Malarial infection is initiated when the sporozoite form of the Plasmodium parasite is inoculated into the skin by a mosquito. Sporozoites invade hepatocytes in the liver and develop into the erythrocyte-infecting form of the parasite, the cause of clinical blood infection. Protection against parasite development in the liver can be induced by injection of live attenuated parasites that do not develop in the liver and thus do not cause blood infection. Radiation-attenuated sporozoites (RAS) and genetically attenuated parasites are now considered as lead candidates for vaccination of humans against malaria. Although the skin appears as the preferable administration route, most studies in rodents, which have served as model systems, have been performed after i.v. injection of attenuated sporozoites. In this study, we analyzed the early response to Plasmodium berghei RAS or wild-type sporozoites (WTS) injected intradermally into C57BL/6 mice. We show that RAS have a similar in vivo distribution to WTS and that both induce a similar inflammatory response consisting of a biphasic recruitment of polymorphonuclear neutrophils and inflammatory monocytes in the skin injection site and proximal draining lymph node (dLN). Both WTS and RAS associate with neutrophils and resident myeloid cells in the skin and the dLN, transform inside CD11b+ cells, and induce a Th1 cytokine profile in the dLN. WTS and RAS are also similarly capable of priming parasite-specific CD8+ T cells. These studies delineate the early and local response to sporozoite injection into the skin, and suggest that WTS and RAS prime the host immune system in a similar fashion.

Malaria is one of the most deadly infectious diseases in the world and is responsible for nearly 700,000 deaths per year. Infection starts when Plasmodium sporozoites are injected into the host skin by a mosquito. Sporozoites, which are highly motile, rapidly reach the liver and invade hepatocytes (1, 2). Each sporozoite multiplies intracellularly, generating thousands new differentiated parasites, called “merozoites.” Merozoites are released from hepatocytes into the blood and invade erythrocytes, thus initiating the cycles of parasite intraerythrocytic replication, during which one merozoite generates 10–30 new merozoites. In contrast to the erythrocytic phase, which causes the symptoms of the disease, the preerythrocytic phase is asymptomatic and rapid. Sporozoites reach hepatocytes from the skin in less than 2 h and multiply into the first generation of merozoites in ∼50–60 h in rodents and ∼7 d in humans.

The preerythrocytic phase of Plasmodium infection is an attractive target for malarial control, primarily because small numbers of sporozoites, typically <100, are transmitted during a mosquito bite (3, 4). Numerous studies have shown that injection of parasites that do not complete preerythrocytic development, and thus do not cause blood infection, can protect against a challenge with infectious sporozoites. In rodents, this is the case of radiation-attenuated sporozoites (RAS) (5) or genetically arrested parasites (GAPs) (610). CD8+ T cells that target infected hepatocytes are the primary effectors of RAS-induced protection (1015); other effectors, including Abs to the sporozoite surface (16, 17) and CD4+ T cells (18), also play a role. In the last 10 y, efforts have been made to translate the attenuated sporozoite model in rodents into an effective vaccination method in humans, and various RAS and GAP formulations have entered clinical trials (1921).

In rodents, most RAS and GAP studies have used i.v. injection of sporozoites, in line with the long-held view that inoculated sporozoites eventually reach the blood and the liver. However, intravital imaging of Plasmodium berghei sporozoites injected into the skin of rodents has shown that the majority of the inoculated sporozoites do not reach the bloodstream; ∼60% stay in the skin, where they can invade and develop inside skin cells (22, 23), and ∼15% actively reach and remain in the proximal draining lymph node (dLN)—that is, only the remaining 25% reach the blood and the liver (1). Injection of RAS into the skin of mice was found to induce a high level of protection (24), via the priming of parasite-specific protective CD8+ T cells in the skin dLN rather than lymph nodes in the liver (25, 26). However, the protective efficacy of RAS immunization in the skin of mice remains unclear. One study found a lower efficacy of intradermal compared with i.v. immunization with P. berghei RAS (27), but other studies using Plasmodium yoelii RAS (24) reported similar protection levels for intradermal and i.v. immunizations. Whether the number of RAS that reach the liver might be sufficient to induce protective responses remains uncertain. The data from Chakravarty et al. (25) clearly indicate that this number is insufficient to induce a CSP-specific CD8+ T cell response. It remains possible, however, that it might be sufficient for inducing protective responses directed at non-CSP Ags.

In humans, most protection studies followed injection of attenuated parasites into the skin by mosquitoes. Early research with Plasmodium falciparum RAS showed that volunteers could be immunized by bites from more than 1000 irradiated infected mosquitoes (28), whereas volunteers exposed three times to 15 infectious mosquito bites and kept under chloroquine cover (to inhibit blood infection) were also protected for a prolonged period (29, 30). More recently, P. falciparum RAS dissected out from mosquito salivary glands and cryopreserved were tested in protection. Five i.v. injections of >105 RAS protected all six tested volunteers against a challenge by bites of five mosquitoes (31). Conversely, immunization in the skin was not found to be protective. In this case, however, the infectivity of cryopreserved sporozoites was clearly not optimal, because 3–4-fold as many cryopreserved sporozoites as compared with freshly isolated RAS were required to achieve a similar level of protection in mice (32).

In this study, we aimed to characterize the host immune response to injection of Plasmodium sporozoites in the skin. Using the P. berghei–C57BL/6 mouse model, we undertook a systematic analysis of the early immune response to intradermal injection of sporozoites in the ear pinna (skin) and of the associated leukocyte-sporozoite interactions. We successively characterized, both in the skin injection site and the proximal auricular dLN, the leukocytes that are recruited, those that associate with sporozoites, the type of host cell-parasite interaction, and the cytokine-chemokine profile in the dLN 24 h and 72 h after injection. Throughout the study, we compared normal and irradiated sporozoites.

Female C57BL/6 mice (5–7 wk old) were purchased from Janvier Laboratories. Female OT-I transgenic mice (6–8 wk old) (PtprcaPepcb/BoyJ [CD45.1] and Tg [TcraTcrb] 1100Mjb [OT-I Rag+/+]) were obtained from The Jackson Laboratory (Bar Harbor, ME) and bred in the Immunobiologie des Cellules Dendritiques unit, Pasteur Institute. All experiments using rodents were approved by the committee of the Pasteur Institute and by the local Ethics Committee on Animal Experimentation (Ethical committee IDF-Paris 1, Paris, France; agreement number: 2012-0015) and performed in accordance with the applicable guidelines and regulations.

Anopheles stephensi (Sda500 strain) mosquitoes were reared at the Centre de Production et d’Infection des Anopheles (CEPIA) at the Pasteur Institute using standard procedures. Mosquitoes were fed on infected mice 3–5 d after emergence and kept as described previously (1, 33). Infected mosquitoes used for transmission experiments (days 18–22 after the infectious blood meal) were deprived of sucrose for 1–2 d before experimentation to enhance the mosquito bite rate.

We used the P.berghei ANKA clone expressing GFP under the control of the hsp70 promoter (34), the P.berghei ANKA clone 676cl1 (Pb GFP-LUCsch) expressing a GFP-luciferase fusion gene via the EEF1α promoter (35), the P. berghei ANKA clone deficient for SPECT2 (Sporozoite micronemal protein essential for cell traversal 2) and expressing GFP under the control of the hsp70 promoter (SPECT2F) (36), and the P.berghei ANKA clone PbA-hsOVA expressing class I and class II OVA epitopes (37).

HepG2 cells, a human hepatoma cell line, were cultured in DMEM plus 10% FBS and maintained at 37°C with 5% CO2. For cell infection, hepatocytes were plated at a density of 5 × 104 in 96-well plates, flat bottom (Falcon) for FACS analysis, and in eight-well μ-slides (Ibidi) for imaging in DMEM plus 10% FBS.

J774 cells, a murine macrophage cell line, were cultured in DMEM plus 10% FBS and 1% MEM nonessential amino acids (Life Technologies) and maintained at 37°C with 5% CO2. For cell infection, J774 macrophages were plated at a density of 4 × 104 in 2 × 9 well μ-slides (Ibidi) in DMEM plus 10% FBS.

XB2 cells, a mouse teratoma keratinocyte cell line, were cultured in DMEM plus 20% FBS and maintained at 37°C with 5% CO2. For cell infection, XB2 cells were plated at a density of 2 × 104 in 2 × 9 well μ-slides in DMEM plus 10% FBS.

Bone marrow–derived macrophages (BMDM) were derived from 8–12-wk-old C57BL/6 mice, as described previously (38). Cells were maintained at 37°C with 5% CO2. For cell infection, BMDM were plated at a density of 4 × 104 in 2 × 9 well μ-slides (Ibidi) or 5 × 105 in eight-well LAB-TEK μ-slides (Nunc; for transmission electron microscopy experiments) in DMEM plus 10% FBS supplemented with 20% L-929 fibroblast-conditioned medium.

The reagents and Abs used in this study were as follows: collagenase from clostridium histolyticum, Type VIII, 0.5–5.0 FALGPA units/mg solid (Sigma-Aldrich); DNase I from bovine pancreas, type IV (Sigma-Aldrich); Liberase TM Research Grade (Roche), Dextran tetramethylrhodamine 10,000 MW, lysine fixable (fluoro-ruby; Molecular Probes/Invitrogen); Anti-upregulated in infectious sporozoites gene 4 (UIS4) polyclonal Ab-AlexaFluor 546 conjugate (Molecular Probes); DAPI (Molecular Probes); anti-mouse CD16/CD32 (2.4G2 clone), CD45 (Ly5 clone), CD11b (M1/70 clone), Ly6G (1A8 clone), Ly6C (AL-21 clone) and CD11c (HL3 clone; BD Biosciences); anti-mouse MHCII, I-A/I-E (M5/114.15.2 clone; eBioscience), Beetle Luciferin (D-luciferin) Potassium Salt (Promega), Life Technologies penicillin (10,000 U/ml)–streptomycin (10,000 μg/ml), Liquid (Invitrogen); Chloroquine (Sigma Aldrich), Recombinant RNAsin RNase Inhibitor (Promega); DNAse I RNAse free (Roche); and Superscript II reverse transcriptase (Invitrogen).

RAS were obtained by irradiation of infected mosquitoes at a dose of 12 krad of γ-irradiation (137Cs source; Institut Pasteur). Mosquitoes kept on ice in a Petri dish were exposed for 69 min at room temperature.

Sporozoites were isolated from infected salivary glands 18–22 d after the infectious blood meal and kept on ice in 1× DPBS.

For intradermal injection, anesthetized mice were injected into the dorsal ear dermis with 1× DPBS, salivary gland extracts (SGE) from uninfected mosquitoes, or 5 × 104 dissected out salivary gland sporozoites in a small volume (0.6 μl) using a 35-gauge needle with a NanoFil syringe (World Precision Instruments) (39). A characteristic papule was observable at the injection site at the end of the operation.

All infection assays were performed at a multiplicity of infection of 0.5, except for transmission electron microscopy experiments (multiplicity of infection of 1). GFP wild-type sporozoites (WTS) or RAS sporozoites freshly dissected in 1× DPBS were added onto plated cells. Culture medium was supplemented with 10% FBS, 100 U/ml penicillin, and 100μg/ml streptomycin. Cells were then centrifuged (500 × g, 5 min, 4°C) and placed at 37°C with 5% CO2 for different times.

For gliding motility assays, GFP WTS or RAS sporozoites freshly dissected in PBS plus 10% FBS were deposited on 18-well flat μ-slides (Ibidi). Parasite gliding activity was recorded by time-lapse microscopy for 5-min periods with intervals of 2 min and for a total of 33 min on an AxioObserver Z1 fluorescence microscope (AxioVision 4.8.2.0; Zeiss). Image files were processed using ImageJ software, version 10.2.

For membrane disruption assays, 1 mg/ml dextran was added to plated HepG2 cells immediately prior to sporozoite addition. SPECT2F parasite mutants were used as a negative control for cell traversal activity. Cells that were mechanically disrupted by scraping cell monolayer with a tip in the presence of 1 mg/ml dextran were used a positive control (40). After centrifugation, cells were incubated for 1.5 h at 37°C. After washing with DPBS, cells were incubated with trypsin for 5 min at 37°C and collected in PBS plus 10% FBS. Cells were then centrifuged (500 × g, 5 min, 4°C) and resuspended in PBS plus 2% FBS before aquisition on a Becton Dickinson FACSCalibur. Data were analyzed with FlowJo version 7.6 (Tree Star).

HepG2 infected cells were imaged 24, 48, and 72 h after contact with parasites, before being processed and analyzed by FACS, as described for the cell traversal and cell invasion assay.

At 5-min, 30-min, 60-min, and 24-h incubation times, infected BMDM cells were kept on ice and CD11b labeled. The percentage of CD11b+GFP+ was determined with a Becton Dickinson FACSCalibur. Data were analyzed with FlowJo version 7.6.

At 24- and 48-h incubation times, J774- and XB2-infected cells were fixed with 4% PFA, labeled with anti-UIS4 Ab (RD-Biotech, Besançon, France) and stained with DAPI.

For the different cell types, imaging of developing parasites was performed with an AxioObserver Z1 fluorescence microscope (AxioVision 4.8.2.0, Zeiss). Image files were processed using ImageJ version 10.2.

To label lysosomes, BMDM were incubated with 16 nm BSA-gold particles for 15 min, followed by a 60-min chase at 37°C (41, 42). BSA gold-loaded BMDM were coincubated with parasites for 60 min and fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer at 4°C for 24 h. After several washes in 0.1 M cacodylate buffer, samples were postfixed for 1 h in the same buffer containing 1% osmium tetroxide (Merck, Darmstadt, Germany). After dehydration in a graded ethanol series, the samples were embedded in Epon resin. Contrasted ultrathin sections (60 nm) were observed with a JEM 1010 transmission electron microscope (Jeol, Tokyo, Japan). A fusion event between lysosomes and phagosomes was based on the presence of one or more gold particles inside phagosomes. At least 100 phagosomes were analyzed for each sample.

In vivo imaging of developing GFP WTS or RAS parasites 24, 48h, 72h and 96h postinjection (p.i.) in the ear of anesthetized mice was performed using a high-speed spinning disk confocal system (Ultraview ERS; PerkinElmer) mounted on an inverted Axiovert 200 microscope (Carl Zeiss) with Optovar option linked to an Orca II ER camera (Hamamatsu, Japan). Image files were processed using ImageJ version 10.2.

For bioluminescence assays, mice were injected i.p. with 100 μl of D-luciferin (150 mg/kg) and anesthetized with 2% isoflurane at different times after inoculation of GFP-LUCsch WTS or RAS. After 5 min, allowing the distribution of D-luciferin in the whole body, mice were transferred to the stage of the intensified charge-coupled device photon-counting video camera. Anesthesia was maintained with 2% isoflurane delivered with nose cones. Imaging was performed using the IVIS Lumina system (Xenogen). As previously described (43), the bioluminescent signal emitted from infected sites is superimposed on a grayscale photograph of the mouse. Relative levels of bioluminescence are depicted as a pseudocolor display, with red and blue representing the highest and lowest amounts of photon flux. After applying a minimum threshold value to the image, a region of interest was manually defined around the light projected from the ear and from the liver. The non-injected ear of injected mice and liver from non-injected mice were used as a negative control of infection. Analysis was performed using the Living Image software (Xenogen Corporation). The level of bioluminescence is expressed on average radiance (photons/second/cm2/steradian). For mice followed up to day 10, a daily chloroquine treatment (40 mg/kg) was established starting from day 1 before sporozoite injection.

dLNs were harvested using minimally invasive surgery 1.5 h after inoculation of GFP WTS or RAS, and they were dehydrated in PBS plus 10% sucrose before being embedded in OCT. Ten-micrometer sections were cut on a CM3050S cryostat (Leica) and adhered to Superfrost Plus Slides (VWR). Sections were observed using an AxioObserver Z1 fluorescence microscope (AxioVision 4.8.2.0; Zeiss). Image files were processed using ImageJ version 10.2.

dLNs were harvested 1.5 h after inoculation of GFP WTS or RAS to quantify the parasite load. Tissues were softly homogenized in 1 ml of cold Trizol using the 2-ml Precellys tubes/Precellys24 tissue homogenizer (6500 rpm, 2 min). Samples were then centrifuged (12,000 rpm, 2 min, at room temperature) and supernatants were treated for RNA extraction (chloroform) and precipitation (isopropanol). Samples were then treated with Recombinant RNAsin RNase Inhibitor (40 U/μl) and DNAse I RNAse-free (10 U/μl) before precipitation. Transcripts from the 18s gene of the P. berghei parasite were targeted specifically for detection using SuperScript III Platinum One-Step quantitative real-time PCR (QRT-PCR) (Invitrogen) with: Forward primer (5′-CGTTTATGTGGGCATATTGA-3′); reverse primer (5′-CATTAAATAAAGCGAATACATCC-3′); probe (5′-6FAM-TGATTCTTTCGTTTACGACATGCCT-BHQ1-3′; Eurogentec). To facilitate comparison between samples, the gene coding for murine GAPDH was used as a reference: Forward primer (5′-TGTGTCCGTCGTGGATCTGA-3′); reverse primer (5′-CCTGCTTCACCACCTTCTTGAT-3′); probe (5′-6FAM-CCGCCTGGAGAAACCTGCCAAGTAT-BHQ1-3′; Eurogentec).

PCR conditions were: one cycle at 50°C for 20 min, one cycle at 95°C for 2 min, and 40 cycles at 95°C for 15 s and at 60°C for 45 s). The detection was performed with a Chromo 4 machine (Bio-Rad Laboratories). Analysis was performed using the comparative ∆Ct method.

For microfluidic-based quantitative PCR analysis, dLNs were harvested 24 h after inoculation of GFP WTS or RAS. Tissues were softly homogenized by TissueLyser-II (Qiagen) in RLT buffer, and RNA was purified using the RNeasy Mini kit with DNase treatment (Qiagen) according to the manufacturer’s instructions. cDNA was generated using ReverAid Reverse Transcriptase (Fermentas) with random primers. cDNA and gene-specific probe and primer pairs (Solaris qPCR gene Expression Assays; ThermoFisher) were loaded onto a 48.48 Dynamic Array Chip (Fluidigm) according to the manufacturer’s protocol, and the chip was processed using the BioMark HD system (Fluidigm). RNA samples with null values in >25% of all genes tested, measurements greater than 3 SD from the mean within a gene, and those with a difference of ≥1 ∆CT between duplicates were excluded from the analysis. Eliminated samples accounted for <5% of all measurements. To facilitate comparison between samples, the gene coding for murine Hprt was used as a reference. Analysis was performed using the comparative 2−∆∆Ct method. The full list of genes analyzed for BioMark analysis is provided in Table I.

Table I.
Cytokine profiling based on multiplex RT-qPCR analysisa
GenesSGE/PBSGenesSGE/PBS
CXCL11 17.1 ± 7.1 IL-18 −0.3 ± 0.1 
IFN-γ 5.3 ± 2.4 CD43 −0.4 ± 0.5 
CXCL-10 5.1 ± 2.4 β-actin −0.6 ± 0.5 
Granzyme B 3.8 ± 1.3 TNF-α −0.7 ± 0.6 
CXCL-9 3.7 ± 1.8 IL-10 −1.4 ± 0.9 
IL-2βR 3.1 ± 0.6 Tbp −1.4 ± 0.4 
TGF-β 3 ± 0.5 IL-1α −1.5 ± 0.7 
IL-2 2.6 ± 0.6 Gusβ −1.7 ± 0.4 
CD69 2.2 ± 0.6 IL-22 −2.9 ± 1.9 
Perforin 1.8 ± 0.5 IFN-α2 ND 
IL-2αR 1.7 ± 0.3 IL-21 ND 
IL-6 1.6 ± 0.4 IFN-β ND 
IL-12 1.5 ± 0.2 IL-23 ND 
FasL 1.4 ± 0.3 IL-17A ND 
TRAIL 1.4 ± 0.3 CCL20 ND 
IL1β 1.3 ± 0.4 Lta ND 
Tfrc 1.3 ± 0.2 IL-9 ND 
Smad3 1.1 ± 0.1 IL-13 ND 
CD27 1.1 ± 0.2 IL-5 ND 
IL-33 1.1 ± 0.2 IL-4 ND 
CCR7 1.1 ± 0.1 IL15 ND 
CD28 1.1 ± 0.1 IL-27 ND 
STAT3 1 ± 0.1 Hprt Reference 
Foxp3 1 ± 0.1   
GenesSGE/PBSGenesSGE/PBS
CXCL11 17.1 ± 7.1 IL-18 −0.3 ± 0.1 
IFN-γ 5.3 ± 2.4 CD43 −0.4 ± 0.5 
CXCL-10 5.1 ± 2.4 β-actin −0.6 ± 0.5 
Granzyme B 3.8 ± 1.3 TNF-α −0.7 ± 0.6 
CXCL-9 3.7 ± 1.8 IL-10 −1.4 ± 0.9 
IL-2βR 3.1 ± 0.6 Tbp −1.4 ± 0.4 
TGF-β 3 ± 0.5 IL-1α −1.5 ± 0.7 
IL-2 2.6 ± 0.6 Gusβ −1.7 ± 0.4 
CD69 2.2 ± 0.6 IL-22 −2.9 ± 1.9 
Perforin 1.8 ± 0.5 IFN-α2 ND 
IL-2αR 1.7 ± 0.3 IL-21 ND 
IL-6 1.6 ± 0.4 IFN-β ND 
IL-12 1.5 ± 0.2 IL-23 ND 
FasL 1.4 ± 0.3 IL-17A ND 
TRAIL 1.4 ± 0.3 CCL20 ND 
IL1β 1.3 ± 0.4 Lta ND 
Tfrc 1.3 ± 0.2 IL-9 ND 
Smad3 1.1 ± 0.1 IL-13 ND 
CD27 1.1 ± 0.2 IL-5 ND 
IL-33 1.1 ± 0.2 IL-4 ND 
CCR7 1.1 ± 0.1 IL15 ND 
CD28 1.1 ± 0.1 IL-27 ND 
STAT3 1 ± 0.1 Hprt Reference 
Foxp3 1 ± 0.1   
a

Average fold increase in gene expression of 46 molecules in the dLN of mice 24 h after intradermal injection of SGE. Relative changes in gene expression were calculated using the 2−ΔΔCT method. The results are presented as the average fold increase or decrease of target genes ± SEM in mice injected with SGE, normalized to the Hprt internal control gene, and compared with PBS injected mice. Genes with significant transcriptional upregulation (>2-fold increase) are in boldface (mean ± SEM; 10 mice; n = 3).

ND, not detectable genes.

dLNs harvested 24 and 72 h after inoculation of GFP WTS or RAS were softly homogenized in 1 ml of cold 1× PBS using the 2 ml Precellys tubes/Precellys24 tissue homogenizer (6500 rpm for 2 min). Samples were then centrifuged (12,000 rpm, 2 min, at room temperature) and supernatants were analyzed for their CXCL9, CXCL10, granzyme B, and IFN-γ content with ELISA, according to the manufacturer’s instructions (R&D Systems).

In vivo depletion of neutrophils was achieved after two i.p. administrations of 0.5 mg anti-Ly6G (clone 1A8; BioXcell) or isotype control (clone 2A3; BioXcell) monoclonal Abs on days 5 and 3 preceding intradermal immunization (44, 45).

At different times after inoculation of GFP WTS or RAS, infected ears and dLNs were collected and digested using a collagenase (400 U/ml) and DNAse (50 μg/ml) treatment at 37°C during 1 h and 15 min respectively. Tissues were then ground on a 70-μm cell strainer to obtain a single-cell suspension. Fc receptors were blocked using the CD32/CD16 Ab, and cells of interest were labeled using the following markers: DAPI, CD45, CD11b, Ly6G, and Ly6C. Samples were then run on a CANTO II (BD Biosciences), and data were analyzed with FlowJo version 7.6.

To identify parasites associated with cell subtypes in the skin and the dLNs, collected tissues at different times were digested using a collagenase treatment and processed as described in the previous section. Cells subpopulations were identified using the CD11b, Ly6G, and Ly6C markers. Acquisition and sorting of CD11b+ cells associated with GFP+ parasites were performed on a MoFlo Astrios (Beckman Coulter), data were analyzed with Summit software version 6.0.4.

To characterize the nature of the parasite-CD11b+ cell interaction, sorted skin cells underwent cytospinning onto glass slides (2000 rpm for 5 min; Cytospin 4; ThermoFisher Scientific) and were fixed with 4% PFA and stained with DAPI. Vectashield mounting medium was used to prevent photobleaching. The preparation was then examined by fluorescence microscopy (AxioObserver Z1, AxioVision 4.8.2.0; Zeiss)

Different amounts of OT-I CD8+ T cells derived from CD45.1 OT-I/Rag-1 mice (stained with CFSE) were adoptively transferred into mice immunized the same day with 1 × 104 WTS or RAS. Four days p.i., dLNs were harvested and labeled with CD8α and CD45.1 Abs, allowing the enrichment of transferred CD8 OT-I T cells by using anti-CD45.1 and CD8 beads. T cell proliferation (CFSE dilution) was measured by flow cytometry (CANTO II; BD Biosciences), and the decrease in CFSE fluorescence intensity and data were analyzed with FlowJo version 7.6.

Splenocytes from naive female CD45.1 C57BL/6 mice were used as in vivo target cells. They were depleted of RBCs and divided into two equal portions. One portion was pulsed for one hour in 1:1000 dilution of SIINFEKL (10 mg/ml) at 37°C and then labeled with a high concentration (5 μM) of CFSE (CFSEhigh). As a control, the other portion was incubated for 1 h at 37°C without peptide and labeled with a low concentration (0.5 μM) of CFSE (CFSElow). After labeling and peptide pulsing, both populations of target cells were washed and mixed 1:1 (1 × 106 total cells) and then injected i.v. into mouse recipients treated daily with 40 mg/kg chloroquine (starting from day 1 before sporozoite injection) to avoid blood infection. A deviation from the 1:1 ratio when the mice are sampled indicates specific lysis of the cells with the relevant epitope on the surface. Twenty-four hours p.i., target cells from the local dLN were collected, stained with an anti-CD45.1 Ab, and measured with flow cytometry. Data were analyzed with FlowJo version 7.6, and the calculation of OVA-specific lysis was determined using the following formula (46):

Percentage of specific kill = (1 −– [Ratio unprimed / Ratio primed) × 100)

where

Ratio = Percentage of CFSElow / Percentage of CFSEhigh

Mice treated with the 1A8 specific mAb or the 2A3 isotype control mAb were immunized in the ear pinna dermis three times at 2-wk intervals with ∼5 × 104 RAS; 1.5 wk after the last immunization, the animals were subjected to a dose of ∼3 × 104 WTS in the footpad. The percentage of protected animals of the total number of immunized mice was assessed with flow cytometry and confirmed with blood smear.

Statistical significance was determined with two-tailed Student t test (Microsoft Excel).

We first compared the infectivity of P. berghei WTS and RAS. In vitro, WTS and RAS displayed comparable gliding motility, host cell traversal, and host cell invasion (Supplemental Fig. 1A–E) capacities. WTS and RAS generated similar numbers of exoerythrocytic forms (EEFs) developing inside hepatoma cells at days 1 and 2 p.i. (Supplemental Fig. 1F). Although WT EEFs progressively increased in size and liberated merozoites at day 2, irradiated EEFs remained small, and their development was blocked (Supplemental Fig. 1G, 1H), as described previously (47).

We next compared the in vivo fate of WTS and RAS inoculated in the skin of C57BL/6 mice. Approximately 5 × 104 sporozoites of the P. berghei GFP-LUC line (35) were injected intradermally and followed by bioluminescence (Fig. 1A). We measured the parasite load immediately after sporozoite deposition (day 0) to control the quality of injection. WT parasites generated a bioluminescent signal peaking at days 2 and 3 in the liver and in the skin, respectively, followed by a diffuse signal of increasing intensity from day 3 onward because of merozoite infection of RBCs. Irradiated parasites induced signals in both the skin and the liver similar to those of WTS at day 1. Signals persisted in the skin beyond day 4, but they rapidly disappeared from the liver. To extend the analysis of bioluminescent signals in the skin, mice were treated with chloroquine, which blocked the diffuse signal emitted by blood-stage parasites without compromising EEF development (Fig. 1B, 1C). At day 2, when WT but not irradiated parasites have multiplied extensively, the bioluminescence signals emitted in the liver by WT EEFs was ∼100-fold higher than that of irradiated EEFs. In the skin, the intensity of the signal caused by both WTS and RAS initially dropped between days 0 and 1, corresponding to sporozoite egress from the skin, clearance by phagocytic cells, or both (Fig. 1C). Between days 1 and 3, the signals obtained from WT and irradiated parasites increased 4-fold and 1.5-fold, respectively, before progressively diminishing until day 10 to reach background levels (Fig. 1C). The possible persistence of some parasites beyond day 10 in the skin or the liver, WT or irradiated, is beyond the sensitivity of the method, particularly in the deep liver organ.

FIGURE 1.

Fate of P. berghei sporozoites in the liver, the skin, and the dLN after intradermal injection. (A) Development of WTS and RAS parasites in the skin and the liver of mice measured by bioluminescence. Mice injected intradermally with ∼5 × 104 WTS or RAS GFP-LUCSCH parasites were monitored daily for luciferase activity. (B and C) To avoid the development of blood infection in the WTS group, animals were treated with chloroquine (40 mg/kg). Average radiance (photons/s/cm2/steradian) of bioluminescent signal emitted from WTS or RAS parasites in the liver (B) compared with naive animals and in the skin (C) compared with the contralateral (Cont.) noninjected ear of mice treated with chloroquine (CQ; mean ± SD, 10 mice per condition with or without CQ; n = 3). (D) Confocal images of developing WTS and RAS GFP-expressing parasite in the skin of mice day 1–4 p.i. (parasites are green, and autofluorescence is shown in red). Images are maximal projections of 30–70 contiguous pictures separated by 5 μm (scale bar, 20 μm). (E) Cumulative numbers of WTS and RAS developing parasites (brightly fluorescent EEF) after intradermal injection at four injection sites and (F) diameter of WTS and RAS developing parasites in the skin, estimated by the EEF maximal projection area (mean ± SD, 12 mice per condition; n = 3). (G) Fluorescent microscopy of WTS and RAS sporozoites (green) within the subcapsular zone of the dLN 1.5 h p.i. (original magnification ×10; scale bar, 20 μm). (H) WTS and RAS parasite load in the dLN estimated by QRT-PCR 1.5 h p.i. Data are shown as ΔCT values relative to GAPDH internal control gene. Each point corresponds to one mouse (n = 3). **p < 0.01, ***p < 0.001.

FIGURE 1.

Fate of P. berghei sporozoites in the liver, the skin, and the dLN after intradermal injection. (A) Development of WTS and RAS parasites in the skin and the liver of mice measured by bioluminescence. Mice injected intradermally with ∼5 × 104 WTS or RAS GFP-LUCSCH parasites were monitored daily for luciferase activity. (B and C) To avoid the development of blood infection in the WTS group, animals were treated with chloroquine (40 mg/kg). Average radiance (photons/s/cm2/steradian) of bioluminescent signal emitted from WTS or RAS parasites in the liver (B) compared with naive animals and in the skin (C) compared with the contralateral (Cont.) noninjected ear of mice treated with chloroquine (CQ; mean ± SD, 10 mice per condition with or without CQ; n = 3). (D) Confocal images of developing WTS and RAS GFP-expressing parasite in the skin of mice day 1–4 p.i. (parasites are green, and autofluorescence is shown in red). Images are maximal projections of 30–70 contiguous pictures separated by 5 μm (scale bar, 20 μm). (E) Cumulative numbers of WTS and RAS developing parasites (brightly fluorescent EEF) after intradermal injection at four injection sites and (F) diameter of WTS and RAS developing parasites in the skin, estimated by the EEF maximal projection area (mean ± SD, 12 mice per condition; n = 3). (G) Fluorescent microscopy of WTS and RAS sporozoites (green) within the subcapsular zone of the dLN 1.5 h p.i. (original magnification ×10; scale bar, 20 μm). (H) WTS and RAS parasite load in the dLN estimated by QRT-PCR 1.5 h p.i. Data are shown as ΔCT values relative to GAPDH internal control gene. Each point corresponds to one mouse (n = 3). **p < 0.01, ***p < 0.001.

Close modal

Next, WTS or RAS of the P. berghei line that constitutively expresses GFP (34) were injected intradermally in the skin and imaged by intravital confocal microscopy over time (Fig. 1D–F). Similar numbers of round fluorescent parasites were observed at day 1 in the skin of mice inoculated with WTS or RAS, indicating a normal capacity of RAS to invade skin cells (Fig. 1D), as previously described for WTS (22). In both cases, the numbers of skin EEFs progressively decreased with time (Fig. 1E). However, whereas WT EEFs rapidly increased in size until day 3 to give rise to merosomes (22), irradiated EEFs remained round-shaped, small, and regular (Fig. 1D, 1F).

To test the capacity of RAS to reach the proximal auricular dLN actively from the skin inoculation site with their own motility, GFP+ WTS or RAS were injected in the skin, and dLN cryosections were prepared 1.5 h p.i. (when sporozoite motility has stopped) and examined by confocal microscopy. Like WTS, RAS were mainly located in the subcapsular zone of the dLN (Fig. 1G). Quantitative RT-PCR analysis indicated that similar amounts of WTS and RAS were present in the dLN 1.5 h p.i. (Fig. 1H). We conclude that RAS and WTS have similar tissue distributions after inoculation in the skin.

We then investigated the sequence of local early inflammatory responses in the skin and the proximal auricular dLN following injection of sporozoites in the skin, starting with WTS. WTS collected from mosquito salivary glands, or the SGE from the same numbers of uninfected salivary glands, were injected intradermally. Tissues from the skin injection site and dLN were collected at different times p.i., digested to obtain a single-cell suspension, labeled, and analyzed by flow cytometry. Myeloid populations were identified as CD45+CD11b+ cells and further differentiated into neutrophils (Ly6CintLy6Ghi) and inflammatory monocytes (Ly6ChiLy6G).

In the skin (Fig. 2A, 2B, top panels), the numbers of neutrophils increased to similar levels 2 h after inoculation of WTS or SGE compared with naive mice, indicating that needle injection and mosquito material caused rapid neutrophil infiltration at the injury site. At 4 h, however, the numbers of neutrophils further increased in WTS-injected mice and decreased in SGE-injected mice to levels in naive mice, showing a sustained neutrophil recruitment specifically caused by the parasite. At 24 h, the number of neutrophils in WTS-injected mice decreased, while remaining higher than those in SGE-injected or naive mice. In parallel, whereas the numbers of inflammatory monocytes were similar in all animals at early time points, they increased sharply, specifically in WTS-injected mice at 24 h. In the dLN (Fig. 2A, 2B, bottom panels), the numbers of neutrophils increased at 2 and 4 h after injection of WTS, but not SGE, and were back at control levels at 24 h. As in the skin, a sharp increase in the numbers of inflammatory monocytes was observed at 24 h in the dLN in WTS-injected but not SGE-injected animals. Therefore, WTS induced a two-wave inflammatory response with a sustained infiltration of neutrophils at early time points followed by a recruitment of inflammatory monocytes at 24 h in both the skin site and associated dLN. As shown in Fig. 2A and 2B, the inflammatory response triggered by RAS was similar to that induced by WTS, in both tissues and at all time points investigated.

FIGURE 2.

Local cellular responses after injection of P. berghei sporozoites into the skin. (A) Representative FACS plots of neutrophils (Ly6CintLy6Ghi, upper region) and inflammatory monocytes (Ly6ChiLy6G lower region) recruited in the skin and the dLN at different time points following intradermal injection of SGE, ∼5 × 104 WTS or RAS parasites. (B) Neutrophils and inflammatory monocytes absolute numbers at 2, 4, and 24 h p.i. (mean ± SEM, nine mice per condition per time point; n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2.

Local cellular responses after injection of P. berghei sporozoites into the skin. (A) Representative FACS plots of neutrophils (Ly6CintLy6Ghi, upper region) and inflammatory monocytes (Ly6ChiLy6G lower region) recruited in the skin and the dLN at different time points following intradermal injection of SGE, ∼5 × 104 WTS or RAS parasites. (B) Neutrophils and inflammatory monocytes absolute numbers at 2, 4, and 24 h p.i. (mean ± SEM, nine mice per condition per time point; n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

We next assessed the proportion of parasites in the skin and dLN that associated with myeloid CD11b+ cells (Fig. 3A–C) and FACS sorted them (Fig. 3D). GFP+ WTS were injected intradermally; single-cell suspensions were prepared from skin and dLN tissues at 4, 24, and 48 h p.i.; and GFP+ cells were analyzed with flow cytometry (Fig. 3A). In the skin, ∼20% of the total GFP+ cells at 4 h were CD11b+ (G2; Fig. 3A, 3B). The remainder of the GFP+ cells (G1, CD11b cells) presumably corresponded to parasites located inside nonprofessional phagocytes, such as keratinocytes or fibroblasts (22), or subpopulations of dermal dendritic cells (DCs) (48). At 24 and 48 h, the total number of GFP+ cells was 4-fold and 5-fold smaller than at 4 h, respectively, but the proportion of GFP+ cells that were CD11b+ was only slightly lower than at 4 h (∼15% compared with 20%; Fig. 3B). Therefore, although many cell-associated parasites disappeared between 4 and 24 h in the skin, as previously reported (22, 23), some skin parasites remained detectable in association with CD11b+ cells. In the dLN, most (∼80%) of the GFP+ cells were CD11b+ (Fig. 3C) at 4 h, and no significant fluorescent signal was detected at later time points. Similar results were obtained with RAS in the skin (Fig. 3B) and in the dLN (Fig. 3C). The GFP+CD11b+ cells at 4 h p.i. in the skin and dLN were then characterized using the Ly6C and Ly6G markers (Fig. 3E). In both tissues, ∼20% of the CD11b+GFP+ cells were neutrophils (Ly6CintLy6G+, gate R1) and ∼70% were resident myeloid cells (Ly6CLy6G, gate R2).

FIGURE 3.

Associations between P. berghei sporozoites and CD11b+ cells. FACS analysis, cell sorting, and fluorescence intensity of CD11b+ cells associated with GFP+ alive parasites in the skin and the dLN 4, 24, and 48 h after intradermal injection of ∼5 × 104 WTS or RAS parasites. (A) Representative FACS plots showing the gating strategy to analyze and sort CD11b+GFP+ cells in both tissues. GFP expressing cells were gated on total live cells and subsequently analyzed for their CD11b expression (G2) or not (G1). (B and C) Absolute numbers of CD11b+GFP and CD11b+GFP+ cells 4, 24, and 48 h p.i. in the skin and 4 h p.i. in the dLN (mean ± SEM; 8 ears and 8 dLN pooled per condition per experiment; n = 3). (D) Wide-field microscopy of sorted CD11b+ skin cells harboring GFP+ WTS or RAS parasite material at 4 h. (E) Representative FACS plots of myeloid cell subsets associated with GFP+ parasites in the skin and the dLN 4 h p.i. with neutrophils (R1), resident myeloid cells (R2), and inflammatory monocytes (R3).

FIGURE 3.

Associations between P. berghei sporozoites and CD11b+ cells. FACS analysis, cell sorting, and fluorescence intensity of CD11b+ cells associated with GFP+ alive parasites in the skin and the dLN 4, 24, and 48 h after intradermal injection of ∼5 × 104 WTS or RAS parasites. (A) Representative FACS plots showing the gating strategy to analyze and sort CD11b+GFP+ cells in both tissues. GFP expressing cells were gated on total live cells and subsequently analyzed for their CD11b expression (G2) or not (G1). (B and C) Absolute numbers of CD11b+GFP and CD11b+GFP+ cells 4, 24, and 48 h p.i. in the skin and 4 h p.i. in the dLN (mean ± SEM; 8 ears and 8 dLN pooled per condition per experiment; n = 3). (D) Wide-field microscopy of sorted CD11b+ skin cells harboring GFP+ WTS or RAS parasite material at 4 h. (E) Representative FACS plots of myeloid cell subsets associated with GFP+ parasites in the skin and the dLN 4 h p.i. with neutrophils (R1), resident myeloid cells (R2), and inflammatory monocytes (R3).

Close modal

The results indicated that some parasites in the skin remained viable in association with CD11b+ cells. To characterize the nature of the parasite-CD11b+ cell interaction, we imaged CD11b+GFP+ FACS-sorted cells extracted from the skin at various time points after infection. Cells were cytospun onto glass slides, fixed with 4% paraformaldehyde, stained with DAPI, and examined with fluorescence microscopy (Fig. 4A–C). Diverse types of CD11b+ cells were observed containing parasite material. At 4 h (Fig. 4A), intracellular GFP signals were frequently small, and more rarely larger (maximum diameter of 5μm) and brightly fluorescent. Surprisingly, some cells contained numerous individual fluorescent structures (up to eight), possibly following the uptake of multiple sporozoites by the same cell. At 24 and 48 h, most of the sorted cells harbored dim and diffuse GFP+ signals (Fig. 4A, 4C), suggesting that most of the parasites internalized by CD11b+ cells in the skin were degraded. However, a few bright GFP+ structures of up to 7 μm in size were still detected at these time points, mainly 24 h p.i. (Fig. 4B), confirming that some parasites survived inside these skin-isolated CD11b+ cells. RAS gave similar infection patterns (Fig. 4A, bottom panel, Fig. 4C), including brightly fluorescent EEFs at 24 h (Fig. 4B, bottom panel).

FIGURE 4.

In vivo and in vitro interactions between P. berghei sporozoites and phagocytic cells. (A) Wide-field microscopy of sorted skin cells harboring GFP+ WTS or RAS parasite material at 4, 24, and 48 h p.i. Cell nuclei are labeled with DAPI (original magnification ×100; scale bar, 5 μm). (B) Wide-field microscopy of sorted skin cells harboring bright WTS or RAS GFP+ parasite material beyond 24 h. Cell nuclei are labeled with DAPI (original magnification ×100; scale bar, 5 μm). (C) Box-and-whisker-plot showing the fluorescence intensity of cells containing GFP+ WTS or RAS intracellular parasite material (n = 18 events per condition and per timepoint). (D) Representative FACS plots of noninfected (NI) or infected CD11b+ BMDM with GFP+ WTS parasites. (E) Kinetic of association of CD11b+ BMDM with GFP+ WTS or RAS parasites 5, 30, and 60 min and 24 h p.i. (mean ± SD, n = 3). (F) Illustration by transmission electron microscopy of phagocytosis, cell traversal, and cell invasion events 1 h after addition of WTS or RAS to BMDM. Phagocytosis: BSA-gold positive compartment containing a degraded parasite (p; scale bar, 1 μm). Cell traversal: BSA-gold negative compartment containing an intact parasite in the nucleus (n; scale bar, 2 μm). Higher magnification: the parasite plasma membrane (PPM) and the inner membrane complex (IMC) were detectable, but not the PV membrane (PVM; scale bar, 500 nm). Cell invasion: BSA-gold negative compartment containing an intact parasite inside a PV (scale bar, 1 μm). Higher magnification: IMC, PPM, and PVM were detectable (scale bar, 500 nm). (G) Percentage of phagocytosis, cell traversal, and cell invasion events 1 h after WTS or RAS parasite contact with BMDM (100 parasites per condition). (H) Percentage of parasites inside a UIS4+ compartment 24 h after WTS or RAS contact with murine XB2 keratinocyte (30 events) or J774 murine macrophage cell-lines (15 events; representative data of thre independent experiments). (I) Wide-field microscopy of XB2 keratinocytes and J774 macrophages harboring WTS or RAS parasites (green) inside a UIS4+ compartment (red). Cell nuclei are labeled with DAPI (blue; original magnification ×40; scale bar, 10 μm). *p < 0.05, **p < 0.01.

FIGURE 4.

In vivo and in vitro interactions between P. berghei sporozoites and phagocytic cells. (A) Wide-field microscopy of sorted skin cells harboring GFP+ WTS or RAS parasite material at 4, 24, and 48 h p.i. Cell nuclei are labeled with DAPI (original magnification ×100; scale bar, 5 μm). (B) Wide-field microscopy of sorted skin cells harboring bright WTS or RAS GFP+ parasite material beyond 24 h. Cell nuclei are labeled with DAPI (original magnification ×100; scale bar, 5 μm). (C) Box-and-whisker-plot showing the fluorescence intensity of cells containing GFP+ WTS or RAS intracellular parasite material (n = 18 events per condition and per timepoint). (D) Representative FACS plots of noninfected (NI) or infected CD11b+ BMDM with GFP+ WTS parasites. (E) Kinetic of association of CD11b+ BMDM with GFP+ WTS or RAS parasites 5, 30, and 60 min and 24 h p.i. (mean ± SD, n = 3). (F) Illustration by transmission electron microscopy of phagocytosis, cell traversal, and cell invasion events 1 h after addition of WTS or RAS to BMDM. Phagocytosis: BSA-gold positive compartment containing a degraded parasite (p; scale bar, 1 μm). Cell traversal: BSA-gold negative compartment containing an intact parasite in the nucleus (n; scale bar, 2 μm). Higher magnification: the parasite plasma membrane (PPM) and the inner membrane complex (IMC) were detectable, but not the PV membrane (PVM; scale bar, 500 nm). Cell invasion: BSA-gold negative compartment containing an intact parasite inside a PV (scale bar, 1 μm). Higher magnification: IMC, PPM, and PVM were detectable (scale bar, 500 nm). (G) Percentage of phagocytosis, cell traversal, and cell invasion events 1 h after WTS or RAS parasite contact with BMDM (100 parasites per condition). (H) Percentage of parasites inside a UIS4+ compartment 24 h after WTS or RAS contact with murine XB2 keratinocyte (30 events) or J774 murine macrophage cell-lines (15 events; representative data of thre independent experiments). (I) Wide-field microscopy of XB2 keratinocytes and J774 macrophages harboring WTS or RAS parasites (green) inside a UIS4+ compartment (red). Cell nuclei are labeled with DAPI (blue; original magnification ×40; scale bar, 10 μm). *p < 0.05, **p < 0.01.

Close modal

We next examined sporozoite interaction with cultured CD11b+ cells in vitro, starting with BMDMs (Fig. 4D, 4E). Using WTS or RAS, ∼5% and ∼7% of the CD11b+ cells were associated with sporozoites after 5 and 30 min incubation, respectively; this proportion did not increase thereafter (Fig. 4E). At 24 h, ∼1% of the CD11b+ cells were still associated with GFP+ WT or irradiated parasites (Fig. 4E), suggesting parasite survival or development inside BMDM. Because these fates predicted initial sporozoite invasion events inside a parasitophorous vacuole (PV), we imaged interactions by high-resolution transmission electron microscopy (Fig. 4F, 4G). BMDM lysosomes were preloaded with 16-nm BSA-gold particles before incubation with parasites, and sporozoites were scored as “phagocytosed” when at least one BSA-gold particle was detected in a parasite-containing vacuole (phagolysosome), “traversing” when lying free in the host cell cytosol, or “invaded” when surrounded by a BSA-gold–negative, tight-fitting PV. After 1 h incubation, ∼94% of the WTS were scored as phagocytosed, 4% as traversing (most frequently observed in the cell nucleus) and 1.6% as invaded (Fig. 4F, 4G). Similar proportions were obtained with RAS (Fig. 4G).

To show that sporozoites were able to invade phagocytic cells inside a PV, sporozoites were incubated with J774 murine macrophages, and after 24 h samples were fixed and stained with DAPI and Abs to UIS4, a parasite protein that specifically accumulates at the PV membrane enclosing the parasite liver stage (6). As a negative control of phagocytic activity, we used the mouse teratoma XB2 cell line, derived from a keratinocyte lineage, which sporozoites efficiently invade in vivo (22). At 24 h, although 20-fold fewer parasites were found inside J774 than XB2 cells (data not shown), developing parasites were detected in both cell types, using both WTS and RAS (Fig. 4H, 4I). Approximately 90% and ∼50% of the intracellular parasites were surrounded by a UIS4-labeled PV membrane in XB2 and J774 cells, respectively. At 48 h, while intense schizogony was observed in WTS but not RAS-infected XB2 keratinocytes (Supplemental Fig. 2), in J774 cells no schizogony was detected in both WTS and RAS, which remained uninucleate (data not shown). Taken together, these data indicate that P. berghei sporozoites are capable of actively invading CD11b+ cells inside a PV and maintaining as transformed parasites at least 24 h before dying.

We investigated cytokine and chemokine patterns in the proximal auricular dLN 24 h after intradermal injection of ∼5 × 104 WTS, SGE, or PBS using a microfluidic-based quantitative PCR approach (Biomark, Fluidigm). mRNA expression was detectable for 34 of 46 genes of interest (Table I). These molecules were classified (Fig. 5A) based on their involvement in inflammation (a), immune regulation (c), or both (b), chemotaxis (d), cell activation, (e) or apoptosis (f). Compared with PBS, SGE induced a significant transcriptional upregulation of the nine genes encoding CXCL11, IFN-γ, CXCL10, Granzyme B, CXCL9, IL-2Rβ, TGF-β, IL-2, and CD69 (Fig. 5A, Table I). Compared with SGE, WTS significantly increased the relative transcript abundance of four of these genes (Fig. 5B): Granzyme B (×6), CXCL9 (×3.5), CXCL10 (×3.3), and to a lesser extent IFN-γ (×2.1). The other genes were expressed at similar levels compared to controls. We then quantified the amounts of Granzyme B, CXCL9, CXCL10, and IFN-γ in extracts of dLN at 24 h p.i. using ELISA (Fig. 5C). Results indicated a significant increase of the four molecules in the dLN after injection of WTS compared with SGE. The absence of detectable proteins in the serum of mice confirmed a local production of the molecules (data not shown). A similar profile was obtained with RAS (Fig. 5A), with the same genes upregulated (Fig. 5B) and the amounts of the same four signaling molecules increased (Fig. 5C). Protein levels remained significantly increased at 72 h p.i. for Granzyme B and CXCL9, but not CXCL10 or IFN-γ.

FIGURE 5.

Molecular signals in the dLN after injection of P. berghei sporozoites in the skin. (A) Average fold increase in gene expression analyzed by QRT-PCR of 34 molecules in the dLN of mice 24 and 72 h after intradermal injection of PBS, SGE, ∼5 × 104 WTS or RAS parasites. Molecules were classified based on their involvement in proinflammation (a), immunoregulation (c) or both (b), chemotaxis (d), cell activation (e) or apoptosis (f). Relative changes in gene expression were calculated by using the 2−ΔΔCT method. The results are presented as the fold increase of target genes in mice injected with SGE, WTS, or RAS, normalized to the Hprt internal control gene and compared with PBS-injected mice. (B) Genes showing a significant transcriptional upregulation following intradermal injection of WTS or RAS parasites compared with PBS in the dLN (mean ± SEM, 10 mice per condition). (C) ELISA of Granzyme B, CXCL9, CXCL10, and IFN-γ production in the dLN 24 and 72 h following intradermal injection of SGE, ∼5 × 104 WTS or RAS parasites compared with naive mice (mean ± SEM, 8 dLN pooled per condition per timepoint per experiment; n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001).

FIGURE 5.

Molecular signals in the dLN after injection of P. berghei sporozoites in the skin. (A) Average fold increase in gene expression analyzed by QRT-PCR of 34 molecules in the dLN of mice 24 and 72 h after intradermal injection of PBS, SGE, ∼5 × 104 WTS or RAS parasites. Molecules were classified based on their involvement in proinflammation (a), immunoregulation (c) or both (b), chemotaxis (d), cell activation (e) or apoptosis (f). Relative changes in gene expression were calculated by using the 2−ΔΔCT method. The results are presented as the fold increase of target genes in mice injected with SGE, WTS, or RAS, normalized to the Hprt internal control gene and compared with PBS-injected mice. (B) Genes showing a significant transcriptional upregulation following intradermal injection of WTS or RAS parasites compared with PBS in the dLN (mean ± SEM, 10 mice per condition). (C) ELISA of Granzyme B, CXCL9, CXCL10, and IFN-γ production in the dLN 24 and 72 h following intradermal injection of SGE, ∼5 × 104 WTS or RAS parasites compared with naive mice (mean ± SEM, 8 dLN pooled per condition per timepoint per experiment; n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001).

Close modal

To complete our study, we assessed activation of and cytotoxicity by parasite-specific CD8+ T cells after a single intradermal injection of sporozoites in the skin. For this assessment, we used the recombinant P. berghei ANKA (Antwerpen-Kasapa) PbA-hsOVA parasite clone expressing class I and class II OVA epitopes, which was named WTSova (37). We also used class I (Kb)-restricted OVA-specific TCR transgenic mice (OT-I mice). Naive mice were inoculated with PBS or ∼104 WTSova in the skin, adoptively transferred the same day with CFSE-labeled OT-I CD8+ T cells, and activation of OT-I CD8+ T cells was analyzed 4 d later using the CFSE dilution profile of CD45.1+ CD8+ cells (Fig. 6A–C). Previous experiments have shown that the transfer of a high number of specific CD8+T cells could affect their proliferative capacity (49); therefore, we titrated the number of transferred OT-I T cells from 103 to 106. As expected, activated OT-I CD8+ T cells were detected in the dLN, but not in the contralateral lymph node (data not shown), after injection of ova-expressing sporozoites, with a level of cell proliferation inversely related to the number of cells transferred (Fig. 6C), as described previously (49, 50). No cell division was observed after injection of PBS (Fig. 6B), SGE, WTS, or RAS (Supplemental Fig. 3). To determine whether these expanded Ag-specific OT-I CD8+ T cells exhibited cytolytic effector activity, we performed an in vivo cytotoxicity assay. Naive mice treated daily with chloroquine to avoid blood infection were inoculated with ∼104 WTSova or PBS and were adoptively transferred the same day with 104 OT-I T cells. Five days later, mice received equal numbers of ova-pulsed (CFSEhi) or nonpulsed (CFSElo) targets cells from the spleen of naive mice and, 20 h later, the frequency of surviving nonpulsed and pulsed cells in the dLN was assessed using FACS (Fig. 6D, 6E). A sharp decrease in the proportion of pulsed (CFSEhi) cells was seen in animals that were immunized with WTS, but not PBS. As shown in Fig. 6D and 6E, similar activation and killing efficiency was observed in mice that had received WTSova or RASova. We conclude that Ags derived from WTS and RAS are cross-presented to T cells with similar efficiencies, and that CD8+ T cells primed after injection of WTS and RAS develop similar killing potentials.

FIGURE 6.

CD8 T cell responses in the dLN after injection of P. berghei sporozoites in the skin. (AC) CFSE dilution assay in the dLN. Mice inoculated intradermally with PBS, 104 WTS, or RAS P.berghei OVA parasites were adoptively transferred the same day with different doses of CFSE-labeled OT-I CD8+ T cells (103, 104, 105, 106). T cell proliferation in the dLN was analyzed 4 d later by flow cytometry. (A) Representative FACS plot showing OT-I CD8+T cells of interest (CD45.1+CD8+). (B) Representative histograms showing CFSE dilution profile of transferred OT-I CD8+T cells after injection of PBS, WTSova, or RASova. (C) Percentage of divided OT-I cells following intradermal injection of WTSova or RASova (mean ± SEM; 6 dLN per condition per cell dilution; n = 2). (D and E) In vivo cytotoxicity assay. Mice treated daily with chloroquine (40 mg/kg) were inoculated intradermally with PBS, 104 WTSova, or RASova and adoptively transferred the same day with 104 OT-I CD8+ T cells. At day 5 p.i., mice received 5 × 105 CFSE-labeled, OVA-pulsed and nonpulsed targets recovered from the spleen of naive mice. (D) Representative histogram profiles showing the frequency of the pulsed (CFSEhi) and nonpulsed (CFSElo) targets. (E) Percentage of Ag-specific killing in the dLN of mice injected with WTSova or RASova (mean ± SEM; 12 dLN per condition; n = 3). The percent specific killing was calculated using the following formula: [1 − (Ratio for PBS-injected mice / Ratio for parasite injected mice)] × 100, where Ratio = (Percent CFSElo / Percent CFSEhi).

FIGURE 6.

CD8 T cell responses in the dLN after injection of P. berghei sporozoites in the skin. (AC) CFSE dilution assay in the dLN. Mice inoculated intradermally with PBS, 104 WTS, or RAS P.berghei OVA parasites were adoptively transferred the same day with different doses of CFSE-labeled OT-I CD8+ T cells (103, 104, 105, 106). T cell proliferation in the dLN was analyzed 4 d later by flow cytometry. (A) Representative FACS plot showing OT-I CD8+T cells of interest (CD45.1+CD8+). (B) Representative histograms showing CFSE dilution profile of transferred OT-I CD8+T cells after injection of PBS, WTSova, or RASova. (C) Percentage of divided OT-I cells following intradermal injection of WTSova or RASova (mean ± SEM; 6 dLN per condition per cell dilution; n = 2). (D and E) In vivo cytotoxicity assay. Mice treated daily with chloroquine (40 mg/kg) were inoculated intradermally with PBS, 104 WTSova, or RASova and adoptively transferred the same day with 104 OT-I CD8+ T cells. At day 5 p.i., mice received 5 × 105 CFSE-labeled, OVA-pulsed and nonpulsed targets recovered from the spleen of naive mice. (D) Representative histogram profiles showing the frequency of the pulsed (CFSEhi) and nonpulsed (CFSElo) targets. (E) Percentage of Ag-specific killing in the dLN of mice injected with WTSova or RASova (mean ± SEM; 12 dLN per condition; n = 3). The percent specific killing was calculated using the following formula: [1 − (Ratio for PBS-injected mice / Ratio for parasite injected mice)] × 100, where Ratio = (Percent CFSElo / Percent CFSEhi).

Close modal

Neutrophils are one of the main phagocytic cells recruited both in the skin and the proximal auricular dLN, and they are found associated with parasites at early times; therefore, we analyzed the effect of neutrophil depletion on parasite distribution, specific CD8+ T cell response in the dLN, and protective immunity (Fig. 7). Mice were treated or not (untreated control group) twice with neutrophil specific (1A8) or isotype control (2A3) mAbs 5 and 3 d prior to intradermal injection of ∼5 × 104 WTS or RAS in the skin. As reported previously (44, 45), a selective depletion of neutrophils (CD11bhiLy6Cint) was observable in the blood 5 (Fig. 7A) to 6 d (data not shown) after the first Ab injection. The depletion was confirmed in the skin and the proximal auricular dLN 4h p.i. (Fig. 7A). As expected, the neutrophil frequency in these three tissues was comparable between the 2A3-treated and untreated groups (Fig. 7A). For both WTS and RAS, no difference in parasite distribution and development was observed following neutrophil depletion, as indicated by a similar level of bioluminescence emitted over time from the liver (Fig. 7B, 7C) and the skin (Fig. 7B, 7D) of mice depleted (1A8) or not (2A3 and untreated group; data not shown in the latter case) in neutrophils. Moreover, neutrophil depletion did not affect the proliferation of transferred OT-I T cell (CD45.1+CD8+; Fig. 7E) in the dLN (Fig. 7F, 7G) in response to intradermal injection of WTSova or RASova in the skin, as well as the capacity of RAS-immunized mice to mount an efficient protective immune response (Fig. 7H).

FIGURE 7.

Effect of neutrophil depletion on P. berghei sporozoite distribution, CD8 T cell response, and protective immune response. (A) Representative FACS plots showing the neutrophil depletion efficiency in the blood, the skin, and the dLN. Mice untreated or treated either with the 1A8 specific mAb or the 2A3 isotype control mAb were intradermally injected with ∼5 × 104 WTS. The blood, the skin injection site, and the dLN were collected at the 4-h time point. Neutrophils were pregated on CD11b+ cells and identified based on their CD11bhiLy6Cint expression. (BD) Development of WTS and RAS parasites in the skin and the liver of mice measured by bioluminescence. (B) Mice depleted (1A8) or not (2A3) in neutrophils were injected intradermally with ∼5 × 104 WTS (upper panels) or RAS (lower panels) GFP-LUCSCH parasites and monitored daily for luciferase activity. (C and D) Average radiance (photons/s/cm2/steradian) of bioluminescent signal emitted from WTS or RAS parasites in the liver (C) compared with naive animals and in the skin (D) compared with the contralateral (Cont.) noninjected ear of mice (mean ± SD; 10 mice per condition per treatment; n = 2). (EG) CFSE dilution assay in the dLN. Mice depleted or not in neutrophils were inoculated intradermally with 104P.berghei WTSova or RASova parasites and adoptively transferred the same day with 105 CFSE-labeled OT-I CD8+ T cells. T cell proliferation in the dLN was analyzed 4 d later using flow cytometry. (E) Representative FACS plot showing OT-I CD8+T cells of interest (CD45.1+CD8+). (F) Representative histograms showing CFSE dilution profiles of transferred OT-I CD8+T cells following intradermal injection of WTSova or RASova in mice depleted (1A8) or not (2A3) in neutrophils. (G) Percentage of divided OT-I CD8+T cells following intradermal injection of WTSova or RASova in mice depleted (1A8) or not (2A3) in neutrophils (mean ± SEM; 6 dLN per condition per treatment; n = 2). (H) Percentage of protected mice following neutrophil depletion (1A8) or not (2A3). The mice were immunized intradermally three times, 2 wk apart with ∼5 × 104 RAS. The challenge was performed 1.5 wk after the last immunization (12 mice per condition; n = 2). **p < 0.01, ***p < 0.001.

FIGURE 7.

Effect of neutrophil depletion on P. berghei sporozoite distribution, CD8 T cell response, and protective immune response. (A) Representative FACS plots showing the neutrophil depletion efficiency in the blood, the skin, and the dLN. Mice untreated or treated either with the 1A8 specific mAb or the 2A3 isotype control mAb were intradermally injected with ∼5 × 104 WTS. The blood, the skin injection site, and the dLN were collected at the 4-h time point. Neutrophils were pregated on CD11b+ cells and identified based on their CD11bhiLy6Cint expression. (BD) Development of WTS and RAS parasites in the skin and the liver of mice measured by bioluminescence. (B) Mice depleted (1A8) or not (2A3) in neutrophils were injected intradermally with ∼5 × 104 WTS (upper panels) or RAS (lower panels) GFP-LUCSCH parasites and monitored daily for luciferase activity. (C and D) Average radiance (photons/s/cm2/steradian) of bioluminescent signal emitted from WTS or RAS parasites in the liver (C) compared with naive animals and in the skin (D) compared with the contralateral (Cont.) noninjected ear of mice (mean ± SD; 10 mice per condition per treatment; n = 2). (EG) CFSE dilution assay in the dLN. Mice depleted or not in neutrophils were inoculated intradermally with 104P.berghei WTSova or RASova parasites and adoptively transferred the same day with 105 CFSE-labeled OT-I CD8+ T cells. T cell proliferation in the dLN was analyzed 4 d later using flow cytometry. (E) Representative FACS plot showing OT-I CD8+T cells of interest (CD45.1+CD8+). (F) Representative histograms showing CFSE dilution profiles of transferred OT-I CD8+T cells following intradermal injection of WTSova or RASova in mice depleted (1A8) or not (2A3) in neutrophils. (G) Percentage of divided OT-I CD8+T cells following intradermal injection of WTSova or RASova in mice depleted (1A8) or not (2A3) in neutrophils (mean ± SEM; 6 dLN per condition per treatment; n = 2). (H) Percentage of protected mice following neutrophil depletion (1A8) or not (2A3). The mice were immunized intradermally three times, 2 wk apart with ∼5 × 104 RAS. The challenge was performed 1.5 wk after the last immunization (12 mice per condition; n = 2). **p < 0.01, ***p < 0.001.

Close modal

In this study, we present a detailed analysis of local host immune response to needle-syringe injection of Plasmodium sporozoites in the skin, using the P. berghei–C57BL/6 model. Results indicate 1) a biphasic inflammatory response to sporozoites; 2) sporozoite association with polymorphonuclear neutrophils and resident myeloid cells in the skin and the dLN; 3) sporozoite invasion, and survival of transformed parasites, inside CD11b+ cells; 4) a Th1 cytokine profile in the dLN 24 h p.i.; and 5) similar innate and adaptive immune responses induced by WTS and RAS.

The primary host response to sporozoite injection in the skin consisted in the successive recruitment of neutrophils followed by inflammatory monocytes in the skin injection site and dLN. The early neutrophil infiltration was in line with previous intravital imaging data that showed the influx, starting at ∼20 min p.i., of brightly fluorescent cells in lys-GFP mice in the skin bitten by P. berghei–infected mosquitoes (36). In agreement with previous reports (51), we found that skin injury inflicted by needle insertion and PBS/SGE was sufficient for a transient neutrophil recruitment. A sustained neutrophil recruitment in the skin and in the dLN, however, was specifically induced by sporozoites, which decreased in both tissues at 24 h. At 4 h, ∼20% of the GFP+CD11b+ cells in the skin were neutrophils, which might be an underestimation of the frequency of sporozoite-neutrophil associations given the speed of sporozoite destruction inside neutrophils.

A rapid infiltration of neutrophils was also reported after inoculation of Leishmania major promastigotes into the ear skin of C57BL/6 mice by needle injection or by the bites of sand flies (51, 52). Surprisingly, the acute neutrophilic response to L. major was found to promote the early infectious process and to suppress immunity. L. major promastigotes first infect and induce apoptosis of neutrophils (52, 53) and are secondarily captured by dermal DCs as they engulf apoptotic, infected neutrophils; this suppresses DC ability to stimulate adaptive CD4+ T cell–mediated responses and the early development of antiparasitic immunity (51). Differently, we observed that the specific depletion of neutrophil at the time of recruitment did not affect sporozoite distribution in the host and did not impair establishment of a protective immune response. Plasmodium sporozoites have powerful weapons to fight neutrophils and other phagocytic cells, with their abilities to glide at high speed (∼2 μm/sec) in the skin (54) and to wound host cell plasma membranes and traverse (i.e., glide through) host cells (55, 56). In the liver sinusoids, normal sporozoites traverse resident macrophages (Kupffer cells), while the sporozoites that lack host cell traversal ability are rapidly cleared by these cells (57). Still, in the skin, many sporozoites are engulfed by neutrophils. Neutrophils might phagocytose only those skin sporozoites that have ceased to move, or might contribute to restrain motile sporozoites using neutrophil extracellular traps (58).

In the skin and the dLN, ∼20% and ∼80% of the GFP+ cells were CD11b+ 4 h after injection respectively. The remainder might be nonhematopoietic cells such as cutaneous fibroblasts or keratinocytes (22, 36), lymphoid podoplanin-expressing cells (1), or hematopoietic cells such as resident dermal CD103-expressing DCs, described to be efficient to cross-present exogenous Ag (48). Given that half of the injected sporozoites remain in the skin and ∼15% reach the dLN (1), it is implied that >20% of the sporozoites injected into the skin in immunization conditions associate with host leukocytes locally.

An interesting finding was that sporozoites actively invaded and survived in CD11b+ cells. Live parasites were found inside CD11b+ cell in the skin until 48 h after injection, and invaded inside a PV, transformed, and survived for up to 3 d in J774 macrophages. Blood-stage parasites of the rodent-infecting P. berghei, P. yoelii, and Plasmodium chabaudii species infect and survive within CD317+ plasmacytoid DCs (pDCs) of the spleen. Parasites were also found to multiply and release infectious merozoites inside pDCs, which have thus been proposed to act as a subclinical reservoir for blood-stage Plasmodium parasites. In addition, infected pDCs do not secrete IL-12 and secrete more IL-10, suggesting that parasite infection of pDCs might have an immune-suppressive effect (59). The effect, if any, of parasite development in CD11b+ cells in the skin and dLN is unknown. Interestingly, after immunization in the skin with RAS or GAPs, Ag continues to be presented for up to 2 months in the dLN, a process that is critical for the complete development and expansion of protective CD8+ T cell responses (50). Parasite persistence inside phagocytes might contribute to or initiate prolonged Ag presentation in the dLN, because DCs or macrophages, or both, were shown to be required to establish Ag persistence (50).

Analysis of the cytokine–chemokine profile in the dLN 24 h p.i. was indicative of a Th1 skewing of the immune response. Of the 46 genes encoding signaling molecules of interest, nine were found to be upregulated after injection of SGE compared with PBS, which encode the following molecules: 1) Granzyme B, a serine protease produced by NK cells and cytotoxic T cells (CTL) (60); 2) CXCL9, CXCL10, and CXCL11, which are IFN-γ–inducible chemokines known to induce chemotaxis of CXCR3-expressing leukocytes (innate immune cells as well as T cells) (61, 62); 3) IFN-γ, which is secreted by NK cells, LTCD4+ activated Th1 T cells, and CTL; it is also produced by other cell types such as NKT cells, B cells, and APCs (63); 4) IL-2/IL-2Rβ, which activates and enhances killing by NK cells and T cells (64); 5) CD69, which is expressed in activated T cells and other hematopoietic cells such as NK cells (65); and 6) TGF-β, a pleiotropic cytokine that can participate in the induction of the inflammatory response (66). Of these nine genes upregulated by SGE, four were further upregulated by the presence of sporozoites compared with SGE alone, which corresponded to the genes encoding Granzyme B, CXCL9, CXCL10, and IFN-γ. These four molecules were found in increased amounts in the dLN after injection of sporozoites in the skin. This profile clearly suggests an early NK cell activation (Granzyme B, IFN-γ) and the establishment of Th1 immune responses (IFN-γ, CXCL9, and CXCL10). Further investigation is necessary to identify the different cell types that produce these proinflammatory cytokines locally.

This Th1 immune response induced by injection of immunizing doses of sporozoites dissected out from mosquito salivary glands differs from the immune response described in response to sporozoite injection by mosquito bites. In this latter case, a smaller number of sporozoites is delivered and in the presence of Anopheles mosquito saliva. Anopheles saliva was shown to cause dermal mast cell degranulation and the accumulation of CD11b+ and CD11c+ leukocytes in the dLN (67). The saliva also increases the levels of immunosuppressive IL-10 in the dLN, which could contribute to downregulation of Ag (OVA)-specific T cell priming in an IL-10–dependent manner (68). Another study reported that 30 min after injection by mosquitoes, P. berghei sporozoites increased the motility of regulatory T cells and decreased expression of MHC class II and CD86 in CD11c+ cells in the skin of mice, corroborating the notion that regulatory T cells might suppress activation of APCs in the skin and dLN (69).

Lastly, we found no significant differences in the in vivo behavior and immunogenicity of WTS and RAS. Previous work that used the P. berghei-BALB/c model and an i.v. injection route of sporozoites reported that RAS primed more robust specific (pb9) T cell responses detected by an ex vivo ELISPOT assay; it also suggested that the difference between WTS and RAS affected early events in T cell priming (70). We failed to detect any significant difference between WTS and RAS in inducing innate immune responses or in priming specific (OVA) CD8+ T cells or in the killing abilities of the respective CD8+ T cells. Our data confirm the notion that the capacity of RAS to induce protection stems only from their defective intracellular development and the lack of blood-stage infection.

Overall, these data show that in immunizing conditions, sporozoites distribute to three tissues and despite their motility and their ability to traverse host cells, many of them end up in cutaneous and lymphoid CD11b+ cells, while some might persist and contribute to prolonged Ag presentation. At 24 h, a Th1 profile and an induction of adaptive immunity with little evidence of immunosuppression was observed; however, many questions remain. Primarily, the parasite forms that induce protection, between sporozoites actively going to the dLN, which do not develop and thus cannot generate effective immunity against liver stage development, and parasites that remain in the skin, which can develop and produce immunogens valuable for anti-liver stage protection, need to be identified. Further study is also needed to better characterize the resident and inflammatory myeloid cell subtypes involved in the uptake and presentation of parasite Ag in the dLN. If early interactions between the parasite and CD11c+ cells have been described previously in the dLN after natural bite (1), their exact nature remain unknown. These questions are essential to guide a rational development of immunizing GAPs.

We thank S. Shorte, P.H. Commere, M.A. Nicola, M.C. Prévost, and the Imagopole team (Institut Pasteur) for the support with confocal microscopy, flow cytometry, bioluminescence and electron microscopy; S. Thiberge for help with labeling and imaging for the ex vivo analysis of myeloid cell-parasite associations; C. Bourgouin, M. Szatanik, and the Center for Production and Infection of Anopheles (CEPIA–Institut Pasteur) for mosquito rearing; and S. Celli, P. Bousso, S. Mecheri, G. Eberl, and G. Milon (Institut Pasteur) for many helpful discussions.

This work was supported by funds from the Pasteur Institute (Programmes Transversaux de Recherche) and the AXA Research Fund. L.M.-D. was supported by private funding from F. Lacoste and the AXA Research Fund.

The online version of this article contains supplemental material.

Abbreviations used in this article:

BMDM

bone marrow–derived macrophage

DC

dendritic cell

dLN

draining lymph node

EEF

exoerythrocytic form

GAP

genetically arrested parasite

pDC

plasmacytoid DC

p.i.

postinjection

PV

parasitophorous vacuole

QRT-PCR

quantitative real-time PCR

RAS

radiation-attenuated sporozoite

SGE

salivary gland extract

UIS4

upregulated in infectious sporozoites gene 4

WTS

wild-type sporozoite.

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The authors have no financial conflicts of interest.

Supplementary data