Name: myeloid cell isolation from mouse skin and draining lymph node following intradermal immunization with live attenuated plasmodium sporozoites
Uploaded: 2016-05-18T13:00:00
Description: Watch this Scientific Journal Video about Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites at JoVE.com

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&#160;and funds from the Laboratoire d&#8217;Excellence &#8220;Integrative Biology of Emerging Infectious Diseases&#8221; (grant no. ANR-10-LABX-62-IBEID).

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.&#160;
1. Materials and Reagents
<ol>
	<li>Use female Anopheles stephensi mosquitoes (Sda500 strain) that feed on infected mice 3-5 days after emergence and rear as described previously21.</li>
	<li>Use parasites <e...

<ol>
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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sporozoite+immunization+of+human+volunteers+under+chemoprophylaxis+induces+functional+antibodies+against+pre-erythrocytic+stages+of+Plasmodium+falciparum.">Sporozoite immunization of human volunteers under chemoprophylaxis induces functional antibodies against pre-erythrocytic stages of Plasmodium falciparum.</a> Malar J. 13, 136 (2014).</li><li>Amino, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+imaging+of+Plasmodium+transmission+from+mosquito+to+mammal.">Quantitative imaging of Plasmodium transmission from mosquito to mammal.</a> Nat Med. 12 (2), 220-224 (2006).</li><li>Yamauchi, L. 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B., Zavala, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T+lymphocytes+protective+against+malaria+liver+stages+are+primed+in+skin-draining+lymph+nodes.">CD8+ T lymphocytes protective against malaria liver stages are primed in skin-draining lymph nodes.</a> Nat Med. 13 (9), (2007).</li><li>Obeid, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin-draining+lymph+node+priming+is+sufficient+to+induce+sterile+immunity+against+pre-erythrocytic+malaria.">Skin-draining lymph node priming is sufficient to induce sterile immunity against pre-erythrocytic malaria.</a> EMBO Mol Med. 5 (2), 250-263 (2013).</li><li>Amino, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+cell+traversal+is+important+for+progression+of+the+malaria+parasite+through+the+dermis+to+the+liver.">Host cell traversal is important for progression of the malaria parasite through the dermis to the liver.</a> Cell Host Microbe. 3 (2), 88-96 (2008).</li><li>Radtke, A. 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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+skin+immunological+disturbance+after+Plasmodium-infected+mosquito+bites.">Early skin immunological disturbance after Plasmodium-infected mosquito bites.</a> Cell Immunol. 277 (1-2), 22-32 (2012).</li><li>Mac-Daniel, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+immune+response+to+injection+of+Plasmodium+sporozoites+into+the+skin.">Local immune response to injection of Plasmodium sporozoites into the skin.</a> J Immunol. 193 (3), 1246-1257 (2014).</li><li>Ribeiro-Gomes, F. L., Peters, N. C., Debrabant, A., Sacks, D. 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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 repo...

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.

<table>
	<tbody>
		<tr>
			<td>Ketamine: Imalgene&#174; 1000</td>
			<td>Merial</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Xylazine: Rompun&#174; 2%</td>
			<td>Bayer</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoFil syringe + 35 gauge needle</td>
			<td>World Precision Instruments&#160;</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Omnican&#174; 50 Insulin syringe 0,5 ml/50 I.U.</td>
			<td>B. Braun Medical&#160;</td>
			<td>9151125</td>
			<td>-</td>
		</tr>
		<tr>
			<td>MultiwellTM 6 well tissue culture plate &#8211; Flat Bottom</td>
			<td>BD Falcon&#160;</td>
			<td>353046</td>
			<td>-</td>
		</tr>
		<tr>
			<td>70 &#181;m cell strainer&#160;</td>
			<td>BD Falcon&#160;</td>
			<td>352350</td>
			<td>-</td>
		</tr>
		<tr>
			<td>2 ml syringe</td>
			<td>Terumo</td>
			<td>SS-02S</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BLUE MAXTM 15ml Polypropylene conical tube</td>
			<td>BD Falcon&#160;</td>
			<td>352097</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BLUE MAXTM 50ml Polypropylene conical tube</td>
			<td>BD Falcon&#160;</td>
			<td>352098</td>
			<td>-</td>
		</tr>
		<tr>
			<td>5ml Polystyrene Round-Bottom Tube with 35&#181;m Cell-Strainer Cap</td>
			<td>BD Falcon&#160;</td>
			<td>352235</td>
			<td>-</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS 1X Cacl2- and MgCl2-free</td>
			<td>Life Technologies</td>
			<td>14190-094</td>
			<td>-</td>
		</tr>
		<tr>
			<td>DMEM 1X + GlutaMAXTM</td>
			<td>Life Technologies</td>
			<td>31966-021</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Collagenase from Clostridium histolyticum, Type IV 0.5-5.0 FALGPA units/mg solid&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>C5138</td>
			<td>400 U/ml&#160;</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas, type IV&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D5025</td>
			<td>50 &#181;g/ml</td>
		</tr>
		<tr>
			<td>EDTA disodium salt</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>E-5134</td>
			<td>10mM or 2.5 mM</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Biowest</td>
			<td>S1810-500</td>
			<td>-</td>
		</tr>
		<tr>
			<td>HEPES buffer solution (1M)</td>
			<td>Gibco</td>
			<td>15630-056</td>
			<td>25 mM</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue Stain (0,4%)</td>
			<td>Life Technologies</td>
			<td>15250-061</td>
			<td>Dilution 1:10&#160;</td>
		</tr>
		<tr>
			<td>Anti-mouse CD16/CD32 (2.4G2 clone)</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td>10&#181;g/ml final (1:50)</td>
		</tr>
		<tr>
			<td>DAPI FluoroPureTM grade</td>
			<td>Life Technologies</td>
			<td>D21490</td>
			<td>1&#181;g/ml final</td>
		</tr>
		<tr>
			<td>Anti-mouse CD45 (30-F11 clone)</td>
			<td>BD Biosciences</td>
			<td>559864</td>
			<td>&#160;Dilution 1:200</td>
		</tr>
		<tr>
			<td>Anti-mouse CD11b (M1/70 clone)</td>
			<td>BD Biosciences</td>
			<td>557657</td>
			<td>&#160;Dilution 1:400</td>
		</tr>
		<tr>
			<td>Anti-mouse CD8&#945; (5H10 clone)</td>
			<td>Life Technologies</td>
			<td>MCD0830</td>
			<td>&#160;Dilution 1:100</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Female C57BL/6JRj mice (7-week-old)&#160;</td>
			<td>Janvier Laboratories</td>
			<td>-</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

myeloid cell isolation from mouse skin and draining lymph node following intradermal immunization with live attenuated plasmodium sporozoites

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.

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 </stron...

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Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

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.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Research

JoVE Journal

Immunology and Infection

Intravital Microscopy of the Inguinal Lymph Node

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Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

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Isolation of Murine Lymph Node Stromal Cells

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Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

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A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-GuÃƒÆ’Ã†'Ãƒâ€šÃ‚Â©rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...