Kinetic Analysis of Ex Vivo Human Blood Infection by Leishmania

The leishmanioses, vector-borne diseases caused by the trypanosomatid protozoan Leishmania, are transmitted to susceptible mammals by infected phlebotomine sand flies that inoculate promastigotes into hemorrhagic pools created in host skin. We assumed that promastigotes are delivered to a blood pool, and analyzed early promastigote interactions (0–5 min) with host components, which lead to parasite endocytosis by blood leukocytes, and to host infection. Promastigotes were incubated with NHS or with heparinized blood in near-physiological conditions, and we used cell radioimmunoassay and flow cytometry to measure the on-rate constants (k+1) of promastigote interactions with natural opsonins and erythrocytes. We obtained quantitative data for parasitized cells to determine the time-course of promastigote binding and internalization by blood leukocytes. In these reactions, promastigotes bind natural opsonins, immune adhere to erythrocytes and activate complement cytolysis, which kills ∼95% of promastigotes by 2 min post-infection. C3-promastigote binding is a key step in opsonization; nascent C3-promastigotes are the substrate for two simultaneous reactions, C3-promastigote immune adherence (IA) to erythrocytes and complement-mediated promastigote killing. The k+1 for IA was 75-fold greater than that for promastigote killing, showing that IA facilitates promastigote endocytosis and circumvents lysis. At 5 min post-infection, when reaction velocity is still linear and promastigote concentration is not limiting, 17.4% of granulocytes and 10.7% of monocytes had bound promastigotes, of which ∼50% and ∼25%, respectively, carried surface-bound (live) or internalized (live and dead) leishmanias. Of other leukocyte types, 8.5% of B cells bound but did not internalize promastigotes, and T cells, NK cells and CD209+ dendritic cells did not bind parasites. These data show that, once in contact with blood, promastigote invasion of human leukocytes is an extremely rapid and efficient reaction, and suggest that the IA reaction constitutes a central strategy for this parasite in subverting host innate immune defenses.


Introduction
The leishmanioses are a group of vector-borne zoonotic diseases caused by trypanosomatid parasites of the genus Leishmania. Leishmanias are heteroxenous protozoa with a life cycle in two different hosts, in the Psychodidae diptera of the genera Phlebotomus and Lutzomyia and in mammals. In the sand fly, Leishmania lives in the digestive tract as an extracellular motile flagellated promastigote; in mammals, it dwells as a sessile aflagellated amastigote inside macrophages [1].
Female phlebotomine sand flies are hematophagous arthropods that require blood proteins for oogenesis. Sand flies feed from hemorrhagic spots created in the host dermis. When feeding on a Leishmania-infected host, flies can ingest amastigotes or amastigoteladen macrophages and become infected. In the vector gut, amastigotes differentiate first into procyclic promastigotes and subsequently into more mature promastigote morphotypes. Promastigote differentiation generates mature, non-dividing parasites termed metacyclic promastigotes, considered the Leishmania forms that infect mammals [2]. In heavily infected flies, the lumen of the food canal appears choked by a promastigote-derived mucin-like gel (PSG) containing large numbers of promastigotes embedded in a filamentous proteophosphoglycan (fPPG) matrix [3]. During sand fly engorgement, PSG limits the food flow and jams the vector feeding system, hampering intake of an adequate blood meal; this prompts sand fly regurgitation and delivery of promastigotes together with saliva and fPPG to the intradermal pool, thus causing infection [4].
Leishmaniosis is transmitted to mammalian hosts when infected sand flies take a second blood meal [5,6]. Cases of Leishmania infection have been reported in humans with no apparent blood uptake by the vector; in this case, promastigotes are presumably deposited into the extracellular matrix in the dermis [7][8][9]. Parasite transmission without blood involvement is also described in experimental rodent infection, in which a considerable fraction of transmitting flies apparently did not ingest blood while feeding [10,11]; nevertheless, one of these studies shows that promastigote transmission was 2.6-fold higher among flies that had taken a second blood meal [10]. Studies of natural sand fly feeding habits showed that a majority (63-68%) of females trapped around animal shelters were blood-fed, and that 58.7% of blood-fed flies were PCR-positive for Leishmania DNA, double the number of positives found in non-blood-fed flies [12]. These data indicate that blood uptake by the vector is frequent in leishmaniosis transmission.
In natural Leishmania infection, promastigotes can therefore be delivered into hematomas or into a bloodless context in the skin. This is not an irrelevant issue, as promastigotes interact in blood with leukocyte populations, whereas in the dermal matrix they interact with fibroblasts, dermal dendritic cells (DDC), mast cells, and macrophages [13,14]. The environment and cell target repertoire in which infection occurs can influence the course of disease development, as well as the type and intensity of immune response induced [15,16].
Recent work in mice explored parasite fate after intradermal promastigote inoculation [13,17], but the physiological and functional differences between the innate immune systems of mouse and man preclude direct extrapolation of results. In humans, comprehensive studies of the initial stages of leishmaniosis transmission in the blood pool are lacking, and most infection studies have been carried out using isolated leukocyte populations [18][19][20][21][22][23][24][25][26]. For a previous study, we designed an ex vivo model of infection in human blood to analyze the early stages of promastigotehost interaction [27]. Opsonisation, binding and internalization of promastigotes by target leukocytes occur within minutes (early infection); ensuing reactions triggered by the cells that endocytose parasites take hours or days to develop or to reach full intensity. Using two Leishmania species with different tropism, L. amazonensis and L. donovani, we studied the kinetics of early Leishmania infection of human blood, and measured the rate constants (k +1 ) of promastigote opsonization reactions and the kinetics of promastigote binding and internalization by blood leukocytes during the very early infection period (0-5 min). Based on these data, we propose a kinetic model of ex vivo human blood infection by Leishmania promastigotes.

Ethics statement
This study was approved by the Ethics Committee (Comité de É tica de la Investigación y Bienestar Animal) of the Instituto de Salud Carlos III (Ref CEI PI 12_2009). All human participants were volunteers and gave written consent.

Human blood and serum collection
Blood was drawn from healthy donors into preservative-free heparin (10 IU/ml), kept at room temperature (20uC), and used in the IA reaction within hours of extraction. Normal human serum (NHS) was collected from clotted blood (20uC, 30 min) and serum aliquots stored in liquid nitrogen.

Measurement of reaction on-rate (k +1 ) constants
The Leishmania opsonization pathway can be depicted as a sequence of four reactions: 1) promastigote+natural antibo-diesRpromastigote-IgM, 2)+complementRpromastigote-C3 (nascent promastigote-C3 are the substrate of two subsequent competing reactions), 3) promastigote-C3+ERpromastigote-E IA and 4) promastigote-C3+C5b-C9Rpromastigote propidium iodide (PI) uptake. In 25% NHS, the concentrations of anti-Leishmania IgM antibodies ([IgM 5 ]), complement components and receptors for complement C3b fragments on erythrocytes ([E-CR1]) are in moderate to great excess over the concentration of promastigote IgM binding sites ([Pm bs ]) and bound C3b with which these molecules interact in reactions (1) to (4). To simplify measurement of on-rate constants, reactions (1) to (4) were considered one-way bimolecular interactions of the type A reactant+B reactantRP (product), and analyzed as described [29,30]. The rate of P formation is described as in which k +1 is the second-order on-rate constant for the reaction.

Author Summary
Leishmania infection is transmitted to mammalian hosts by phlebotomine sand flies. During the vector's bloodmeal, promastigotes are inoculated into hemorrhagic spots in the skin or are delivered into the extracellular matrix of the dermis. In the first case, blood is involved in transmission; in the second, it apparently is not. This is important, as the cellular milieu of infection can be critical for induction of the anti-parasite immune response and the subsequent course of disease. In humans, there are few comprehensive studies of the initial stages of Leishmania transmission in blood. Using blood to mimic the skin hematoma, we carried out kinetic and quantitative analyses of the reaction with serum and blood components of promastigotes from two Leishmania species with different tropism. We describe the kinetics of the promastigote reaction pathway that leads to blood infection and provide quantitative data for the cell types infected in the first five minutes of leishmaniosis transmission.

Promastigote-IgM binding
Aliquots (50 ml) of L. donovani or L. amazonensis promastigotes (10 7 /ml) were mixed with 50 ml 50% PBS-diluted pooled NHS and incubated for varying time periods between 0 and 60 sec. The reaction was terminated by dilution with 1 ml cold (4uC) PBS containing 2.5% FCS and 0.05% NaN 3 (PFS buffer), followed by centrifugation (11,0006g, 1 min). To avoid cell loss, untreated promastigotes (5610 6 ) were then added to the pellet and the samples washed twice in cold PFS (11,0006g, 1 min) to remove traces of serum IgM that could block 125 I-goat anti-m binding. Parasites were resuspended in 0.2 ml PFS containing 2610 5 cpm 125 I-goat anti-m (10 7 cpm/mg), incubated 1 h on ice, washed twice as above, and promastigote-bound antibody was determined.

Determination of complement-resistant parasite numbers in stationary promastigote inoculum
To estimate the number of live parasites after opsonization in different NHS concentrations, single aliquots (0.3 ml) containing L. amazonensis promastigotes (2610 5 ), 10 mg/ml PI, and serially diluted (50% to 0.78%) NHS were incubated (37uC) for 1 to 9 min. Leishmania killing was determined by measuring promastigote PI uptake in real-time by flow cytometry (FACSCalibur). The precise percentage of complement-killed promastigotes cannot be determined by flow cytometry due to background signal from small particles and debris in stationary cultures; to reduce background, promastigotes were gated (SSC vs. FSC) and PI uptake emission measured in a dot plot of FL-2 (585/42 nm) vs. time (sec); data were analyzed with CELLQuest software (Becton Dickinson).
To measure promastigote complement-resistance in 50% NHS, aliquots (50 ml) of promastigotes (2610 6 /ml) of six L. amazonensis stationary cultures (96.6% viable) were incubated in 50% pooled NHS (37uC, 5 min), after which promastigote PI uptake was measured by flow cytometry. Additional experiments (n = 3) compared PI uptake by stationary promastigotes and promastigotes enriched in metacyclic forms by centrifugation in a 10% to 30% Ficoll density gradient (''top 10'' promastigotes) [31,32]. Briefly, 1 ml of stationary-phase promastigotes (2610 8 /ml) was layered onto a discontinuous gradient of 2 ml 10% and 2 ml 30% Ficoll solutions, and centrifuged (13006g or 3656g, no brake; 22uC, 10 min). Promastigotes at the 10% Ficoll interface and the upper part of the 10% Ficoll cushion were pooled, diluted with RPMI 1640 complete medium and washed by centrifugation (15006g, 15 min). The pellet, resuspended in complete RPMI 1640, was washed again and adjusted to 2610 7 cells/ml. Aliquots (50 ml) of stationary-phase and ''top 10'' promastigotes were incubated (37uC, 5 min) in 50% NHS; for controls, NHS was replaced by PBS. The reaction was terminated by dilution with complete RPMI 1640 and centrifugation. Promastigote PI incorporation was determined by flow cytometry after incubating a 10 ml aliquot of each sample in 0.2 ml of PBS containing 2 ml PI (1 mg/ml). Promastigotes were gated (SSC vs. FSC) to eliminate small particles and debris, and PI uptake emission was collected in the FL2 detector through a 585/42 nm band pass filter.
After opsonization in different NHS concentrations, the percentage of live promastigotes was quantitated by light microscopy. Aliquots (50 ml) of stationary-phase L. amazonensis promastigotes were incubated (37uC, 5 min) in serially diluted NHS (1:2; 50% to 0.78% concentration). The reaction was terminated by dilution with PBS, and live parasites counted under a microscope. It should be noted that after parasite incubation in $3% serum, motile promastigotes with a slender shape are observed very infrequently. Most parasites registered as viable undergo marked changes in cell geometry, lose the flagellum and become ellipsoidal or small round refractile bodies with limited but noticeable drifting; such changes complicate identification.

Determination of leukocyte binding of promastigotes
To measure initial blood leukocyte binding of opsonized leishmanias, we incubated CMFDA-labeled promastigotes with heparin-treated blood and used flow cytometry to determine the percentage of each leukocyte subpopulation that bound parasites after 5 min. We mixed 1 ml CMFDA-labeled L. amazonensis or L. donovani promastigotes (10 7 /ml) with 1 ml heparinized blood, followed by incubation (37uC, 5 min, waterbath). The mixture was divided into 200 ml aliquots and E were lysed by incubation with 2 ml E-lysing reagent (EasyLyse, DakoCytomation; 10 min). Cells were washed in PBS by centrifugation (5006g, 10 min, 20uC). Each pellet was incubated (30 min, 20uC) with one of the following mAb: anti-CD14-APC, -CD15-PE, -CD3-PE-Cy5, -CD56-APC, -CD19-APC or -CD209-PE. Cells were washed in PBS by centrifugation (5006g, 10 min). Leukocyte subpopulations were identified by flow cytometry and represented in a dot plot as side scatter (SSC) vs. the specific fluorescent label of each population. The percentage of each subpopulation that bound promastigotes was analyzed in a secondary plot by independently representing the fluorescence intensity of each gated mAb-labeled population vs. that of cell-bound CMFDA-labeled promastigotes (FL1, green 530 nm) following excitation with a 488 nm argon ion laser. To account for nonspecific binding, we subtracted promastigote binding at time zero from all values registered. In heat-inactivated serum (HIS), promastigote binding by leukocytes was negligible at the times tested (Fig. S1). Use of HIS as control would require cell separation from plasma and its replacement with HIS, a less physiological approximation of infection conditions. The number of events acquired by the cytometer varied from 5610 4 to 2610 5 . A total of 5610 3 events were counted for each leukocyte population, except in the case of CD209 + cells, for which 2610 5 leukocytes were acquired and 150 events counted.

Time-course analysis of promastigote binding and internalization by granulocytes and monocytes
To measure the kinetics of promastigote binding and internalization by granulocytes and monocytes, 50 ml aliquots of heparinized human blood were mixed with 50 ml CMFDA-labeled Leishmania promastigotes (10 7 /ml) and incubated (37uC) for various times (0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 10, 30 and 60 min). The reaction was terminated by addition of 2 ml E-lysing reagent. After 10 min incubation, 3 ml of sheath fluid were added to each tube and centrifuged (5006g, 5 min); the pellet was washed with 5 ml sheath fluid as above and resuspended in 200 ml sheath fluid. Samples were divided in two 100 ml aliquots. To quench extracellular fluorescence, 10 ml trypan blue (TB) solution (10 mg/ml; final concentration 1 mg/ml) were added to one sample. To register the percentage of leukocytes that bound and internalized promastigotes, granulocytes and monocytes were gated by SSC vs. forward scatter (FSC) and plotted independently in a secondary plot of SSC vs. FL1 (green, 530 nm) following excitation with a 488 nm argon ion laser. Quenched samples were acquired for 10 min after TB addition. A total of 2610 4 events were acquired for each measurement and analyzed with CELLQuest software (BD Biosciences).

Results
For kinetic analysis of the early mechanisms of ex vivo human blood infection by Leishmania, we studied promastigote interactions with NHS and with heparin-treated blood. Experiments were performed in conditions of near-physiological time (0-360 sec), temperature (37uC) and serum or blood concentrations (25% and 50%, respectively), with a constant reaction volume (0.1 ml) and promastigote inoculum (5610 5 ) throughout the study. This inoculum size corresponds to ,5000 promastigotes delivered in a blood meal of 0.5 ml, well within the range of experimentally determined values [6,11,33]. All kinetic data were obtained from initial rate measurements, when reaction velocity is linear with time.
To infect blood, we used early stationary culture promastigotes taken not later than two days after they had reached maximum growth. Promastigote populations from early stationary cultures are heterogeneous; nevertheless, after contact with human blood, whose complement is cytolytic for Leishmania, only serum-resistant metacyclic promastigotes are alive [34]. We assume that promastigotes that survive .3 min in blood constitute the parasite population with infective capacity. Alternatively, one might use parasite inocula enriched in metacyclic promastigotes by negative selection methods, but these procedures also render heterogeneous populations [31,[35][36][37][38], and inoculum enrichment with metacyclic promastigotes would boost infection artificially.
To ascertain the number of complement-resistant promastigotes in the inocula, we measured PI uptake in real time by L. amazonensis parasites incubated with serially diluted pooled NHS. The extent of promastigote killing by human complement is dependent on serum dilution (Fig. S2). At $25% NHS, ,90% of promastigotes incorporated PI very rapidly; at higher serum dilutions, promastigote PI uptake showed a gradual delay, although the percentage of promastigotes killed by the end of incubation reached maximum levels and was similar to that observed at 50% NHS. In 3% NHS and in more dilute serum conditions, most promastigotes were alive.
We used flow cytometry to assess the percentage of complement-resistant parasites after incubation in 50% pooled NHS (37uC, 5 min). Complement-mediated PI uptake by promastigotes was studied in parasites of six L. amazonensis stationary cultures. In three cultures, we compared PI uptake by stationary promastigotes and stationary promastigotes enriched in ''top 10'' metacyclic forms on a Ficoll density gradient. Promastigote aliquots were incubated in 50% NHS and PI incorporation determined by flow cytometry. Promastigotes from stationary cultures showed 4.4%61.0% (range 2.8% to 6.8%) live parasites and controls, 96.5%61.3%. Complement resistance was similar in stationary promastigotes and ''top 10'' stationary promastigotes in 50% NHS; the former showed 4.8%61.1% and the latter, 4.6%61.1. ''Top 10'' stationary promastigotes were thus not more resistant to human complement. Average recovery of ''top 10'' promastigotes was 16% and 35% after centrifugation at 13006g and 3656g, respectively; this indicates that the small percentage of complement-resistant promastigotes in stationary cultures is not a function of low levels of ''top 10'' metacyclic forms, but that promastigotes resistant to lysis in 50% NHS are very infrequent.
To calculate the number of live promastigotes by a non-flow cytometric method, we used light microscopy. The percentage of live parasites in serum concentrations from 50% to 0.78% is indicated (Fig. S3). This method shows an average percentage of live promastigotes of 5.0%60.7% (range 3.2% to 6.9%).
Infection of blood with promastigotes calls for the use an antihemostatic agent such as heparin. To determine the inhibitory capacity of heparin in Leishmania opsonization, we studied realtime PI uptake (i.e., killing) by L. amazonensis promastigotes opsonised with NHS, normal plasma, normal plasma treated with 50 mg/ml lepidurin (Refludin; Pharmion) or with heparin at concentrations of 10, 12.5, 15, 20, 40 or 80 IU/ml (Fig. S4). The velocity of promastigote PI uptake in plasma treated with 10 to 40 IU/ml heparin lagged by ,10 sec relative to the velocity of promastigote PI uptake in NHS. To observe a ,40-sec delay compared to NHS or to lepirudin-treated plasma, 80 IU/ml heparin was needed. Reaction intensity was identical in NHS, in lepirudin-treated plasma, and in plasma treated with different heparin concentrations; after 2 min incubation, all promastigotes had incorporated PI to the same extent. These data indicate that heparin does not interfere appreciably with parasite opsonisation in heparin-treated (10 IU/ml) human blood.
We also analyzed the influence of heparin on promastigote binding to granulocytes and monocytes in heparin-treated (10 IU/ ml) human blood. Binding of L. amazonensis promastigotes by granulocytes and monocytes after 1, 3, and 5 min incubation in heparin-treated blood was indistinguishable from that in lepidurintreated (50 mg/ml) blood (Fig. S5). At a 10 IU/ml concentration, heparin does not affect the course of human blood infection by Leishmania. Kinetics of natural anti-Leishmania IgM binding to promastigotes Non-immune serum from most vertebrates contains natural antitrypanosomatid antibodies that act as an innate recognition system in the host [28,39]. To determine the velocity of IgM binding to Leishmania parasites, we measured the association rate constant (k +1 ) by incubating promastigotes in NHS for varying times. The reaction was terminated by sample dilution with PFS, and promastigotebound IgM was measured using 125 I-goat anti-m. Pentameric IgM (IgM 5 ) binding reached maximum after 20 sec incubation for both L. donovani (Fig. 1A) and L. amazonensis (Fig. 1B). [IgM 5 ] in adult NHS is ,1.3 g/L; if the M r of IgM 5 is considered to be 950,000, then serum [IgM 5 ] is ,1.4610 26 M. Exhaustive adsorption of 25% NHS with L. amazonensis and L. donovani promastigotes removed ,15% and ,30% of IgM 5 , respectively [28], indicating that [IgM 5 ] anti-L. amazonensis in unadsorbed serum is ,0.5610 27 M and that of anti-L. donovani, ,1610 27 M. At equilibrium, L. amazonensis and L. donovani promastigotes bind ,5,000 and ,2,500 IgM molecules/ cell, respectively. Early in the promastigote-IgM 5 interaction, antibody binding is assumed to be monovalent [29], which would render [Pm bs ] on L. amazonensis promastigotes of Time course of C3 deposition on Leishmania promastigotes Early in Leishmania infection, human complement has effects both advantageous for and harmful to the promastigote. Complement activation triggers binding of C3 fragments to the parasite, which mediates IA and Leishmania internalization by host leukocytes; however, promastigote-C3 binding also nucleates assembly of the C5 convertases, activating the complement lytic cascade that kills the parasites.
To establish how Leishmania circumvents complement effector activity, the kinetics and velocity of complement activation must be understood. We incubated promastigotes with NHS and measured the reaction time course and k +1 of promastigote-C3 binding. Promastigote-C3 binding follows a sigmoidal course, with an initial ,30-sec lag during which 10 to 20% of total C3 ligands are fixed; C3 molecules that bind during this lag period probably attach to IgM. This amount of C3 is insufficient to activate the alternative pathway and the lytic cascade, but is sufficient to establish multipoint contacts with clustered E-CR1, triggering the IA reaction [40][41][42]. Thereafter, C3 binds at an exponential rate for ,1 min, during which C3 is bound at an average rate of

Promastigote-erythrocyte immune adherence reaction
In human blood, C3-opsonized promastigotes immune adhere to erythrocytes [27]. IA is a C3-mediated innate immune mechanism, almost ubiquitous in mammals, that enhances phagocytosis and clearance of opsonized microorganisms from blood [43]. To study the velocity of IA in the reaction sequence of Leishmania infection, we followed the kinetics of IA complex formation and determined the on-rate constant of the interaction between promastigote-C3 and erythrocytes.
Within seconds of serum contact, nascent C3-opsonized promastigotes bind to CR1 on E and form promastigote-E IA complexes. This interaction has an initial lag time of ,15 sec followed by a period in which IA complexes are formed at an exponential rate until the reaction reaches completion at 30 to 40 sec of incubation. The IA reaction is so rapid that it gives the impression that promastigote-E binding proceeds before C3 opsonization (Fig. 3A, B). Considering the average number of CR1 molecules per erythrocyte to be 500 [44], the [E-CR1] in this assay is ,2.1610 29 M. At equilibrium, L. donovani promastigotes bind ,180,000 C3 molecules/cell [28]; at the onset of the IA reaction in L. donovani

Complement-mediated PI uptake by promastigotes
Complement activation leads to assembly of the C5 convertases (C4b3b2a, C3b 2 Bb), triggering the cytolytic complex (C5b-C9) that causes promastigote death. We measured the time course of parasite membrane damage by complement as PI uptake and determined the on-rate constant of the reaction. The kinetics of promastigote PI uptake is very rapid; it begins at ,50 sec after complement activation and by ,100 sec after serum contact, most leishmanias have incorporated PI (Fig. 4A, B). During that period, the [Pm-C3] varies from ,6610 210 to ,1610 29 M in L. amazonensis and from ,5610 210 to ,1.5610 29 M in L. donovani.
In 25% human serum, the concentration of C5b-C9 components ranges from 9.2610 28 M for C5 to 2.1610 27 M for C9. C5b deposition is considered the rate-limiting step; at the onset of the lytic reaction, [C5b-C9] is greater than [promastigote-C3] by ,150-fold (L. amazonensis) and ,180-fold (L. donovani). The rate of complement-mediated PI uptake by promastigotes was calculated by plotting the percentage of PI incorporation as L (PI max / PI max 2PI ti ) against incubation time (t i ). k app values for L. amazonensis and L. donovani PI uptake were 0.13 sec 21 and 0.083 sec 21 , respectively, and the k +1 constants were ,1. The on-rate constant values of opsonization, immune adherence, and PI uptake reactions of L. donovani and L. amazonensis promastigotes in human blood are summarized in Table 1.

Time-course of granulocyte and monocyte binding to and internalization of promastigotes in human blood
We infected heparin-treated blood with CMFDA-labeled L. amazonensis or L. donovani promastigotes. At various times postinfection, granulocytes and monocytes were gated by SSC vs. FSC, and the percentage of each subpopulation that bound parasites was calculated in a secondary dot plot by representing SSC-gated cells against fluorescence intensity of granulocyte-and monocyte-bound CMFDA-labeled promastigotes (FL1). We simultaneously determined the percentage of granulocytes and monocytes that internalized promastigotes by quenching fluorescence emitted by cell-bound complement-killed CMFDA-labeled promastigotes, using trypan blue [45].
The time course of granulocyte and monocyte binding of promastigotes was linear in the first 5 to 10 min of infection. Promastigote concentration subsequently became limiting and the kinetics followed a hyperbolic course; the binding reaction was complete by 60 min (Fig. 6). After 30 min incubation, 39.2% of granulocytes (,94% of total binding) had already bound L. amazonensis and 45.1% (,90% of total binding) had bound L. donovani promastigotes. At this time, 23.5% of monocytes (,76% of total binding) had bound L. amazonensis and 20.2% (,75% of total binding), L. donovani promastigotes. We analyzed leukocyte binding and promastigote internalization in the period between 0 and 5 min, when the velocity of promastigote binding is proportional with time and the cytolytic activity of complement on Leishmania is probably not yet complete (Fig. 6A,B; insets). After 3 min incubation, the ratio of cells that bound:internalized promastigotes was 12.9:5.4% for granulocytes and 8.9:2% for monocytes (mean value of L. amazonensis and L. donovani); after 5 min, these values were 17.4:8.4% for granulocytes and 10.7:2.6% for monocytes.

Discussion
Phlebotomine sand flies transmit promastigotes to mammalian hosts in two ways, by direct inoculation of parasites into a blood pool in the skin or by their delivery into the interstitial space of the dermis [4,14]. Here we used human blood as a surrogate model of skin hematoma for a comprehensive kinetic analysis of promastigote interactions with host blood components in the first minutes of infection. As the time course of L. amazonensis and L. donovani promastigote reactions were similar, we compiled only the data for L. amazonensis (Fig. 7). Immediately after promastigote interaction with human blood, natural anti-trypanosomatid IgM/IgG antibodies bound the parasite with a hyperbolic course characteristic of first-order or pseudo-first order reactions; in contrast, the kinetics of promastigote-C3 deposition, promastigote IA, and promastigote PI uptake showed S-shaped curves, indicating cooperative (IA) or multimolecular (C3 deposition, PI uptake) mechanisms. Once opsonization was triggered, all reactions proceeded simultaneously, and by ,2 min most parasites had been killed by complement activity. In human blood, Leishmania survival probably depends on the relative rate by which newly C3-opsonized promastigotes are incorporated into IA and PI uptake reactions. The 75-fold greater velocity constant of the promastigote IA (k +1, Pm IA ,9610 7 M 21 sec 21 ) than that of promastigote PI uptake (k +1, Pm PI ,1.2610 6 M 21 sec 21 ) permitted the IA reaction to be 50% complete by ,30 sec before parasite PI uptake began. PI incorporation into promastigotes started ,50 sec after serum contact, and required an additional 20 sec to reach 90% maximum; due to these differences in reaction times, complement killing begins at ,70 sec after serum contact. In this interval, Leishmania IA can ferry promastigotes to blood leukocytes.
Leukocyte populations that express receptors for opsonic ligands, principally C3 complement fragments, bound promasti- Figure 6. Kinetics of leukocyte binding and internalization of L. amazonensis and L. donovani promastigotes in human blood. Aliquots of heparinized human blood were infected with CMFDA-labeled L. amazonensis or L. donovani promastigotes and incubated (37uC) for various times (0-60 min). Samples were then processed as indicated in Methods. To calculate the percentage of granulocytes and monocytes that bound and internalized parasites, cells were gated by SSC vs. FSC and plotted independently in a secondary plot of SSC vs. FL1 (green, 530 nm). The percentage of cells that internalized promastigotes was determined using trypan blue to quench the extracellular fluorescence emitted by cell-bound complement-killed CMFDA-labeled promastigotes.  gotes in proportion to the concentration of each subpopulation in the blood pool. In human blood, granulocyte concentration exceeds that of monocytes, B cells and NK cells by 6-to 8-fold, and that of natural killer (NK) and CD209 + dendritic cells (DC-SIGN) by two to three orders of magnitude; the latter constitute a very small cell population (0.01-0.05%) that binds diverse microorganisms in vitro, including promastigotes and axenic amastigotes of Leishmania [46][47][48].
At 5 min post-infection, 13% of blood leukocytes bound promastigotes in the proportion granulocytes (76.3%).monocytes (10.7%).B lymphocytes (8.5).CD3 + cells (2.7%).CD3 2 CD56 + cells (1.2%) cells.CD-209 + cells (0.5%) (%Fig. 5C). This shows that CD3 + , NK and DC-SIGN + cells have no relevant role in early blood infection. In addition to granulocytes and monocytes, ,8.5% of B lymphocytes bound promastigotes; to our knowledge, this interaction has not been previously reported. B cell binding of opsonized promastigotes was confirmed with Raji lymphoblastoid B cells, which bound C3-opsonized promastigotes to the same extent as U937 monocytes (unpublished data). Promastigote-C3 binding by B cells is probably mediated by the IA reaction. In autologous serum and in PBS/RPMI 1640 medium, human erythrocytes transfer CR1-bound C3-opsonized promastigotes or antigen-antibody immune complexes (IC) to granulocytes, monocytes, B lymphocytes, and U937 cells [27,[49][50][51]. Binding of C3opsonized promastigotes allows B lymphocytes to present antigens to monocytes and macrophages through a CR2-mediated reaction similar to the transfer of E-bound C3-IC to B cells [52]. B lymphocytes have a still-undefined role in host immune response to Leishmania. Early data showed that BALB/c mice depleted of B cells by anti-IgM treatment had enhanced resistance to L. major infection [53] and subsequent studies indicated that B lymphocytes are involved in mouse susceptibility to Leishmania infection and disease pathogenesis [54][55][56][57]. Our results confirm that B lymphocytes have an early role in Leishmania immunity.
In the ex vivo Leishmania blood infection experiments, promastigote inoculum was 5610 3 parasites/0.5 ml blood, a 1.8:1 promastigote:leukocyte ratio. Assuming that the number of live opsonized promastigotes in the inoculum is 5%, 0.5 ml of host blood would contain ,250 live parasites. In the first minutes after infection, granulocytes and monocytes bound parasites at a constant rate, and promastigote binding and ingestion was detectable from 3 min incubation (Fig. 6, insets). At 5 min postinfection, 17.4% of granulocytes bound promastigotes and 8.4% carried TB-unquenched parasites; for monocytes these figures were 10.7% and 2.6%, respectively. At that time, 8.5% of B cells bound promastigotes, but did not internalize them (unpublished data). To illustrate the number of Leishmania-infected leukocytes, after 5 min incubation a 0.5 ml blood pool with 5.6610 6 leukocytes/ml (66.9% granulocytes, 6.9% monocytes) would have ,330 granulocytes with bound promastigotes, ,160 of which would have surface-bound (live) and internalized (live and dead) parasites. In the case of monocytes, there would be ,20 cells with surface-bound promastigotes and ,5 cells with surface-bound and internalized parasites.
The early period of Leishmania infection was recently addressed in the mouse using intravital two-photon microscopy. Ng et al. highlight the role of DDC in invasion, and showed that between 55 and 70% of DDC had ingested parasites by 2 to 3 h postinfection [13]. Host neutrophil depletion before infection did not affect the number of DDC that internalized promastigotes, suggesting that these cells act independently of neutrophils. This apparent lack of neutrophil involvement in infection control could be due to the method of parasite inoculation or to the high intradermal promastigote:DDC ratio used in these experiments. In any case, the intradermal cell compartment is very different from the hematoma environment; this experiment probably mimics promastigote transmission in a bloodless context, and it is not appropriate to compare it with the blood pool. In another study, Peters et al. transmitted infection by intradermal injection of large numbers of promastigotes or with infected flies fed ad libitum on restrained mice [17]. Both inoculation methods are likely to have caused dermic hematomas and to have induced substantial neutrophil infiltrate at the injection site. One day post-infection, ,90% of infected neutrophils harbored live promastigotes; after a week, all leishmanias were inside macrophages and DDC. This change in host cells could be explained by the neutrophil Trojan horse model, which proposes that macrophages are infected when they dispose of apoptotic leukocytes [58]. Peters et al. [17] and others [59] nonetheless consider that when infected neutrophils become apoptotic, they release live promastigotes or amastigotes that are phagocytosed by macrophages and DDC (''Trojan rabbit strategy'').
To compare early Leishmania infection reactions in mouse and man, we must consider that the earliest host-protective mechanisms, serum complement and professional blood phagocytes, differ in their anti-promastigote activity and in cell proportion in these species. In human blood, the granulocyte:monocyte ratio is 6-7:1, whereas in mouse it is from 2-3:1 [60]. This is important, as these cells are the main parasite targets during infection and thereafter, when they are recruited to the inoculation site. Persistence of neutrophils harboring promastigotes is considered of paramount importance for subsequent disease development [24,[61][62][63].
We anticipate that the size of such a neutrophil reservoir would differ between mouse and man. Human complement is highly cytotoxic to promastigotes, whereas mouse complement is not [28,64]. After inoculation, we estimate the number of live promastigotes in human blood to be one twentieth of that in mouse, which would considerably reduce the parasite load of infected neutrophils.
Based on these data, we outline a kinetic model of Leishmania infection in human blood that incorporates rate constants for promastigote interactions, which measure the speed of these reactions (Fig. 8). At 50 sec post-Leishmania inoculation, promastigote opsonization reactions are terminated and complement-dependent parasite killing has not begun, indicating that rapid leukocyte uptake of promastigotes promotes Leishmania survival in the host [23,27]. We suggest that a crucial step in the infection pathway is determined by the high on-rate constant of the Leishmania IA reaction. This mechanism competes with complement-mediated parasite lysis by promoting promastigote binding and internalization by granulocytes and monocytes, and binding by B cells (Fig. 8). The substantial number of promastigote-infected leukocytes in the blood pool is probably sufficient to establish host infection. Nevertheless, subsequent macrophage and DDC phagocytosis of promastigotes released from neutrophils, or of apoptotic neutrophils, would boost the parasite load during the early silent phase post-infection.
During natural transmission, sand flies deliver salivary bioactive components to the host, two of which could potentially interfere with early infection mechanisms: saliva and fPPG (PSG). Sand fly saliva inhibits hemostasis and facilitates feeding [65]. Mice inoculated with sand fly salivary gland extracts show exacerbated lesion development, which is associated with early (6 h) increase in type-2 cytokine production and with early (2 to 4 h) expression of macrophage-recruiting chemokines that promote inflammatory cell influx to the injection site [66][67][68].
Time Course of Human Leishmania Infection www.plosntds.org fPPG inoculated during sand fly bite causes substantial disease exacerbation in mice [6]. fPSG facilitates early parasite establishment by two mechanisms, macrophage recruitment to the infection site and enhancement of alternative macrophage activation, which upregulates arginase activity and promotes amastigote growth [69]. Early leukocyte recruitment to the infection site is detected in vivo from 40 min to 6 h [17] and in vitro after 4 h; the effect of enhanced alternative macrophage activation takes 24-48 h to develop [69]. In mouse, induction of arginase mRNA peaks at day 3 post-stimulation [70] and in man, leukocyte arginase is constitutively expressed only in granulocytes, independently of proinflammatory or anti-inflammatory stimuli [71]. In addition, neither PSG nor sand fly saliva appear to affect macrophage phagocytosis of L. mexicana metacyclic promastigotes [69]. We did not have access to PSG material and thus did not examine PSG activity in our system. We therefore cannot assert that PSG does not affect Leishmania early infection; nevertheless, the data cited above strongly suggest that PSG and bioactive salivary components exert their effects at a later stage of early infection reactions. Future experiments will help clarify the PSG effect on Leishmania ex vivo blood infection.
The most effective innate mechanism against pathogens is said to be denial of access [72]. Leishmania has developed an extremely rapid and effective infection strategy, and the prospect of blocking initial promastigote access to a host seems highly improbable. Research efforts should focus on development of therapeutic approaches to prevent Leishmania establishment of permanent infection through enhancement of macrophage leishmanicidal mechanisms. Figure S1 Leukocyte binding of promastigotes opsonized with NHS or heat-inactivated serum. Aliquots (50 ml) aliquots of heparinized blood were centrifuged (12006g, 10 min, 20uC) to separate plasma from cells. Cells were washed twice by centrifugation in PBS and adjusted to the initial blood volume with NHS or heat-inactivated serum (56uC, 60 min; HIS). Reconstituted blood aliquots were incubated (37uC, 5 min) with CMFDA-labeled L. amazonensis promastigotes, and leukocytepromastigote interaction measured as in Fig. 2. Results are expressed as percent (mean 6 SEM) of granulocytes (&) and monocytes (%) that bound promastigotes. Data are derived from three experiments, with blood of different donors. Found at: doi:10.1371/journal.pntd.0000743.s001 (0.06 MB TIF) Figure S2 Time-course of PI uptake by L. amazonensis promastigotes incubated with different concentrations of NHS. Single aliquots (0.3 ml) containing L. amazonensis promastigotes (2610 5 ), 10 mg/ml PI, and serially diluted (50% to 0.78%) NHS were incubated (37uC) for 1 to 9 min. PI uptake by parasites was measured in real-time flow cytometry (FACSCalibur). Promastigotes were identified and gated by SSC vs. FSC. PI emission was measured in a dot plot of FL-2 (585/42 nm) vs. time (sec) and data were analyzed with CELLQuest software (Becton Dickinson). Time-course of percent promastigote PI uptake in different NHS concentrations: 50% ( N ), 25% (#), 12.5% (.), 6.25% (n), 3.12%