We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.
Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.
Malaria is one of the deadliest infectious diseases in the world, killing more than half a million people per year. Infection by Plasmodium, the causal agent of the disease, begins by a pre-erythrocytic (PE) phase. During this phase, sporozoites injected into the host skin by a female Anopheline mosquito reach the liver via the bloodstream and differentiate inside hepatocytes into the parasite forms that infect red blood cells and cause the symptoms of the disease.
The PE stages of Plasmodium represent a privileged target for anti-malaria vaccination. Indeed, live attenuated vaccines against these stages, such as radiation attenuated sporozoites (RAS), genetically arrested parasites (GAP) or chemoprophylaxis and sporozoites (CPS) have demonstrated their capacity to protect both rodent and human hosts1-9. In the rodent model, most vaccination studies are conducted utilizing intravenous immunization, which is the gold standard in terms of protective efficacy. However, the description of a skin stage and the importance of the skin-associated draining lymph node (dLN) in eliciting protection has changed our perception of the PE phase and emphasized the importance of the intradermal route of injection. Intravital imaging of P. berghei sporozoites injected into the skin of rodents has shown that only ~25% of the inoculum reaches the liver via the bloodstream. The remaining ~75% distributes between the proximal dLN (~15%) and the skin (~50%)10,11, where a small proportion can transform and remain alive for weeks inside skin cells12,13. Moreover, subsequent studies described that the establishment of effective protective immunity after intradermal immunization mainly takes place in the skin-dLN, where parasite specific CD8+ T cells are activated, and only marginally in the spleen or liver-dLNs14,15.
While most studies have concentrated on the characterization of the effector cells implicated in the establishment of protective immune response, much less is known about the fate of live attenuated parasites injected into the skin, especially their interactions with the innate immune system. In particular, characterization of antigen-presenting cells involved in parasite antigen uptake, processing and presentation to CD8+ T cells is of critical importance, knowing that PE antigen acquisition can occur both in the skin and dLN compartments. Previous intravital imaging studies described an early influx of brightly fluorescent Lys-GFP positive cells in the skin following an infectious mosquito bite16 while early interactions between sporozoites and dendritic cells were observed in the dLN10,17. More recently, it has been reported that sporozoites inoculated in the skin by mosquitoes increases the motility of both dendritic and regulatory T cells in the skin of mice, while a decrease number of antigen presenting cells was observed in the dLN18.
We aimed to identify and quantify more precisely the leukocyte subsets recruited in the skin and corresponding dLN as well as those interacting with the parasite following intradermal injection of immunizing doses of RAS19. In this context, we isolated myeloid cells (CD45+CD11b+) from both tissues and characterized subpopulations of interest by multi=parametric flow cytometry. Consistent with the immune response described in the early stage of Leishmania major skin infection20, the primary host response to sporozoite injection consists of a successive recruitment of polymorphonuclear neutrophils (CD45+CD11b+Ly6G+Ly6Cint) followed by inflammatory monocytes (CD45+CD11b+Ly6G–Ly6C+) which are identified on the basis of differential expression of the Ly6G and Ly6C surface markers.
We describe here a protocol for isolating myeloid cells from the mouse skin and dLN following intradermal injection of immunizing doses of RAS extracted from infected mosquito salivary glands. Reproducible intradermal injections and tissue processing are critical steps to quantify phenotypic changes of infiltrating cell population within infected tissues. The approach detailed below provides a reliable assay to assess the skin and dLN inflammatory response to Plasmodium parasite and can be extended to various experimental systems.
All procedures 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.
1. Materials and Reagents
2. Radiation-attenuated Sporozoite Isolation from Mosquito Salivary Glands
3. Injection of Sporozoites into the Dermal Layer of the Ear
4. Skin and Draining Lymph Node Dissection
5. Cell Isolation from Lymph Nodes
6. Cell Isolation from Skin of the Ear
7. Count and Viability of Collected Cells
8. Blocking of Non Antigen-specific Binding
9. Staining Skin and Lymph Node Myeloid Cells
We recently demonstrated that needle-syringe injection of immunizing doses of P. berghei sporozoites in the mouse skin induces a successive recruitment of polymorphonuclear neutrophils followed by inflammatory monocytes in the skin and dLN19. The Protocol Section described above details the procedure used to successfully isolate live myeloid cells from both tissues following multiple injections of large number of sporozoites in the ear dermis (Figures 1 and 2). Within the first 24 hr, the dLN (Figure 3) and the skin injection site (Figure 4) were isolated and processed as summarized in Figure 5 to analyse the cellular infiltration by multi-parametric flow cytometry. Overall, this technique allowed us to isolate an average 104 CD45+CD11b+ and 2.104 CD11b+ live single cells for the skin and dLN respectively. The acceptable viability of non-debris single cells ranged from 40-70% for the skin and 70-90% for the dLN. As shown in Figure 6, the frequency of myeloid cells in the skin (CD45+CD11b+) and the dLN (CD8α–CD11b+) strongly increases following intradermal injection of RAS compared with non-infected mice.
Figure 1: Intradermal Injection of Sporozoites into the Ear of Mouse. (A) Image showing intradermal injection of sporozoites in the ear pinna of an anesthetized mouse. The ventral side of the ear is stabilized with clear tape previously placed under a dissecting microscope. The needle is carefully inserted on the dorsal side of the ear, beneath the epidermis, with the bevel up. (B–C) Pictures showing the dorsal side of the ear pinna prior (B) and after (C) intradermal injection of 0.1-0.2 μl of parasite suspension. A characteristic papule (black arrow) is observable at the injection site at the end of the operation. Please click here to view a larger version of this figure.
Figure 2: Imaging of Sporozoites Deposited in the Ear Dermis of Mouse. (A) Fluorescence microscopy showing RAS migrating from the injection site (indicated by the dashed lines) in the mouse skin 15 min post-injection (Scale bar = 60 μm; 10X magnification). The path of sporozoites is represented by the maximum intensity projection of the fluorescent signal. Green and red fluorescence respectively show parasite position in the skin at the beginning and after 10 sec of acquisition. (B) Higher magnification of RAS (green) injected in the skin (Scale bar = 20 μm; 25X magnification). Please click here to view a larger version of this figure.
Figure 3: Draining Lymph Node Processing. (A) Picture showing the dissection of the proximal auricular dLN following intradermal injection of Evans blue for demonstration purposes. After cervical dislocation of the mouse, the neck is disinfected with 70% ethanol. A first incision is made in the jugular-carotid area with sharp scissors and the skin is removed from the operating field. The incision is stretched with 2 forceps to expose the dLN that appears grayish compared to the surrounding tissue. The connective tissues are then carefully pinched with forceps on the top of the dLN and progressively separated from it. Finally, the dLN is extracted by placing a forceps underneath the lymphoid tissue. (B) Picture showing the extracted dLN in a drop of PBS. Please click here to view a larger version of this figure.
Figure 4: Ear Skin Processing. Pictures showing the successive steps to process the inoculated ear. (A) After cervical dislocation of the mouse, the ear is disinfected with 70% ethanol and removed using sterile sharp scissors and forceps. (B–D) The dorsal and ventral aspects of the ear are lightly separated by pinching and spacing apart the cut ends with two forceps. (E) Picture showing the dorsal and ventral aspects of the ear prior enzymatic treatment. Please click here to view a larger version of this figure.
Figure 5: Protocol Summary. Schematic representation of the key steps to isolate total cells from the skin and dLN tissues. Please click here to view a larger version of this figure.
Figure 6: Myeloid Cell Population Isolated From the Ear Skin and the Proximal dLN. Representative FACS plots showing the gating strategy to analyse the myeloid compartment in the skin (A) and the dLN (B). CD45+CD11b+ and CD8α–CD11b+ cells were gated on total live singlet events for the skin and dLN respectively. For each condition, 5 x 105 total events were acquired (2 ears and 2 dLN pooled per sample). Please click here to view a larger version of this figure.
In the perspective of large-scale vaccination of humans using a whole sporozoite malaria vaccine, one of the main challenges to overcome is to develop optimized routes and methods of parasite administration to ensure successful immunization and protection24,25. In humans, the evaluation of protective efficacy mediated by live attenuated parasites (LAP) has been performed following natural mosquito bites2, as well as intradermal, subcutaneous25,26 and IV immunizations27. As reported in rodents28 and non-human primates25, IV administration of LAP induces stronger protective immune responses compared to the above-mentioned routes. Nevertheless, non-IV routes of administration using needle and syringe still remains more suitable for human mass vaccination and efforts are currently being made to optimize their protective efficacy.
Recent studies conducted in mice demonstrated, especially in the case of the ID route, that the deposition of a small volume of sporozoite suspension (range of 1 µl) in multiple injection sites (4 points) significantly increased parasite infectivity compared to a single inoculation of parasites diluted in larger volumes (50 µl)24. In our experimental system (C57BL/6 mouse – P. berghei), complete protection was achieved after multiple injections in the ear dermis of highly concentrated suspension of parasites (50,000 RAS in 0.6 µl, 4 injection sites)19. In this context, we established the protocol described above to analyze more precisely the local host immune response to immunizing doses of RAS that allowed us to identify 2 major myeloid cell subsets recruited in the skin and dLN in response to the parasite19.
Multi-parametric flow cytometry allows accurate identification and quantification of immune cell changes in the context of host response to infection19,20. Isolation of large number of viable cells is therefore critical to perform reproducible phenotypic analysis. For this purpose, the enzyme concentration and the duration of tissue digestion must be optimized. Indeed, low concentration of enzymes or insufficient incubation time may lead to incomplete digestion with low yield of cells while prolonged tissue digestion may result in decrease cell viability and alteration of cell surface antigen expression29. Nevertheless, well-established protocols using collagenase treatment have been shown to have marginal impact on the detectability of relevant surface molecules frequently used for phenotypic analyses30.
Among the several parameters that can optimize the quality of cell isolation, the separation of the dorsal and ventral sections of the ear skin is essential, since it allows the collagenase/DNAse to access the dermis. In addition, mincing the skin into small pieces increases the surface area accessible to the enzymes, therefore allowing shorter duration of digestion. Finally, the use of mechanical dissociation helps to increase the yield in cell number for both the skin and dLN. However, excessive dissociation can cause additional stress that could negatively impact cell viability.
We favoured acquisition of live cells since fixation leads to suboptimal staining, complicating distinction of rare positive events of interest (data not shown). Moreover, the discrimination of dead cells was more problematic. In cases where cell fixation is required, we would suggest the use of LIVE/DEAD fixable dead cell stains.
Using the defined protocol, we successfully isolated myeloid cells infiltrating into the skin and dLN in response to parasite injection (Figure 6A and B). We usually obtain higher levels of viable single cells from the dLN (70-90%) compared to the skin (40-70%). This difference can be explained by the longer incubation time needed for efficient enzymatic digestion together with additional steps of mechanical dissociation to isolate skin cells (skin layer separation, mincing and stronger mashing).
Finally, an important parameter to take into account when analysing the host response to ID injection of sporozoites is the level of inflammation induced by both needle insertion and mosquito salivary gland extract deposition in the skin19,20. We therefore suggest to inject additional mice with either 1x PBS alone or salivary gland extracts from the same number of non-infected mosquitoes to evaluate the immune response specifically induced by the parasite. Altogether, we advise to dissect infected mosquitoes with high parasite load in the salivary glands and to filter the parasite suspension to limit the amount of mosquito material that could be deposited in the skin.
In summary, the isolation of myeloid cells from the skin and dLN through the described protocol provides a reliable assay to assess dynamic cellular changes during inflammatory response to Plasmodium infection and can be extended to other pathogens transmitted into the skin of the host.
The authors have nothing to disclose.
The authors thank Patricia Baldacci, Vanessa Lagal and Sabine Thiberge for critical reading, Irina Dobrescu and Sabine Thiberge for help in taking pictures and Pauline Formaglio for teaching in vivo imaging of sporozoite motility in the mouse skin. We also would like to thank Marek Szatanik and the Center for Production and Infection of Anopheles (CEPIA-Institut Pasteur) for mosquito rearing. This study was supported by the AXA Research Fund and funds from the Laboratoire d’Excellence “Integrative Biology of Emerging Infectious Diseases” (grant no. ANR-10-LABX-62-IBEID).
Ketamine: Imalgene® 1000 | Merial | – | – |
Xylazine: Rompun® 2% | Bayer | – | – |
NanoFil syringe + 35 gauge needle | World Precision Instruments | – | – |
Omnican® 50 Insulin syringe 0,5 ml/50 I.U. | B. Braun Medical | 9151125 | – |
MultiwellTM 6 well tissue culture plate – Flat Bottom | BD Falcon | 353046 | – |
70 µm cell strainer | BD Falcon | 352350 | – |
2 ml syringe | Terumo | SS-02S | – |
BLUE MAXTM 15ml Polypropylene conical tube | BD Falcon | 352097 | – |
BLUE MAXTM 50ml Polypropylene conical tube | BD Falcon | 352098 | – |
5ml Polystyrene Round-Bottom Tube with 35µm Cell-Strainer Cap | BD Falcon | 352235 | – |
DPBS 1X Cacl2- and MgCl2-free | Life Technologies | 14190-094 | – |
DMEM 1X + GlutaMAXTM | Life Technologies | 31966-021 | – |
Collagenase from Clostridium histolyticum, Type IV 0.5-5.0 FALGPA units/mg solid | Sigma-Aldrich | C5138 | 400 U/ml |
Deoxyribonuclease I from bovine pancreas, type IV | Sigma-Aldrich | D5025 | 50 µg/ml |
EDTA disodium salt | Sigma-Aldrich | E-5134 | 10mM or 2.5 mM |
FBS | Biowest | S1810-500 | – |
HEPES buffer solution (1M) | Gibco | 15630-056 | 25 mM |
Trypan blue Stain (0,4%) | Life Technologies | 15250-061 | Dilution 1:10 |
Anti-mouse CD16/CD32 (2.4G2 clone) | BD Biosciences | 553142 | 10µg/ml final (1:50) |
DAPI FluoroPureTM grade | Life Technologies | D21490 | 1µg/ml final |
Anti-mouse CD45 (30-F11 clone) | BD Biosciences | 559864 | Dilution 1:200 |
Anti-mouse CD11b (M1/70 clone) | BD Biosciences | 557657 | Dilution 1:400 |
Anti-mouse CD8α (5H10 clone) | Life Technologies | MCD0830 | Dilution 1:100 |
Female C57BL/6JRj mice (7-week-old) | Janvier Laboratories | – | – |