Parasites, Probiotics, and Piglets: A Novel Approach to IBD
Authors: Eileen Devaney
Journal: Molecular Therapy (2014)

Helminth parasites are among the most potent modulators of mammalian immune responses. Their ability to suppress specific immune responses characteristically results in chronic long-lasting infections and reflects their long co-evolutionary history with their hosts. Indeed, studies in mouse models have shown that infection with live parasites or exposure to defined worm components can downregulate pathogenic proinflammatory or allergic responses. In this issue of Molecular Therapy, Whelan et al.1Whelan RA Rausch S Ebner F Günzel D Richter JF Hering NA et al.A transgenic probiotic secreting a parasite immunomodulator for site-directed treatment of gut inflammation.Mol Ther. 2014; 22: 1730-1740Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar describe the modification of a probiotic bacterium to express cystatin (AvCys), a potent immunomodulator from the filarial nematode Acanthocheilonema viteae, which, when administered to piglets, suppressed the pathology associated with post-weaning gut inflammation. The authors suggest that the modified probiotic may provide a potential therapy for the management of inflammatory bowel disease (IBD) in humans. Over the past 20–30 years, many studies have described the strong T helper type 2 cell (Th2)-polarizing impact of helminth infection, as well as the induction of various immune-regulatory networks (reviewed in ref. 2Maizels RM Infections and allergy—helminths, hygiene and host immune regulation.Curr Opin Immunol. 2005; 17: 656-661Crossref PubMed Scopus (183) Google Scholar). In areas of the world where parasitic helminths and other infectious diseases remain common, the incidence of various allergic and autoimmune conditions is low.3Yazdanbakhsh M Kremsner PG van Ree R Allergy, parasites, and the hygiene hypothesis.Science. 2002; 296: 490-494Crossref PubMed Scopus (1194) Google Scholar By contrast, countries where helminth infections have largely been controlled have an higher incidence of inflammatory conditions such as asthma, type 1 diabetes, and IBD. The precise mechanisms underlying helminth immunomodulation have been the source of much study, with recent focus on molecules that are excreted or secreted by parasites (ES). Of this class of molecule, several have been particularly well studied, including ES-62, a phosphorylcholine-containing molecule from A. viteae,4Harnett W Harnett MM Immunomodulatory activity and therapeutic potential of the filarial nematode secreted product, ES-62.Adv Exp Med Biol. 2009; 666: 88-94Crossref PubMed Scopus (27) Google Scholar the transforming growth factor-β homologue secreted by the murine gastrointestinal nematode Heligomosoides polygyrus,5Grainger JR Smith KA Hewitson JP McSorley HJ Harcus Y Filbey KJ et al.Helminth secretions induce de novo T cell Foxp3 expression and regulatory function through the TGF-beta pathway.J Exp Med. 2010; 207: 2331-2341Crossref PubMed Scopus (367) Google Scholar the omega-1 molecule released by the eggs of the trematode Schistosoma mansoni,6Everts B Perona-Wright G Smits HH Hokke CH van der Ham AJ Fitzsimmons CM et al.Omega-1, a glycoprotein secreted by Schistosoma mansoni eggs, drives Th2 responses.J Exp Med. 2009; 206: 1673-1680Crossref PubMed Scopus (284) Google Scholar and A. viteae cystatin, the focus of the paper by Whelan et al. Each of these well-characterized helminth products appears to influence the immune response via multiple independent mechanisms, underscoring the potential complexity of the host–parasite interaction. Cystatins first came to light in the early days of parasitic nematode molecular biology, when a common approach to identifying potential antigens was the screening of complementary DNA libraries with serum from infected individuals or animals. Using this approach, genes with homology to cystatins were cloned from the filarial nematodes Onchocerca volvulus, Brugia malayi, and A. viteae. In 1997, it was demonstrated that the cystatin from A. viteae (then known as Av-17) inhibited T-cell proliferation in response to polyclonal and antigen-specific stimulation via the induction of the immunoregulatory cytokine interleukin-10 (IL-10).7Hartmann S Kyewski B Sonnenburg B Lucius R A filarial cysteine protease inhibitor down-regulates T cell proliferation and enhances interleukin-10 production.Eur J Immunol. 1997; 27: 2253-2260Crossref PubMed Scopus (123) Google Scholar In keeping with these observations, high levels of IL-10 are frequently associated with reduced proliferative responses in humans infected with filarial parasites. Some cystatins are involved in the regulation of molting, whereas the molecules, represented by AvCys, B. malayi CPI-2, and others, are expressed during the mammalian stages of the parasite life cycle, are secreted, and have evolved immunomodulatory activity (reviewed in ref. 8Gregory WF Maizels RM Cystatins from filarial parasites: evolution, adaptation and function in the host–parasite relationship.Int J Biochem Cell Biol. 2008; 40: 1389-1398Crossref PubMed Scopus (57) Google Scholar). In keeping with these findings, the cystatin from the free-living nematode Caenorhabditis elegans does not possess an equivalent modulatory activity. Work from the Maizels group on B. malayi CPI-2 (ref. 8Gregory WF Maizels RM Cystatins from filarial parasites: evolution, adaptation and function in the host–parasite relationship.Int J Biochem Cell Biol. 2008; 40: 1389-1398Crossref PubMed Scopus (57) Google Scholar) and the Hartmann group on AvCys9Hartmann S Lucius R Modulation of host immune responses by nematode cystatins.Int J Parasitol. 2003; 33: 1291-1302Crossref PubMed Scopus (165) Google Scholar suggested several ways in which cystatins interfere with immune cell function. Elegant in vitro studies demonstrated that B. malayi CPI-2 blocked the endosomal/lysosomal proteases involved in antigen processing in a B-cell line and also inhibited presentation of a model antigen in a peptide-specific manner,10Manoury B Gregory WF Maizels RM Watts C Bm-CPI-2, a cystatin homolog secreted by the filarial parasite Brugia malayi, inhibits class II MHC–restricted antigen processing.Curr Biol. 2001; 11: 447-451Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar suggesting that cystatins act at multiple points to suppress the development of an effective immune response. The main target of AvCys is the macrophage, and studies in a mouse model of asthma demonstrated the potent in vivo activity of AvCys in regulating airway hypersensitivity or in modulating colonic inflammation induced by treatment of mice with dextran sodium sulfate (DSS). Blocking IL-10 or depletion of macrophages reversed the protective effect of cystatin on airway hypersensitivity.11Schnoeller C Rausch S Pillai S Avagyan A Wittig BM Loddenkemper C et al.A helminth immunomodulator reduces allergic and inflammatory responses by induction of IL-10–producing macrophages.J Immunol. 2008; 180: 4265-4272Crossref PubMed Scopus (202) Google Scholar These and other studies in animal models of allergy and autoimmunity paved the way for clinical trials using helminth therapy. Several studies have used live nematodes to modulate pathogenic responses in humans. To date, most studies have focused on eggs of the pig whipworm, Trichuris suis, which produce a self-limiting infection in humans, or infective larvae of the human hookworm, Necator americanus. In a randomized controlled trial, administration of T. suis ova resulted in an improvement in clinical scores in several individuals suffering from IBD.12Summers RW Elliott DE Urban Jr, JF Thompson RA Weinstock JV Trichuris suis therapy for active ulcerative colitis: a randomized controlled trial.Gastroenterology. 2005; 128: 825-832Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar Studies such as these sparked significant interest in using nematodes to control IBD and other pathologies, and clinical trials are now under way using T. suis ova to treat a variety of inflammatory conditions. In addition, some individuals are sufficiently motivated to self-treat: for example, a patient with ulcerative colitis ingested eggs of T. trichiura (the human-infective species), resulting in an improvement in symptoms, an effect that was associated with elevated levels of mucosal CD4 cells secreting IL-22 (ref. 13Broadhurst MJ Leung JM Kashyap V McCune JM Mahadevan U McKerrow JH et al.IL-22+ CD4+ T cells are associated with therapeutic Trichuris trichiura infection in an ulcerative colitis patient.Sci Transl Med. 2010; 2: 60ra88Crossref PubMed Scopus (170) Google Scholar). However, as might be predicted, the modulatory effect of worms varies with the specific condition under study, as well as the worm species employed. For example, experimental infection with ten L3 of N. americanus had no effect on the symptoms of asthma,14Feary JR Venn AJ Mortimer K Brown, AP Hooi D Falcone FH et al.Experimental hookworm infection: a randomized placebo-controlled trial in asthma.Clin Exp Allergy. 2010; 40: 299-306Crossref PubMed Scopus (145) Google Scholar whereas more recent studies investigating the interaction between N. americanus and gluten intolerance demonstrated that the presence of worms modified the immune response to gluten but did not improve clinical scores.15Croese J Gaze ST Loukas A Changed gluten immunity in celiac disease by Necator americanus provides new insights into autoimmunity.Int J Parasitol. 2013; 43: 275-282Crossref PubMed Scopus (28) Google Scholar Thus, although helminth therapy can produce many interesting data, the focus for the future should be on defining the parasite molecules that modulate specific pathogenic responses. In the study by Whelan et al.,1Whelan RA Rausch S Ebner F Günzel D Richter JF Hering NA et al.A transgenic probiotic secreting a parasite immunomodulator for site-directed treatment of gut inflammation.Mol Ther. 2014; 22: 1730-1740Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar AvCys was cloned into a probiotic bacterium, Escherichia coli Nissle 1917 (EcN), for ease of delivery, and the immunomodulatory potential of AvCys was assessed in both piglet and mouse models of gut inflammation. Because the intestinal tract of pigs is anatomically and physiologically more similar to humans than that of mice, the pig offers an interesting "halfway house" between mouse and human for such proof-of-concept studies. Colitis was induced in mice via administration of DSS and the effects of treatment compared in mice fed EcN alone, EcN expressing AvCys, and untreated controls. Mice receiving the modified probiotic showed lower pathology scores along with a significant decrease in macrophage numbers, which correlated with the reduced expression of selected chemokines. The frequency of regulatory T cells in draining lymph nodes was increased in animals receiving both the probiotic and the transgenic probiotic, and IL-17 production was significantly reduced compared with DSS control animals—but only in the animals receiving the cystatin-expressing probiotic. In contrast to the murine studies, the pig model relied on a naturally occurring post-weaning syndrome, an inflammatory reaction in the gut associated with the change of diet and/or stress. The authors set out to investigate whether the transgenic probiotic would confer additional protection over and above that of EcN alone, which is known to have various anti-inflammatory effects. The results are more complex than those obtained using inbred mice: when compared with piglets fed EcN or untreated controls, those fed the probiotic expressing cystatin exhibited a significant reduction in colonic inflammation. However, mechanistic studies failed to identify the immunological basis of the improvement in pathology scores; although there was a reduction in inflammation compared with saline-fed controls, the frequency of macrophages or of T cells with a regulatory phenotype did not differ between groups. These data suggest that the underlying immunological mechanisms that ameliorate pathology may differ between mouse and piglet. A more physiological assay, in which the transepithelial resistance of tissue explants from the pig colon was measured, demonstrated that the transformed probiotic enhanced epithelial barrier function when compared with the saline control group, an effect that could be mimicked by simply exposing the excised tissue to culture supernatant from the modified bacterium. Although the focus of the study by Whelan and colleagues was on possible use of the transgenic probiotic in humans afflicted with IBD, the work will also be of interest to the pig industry, where post-weaning syndrome can cause serious economic and welfare issues, as exemplified in the study by morbidity in the control groups. Although this study demonstrates that a modified probiotic has beneficial effects on gut inflammation in piglets, additional work is required to confirm the immunological mechanisms underlying the mitigation of pathology in animals fed the transgenic probiotic and to further investigate the apparent differences between the two model systems. In the murine model, gut inflammation is induced using a chemical stimulus (DSS), whereas in the piglet, inflammation is a consequence of weaning or stress, and the differences in results could reflect subtleties of the inflammatory insult. The disparate results may reflect differences in the basic mechanisms of disease and/or immunomodulation in the respective models and may also be influenced by temporal differences in the immune response elicited in either model (piglets were sampled at a single time point of 42 days). The gut microbiota is altered in diseases such as IBD and can be influenced by both probiotic bacteria and, as shown in some studies, intestinal helminths, so it would be interesting to determine whether the effects of the transgenic probiotic relate to changes in the intestinal microbiome. Whatever the case, the results of Whelan and colleagues are very promising and provide proof of concept that a probiotic bacterium transformed with a single parasite protein may provide a novel method of controlling gut inflammation. Still, IBD is a complex condition influenced by many factors, both genetic and environmental, and further studies aimed at defining the safety and the specificity of action of the transgenic bacterium will be essential. The author thanks Vicky Gillan and Roz Laing (University of Glasgow) for helpful comments on the manuscript.