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

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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

1. Use female Anopheles stephensi mosquitoes (Sda500 strain) that feed on infected mice 3-5 days after emergence and rear as described previously²¹.
2. Use parasites Plasmodium berghei ANKA clone expressing the Green Fluorescent Protein (GFP) gene under the control of the constitutive Heat Shock Protein 70 (....

Results

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

Discussion

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 protection^24,25. In humans, the evaluation of protective efficacy mediated by live attenuated parasites (LAP) has been performed following natural mosquito bites², as well as intradermal, subcutaneous^25,26 and IV immunizations²⁷. As repo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ketamine: Imalgene® 1,000	Merial	-	50 mg/kg: prior sporozoite injection 125 mg/kg: prior sacrifice
Xylazine: Rompun® 2%	Bayer	-	5 mg/kg: prior sporozoite injection 12.5 mg/kg: prior sacrifice
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 15 ml Polypropylene conical tube	BD Falcon	352097	-
BLUE MAXTM 50 ml Polypropylene conical tube	BD Falcon	352098	-
5 ml Polystyrene Round-Bottom Tube with 35 µm 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	10 mM 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	10 µg/ml final (1:50)
DAPI FluoroPureTM grade	Life Technologies	D21490	1 µg/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	-	-