1Institute of Immunology, Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, 2Pathobiology, School of Veterinary Medicine, University of Pennsylvania
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Joyce, K. L., Morgan, W., Greenberg, R., Nair, M. G. Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation. J. Vis. Exp. (64), e3905, doi:10.3791/3905 (2012).
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.
1. Purifying Schistosoma mansoni Eggs
2. Intraperitoneal Sensitization with S. mansoni Eggs: Day 0
3. Retro-orbital Intravenous Challenge with S. mansoni Eggs: Day 14
All of these procedures must be carried out with care, and mice must be monitored until they recover from anesthesia. These protocols were approved by the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC).
4. Experimental Harvest: Day 22
5. Preparation of Recovered Tissues
6. Representative Results
This protocol details all the steps necessary to (i) prepare purified Schistosoma mansoni eggs for in vivo use (Fig. 1), (ii) sensitize and challenge mice with these eggs and (iii) recover organs for examination of the lung inflammatory response. This in vivo model reproducibly drives a potent Th2 lung inflammatory response. This is shown by airway inflammation and eosinophilia as visualized by BAL cell counts and cytocentrifuge preparations (Fig. 2). H&E-stained lung sections can be examined for egg-induced granulomas (Fig. 3a), and immunofluorescent staining of lung sections allows visualization of alternatively activated macrophages and Th2 cytokine-induced genes such as Resistin-like molecule (RELM) α (Fig. 3b). The antigen-specific CD4 Th2 cytokine response is examined by ELISAs for IL-5, IL-4 and IL-13 of antigen-stimulated parathymic lymph node cells (Fig. 4).
Figure 1. Schistosoma mansoni egg injection model. Representative pictures of granulomatous livers, characteristic of high S.mansoni-egg burdens, and of S.mansoni eggs are shown - average size (L) 120 μm by (W) 50 μm.
Figure 2. S. mansoni egg injection drives airway inflammation. A. BAL cell numbers (x105) from naïve (N) or S.mansoni (Sm) egg-injected mice. *P<0.05. B. Cytocentrifuge preparation of BAL cells. Mac, MACROPHAGE; Eos, EOSINOPHIL; LYMPH, lymphocyte. Bar, 10 μm.
Figure 3. Visualization of pulmonary granulomas and alternatively activated macrophages surrounding the S.mansoni egg. A. Pulmonary granuloma surrounding the Sm egg (black arrow) was visualized in H&E-stained lung sections. B. Immunofluorescence staining for RELMα (green), mannose receptor (red) and DAPI (blue) reveals alternatively activated macrophages (white arrow) in the granuloma surrounding the autofluorescent Sm egg (black arrow). Bar, 50 μm.
Figure 4. S.mansoni egg antigen-specific Th2 cytokine response. Draining parathymic lymph node cells from naïve (N) or Sm egg-injected mice were stimulated with Sm egg antigen for 72 hours, followed by ELISA of supernatants for IL-4, IL-5 and IL-13. Scale, ng/mL.*P<0.05.
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, analysis of the cell infiltration in the airways, and size of the pulmonary granulomas. An additional parameter that can be measured is egg-induced fibrosis 12. This is measured by staining egg-injected lung sections with Masson's trichrome, which allows visualization of collagen deposition that occurs surrounding the vessels, the airways, and within the granuloma (shown in 6).
Although this protocol employs S. mansoni eggs, one limitation of this model is that it does not reproduce S. mansoni infection, which is performed by infecting mice with infectious cercariae. However, advantages of this model is the convenience for setting up, and that there is no risk of infection of the investigator. Following purification, the eggs can be conveniently frozen and used several months later. Once thawed, the eggs are not infectious, and can therefore be handled more easily than live eggs, or infectious cercariae. When preparing eggs for injection, one must ensure that eggs are intact by inspection under the microscope. To minimize egg disruption, wide-bore pipette tips and needles must be used when handling, and pipetting of eggs only when necessary. Since eggs settle rapidly, it is also critical to re-suspend by gentle rocking before counting or injecting.
Although the purified eggs can be frozen, infected livers must be processed immediately for successful purification of eggs. Since the severity of the infection, and the number of eggs present in the liver are mouse strain-dependent, the time-points for egg recovery and egg yields will vary dependent on the mice used. Here Swiss-Webster females were used, and sacrificed at 6-7 weeks post-infection for the generation of 10,000-30,000 eggs/liver. As a useful reference, Cheever et al. detail the liver egg burdens in several mouse strains 13.
Egg injection does not induce mortality. However, mice may succumb if over-anesthetized, if the egg preparation is not washed thoroughly (as detailed in the protocol), or if egg injection causes severe bleeding. An alternative to retro-orbital injection is tail vein injection of the eggs. This also results in a potent egg-induced lung inflammatory response 3. All of these procedures must be carried out with care, and mice must be monitored until they recover from anesthesia.
In conclusion, this protocol details injection of S. mansoni eggs for the induction of lung inflammation and Th2 cell responses, and is applicable for the study of helminth-induced inflammation, asthma, allergy and fibrotic diseases.
No conflicts of interest declared.
This work was supported by the National Institutes of Health (NIH) AI091759 and the Crohn's and Colitis Foundation of America's William and Shelby Modell Family Foundation Research Award (to MN). Schistosome-infected mice were supplied by the NIAID Schistosome Resource Center (NIAID Contract N01 A130026).
|Collagenase/Dispase||Roche Applied Science||11 097 113 001 (500mg)|
|Diff Quik Hema 3 Stain||Pack||Fisher Scientific Co. LLC||122-911|
|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)|
|RELM╬▒ antibody||Peprotech||500-P214 (50┬μg)|
|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.||150┬μm, 0.0059"
ASTME-11, Spec. No. 100