slits of the open, slow red pulp microcirculation. Ten percent of rings were retained at each spleen passage, a rate matching the proportion of blood flowing through the slow circulatory compartment established in parallel using spleen contrast-enhanced ultrasonography in healthy volunteers. Rings displayed a mildly but significantly reduced elongation index, consistent with a retention process, due to their altered mechanical properties. This raises the new paradigm of a heterogeneous ring population, the less deformable subset being retained in the spleen, thereby reducing the parasite biomass that will sequester in vital organs, influencing the risk of severe complications, such as cerebral malaria or severe anemia. Cryptic ring retention uncovers a new role for the spleen in the control of parasite density, opening novel intervention opportunities.

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.

A Natural Model of Leishmania major Infection Reveals a Prolonged “Silent” Phase of Parasite Amplification in the Skin Before the Onset of Lesion Formation and Immunity

A model of Leishmania major infection in C57BL/6 mice has been established that combines two main features of natural transmission: low dose (100 metacyclic promastigotes) and inoculation into a dermal site (the ear dermis). The evolution of the dermal lesion could be dissociated into two distinct phases. The initial “silent” phase, lasting 4–5 wk, favored establishment of the peak load of parasites in the dermis in the absence of lesion formation or any overt histopathologic changes in the site. The second phase corresponds to the development of a lesion associated with an acute infiltration of neutrophils, macrophages, and eosinophils into the dermis and was coincident with the killing of parasites in the site. The onset of immunity/pathology was correlated with the appearance of cells staining for IL-12p40 and IFN-γ in the epidermal compartment, and an expansion of T cells capable of producing IFN-γ in the draining lymph node. Parasite growth was not enhanced over the first 4.5 wk in anti-CD4-treated mice, SCID mice, or C57BL/6 mice deficient in IL-12p40, IFN-γ, CD40 ligand, or inducible NO synthase. These mice all failed to ultimately control infection in the site, but in some cases (anti-CD4 treated, IL-12p40−/−, CD40 ligand−/−, and SCID) high dermal parasite loads were associated with little or no pathology. These results extend to a natural infection model a role for Th1 cells in both acquired resistance and lesion formation, and document the remarkable avoidance of this response during a prolonged phase of parasite amplification in the skin.

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.

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).

An Immunomodulatory Function for Neutrophils During the Induction of a CD4+ Th2 Response in BALB/c Mice Infected with Leishmania major

The possible immunomodulatory role of polymorphonuclear leukocytes (PMN) in CD4+ T lymphocyte differentiation in mice was examined by studying the effect of transient depletion of PMN during the early phase after Leishmania major delivery. A single injection of the PMN-depleting NIMP-R14 mAb 6 h before infection with L. major prevented the early burst of IL-4 mRNA transcription otherwise occurring in the draining lymph node of susceptible BALB/c mice. Since this early burst of IL-4 mRNA transcripts had previously been shown to instruct Th2 differentiation in mice from this strain, we examined the effect of PMN depletion on Th subset differentiation at later time points after infection. The transient depletion of PMN in BALB/c mice was sufficient to inhibit Th2 cell development otherwise occurring after L. major infection. Decreased Th2 responses were paralleled with partial resolution of the footpad lesions induced by L. major. Furthermore, draining lymph node-derived CD4+ T cells from PMN-depleted mice remained responsive to IL-12 after L. major infection, unlike those of infected BALB/c mice receiving control Ab. PMN depletion had no effect when the NIMP-R14 mAb was injected 24 h postinfection. The protective effect of PMN depletion was shown to be IL-12 dependent, as concomitant neutralization of IL-12 reversed the protective effect of PMN depletion. These results suggest a role for an early wave of PMN in the development of the Th2 response characteristic of mice susceptible to infection with L. major.

Real-Time PCR for Detection and Quantitation of Leishmania in Mouse Tissues

ABSTRACT Leishmania spp. are intracellular protozoan parasites that cause a wide spectrum of diseases in humans and dogs worldwide. However, monitoring of the Leishmania burden in its different hosts is still based on cumbersome and poorly sensitive methods. Here we have developed a highly accurate real-time PCR assay with which to reproducibly detect and quantify the relative Leishmania major burden in mouse tissue samples. The assay is performed with the LightCycler system using SYBR Green I and primers amplifying a ca. 120-bp fragment from minicircles of the kinetoplast DNA (kDNA). The assay was able to detect as little as 100 fg of L. major DNA per reaction, which is equivalent to 0.1 parasite. The standard curve designed for quantitation of parasites showed linearity over an at least 6-log DNA concentration range, corresponding to 0.1 to 104 parasites per reaction, with a correlation coefficient of 0.979. The assay also proved to have a detection range of the same magnitude as that used for detection of L. donovani and L. amazonensis, but it was 100-fold less sensitive for L. mexicana. When applied to tissues from experimentally infected mice, the real-time PCR assay is not only as sensitive as a conventional PCR assay for detection of Leishmania kDNA but also more rapid. Results indicate that this assay is compatible with the clinical diagnosis of leishmaniasis and will be a great help to scientists who use animals to monitor the efficacy of antileishmanial drugs or vaccines or decipher the unique properties of the life cycle of Leishmania spp .

Isolation and flow cytometric analysis of the free lymphomyeloid cells present in murine liver

Recruitment of circulating lymphomyeloid cells in the liver during infection often plays a critical role, mediating control or exacerbation of the pathogen growth. This paper describes a simple and rapid technique to recover these lymphomyeloid cells from a normal or an infected liver. After portal perfusion with saline buffer, the liver is gently dissociated on steel screens and the resulting cell population spun in 35% Percoll in 100 IU/ml Calciparine to remove all nuclei and cell debris: the recovery of a pure liver lymphomyeloid cell population is usually achieved in 40-60 min. Phenotypic and functional analysis could then be easily carried out on this cell population. This methodology was applied to normal mouse liver: flow cytometric analysis of the purified free lymphomyeloid cells showed the presence of T lymphocytes (46% +/- 3 with a CD4/CD8 ratio of 2.8), B lymphocytes (20% +/- 2 IgG and 30% IgM positive) and myelomonocytic cells (14% +/- 2 complement receptor type III positive).

Dendritic cells are early cellular targets of Listeria monocytogenes after intestinal delivery and are involved in bacterial spread in the host

We studied the sequence of cellular events leading to the dissemination of Listeria monocytogenes from the gut to draining mesenteric lymph nodes (MLNs) by confocal microscopy of immunostained tissue sections from a rat ligated ileal loop system. OX‐62‐positive cells beneath the epithelial lining of Peyers patches (PPs) were the first Listeria targets identified after intestinal inoculation. These cells had other features typical of dendritic cells (DCs): they were large, pleiomorphic and major histocompatibility complex class IIhi. Listeria were detected by microscopy in draining MLNs as early as 6 h after inoculation. Some 80–90% of bacteria were located in the deep paracortical regions, and 100% of the bacteria were present in OX‐62‐positive cells. Most infected cells contained more than five bacteria each, suggesting that they had arrived already loaded with bacteria. At later stages, the bacteria in these areas were mostly present in ED1‐positive mononuclear phagocytes. These cells were also infected by an actA mutant defective in cell‐to‐cell spreading. This suggests that Listeria are transported by DCs from PPs to the deep paracortical regions of draining MLNs and are then transmitted to other cell populations by mechanisms independent of ActA. Another pathway of dissemination to MLNs was identified, probably involving free Listeria and leading to the infection of ED3‐positive mononuclear phagocytes in the subcapsular sinus and adjacent paracortical areas. This study provides evidence that DCs are major cellular targets of L. monocytogenes in PPs and that DCs may be involved in the early dissemination of this pathogen. DCs were not sites of active bacterial replication, making these cells ideal vectors of infection.

Bioluminescent Leishmania expressing luciferase for rapid and high throughput screening of drugs acting on amastigote‐harbouring macrophages and for quantitative real‐time monitoring of parasitism features in living mice

In this study, we have established conditions for generating Leishmania amazonensis recombinants stably expressing the firefly luciferase gene. These parasites produced significant bioluminescent signals for both in vitro studies and the development of an in vivo model, allowing the course of the parasitism to be readily monitored in real time in the living animals such as laboratory mice. First, a model was established, using parasite‐infected mouse macrophages for rapidly determining the activity of drugs against intracellular amastigotes. Results indicated that recombinant Leishmania can be reliably and confidently used to monitor compounds acting on intracel‐lular amastigote‐harbouring macrophages. Secondly, temporal analyses were performed following inoculation of metacyclic promastigotes into the ear dermis of BALB/c mice and the bioluminescent light transmitted through the tissue was imaged externally using a charge coupled device (CCD) camera. Bioluminescent signals, measured at the inoculation site and in the draining lymph node of mice containing these parasites correlated well with the more classical quantification of parasites. These assays prove that the real‐time bioluminescent assay is not only sensitive but also more rapid than culture‐base techniques allowing to monitor parasite‐load before any clinical signs of leishmaniasis are detectable. In short, this luciferase imaging study is useful to monitor the efficacy of anti‐leishmanial drugs on live cell culture and to trace leishmanial infection in animal models.