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Production and analysis of immunomodulatory excretory-secretory products from the mouse gastrointestinal nematode Heligmosomoides polygyrus bakeri

Nature Protocols volume 9, pages 2740–2754 (2014)Cite this article

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Abstract

Heligmosomoides polygyrus bakeri (Hpb) infection in mice is a convenient model for studying the pathophysiology and immunology of gastrointestinal (GI) helminth infection. Hpb infection suppresses immune responses to bystander antigens and unrelated pathogens, and it slows the progression and modifies the outcome of immune-mediated diseases. Hpb-derived excretory-secretory (ES) products potently modulate CD4+ helper T cell (TH) responses by inducing regulatory T cells, tolerogenic dendritic cells (DCs) and immunoregulatory cytokines. This observation has spiked interest in identifying the immunomodulatory molecules, especially proteins, in ES products from Hpb and other GI nematodes for development as novel therapies to treat individuals with immune-mediated diseases, such as inflammatory bowel diseases (IBDs). In this protocol, we describe how to (i) maintain Hpb in the laboratory for experimental infections, (ii) collect adult worms from infected mice to generate ES products and (iii) evaluate the modulatory effects of ES products on toll-like receptor (TLR) ligand–induced maturation of CD11c+ DCs. The three major sections of the PROCEDURE can be used independently, and they require ∼6, 10 and 27 h, respectively. Although other methods use a modified Baermann apparatus to collect Hpb adult worms, we describe a method that involves dissection of adult worms from intestinal tissue. The protocol will be useful to investigators studying the host-parasite interface and identifying and analyzing helminth-derived molecules with therapeutic potential.

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Figure 1: Life cycle of H. polygyrus bakeri.
Figure 2: Hpb-derived ES products analyzed by denaturing SDS-PAGE followed by silver staining.
Figure 3: Culture of feces from Hpb-infected mice to generate L3-stage larvae (Steps 8–12).
Figure 4: Typical ELISA data from in vitro stimulation of spleen cells with Hpb AWH.
Figure 5: Typical ELISA result of suppression of CpG-ODN-induced BMDC IL-12 secretion by Hpb-derived ES products.

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Acknowledgements

R.M.V. is a recipient of a Studentship (2013–2014) from the Research Institute of the McGill University Health Centre. The work in our laboratories is supported by funding from the Natural Sciences and Engineering Research Council of Canada (NSERC) to M.S.; the Canadian Institutes of Health Research (CIHR) (MOP 94589) to A.J.; NSERC and Canada Research Chairs grants to T.G.G.; and CIHR (MOP 81169 and MOP 130369) to M.M.S. The Centre for Host-Parasite Interactions is supported by funding from the Fonds de Québec de recherche sur la nature et les technologies.

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Author notes

  1. Mariela Segura

    Present address: Present address: Faculty of Veterinary Medicine, Laboratory of Immunology, University of Montreal, St.-Hyacinthe, Quebec, Canada.,

  2. Rajesh M Valanparambil and Mariela Segura: These authors contributed equally to this work.

Authors and Affiliations

  1. Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada

    Rajesh M Valanparambil, Mariela Segura, Mifong Tam & Mary M Stevenson

  2. Department of Medicine, McGill University, Montreal, Quebec, Canada

    Rajesh M Valanparambil & Mary M Stevenson

  3. Centre for Host-Parasite Interactions, Institute of Parasitology, McGill University, Ste.-Anne de Bellevue, Quebec, Canada

    Rajesh M Valanparambil, Mariela Segura, Mifong Tam, Armando Jardim, Timothy G Geary & Mary M Stevenson

  4. Institute of Parasitology, McGill University, Ste.-Anne de Bellevue, Quebec, Canada

    Armando Jardim & Timothy G Geary

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Contributions

All authors contributed extensively to this manuscript. R.M.V. wrote the first draft and provided examples of original data for the ANTICIPATED RESULTS section. M.S. contributed to the development of the procedures for the adult worm collection and ES production, the generation of BMDCs and the evaluation of ES products on DC function. M.T. contributed to the development of the procedures for maintaining the Hpb parasite, for egg collection to obtain larvae for infection and to obtain ES products, and for collection of adult worms. A.J. and T.G.G. provided expert advice on the procedures and contributed significantly to finalizing the submitted manuscript. A.J., T.G.G. and M.M.S. jointly conceived the idea for the manuscript. M.M.S. provided supervision to R.M.V. in preparing the draft and wrote and revised the final manuscript.

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Correspondence to Mary M Stevenson.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Protein production by Hpb adult worm in culture. (PDF 84 kb)

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Valanparambil, R., Segura, M., Tam, M. et al. Production and analysis of immunomodulatory excretory-secretory products from the mouse gastrointestinal nematode Heligmosomoides polygyrus bakeri. Nat Protoc 9, 2740–2754 (2014). https://doi.org/10.1038/nprot.2014.184

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