Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Summary

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

Abstract

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide ¹. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself ². Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation.

Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages ^3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from ⁷), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis.

Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Protocol

1. Purifying Schistosoma mansoni Eggs

1. Infect Swiss-Webster mice with schistosome cercariae, which is the infectious stage in the Schistosoma life cycle ⁸. Alternatively, obtain schistosome-infected mice from the NIAID Schistosomiasis Resource Center (http://www.schisto-resource.org/). Leave mice for 6-7 weeks following infection, by which time-point, eggs are present in the liver and can be recovered as described below (See Fig. 1).
2. Euthanize mice with CO₂, and wet with 70% ethanol. Place mouse on back. Use blunt forceps and sci....

Discussion

Here, a model employing Schistosoma mansoni eggs to induce type 2 lung inflammation is described. Characteristic features of this model include the potent Th2 immune response, airway inflammation and pulmonary granuloma formation. Since these parameters are dependent on CD4 Th2 cell responses ¹¹, this is a useful model to investigate the effects of protein- specific or lineage-specific deletions on Th2 cell responses. Here the readouts would include quantification of antigen-specific Th2 cytokines, an.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Collagenase/Dispase		Roche Applied Science	11 097 113 001 (500mg)
Diff Quik Hema 3 Stain	Pack	Fisher Scientific Co. LLC	122-911
DMEM	Liquid	Gibco	11965
Falcon 50mL	Pack	Falcon	352070
Intramedic PE Tubing		Becton Dickinson and Co.	427410 (0.58mm)
Isoflurane		Abbot Animal Health	52600405 (250mL)
Ketamine		Fort Dodge, Animal Health	100mg/mL
Mannose receptor antibody	Biotin	AbD Serotec	MCA2235B
Metallic Strainer	Standard kitchen strainer	Target	Laurentiis
Paraformaldehyde		Electron Microsc. Sciences	15710 (16%)
Penicillin/ Streptomycin		Gibco	15070 (100x)
Percoll		GE Healthcare Biosciences	17 0891 01 (1 Liter)
RELMα antibody		Peprotech	500-P214 (50μg)
Surgical Thread		Surgical Specialties Corp.	SP118 (2.0 USP 100yds)
Syringe Needle		BD Vacutainer Lab. Med.	305143 (23g, 3/4 inch)
USA Standard Testing Sieve		W.S. Tyler, Inc.	150μm, 0.0059" ASTME-11, Spec. No. 100
Xylazine		Butler	20mg/mL