of worms, mice and man

Pharmacology & Therapeutics xxx (2014) xxx–xxx

JPT-06663; No of Pages 15

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Pharmacology & Therapeutics

j ourna l homepage: www.e lsev ie r .com/ locate /pharmthera

Associate editor: P. Holzer

Of worms, mice and man: An overview of experimental and clinicalhelminth-based therapy for inflammatory bowel disease

Marthe Heylen a,1, Nathalie E. Ruyssers a,1, Els M. Gielis a, Els Vanhomwegen a, Paul A. Pelckmans a,b,Tom G. Moreels a,b, Joris G. De Man a, Benedicte Y. De Winter a,⁎a Laboratory of Experimental Medicine and Pediatrics, Division of Gastroenterology, University of Antwerp, Antwerp, Belgiumb Antwerp University Hospital, Division of Gastroenterology & Hepatology, Antwerp, Belgium

Abbreviations:CDAI, Crohn's disease activity index; DC,zene sulfonic acid; DSS, dextran sulfate sodium; GMP, goodflammatory bowel disease; IBDQ, inflammatory bowelinterferon gamma; Ig, immunoglobulin; IL, interleukin; ILC,natively activatedmacrophage; NK, natural killer; T cell, T lygrowth factor beta; Th, T helper lymphocyte; TNBS, trinitrtumor necrosis factor alpha; Treg, regulatory T cell; UCDAI,index.⁎ Corresponding author at: Laboratory of Experime

University of Antwerp, Universiteitsplein, 12610Antwerp,E-mail address: [email protected] (

1 Both authors have equally contributed.

http://dx.doi.org/10.1016/j.pharmthera.2014.02.0110163-7258/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Heylen, M., et al.,inflammatory bowel disease, Pharmacology &

a b s t r a c t
a r t i c l e i n f o
Keywords:
HelminthsHelminth-derived moleculesTherapyInflammatory bowel diseaseColitisInnate and adaptive immunity
The incidence of inflammatory and autoimmune disorders is highest inwell-developed countrieswhich is direct-ly related to their higher hygienic standards: it is suggested that the lack of exposure to helminths contributes tothe susceptibility for immune-related diseases. Epidemiological, experimental and clinical data support the ideathat helminths provide protection against immune-mediateddiseases such as inflammatory bowel disease (IBD).The most likely mechanism for the suppression of immune responses by helminths is the release of helminth-derived immunomodulatory molecules. This article reviews the experimental and clinical studies investigatingthe therapeutic potential of helminth-based therapy in IBD and also focuses on the current knowledge of itsimmunomodulatory mechanisms of action highlighting innate as well as adaptive immune mechanisms.Identifying the mechanisms by which these helminths and helminth-derived molecules modulate the immunesystem will help in creating novel drugs for the treatment of IBD and other disorders that result from anoveractive immune response.

© 2014 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02. Short overview of the immunological changes during IBD . . . . . . . . . . . . . . . . . . . . . . . . . . . 03. Animal models to mimic human intestinal inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . 04. Helminths and helminth-derived molecules as therapeutic agents in IBD . . . . . . . . . . . . . . . . . . . . 05. What do we know about the underlying immunological mechanisms of protection? . . . . . . . . . . . . 06. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
dendritic cell; DNBS, dinitroben-manufacturing practice; IBD, in-disease questionnaire; IFN-γ,innate lymphoid cell; M2, alter-mphocyte; TGF-β, transformingobenzene sulfonic acid; TNF-α,ulcerative colitis disease activity

ntal Medicine and Pediatrics,Belgium. Tel.:+32 3 265 27 10.B.Y. De Winter).

Of worms, mice and man: ATherapeutics (2014), http://

1. Introduction

Worldwide, about 4 to 5 million people suffer from Crohn's diseaseor ulcerative colitis, together known as inflammatory bowel disease(IBD). IBD is a group of chronic inflammatory disorders of the gastroin-testinal tract, characterized by remitting and relapsing episodes of intes-tinal inflammation, often resulting in symptoms such as intermittentabdominal pain, rectal bleeding, fever, weight loss, fatigue and diarrhea(Braus & Elliott, 2009). There is a broad arsenal of therapeutic optionsfor IBD, including 5-aminosalicylates (e.g. sulfasalazine, mesalamine),corticosteroids (e.g. prednisone, budesonide), immunomodulators(e.g. azathioprine, methotrexate), antibiotics (e.g. ciprofloxacin,

n overview of experimental and clinical helminth-based therapy fordx.doi.org/10.1016/j.pharmthera.2014.02.011

http://dx.doi.org/10.1016/j.pharmthera.2014.02.011

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2 M. Heylen et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx

metronidazole) and/or biologicals (e.g. infliximab, adalimumab)(Morrison et al., 2009; De Vroey & Colombel, 2011). However,no curative treatment is currently available (McSorley et al., 2013;Pedersen et al., 2014). The impact of IBD on the patient's quality of

Please cite this article as: Heylen, M., et al., Of worms, mice and man: Ainflammatory bowel disease, Pharmacology & Therapeutics (2014), http://

life is high, as the onset of IBD typically occurs in the second and thirddecades of life, i.e. in the active socioeconomic years of one's life, andoften leads to absenteeism (Xavier & Podolsky, 2007). The etiology ofIBD still remains unknown, but a loss of immune tolerance to normal



3M. Heylen et al. / Pharmacology & Therapeutics xxx (2014) xxx–xxx

commensal enteric flora in genetically susceptible individuals is postulat-ed as the underlying pathogenic mechanism (Podolsky, 2002; Braus &Elliott, 2009; Scharl & Rogler, 2012).

A rapid increase of IBDwas seen during the second half of the twen-tieth century, especially in the industrialized, developedWestern coun-tries (Lapidus, 2001). Industrialization was accompanied by improvedhygiene, sanitation and medical conditions and regulated food indus-tries which have led to the eradication of infectious agents such asparasitic worms (helminths) (Feillet & Bach, 2004; Elliott et al., 2007).For example, the prevalence of hookworm infections, an intestinal nem-atode, inNorth American schoolchildren dropped from65% in the 1910sto less than 2% in the 1980s (Elliott &Weinstock, 2012a). It is suggestedthat the lack of exposure to helminths contributes to the susceptibilityfor immune-related diseases such as IBD (Elliott et al., 2000), asthma,atopic eczema, allergic rhinoconjunctivitis (van den Biggelaar et al.,2000; Cooper et al., 2003; Rautava et al., 2004; van den Biggelaar et al.,2004; Yazdanbakhsh &Wahyuni, 2005;Wordemann et al., 2008), mul-tiple sclerosis (Fleming, 2013), cardiovascular diseases (Magen et al.,2005), rheumatoid arthritis (Harnett &Harnett, 2009) and type 1 diabe-tes mellitus (Cooke et al., 2004; Zaccone et al., 2009). Epidemiological,experimental and clinical data now support the idea that helminthsprovide protection against immune-mediated diseases. However, themechanism by which helminths provide this protection is not yetfully understood. It is known that helminths release helminth-derivedimmunomodulatory molecules which may be responsible for thesuppression of immune responses (Ruyssers et al., 2008).

2. Short overview of the immunological changes during IBD

The clinical manifestations of IBD result from complex interactionsbetween genetic factors, environmental factors and the immune system(Fiocchi, 1998, 2012; Speight &Mansfield, 2013). Under normal circum-stances, mucosal immune responses are tightly regulated to ensure guthomeostasis. The mucosal immune system balances between a protec-tive response to pathogenic antigens and tolerance to antigens fromfood and common bacterial luminal flora (Neuman, 2007). However,it is postulated that in IBD this important immunological homeostasisis disrupted leading to uncontrolled chronic inflammation towardsintraluminal antigens of bacterial origin (Plevy, 2002; Bamias et al.,2005; Jeon et al., 2013). The precise role of bacteria in the etiology ofIBD remains elusive but three theories, not necessarily mutually exclu-sive, have been proposed: (1) an unidentified persistent pathogen,(2) an abnormally permeable mucosal barrier leading to excessive bac-terial translocation and (3) a breakdown in the balance of the gutmicrobiome called dysbiosis (De Hertogh et al., 2008; Shim, 2013).Published data suggest thatmucosal and fecalmicroflora differ betweenIBD patients and healthy controls (DeHertogh et al., 2008; Knights et al.,2013). Evidence exists in favor of an intestinal barrier dysfunction in IBDproviding access of luminal antigens to the underlying tissue (Baumgart& Carding, 2007; Pastorelli et al., 2013). The perturbations in the luminalbarrier function in IBD include a reduction of secretions, a reducednumber of secretory cells and disabled tight junctions (McGuckin et al.,

Fig. 1. Immunology of the mucosa of the small intestine in a healthy (A) and inflammatory condimmune system (e.g. DCs and macrophages) and the adaptive immune system (e.g. T and B lymPeyer's patch is antigen sampling, as Peyer's patches have a thin mucus layer and contain spebarrier. DCs and macrophages present in the matrix of the Peyer's patch sample antigens thatto Th0 cells in the T cell zone of the Peyer's patch or mesenteric lymph nodes. In healthy condof Treg cells results in an anti-inflammatory response, as Treg cells migrate back to the lamina pecules such as IL-10 and TGF-β, which exert a suppressive action on the immune cells within thand preventing unnecessary inflammation.Defects in the function of Treg cells are associatedwisusceptibility, can lead to intestinal inflammation. In this case, epithelial cells which are in contpatches. DCs and macrophages consider these bacteria as foreign and are activated and convertmatory molecules such as IL-1β, IL-6, IL-12, IL-18, IL-23 and IL-27. Due to this inflammatory enfrom Th0 cells. The Th1 and Th17 cells migrate via the efferent lymphatic vessels and the bloodsponse by secreting their own inflammatory molecules such as IFN-γ, IL-2 and IL-17 which calymphocyte; IFN-γ: interferon-γ; IL: interleukin; ILC: innate lymphoid cell; M1: classically activT lymphocyte; Th1: T helper 1 lymphocyte; Th17: T helper 17 lymphocyte; Treg: regulatory T


2009). Increased permeability of both the inflamed and non-inflamedmucosa has been described in IBD patients (Soderholm et al., 2002).

Antigens breaking through this disturbed barrier are subsequentlyphagocytosed by antigen presenting cells such as dendritic cells (DCs)and macrophages. DCs and macrophages are key cells in controllingimmune responses presenting antigenic peptides via the major his-tocompatibility complex class II molecules to lymphocytes (Cader &Kaser, 2013). It is assumed that DCs and macrophages in healthy indi-viduals stimulate naive T cells to differentiate into regulatory T (Treg)cells which play an important role in controlling immune homeostasisand maintaining tolerance (Fig. 1A). On the contrary, activated DCsand macrophages in IBD patients stimulate T cells to differentiateinto proinflammatory T helper (Th) 1 and Th17 effector cells (Fig. 1B)(Baumgart & Carding, 2007; Rutella & Locatelli, 2011). Patients sufferingfrom IBD have disturbed clearance of these proinflammatory Th1 andTh17 cells which might overwhelm regulatory control mechanismsand can result in inflammation. On the other hand, IBD may developdue to a failure of Treg cells and their corresponding regulatory cyto-kines, such as interleukin (IL)-10 and tissue growth factor (TGF)-β, tocontrol inflammation and effector pathways (Eksteen et al., 2005;Baumgart & Sandborn, 2012). Proinflammatory cytokines such as IL-2,IL-12 and interferon (IFN)-γ produced by activated Th1 cells stimulatemacrophages to secrete large amounts of other proinflammatorycytokines e.g. tumor necrosis factor (TNF)-α, IL-1 and IL-6 (Moreels &Pelckmans, 2005; Neuman, 2007). These cytokines amplify the proin-flammatory immune response by promoting the proliferation of effec-tor Th1 and Th17 cells and by stimulating the release of chemokineswhich will attract more inflammatory cells to the site of inflammation(Baumgart & Carding, 2007; Neuman, 2007). Overall, a disruption ofthe immune balance whereby proinflammatory Th1 and Th17 cellsand their corresponding proinflammatory cytokines outnumber theregulatory T cell responses can lead to inflammation (Fig. 2A). Findingways to influence the immunological process during inflammationmight contribute to the development of new therapeutic options in IBD.

3. Animal models to mimic human intestinal inflammation

Helminths and helminth-derived molecules provide a promisingnew therapy against IBD. The underlying mechanisms of how theseorganisms influence the immune system to attenuate inflammationare a topic of increasing research interest.

Animalmodels of intestinal inflammation provide powerful tools forthe investigation of the pathogenesis of IBD (Wirtz & Neurath, 2007).Although these models do not exactly represent the complexity ofhuman disease, they have similar immunological and histopathologicalfeatures (Elson et al., 2005; Mizoguchi & Mizoguchi, 2008). In additionto the study of the pathogenesis, animal models also allow the testingof new therapeutic strategies in the preclinical phase (Wirtz &Neurath, 2000, 2007). Several experimental animalmodels are availableto investigate intestinal inflammation. They can be divided into four dif-ferent categories: spontaneous models, inducible models, geneticallyengineered models (transgenic mice, knockout mice) and adoptive

ition (B).Within the Peyer's patchmatrix lies a mix of immune cells from both the innatephocytes), which forms a barrier against microbial invasion. An important function of thecialized phagocytic cells called M cells, which can transport material across the epithelialare then broken down. These antigen-loaded DCs and macrophages present the antigensitions (A), the Th0 cells differentiate into immune modulatory Treg cells. The activationropria of the villi via the efferent lymphatic vessels and the bloodstream, and secrete mol-e lamina propria. IL-10 and TGF-β are therefore critical in maintaining immune toleranceth IBD (B). It is assumed that barrier disruption, due to enteric bacterial triggers and geneticact with commensal enteric bacteria are activated leading to bacterial influx in the Peyers'ed into inflammatory DCs or M1macrophages. These DCs andM1s start to release inflam-vironment, proinflammatory effector T cells such as Th1 and Th17 cells are differentiatedstream back to the lamina propria where they coordinate an escalation of the immune re-n lead to intestinal inflammation. B: B lymphocyte; DC: dendritic cell; IEL: intraepithelialatedmacrophage; M cell: microfold cell; TGF-β: transforming growth factor-β; Th0: naivelymphocyte.



Fig. 2. Immune profiles duringCrohn's disease (A) and thehypothesized immune profile of helminth-based therapy inCrohn's disease (B). The immunebalance inCrohn's disease patientsis disrupted (A), as it is primarily driven by Th1 and Th17 cells, M1 andDCs and their corresponding proinflammatory cytokines such as TNF-α, IFN-γ, IL-17, IL-1, IL-2, IL-6, IL-12 and IL-23.This proinflammatory response outnumbers the Treg cell response, responsible for controlling immunity and maintaining tolerance. Therefore the immune balance shifts towards theproinflammatory side resulting in inflammation. Thanks to the immune regulatory mechanisms of helminths, it is hypothesized that helminth-based therapy in Crohn's disease patients(B) can restore the immune balance by creating a regulated environment inwhich the proinflammatory Th1 and Th17 responses are suppressed. To obtain that goal, Treg activity needs tobe restored and Th2, M2macrophages and tolerogenic DC responses are induced with an increase in corresponding regulatory cytokines such as IL-10 and TGF-β, IL-4, IL-5 and IL-13. DC:dendritic cell; IFN-γ: interferon-γ; IL: interleukin; M1: classically activated macrophage; M2: alternatively activated macrophage; TGF-β: transforming growth factor-β; Th1: T helper 1lymphocyte; Th2: T helper 2 lymphocyte; Th17: T helper 17 lymphocyte; TNF-α: tumor necrosis factor-α; tolDC: tolerogenic DC; Treg: regulatory T lymphocyte.


transfer models (Wirtz & Neurath, 2000, 2007; Dothel et al., 2013).Models of spontaneous colitis include C3H/HeJBir mice, SAMP1/Yitmice and the cotton-top tamarin model (Wirtz & Neurath, 2000;Jurjus et al., 2004). Experimental inflammation of the gut can also be in-duced by variousmechanical or chemicalmethods leading to disruptionof the mucosal barrier. Examples of inducible colitis models areacetic-acid colitis, dextran sulfate sodium (DSS) colitis and dinitro-benzene and trinitrobenzene sulfonic acid (DNBS and TNBS) colitis(Wirtz & Neurath, 2000; Kawada et al., 2007; Wirtz et al., 2007).The use of genetically engineered models allows the study of immu-noregulatory pathways and they include the IL-2 and IL-10 knock-out (IL-2−/− and IL-10−/−) mice and the IL-7 and signal transducerand activating transcription (STAT) 4 transgenic mice (Wirtz &Neurath, 2000, 2007). The pioneering work of Powrie et al. led tothe adoptive transfer models where inflammation is induced by theselective transfer of certain immune cell types (e.g. IL-10−/− Tcells, CD4+CD25−CD62L+ T cells, CD45RBhiCD4+ T cells) to immu-nocompromised host animals (Powrie et al., 1993; Read & Powrie,2001, chap. 15; Ostanin et al., 2006). The models most widely usedfor the study of helminthic therapy during intestinal inflammationare the DSS, DNBS and TNBS model, and the T cell transfer colitismodel (Elliott & Weinstock, 2012b).

4. Helminths and helminth-derivedmolecules as therapeutic agents in IBD

4.1. Why helminths can be used to ameliorate gut inflammation

Helminths have always colonized humans and consequently haveco-evolved with their hosts (Dunne & Cooke, 2005). It is thought thathelminths were free-living organisms that adapted to parasitic lifecycles to ensure their survival when environmental conditions werepoor (Mulcahy et al., 2004). Many of the various helminth specieshave different life cycles and occupy different niches in their hosts,e.g. the intestinal lumen, blood stream or lymphatics (Weinstock et al.,2005). Although different helminth species may evoke somewhatdifferent host immune responses due to different life cycles, there aremany similarities in these reactions (Meeusen, 1999; Jackson et al.,


2009). During helminth infection the host evokes a strong Th2 immuneresponse that involves IL-4, IL-5, IL-9, IL-10 and IL-13 cytokine release,the production of immunoglobulins (Ig) IgG1, IgG4 and IgE and the ac-tivation of dendritic cells, eosinophils, basophils, mast cells and alterna-tively activated macrophages (M2) to provide protection against wormcolonization (Fig. 3) (Maizels et al., 2004; Allen & Maizels, 2011;Salgame et al., 2013).

To ensure survival within their host, helminths developed immunemechanisms to prevent evasion such as migratory strategies togetherwith the induction of Treg cells and the production of their regulatorycytokines IL-10 and TGF-β which leads to a more immunosuppressivestate in the host (Fig. 3) (Elliott et al., 2005; Maizels et al., 2012;Taylor et al., 2012). More recently attention is being paid to the innateimmune cells. As part of the innate immune system, phagocyteslike monocytes, macrophages and DCs, and cytotoxic cells like naturalkiller cells will respond to pathogen associated molecular patterns(Medzhitov & Janeway, 2000; Pulendran & Artis, 2012). These cellswill be the ‘first responders’ towards helminths and their productsand they will subsequently skew the adaptive immune responsetowards Th2 and Treg responses which is suggested to suppress thedamaging Th1 and Th17 effector cells, responsible for maintaining theinflammation in IBD (Ruyssers et al., 2008; McSorley & Loukas, 2010;Khan & Fallon, 2013) (Fig. 2B).

4.2. Helminthic therapy: animal studies

A vast amount of experimental data supports the hypothesis thathelminths are able to modulate IBD. The possible beneficial effects ofhelminth infections on the development and the course of colitis havebeen investigated in different animal models (Weinstock & Elliott,2013). Preliminary data of Elliott et al. (2000) first confirmed thatthe loss of exposure to helminths increased the risk of intestinal inflam-mationwhen they found thatmice pre-exposed to Schistosomamansoniwere protected fromdeveloping TNBS-induced colitis and that IL-10−/−

mice treated with Heligmosomoides polygyrus bakeri third stage larvaedeveloped significantly less intestinal inflammation. Furthermore, thisgroup showed that Trichuris muris eggs significantly attenuatedintestinal inflammation in IL-10−/− mice and protected mice from



Fig. 3. Immune profiles during helminth infection. During helminth infection, helminthsattach to or traverse through the epithelial cell layer. Damaged epithelial cells release“alarmins” such as IL-25, IL-33 and TSLP. These alarmins together with helminth-derivedproducts promote a Th2 and Treg response through the tolerogenic activation of DCs.The evoked Th2 response results in IL-4, IL-5, IL-13, IL-9, IL-10 cytokine release and theproduction of immunoglobulins IgG1, IgG4 and IgE which on their turn drive the activa-tion of M2macrophages, mast cells, eosinophils and basophils. B: B lymphocyte; DC: den-dritic cell; Ig: immunoglobulin; IL: interleukin; M2: alternatively activated macrophage;TGF-β: transforming growth factor-β; Th2: T helper 2 lymphocyte; tolDC: tolerogenicDC; Treg: regulatory T lymphocyte; TSLP: thymic stromal lymphopoietin.


TNBS colitis (Elliott et al., 2000). These preliminary data have led to afirst complete report on the beneficial effect of helminths on the courseof DSS-induced colitis (Reardon et al., 2001). They were able to showthat Hymenolepis diminuta infection, either prophylactic or therapeutic,caused a significant amelioration of DSS associated ion transport abnor-malities in the colon. However, no improvements in colonic histopa-thology were observed (Reardon et al., 2001). Since then, severalstudies showed that helminth infections of different species have bene-ficial preventive and therapeutic effects on experimental colitis, al-though the underlying mechanisms to reach the positive effects maydiffer between different helminth species. What follows is an overviewof all the experimental studies conducted so far. Table 1 summarizes thestudies explicating the helminths tested, the animal models used, theoutcomes measured and the suggested mechanisms of action.

We and others demonstrated that an infection with S. mansoni cer-cariae exerted preventive effects on the course of TNBS-induced colitisin rats and DSS-induced colitis in mice (Moreels et al., 2004; Smithet al., 2007; Bodammer et al., 2011). Furthermore, S. mansoni eggsprevented mice from developing TNBS-induced colitis, but did not pre-vent mice from developing DSS colitis (Elliott et al., 2003; Smith et al.,2007). Schistosoma japonicum eggs also exerted preventive effects onTNBS-induced colitis in mice (Zhao et al., 2009; Xia et al., 2011). Aprior infection of mice with Trichinella spiralis larvae and Trichinellapapuae larvae reduced the severity of DNBS- and DSS-induced colitis re-spectively (Khan et al., 2002; Adisakwattana et al., 2013). H. polygyrusbakeri larvae had different effects on different animal models of colitis.For example, they suppressed established colitis in piroxicam treatedIL-10−/− mice (Elliott et al., 2004, 2008), they exerted both preventiveand therapeutic effects on colitis in the IL10−/− T cell transfer modelin mice (Metwali et al., 2006; Hang et al., 2010; Blum et al., 2012) andthey protected mice from TNBS-induced, DSS-induced and antigen-


driven colitis (Setiawan et al., 2007; Sutton et al., 2008; Donskow-Lysoniewska et al., 2012; Leung et al., 2012). However, H. polygyrusbakeri larvae enhanced Citrobacter rodentium-induced infectious colitisin mice (Chen et al., 2005, 2006) and failed to prevent colitis in TGF-βRII DN mice (i.e. mice with interrupted T cell TGF-β signaling), sug-gesting the requirement of TGF-β signaling through mucosal T cellsfor the control of colitis (Ince et al., 2009). InfectionwithH. diminuta lar-vae in DNBS mice had a profound anti-colitis effect (both prophylacti-cally and as a treatment), which was not seen in semipermissive rats(Hunter et al., 2005, 2010; Melon et al., 2010). In contrast, H. diminutainfection caused an exacerbation of oxazolone-induced colitis in mice(Hunter et al., 2007; Wang et al., 2010). T. muris-infected IL-10−/−

mice were not protected from developing a severe colitis (Wilsonet al., 2011). Worthwhile mentioning is also the study of Broadhurstet al. (2012) who used macaque monkeys with idiopathic chronic diar-rhea, a preclinical model for ulcerative colitis. Treatment of these non-human primates with Trichuris trichiura eggs resulted in clinical im-provement in fecal consistency and weight gain.

As shown in Table 1, most of these studies clearly indicate that sev-eral species of helminths exert both prophylactic and therapeutic effi-ciency in different models of experimental colitis in animals, althoughthe studies also indicate that helminthic therapy is both disease- andhelminth-specific (McKay & Wallace, 2009).

4.3. Helminthic therapy: clinical studies

Soon after the first promising findings of helminth infections on ex-perimental colitis were published, clinical trials were started to explorewhether helminths could alter the course of disease in IBD patients.These studies are summarized in Table 2 describing the study protocol,parameters measured and main outcomes. Summers et al. (2003,2005a,b,c) were the first to conduct three small clinical trials, in whichpatients with ulcerative colitis or Crohn's disease consumed viable em-bryonated eggs (ova) of the pig whipworm Trichuris suis. In the firstsmall open-label trial, a single dose of 2500 T. suis ova was tested in 4Crohn's disease and 3 ulcerative colitis patients and patients werefollowed every 2 weeks for 12 weeks. Furthermore, repeated doses of2500 T. suis ova, at 3-week intervals for 28 weeks, were tested in 2Crohn's disease and 2 ulcerative colitis patients. During both treatmentregimens, all patients had improvement in their symptoms withoutside effects. After a single dose of T. suis ova, 3 of the 4 patientswith Crohn's disease achieved remission according to the Crohn'sDisease Activity Index (CDAI b= 150) and all ulcerative colitis patientsachieved remission according to the Simple Clinical Colitis ActivityIndex (SCCAI b= 4). Although this beneficial effect was temporary, itwas shown that repeated doses of T. suis ova sustained clinical improve-ment in all patients (Summers et al., 2003). In the second small open-label trial including 29 active Crohn's disease patients, 79.3% of thepatients responded to repeated doses of 2500 T. suis ova given every3 weeks for 24 weeks (decrease in CDAI N 100 points), while 72.4%of the patients achieved remission (CDAI b= 150) (Summers et al.,2005a). The third trial was a randomized double-blind placebo-controlled trial including 54 active ulcerative colitis patients. It revealedthat a significant number of ulcerative colitis patients (43.3%) receivingrepeated doses of 2500 T. suis ova every 2 weeks for 12 weeksresponded (Ulcerative Colitis Disease Activity Index (UCDAI) b 4) com-pared to placebo-treated ulcerative colitis patients (16.7%). However,no significant differences in remission rates were observed betweenboth treatment groups (UCDAI b 2) (Summers et al., 2005b).

After these promising results, the United States Food and DrugAdministration requested the development of T. suis ova under goodmanufacturing practice (GMP) and appropriate safety testing in orderto continue clinical tests. Consequently, a small randomized double-blind placebo-controlled trial was initiated, testing the tolerabilityand safety of different doses of GMP-approved T. suis ova (http://clinicaltrials.gov/ trial identifier NCT01434693) (Sandborn et al.,


http://clinicaltrials.gov/

http://clinicaltrials.gov/


Table 1

Effects of helminths on colitis in experimental animal studies.

Authors & year Helminth Class Host Colitismodel

Outcome Mechanisms of action

Reardon et al., 2001 Hymenolepisdiminuta larvae

Cestode Mouse DSS colitis Preventive andcurative treatment: normalization ofcolonic ion transport, but noimprovement in colonichistopathology

Assumed Th2 response

Khan et al., 2002 Trichinellaspiralis larvae

Nematode Mouse DNBS colitis Preventive treatment: attenuationof colitis

Augmented Th2 response (↑IL-4, ↑IL-13)

Elliott et al., 2003 Schistosomamansoni eggs

Trematode Mouse TNBS colitis Preventive treatment: attenuationof colitis

Diminished Th1 response (↓IFN-γ), augmented Th2response (↑IL-4), Treg response (↑IL-10 mRNA expression),STAT6-mediated response required for protection

Moreels et al., 2004 Schistosomamansoni larvae

Trematode Rat TNBS colitis Preventive treatment: attenuationof colitis

Augmented Th2 response (↑IL-4)

Elliott et al., 2004 Heligmosomoidespolygyrus bakerilarvae

Nematode Mouse IL-10−/−

colitisCurative treatment: suppressionof established colitis

Diminished Th1 response (↓IL-12, ↓IFN-γ), augmented Th2response (↑IL-13), Treg response (↑Foxp3 mRNAexpression)

Hunter et al., 2005 Hymenolepisdiminuta larvae

Cestode Mouse DNBS colitis Preventive treatment: attenuationof colitis

Augmented Th2 response (↑IL-4, ↑IL-10 mRNA expression),

Curative treatment: enhancedrecovery of colitis

IL-10 required for protective effect


Cestode Rat DNBS colitis Preventive treatment: no effect oncolitis

Not specified

Chen et al., 2005 Heligmosomoidespolygyrus bakerilarvae

Nematode Mouse Citrobacterrodentium-inducedcolitis

Preventive treatment: worseningof Citrobacter rodentium-inducedinfectious

Augmented Th2 response (↑IL-4, ↑ IL-5, ↑IL-10), augmentedTreg response (↑IL-10), changes in Th1 response (↓IFN-γ,↑TNF-α), STAT6-mediated mechanism

Chen et al., 2006 Heligmosomoidespolygyrus bakerilarvae

Nematode Mouse Citrobacterrodentium-inducedcolitis

Colitis preventive treatment:worsening of Citrobacter rodentium-induced infectious colitis

CD11c+ DC expansion and ↑IL-10 mRNA expression

Metwali et al., 2006 Heligmosomoidespolygyrus bakerilarvae

Nematode Mouse IL10−/− Tcell transfercolitis

Curative treatment: reversal ofestablished colitis

Appearance of CD8+ regulatory T cells


Cestode Mouse Oxazolonecolitis

Preventive treatment: worseningof colitis

Augmented Th2 response (↑IL-4, ↑IL-5, ↑IL-13, ↑IL-10),changes in Treg response

Smith et al., 2007 Schistosomamansoni larvae

Trematode Mouse DSS colitis Preventive treatment: protectionfrom colitis

Induction of F4/80+CD11b+CD11c− macrophages

Smith et al., 2007 Schistosomamansoni eggs

Trematode Mouse DSS colitis Preventive treatment: worseningof colitis

Not specified

Setiawan et al.,2007

Heligmosomoidespolygyrusbakerilarvae

Nematode Mouse TNBS colitis Preventive treatment: protectionfrom colitis

Diminished Th1 response (↓IL-12p40, ↓IFN-γ), augmentedTreg response (↑IL-10)

Elliott et al., 2008 Heligmosomoidespolygyrus bakerilarvae

Nematode Mouse IL-10−/−

colitisCurative treatment: improvementof established colitis

IL-4 and IL-10mediated inhibition of Th17 response (↓IL-17)

Sutton et al., 2008 Heligmosomoidespolygyrus bakerilarvae

Nematode Mouse TNBS colitis Preventive treatment: attenuationof colitis

Diminished Th1 response (↓TNF-α, ↓IFN-γ mRNAexpression), augmented Th2 response (↑IL-4, ↑IL-13 mRNAexpression), mast cell-mediated effect

Zhao et al., 2009 Schistosomajaponicum eggs


Changes in Th1 response (↓IFN-γ,=TNF-α), augmentedTh2response (↑IL-10 mRNA expression), downregulation ofTLR4 mRNA expression related to ↓IFN-γ and ↑IL-10

Ince et al., 2009 Heligmosomoidespolygyrus bakerilarvae

Nematode Mouse TGF-βRII DNcolitis

Preventive treatment: no effect oncolitis

IL-10 secretion requires intact T cell TGF-β signaling

Hang et al., 2010 Heligmosomoidespolygyrus bakerilarvae


Preventive and curative treatment:suppression of colitis

Alteration of intestinal DC function resulting in diminishedIFN-γ and IL-17 responses

Wang et al., 2010 Hymenolepisdiminuta larvae

Cestode Mouse Oxazolonecolitis

Preventive treatment: worseningof colitis

Involvement of Th2 response (↑IL-5 and ↑recruitment ofeosinophils)

Melon et al., 2010 Hymenolepisdiminuta larvae

Cestode Mouse DNBS colitis Preventive treatment: protectionfrom colitis

Diminished Th1 response (↓TNF-α, ↓IFN-γ), augmented Th2response (↑IL-4, ↑ IL-10, ↑eosinophils), augmented Tregresponse (↑IL-10)


Please cite this article as: Heylen, M., et al., Of worms, mice and man: An overview of experimental and clinical helminth-based therapy forinflammatory bowel disease, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.011


Table 1(continued)

Authors & year Helminth Class Host Colitismodel

Outcome Mechanisms of action


Cestode Mouse DNBS colitis Preventive treatment: attenuationof colitis

↑Mobilization of alternatively activated macrophages

Bodammer et al.,2011

Schistosomamansoni larvae

Trematode Mouse DSS colitis Preventive treatment: attenuationof colitis

Diminished Th1 response (↓TNF-α, ↓IL-2mRNA expression),diminished Th2 response (↓IL-4 mRNA expression)

Xia et al., 2011 Schistosomajaponicum eggs


Diminished Th1 response (↓TNF-α, ↓IFN-γ), maintainingepithelial barrier function trough augmented tight junctionproteins

Wilson et al., 2011 Trichuris muriseggs

Nematode Mouse L-10−/−

colitisExacerbation of colitis Augmented Th1 response (↑IFN-γ), augmented Th17

response (↑IL-17A), ↑IL-13 decoy receptor (IL-13Rα2)resulting in ↓IL-13 activity

Blum et al., 2012 Heligmosomoidespolygyrus bakerilarvae


Preventive treatment: protectionfrom colitis

Induction of tolerogenic DCs

Donskow-Lysoniewskaet al., 2012

Heligmosomoidespolygyrus bakerilarvae

Nematode Mouse DSS colitis Curative treatment: protectionfrom colitis

Augmented macrophage infiltration (↑IL-1β, TNF-α, IL-6),augmented expression of MOR1, POMC and β-endorphin

Leung et al., 2012 Heligmosomoidespolygyrus bakerilarvae

Nematode Mouse Antigen-drivencolitis

Preventive treatment: protectionfrom colitis

Diminished Th1 response (↓IFN-γ), diminished Th17response (↓IL-17), induction of Foxp3+ Treg cells, ↑IL-10from non-T cells

Broadhurst et al.,2012

Trichuristrichiura eggs

Nematode Macaquesmonkeys

Idiopathicchronicdiarrhea

Curative treatment: clinicalimprovement in fecal consistencyand weight gain

Augmented Th2 response (↑CD4+ T cells producing IL-4),diminished fraction of Ki67+ CD4+ T cells (which areindicators of ongoing inflammation), diminished Th1-typeinflammatory gene expression, augmented gene expressionassociated with IgE signaling, mast cell, eosinophil andalternatively activated macrophage activation, reducedbacterial attachment to the intestinal mucosa and changesin composition of attached bacteria

Adisakwattanaet al., 2013

Trichinellapapuae larvae

Nematode Mouse DSS colitis Preventive treatment: attenuationof colitis

Changes in Treg response

CD: cluster of differentiation; DCs: dendritic cells; DNBS: dinitrobenzene sulfonic acid; DSS: dextran sulfate sodium; Foxp3: forkhead box p3; IFN-γ: interferon-γ; IgE: immunoglobulinE; IL: interleukin; IL10−/−: IL-10 deficient; MOR1: μ-opioid receptor; POMC: proopiomelanocortin; STAT6: signal transducer and activator of transcription 6; TGF-β: transforming growthfactor-β; TGF-βRII DN: interrupted T cell TGF-β signaling; Th: T helper cells; TLR4: toll-like receptor 4; TNBS: trinitrobenzene sulfonic acid; TNF-α: tumor necrosis factor-α; Treg:regulatory T cells.


2013). Crohn's disease patients receiving a single dose of 500, 2500,7500 GMP-approved ova or placebo did not show short-term (2 weeks)or long-term (6 months) treatment-related adverse effects, and a singledose of T. suis ova up to 7500 was well tolerated (Sandborn et al., 2013).In the meantime, a small open-label trial with the human hookwormNecator americanus was conducted in Crohn's disease patients (Croeseet al., 2006). Twenty weeks postinfection, the average CDAI and theaverage Inflammatory Bowel Disease Questionnaire (IBDQ) improved(CDAI b= 150 and IBDQ N= 170). However, 2 out of 9 patients withmoderately active disease showed a worsening in symptom scores afterthey received 50 infective larvae (L3i) (Croese et al., 2006). Recently,twomulticenter randomizeddouble-blindplacebo-controlled Phase II tri-als were conducted in the USA (TRUST-I: trial identifier NCT01576471(Coronado Biosciences)) and in Europe (TRUST-II: trial identifierNCT01279577 (Dr. Falk Pharma)), testing the safety and efficacy of a 12week treatmentwithGMP-approved T. suisova inpatientswithmoderateto severe Crohn's disease. In a recent press release Coronado Biosciencesannounced the results of the TRUST-I trial. Although theprimary endpointof the study was not met (there was no difference between the responserate of patients on T. suis ova versus placebo), they postulate thatthere was a non-significant improved response after treatment withT. suis ova in patients with more severe Crohn's disease (CDAI N 290)(Coronado Biosciences Press Release, 2013). The results of the TRUST-IIstudy are expected by the end of March 2014. In two additional trials,the safety and effectiveness of T. suis ova in ulcerative colitis patientswill be tested and the changes in the mucosal immune response will beevaluated (trial identifier NCT01953354 andNCT01433471). Both studiesare still recruiting participants. Helminthic therapies are not only beingtested in IBD patients, but also in patients with other inflammatory


diseases. Helminthic therapy in multiple sclerosis showed a trend to-wards reduced numbers of new lesions (Correale & Farez, 2007;Correale et al., 2008; Correale & Farez, 2011; Fleming, 2011; Fleminget al., 2011; Benzel et al., 2012) whereas no changes in clinical symptomsare observed in allergic rhinitis, allergic rhinoconjunctivitis and asthma(Blount et al., 2009; Bager et al., 2010; Feary et al., 2010; Bager et al.,2011). Helminthic therapy in celiac disease showed a suppression of in-testinal inflammatory cytokines without a clinical benefit (Davesonet al., 2011; McSorley et al., 2011). A clinical trial studying the effectof T. suis ova for allergies to peanuts and tree nuts (trial identifierNCT01070498) has been completed, but no data are available yet. Clinicaltrials to test the effect of T. suis ova in autism spectrum disorders (trialidentifier NCT01040221) and chronic plaque psoriasis (trial identifierNCT01948271) will soon be launched. In addition, helminthic therapy isalso under consideration in transplantation (Johnston et al., 2014).

A new emerging phenomenon is patients self-treating with hel-minths. Some IBD patients do not do well on conventional therapyand seek their own alternative solutions, rather than waiting for thetime-consuming approval processes of new therapies (Weinstock,2012). For example, there are reports documenting that Crohn's diseasepatients buy helminths on the internet, infect themselves and sharetheir clinical experience through online forums with other patients(Broadhurst et al., 2010; Flowers & Hopkins, 2013).

4.4. Helminth-derived molecules as therapeutic agents

As described above, T. suis was shown to be a safe therapeuticapproach showing no short- or long-term treatment related adverseeffects. This agent has received GMP approval and is now licensed to




the pharmaceutical industry. Although helminth infections have provento be effective against IBD, introducing living helminthic therapy intothe clinic may still encounter some problems. For example, patientsmight find it hard to accept being infected with a living helminth,leading to poor treatment adherence (Ruyssers et al., 2008). It alsoremains to be seen whether large scale production of living helminths(e.g. larvae or embryonated eggs) under GMP can satisfy the demandfrom large numbers of patients (McSorley et al., 2013). Furthermore,little is known about the variation in human responses to helminths:persistent infection and/or invasion of the helminth in other tissuesin the human host cannot be excluded and might cause pathology(Ruyssers et al., 2008; McSorley et al., 2013). Garg et al. (2014) recentlyshowed that there is currently not enough data available to draw con-clusions regarding the efficacy and safety of helminthic therapy totreat patients with IBD. As long as the risk/benefit ratio in patients isnot fully understood, caution is advised when using living helminths

Table 2Clinical studies of helminthic therapy in Crohn's disease and ulcerative colitis patients.

Authors &year

Study protocol and number of subjects Clinical evaluation paramete

Summerset al.,2003

Single dose testing of 2500 T. suis ova p.o.,monitoringpatients every 2 wk for N= 12wk and repeated dosetesting of 2500 T. suis ova p.o. at 3-wk intervals,monitoring patients every 3 wk for 28 wk:– 4 CDpatients ofwhich 2 given repeated doses– 3 UCpatients ofwhich 2 given repeated doses

Safety: clinical and laboratoIBDQ N= 170 pointsCDAI b= 150 pointsSCCAI b= 4 points

Summerset al.,2005a

Repeated dose testing of 2500 T. suis ova p.o.at 3-wk intervals for 24 wk:– 29 active CD patients

Safety: patient diaries for adEfficacy (response): decreaspoints Efficacy (remission):

Summerset al.,2005b

Repeated dose testing of 2500 T. suis ova orplacebop.o. at 2-wk intervals for 12 wk:– 54 active UC patients

Safety: patient diaries for adEfficacy (response): UCDAIEfficacy (remission): UCDAI

Croese et al.,2006

Single dose testing of 25–50 N. americanusinfectivelarvae (L3i) s.c. at wk 0 and repeated dosetestingof 25–50N. americanus infective larvae (L3i) s.c.atwk 0 and wk 27 or 30:– 9 CDpatients ofwhich 5 given repeated doses

Efficacy (remission):CDAI b= 150 points IBDQ N

Sandbornet al.,2013

Single dose testing of 3 different doses(500, 2500 or 7500) GMP-approved T. suis ovaorplacebo p.o., monitoring patients 1, 3, 5, 7, 9, 11and 14 days postinfection and 1, 2 and6 months postinfection:36 CD patients → 3 cohorts of 12 patients(9 treated with 500, 2500 or 7500 T. suis ovaand 3 treated with placebo)

Safety and tolerability:– Telephone calls and patienfollow-up adverse effects, chconcomitant medications ansigns and symptoms– Physical examination, asseeffects and review of reportsigns and symptoms at day– Stool sample at month 6 p

CD: Crohn's disease; CDAI: Crohn Disease Activity Index; GMP: Good Manufacturing Practicep.o.: per os; SCCAI: Simple Clinical Colitis Activity Index; s.c.: subcutaneous; T. suis: Trichuris su


(Levison et al., 2010; Hernandez et al., 2013). Therefore, the identifi-cation, the characterization and the synthetic engineering of helminth-derived immunomodulatory molecules responsible for the anti-inflammatory effect might overcome these possible drawbacks andmight lead to new therapeutic approaches in IBD without the needfor living helminth infection.

Several experimental animal studies with helminth-derived immu-nomodulatory molecules, with or without beneficial effects on colitis,have already been published and are summarized in Table 3.

Attenuation or suppression of DSS-induced colitis in mice was dem-onstrated by recombinant Acanthocheilonema viteae cystatin, a secretedcysteine protease inhibitor, recombinant Toxascaris leonine galectin-9homologue (rTl-GAL), recombinantAnisakis simplexmacrophagemigra-tion inhibitory factor-like protein (rAs-MIF), recombinant type I cystatinof Clonorchis sinensis (rCsStefin-1) and Ancylostoma ceylanicum excretory/secretory products (AcES) and adult worm crude extracts (AcAw)

rs Main effects

ry tests Efficacy (remission): • No adverse effects• Single dose testing:– 75% CD patients achieved remission, 67%relapsed within 12 wk– 100% UC patients achieved remission, 33%relapsed within 12 wk• Repeated dose testing:– 100% CD and UC patients achieved remis-sion,50% CD patients and 100% UC patientsremained in remission for N= 1 yr

verse effectse in CDAI N 100CDAI b= 150 points

• No adverse effects• Repeated dose testing:– 75.9% CD patients responded within 12 wk– 79.3% CD patients responded within 24 wk– 65.5% CD patients remitted within 12 wk– 72.4% CD patients remitted within 24 wk

verse effectsb 4 pointsb 2 points

• No adverse effects• Repeated dose testing:– 43.3% UC patients treated with T. suis ova vs.16.7% UC patients treated with placeboresponded within 12 wk (p = 0.04)– 10% UC patients treated with T. suis ova vs.4.2% UC patients treated with placebo remit-ted(not significant) within 12 wk

= 170 points• Adverse effects: mild itch, painful transiententeropathy, eosinophilia• CDAI improved 20 weeks and 45 weekspostinfection compared to baseline– Mean 165 (wk 1) vs. 64 (wk 20)(p = 0.132)– Mean 165 (wk 1) vs. 75 (wk 45)(p = 0.246)• IBDQ improved 20 weeks postinfection:– Mean 151 (wk 14) vs 179 (wk 20)

ts diaries toanges tod gastrointestinal

ssment of adverseed gastrointestinal14 postinfectionostinfection

• Each T. suis ova dose was well tolerated• 25.9% of T. suis ova-treated CD patients vs.33.3% placebo-treated CD patients reportedgastrointestinal disturbances• No dose-dependent relationship for adverseeffects was observed:– 33.3% placebo-treated patients, 44.4% 500T. suisova-treated patients, 0% 2500 T. suis ova-treatedpatients and 33.3% 7500 T. suis ova-treatedpatients experienced at least 1 gastrointestinalevent

; IBDQ: Inflammatory Bowel Disease Questionnaire; N. americanus: Necator americanus;is; UC: ulcerative colitis; UCDAI: Ulcerative Colitis Disease Activity Index; wk: week.



Table 3Effects of helminth-derived molecules on colitis in experimental animal studies.

Authors & year Helminth-derived molecule Class Host Colitis model Outcome Mechanisms of action

Schnoeller et al.,2008

Acanthocheilonema viteaecystatin (recombinant)

Nematode Mouse DSS colitis Preventive treatment:reduction of theepithelialdamage andinflammatorycell infiltrates in thecolon

Induction of IL-10 producingmacrophages

Ruyssers et al., 2009 Schistosoma mansoni solubleworm proteins (SmSWP)

Trematode Mouse TNBS colitis Curative treatmentamelioration of colitis

Diminished Th1 response(↓IFN-γ mRNA expression), diminished Th17response (↓IL-17 mRNA expression), augmentedTreg response (↑IL-10,↑TGF-β mRNA expression)

Ruyssers et al., 2009 Ancylostoma caninumexcretory/secretory proteins(AcES)

Nematode Mouse TNBS colitis Curative treatment:amelioration of colitis

Not specified

Motomura et al.,2009

Trichinella spiralis antigen Nematode Mouse DNBS colitis Preventive treatment:attenuation of colitis

Augmented Th2 response (↑IL-13),augmented Treg response (↑TGF-β),downregulation of IL-1β and iNOS

Johnston et al., 2010 Hymenolepis diminuta high-molecular-mass extract(HdHMW)

Cestode Mouse DNBS colitis Preventive and curativetreatment: attenuationof colitis

Diminished Th1 response (↓TNF-α),augmented Th2 response(↑IL-4, ↑IL-10), augmented Tregresponse (↑IL-10)

Kim et al., 2010 Toxascaris leonine galectin-9homologue (recombinant)(rTl-GAL)

Nematode Mouse DSS colitis Preventive treatment:attenuation of colitis

Augmented Treg response(↑TGF-β, ↑IL-10)

Du et al., 2011 Trichinella spiralis 53-kDa protein(recombinant) (rTs-P53)

Nematode Mouse TNBS colitis Preventive treatment:attenuation of colitis

diminished Th1 response(↓TNF-α mRNA expression), diminished Th17response (↓IL-6 mRNA expression), augmentedTreg response (↑IL-10,↑TGF-β mRNA expression), activation ofalternatively activated macrophages

Cancado et al., 2011 Ancylostoma ceylanicumexcretory/secretory products(AcES)


Diminished Th1 response (↓IFN-γ,↓TNF-α), diminished Th17 response(↓IL-17), no changes in Th2 andTreg responses

Cancado et al., 2011 Ancylostoma ceylanicum adultworm crude extract (AcAw)


Diminished Th1 response(↓IFN-γ, ↓TNF-α), diminished Th17response (↓IL-17), no changes inTh2 and Treg responses

Cho et al., 2011 Anisakis simplex macrophagemigration inhibitory factor-likeprotein (recombinant) (rAs-MIF)

Nematode Mouse DSS colitis Preventive treatment:amelioration of colitis

Diminished Th1 response(↓IFN-γ, ↓TNF-α), diminished Th17response (↓IL-6), diminished Th2response (↓IL-13), augmented Tregresponse (↑IL-10, ↑TGF-β),requires recruitment of Treg cells andbinding with TLR2

Bodammer et al.,2011

Schistosoma mansoni solubleegg antigen (SEA)

Trematode Mouse DSS colitis Curative treatment:no effect on colitis

No remarkable effects on cytokines

Jang et al., 2011 Clonorchis sinensis type I cystatin(recombinant) (rCsStefin-1)

Trematode Mouse DSS colitis Curative treatment:amelioration of colitis

Diminished Th1 response (↓TNF-α),no changes in Treg response, ↑IL-10secreting F4/80+ macrophages

Ferreira et al., 2013 Ancylostoma ceylanicumexcretory/secretory products(AcES)

Nematode Mouse DSS colitis Preventive treatment:protection from colitis

Diminished Th1 response (↓IFN-γ),diminished Th17 response (↓IL-17A),↑ IL-4/IL-10 double positiveCD4+ T cells, ↑alternativelyactivated macrophages and eosinophils

Heylen et al., 2013 Schistosoma mansoniworm adultproteins (SWAP)

Trematode Mouse CD4+CD25−

CD62L+

T cell transfer coli-tis

Curative treatment:amelioration of colitis

Diminished Th1 response (↓IFN-γ mRNAexpression),diminished Th17 response (↓IL-17A mRNAexpression),augmented Th2 response (↑IL-4 mRNA expression)

Heylen et al., 2014 Schistosoma mansoni solubleegg antigen (SEA)

Trematode Mouse CD4+CD25−

CD62L+

T cell transfer coli-tis

Curative treatment:amelioration of colitis

Diminished Th17 response (↓IL-17),augmented Th2 response (↑IL-4)

CD: cluster of differentiation; DNBS: dinitrobenzene sulfonic acid; DSS: dextran sulfate sodium; IFN-γ: interferon-γ; IL: interleukin; iNOS: inducible nitric oxide synthase; kDa: kilodalton;TGF-β: transforming growth factor-β; Th: T helper cells; TLR2: toll-like receptor 2; TNBS: trinitrobenzene sulfonic acid; TNF-α: tumor necrosis factor-α


Please cite this article as: Heylen, M., et al., Of worms, mice and man: An overview of experimental and clinical helminth-based therapy forinflammatory bowel disease, Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pharmthera.2014.02.011



(Schnoeller et al., 2008; Kim et al., 2010; Cancado et al., 2011; Cho et al.,2011; Jang et al., 2011; Ferreira et al., 2013). However, in a study per-formed by Bodammer et al. (2011), S. mansoni soluble egg antigen failedto improve DSS colitis. Next to the DSS model the effect of helminth-derived molecules was also studied in the DNBS and TNBS model andthe CD4+CD25−CD62L+ T cell transfer model. TNBS-induced colitis inmice was attenuated by S. mansoni soluble worm proteins (SmSWP)and Ancylostoma caninum excretory/secretory proteins (AcES) and byrecombinant T. spiralis 53-kDa glycoprotein (rTs-P53), a defined com-ponent of excretory/secretory proteins (Ruyssers et al., 2009; Du et al.,2011). In addition, T. spiralis antigen and a high-molecular-mass frac-tion ofH. diminuta (HdHMW) reduced the severity of DNBS-induced co-litis inmice (Motomura et al., 2009; Johnston et al., 2010). Furthermore,we demonstrated that a curative treatmentwith S. mansoniworm adultproteins (SWAP) or S. mansoni soluble egg antigens (SEA) reduced theseverity of colitis induced by the adoptive transfer of CD4+CD25−

CD62L+ T cells in immunocompromised mice (Heylen et al., 2013, inpress).

Thanks to genomics and proteomics, the genomes of helminths arenow being unraveled and new helminth-derived proteins with poten-tially anti-inflammatory effects are being identified at an increasingpace (Cantacessi et al., 2011). In addition to the above mentioned mol-ecules tested in animal models of IBD, helminth-derived products arealso being tested extensively in other disease models. For example,omega-1 and IPSE/α-1, two glycoproteins secreted by S. mansoni eggs,were evaluated in a mouse model of type 1 diabetes, whereas ES-62, aphosphorylcholine-containing glycoprotein secreted by A. viteae, wastested in a mouse model of rheumatoid arthritis (Harnett & Harnett,2009; Zaccone et al., 2011). Recently McSorley et al. (2013) extensivelyreviewed a whole range of helminth-derived mediators and their asso-ciated immunoregulatory mechanisms. Because it is clear that somehelminth-derived molecules have a similar beneficial effect as livinghelminths, there is an increasing interest in these molecules as newpromising therapeutics for inflammatory and autoimmune diseases.However, there is still no consensus on the exact mechanism of actionthat underlies this protective effect of helminths or helminth-derivedmolecules (McSorley et al., 2013; Weinstock & Elliott, 2013).

5. What do we know about theunderlying immunological mechanisms of protection?

Unraveling the mechanisms of action underlying the helminth-mediated anti-inflammatory properties is an absolute requirement.It seems that helminths activate several distinct regulatory pathwaysinvolving cellular components of both the innate and adaptiveimmune system to control gut inflammation (McSorley & Maizels,2012).

5.1. Effect of helminths and their products on innate immunity ininflammatory bowel disease models

5.1.1. Dendritic cellsDCs, a specialized antigen presenting cell population of the innate

immune system, are known as centralmediators of immunity and toler-ance since they are able to orchestrate other immune cells including Tcells, B cells and macrophages (Niess & Reinecker, 2006; Farache et al.,2013). DCs originate in the bone marrow and are present in blood andin tissue. Interestingly, they markedly accumulate in diseased tissue ofIBD patients (Silva, 2009). This makes DCs an important cell populationto investigate, not only from an immunopathogenic point of view, butalso in a therapeutic perspective. Hart et al. (2005) reported that DCsare altered in IBD. More specifically, DCs are activated, the expressionof pathogen receptors is upregulated and there aremore DCs producingproinflammatory cytokines (Hart et al., 2005). These changes supportthe notion that DCs contribute to inflammation in IBD. As DCs intervenehigh in the cascade of the immune response much attention is being


paid to these antigen presenting cells. In IBD, DCs may act by primingabnormal T cell responses to the enteric flora in organized lymphoidtissues, by sustaining T cell reactivity within the inflamed mucosathrough interaction with T cells, and by functioning as effector cellsvia the release of proinflammatory cytokines (Niess & Reinecker,2006). There is evidence in mice that DCs can also cause gut inflamma-tion in the absence of B and T cells (Abe et al., 2007). On the other handit was shown that if DCs are ablated before DSS treatment in mice, thecolitis was exacerbated (Abe et al., 2007). This suggests that DCs mayplay a protective role in the initial phases of colitis but can play a path-ogenic role at a later time in the disease course (Mann et al., 2013). Be-cause altering DC activity can influence inflammation, it might beattractive, from a therapeutic point of view, to try modulating the DCmaturation process. In this way we might be able to induce DC subsetswith a more protective/tolerogenic role against inflammation.

As reviewed byMcSorley et al. (2013) there is evidence that DCs be-come anergic or tolerogenic after stimulation with different helminthproducts. It was shown (in vitro and in vivo) that treatment of DCswith nematode products suppresses their production of IL-12 and de-creases the expression of co-stimulatory molecules and major histo-compatibility complex class II molecules (Whelan et al., 2012). Recentstudies on intestinal DCs and on DCs in gut-draining mesentericlymph nodes report an increase of protolerogenic DCs after helminth in-fection (Balic et al., 2009; Cruickshank et al., 2009; Smith et al., 2011).Thus, there is good evidence that helminthic infectionmodulates DC ac-tivity enabling helminths to control or prevent overt inflammation.With regard to IBD, it was recently shown byWeinstock and colleaguesthat infection with H. polygyrus bakeri alters the function of DCs in thegut, rendering these cells highly tolerogenic, which suppressed inflam-mation in amurinemodel of colitis (Hang et al., 2010; Blumet al., 2012).They showed that the transfer of DCs isolated from the intestines ormesenteric lymph nodes from H. polygyrus bakeri-infected miceprotected immunocompromised mice from the development of colitisinduced by the injection of IL-10−/− T cells (Hang et al., 2010; Blumet al., 2012). As mentioned, the DCs isolated from H. polygyrus bakeri-infected mice had a tolerogenic phenotype as they lost their ability toproduce proinflammatory colitogenic cytokines such as IFN-γ and IL-17 after ovalbumin stimulation (Hang et al., 2010; Blum et al., 2012).To our knowledge, this is the only study that reported on the effect ofhelminthic therapy on DCs in a mouse model of IBD. Intestinal DCs areconstantly exposed to the microflora and to food antigens and can beexposed to harmful pathogens. As such, it is essential that they immedi-ately react on subtle changes in their microenvironment hence theirspecialization and plasticity. In addition, recent evidence points towardsdifferent DC phenotypes in the ileum and colon with even differencesdemonstrated between the ascending and descending colon (Mannet al., 2013). Therefore, investigating DCs in the gastrointestinal tractremains a challenge and more profound studies on the influence ofhelminthic therapy on DCs in IBD are necessary.

5.1.2. MacrophagesThe small and large intestines contain the largest number of macro-

phages in the body and these cells are strategically located directly un-derneath the epithelial layer, enabling them to sample the intestinallumen (MacDonald et al., 2011). In steady state, the phenotype of the in-testinalmacrophages differs fromother tissuemacrophages in that theycan ingest and kill microbes but they do not initiate proinflammatoryresponses, rather they display an anti-inflammatory signature produc-ing the immune-regulatory cytokine IL-10 (Denning et al., 2007; Bar-On et al., 2011). These properties are essential for maintaining a healthyintestine and immune homeostasis.

In IBD, recentlymuch attention is beingpaid toDCs but it is clear thatalso macrophages play an essential role in inflammation and protectiveimmunity (Qualls et al., 2006; Mowat & Bain, 2011). Macrophagescontribute to intestinal inflammation during IBD by the release of proin-flammatory cytokines (Ince & Elliott, 2007).Whether the inflammatory




macrophages that occur in inflammation are resident cells with changedbehavior or whether they are newly recruited cells remains to beestablished (Mowat& Bain, 2011).Macrophages can exist in different ac-tivation states (effector M1 or regulator M2macrophages) which makesthem an attractive target for treatment of IBD. It is well known that hel-minths and their products induce alternatively activatedmacrophages inresponse to IL-10 and Th2 cytokines IL-4 and IL-13 (Siracusa et al., 2008;Elliott &Weinstock, 2012b).M2 have regulatory and inhibitory functionsand are strong producers of regulatory TGF-β and IL-10 that helpskewing the immune response away from the proinflammatory cascade(Gordon & Martinez, 2010).

Several studies focusing on macrophages have been conducted inIBD animal models. Smith et al. (2007) first reported that infectionwith S. mansoni prevented DSS-induced colitis in mice by a mechanismdependent on macrophages. Transfer of colon lamina propria macro-phages isolated from S. mansoni infected mice protected recipientmice from DSS-induced colitis (Smith et al., 2007). Hunter and col-leagues (2010) reported that the severity of DNBS-induced colitis inmice was significantly reduced by injection of M2 macrophages andthat infection with H. diminuta induces M2 macrophages that protectmice from colitis. Other helminth-based studies also identified the M2macrophages to be responsible for the relief of experimental colitis inanimals (Schnoeller et al., 2008; Du et al., 2011; Jang et al., 2011;Broadhurst et al., 2012; Ferreira et al., 2013).

As such it is believed that helminths and their products ameliorategastrointestinal inflammation through stimulation of M2 macrophages.This notion is supported by the finding that M2 macrophages are moreabundant in colonic biopsies frompatientswith inactive Crohn's diseasecompared to patients with active Crohn's disease (Hunter et al., 2010).

In general, tolerogenic DCs and alternatively activatedmacrophagesfunction to block antigen-specific T cell responses, preventing the differ-entiation of dangerous effector T cells which lead to inflammation anddisease (Weinstock, 2012; Weinstock & Elliott, 2013).

5.1.3. Other components of the innate immune system: epithelial barrierand innate lymphoid cells

Within this review we would like to touch briefly on some of theother important players of the innate immune system that may play arole in the prevention of intestinal inflammation by helminths but cur-rently represent somewhat of a caveat in our research field.

As described in a previous paragraph, intestinal epithelial barrierfunction is severely disrupted during IBD (Baumgart & Carding, 2007;Laitman & Dahan, 2012). It was proposed by Wolff et al. (2012) that in-testinal helminths may also protect against colitis by enhancement ofthe mucosal barrier function. The immune response against intestinalhelminths (which is aimed at expelling the parasite from the gastrointes-tinal tract) includes increased mucus secretion, increased epithelial cellturnover and changed composition of the mucus secreted by gobletcells. All these effects can promote mucosal healing and as such restorenormal barrier function leading to amelioration of inflammation (Wolffet al., 2012). A few studies also show that intestinal helminths alter thebacterial composition of the intestinal flora, which can impact host im-munity and can help to maintain intestinal health (Dwinell et al., 1997;Andersson et al., 2003; Hunter & McKay, 2004; Walk et al., 2010;Weinstock, 2012; Wolff et al., 2012; Weinstock & Elliott, 2013).Although this sounds compelling for intestinal helminths, there arehelminth species that protect against colitis (e.g. S. mansoni) whileliving outside the gastrointestinal tract. Furthermore, it still remains tobe elucidated whether helminth-derived products that protect againstcolitis also do so by directly affecting themucosal barrier. This interestingtopic may provide an additional novel mechanism by which helminthsand their products protect against intestinal inflammation but it definite-ly calls for more research.

Natural killer (NK) cells are part of the innate immune system, be-cause they lack antigen specific receptors on their membrane, but theyhave been classified as lymphocytes based on their morphology (Vivier


et al., 2011). They are considered the killer cells of the innate immune sys-tem and play a role as cytotoxic cells against viral infections and are im-portant in tumor surveillance (Mortha & Diefenbach, 2011). However,the role of NK cells in IBD is less well understood. They are thoughtto possess both proinflammatory and regulatory functions. There isincreasing evidence that distinct subsets of NK cells (with changesin activation and cytotoxic activity) are important during the patho-genesis of IBD (Takayama et al., 2010; Yadav et al., 2011). A recentstudy by Hall et al. (2013) showed that a depletion of NK cells im-paired the survival of mice with DSS colitis and this was linkedwith dramatic increases in colonic damage, leukocyte infiltration,and proinflammatory profiles.

The innate lymphocyte lineage is continuously expanding. Thefamily of innate lymphoid cells (ILC) not only comprises NK cells andlymphoid tissue inducer cells (Koyasu & Moro, 2013), but now also in-corporates ILC type 1, ILC type 2 and ILC type 3 (see Spits et al., 2013for an extensive review on these ILC types). Importantly, these differentsubsets of ILC have cytokine patterns that are similar to the cytokine se-cretion profiles of the different T helper cell subsets (Spits et al., 2013).Changes in ILC responses have been associated with IBD (Hepworthet al., 2013) and ILC producing IL-17 and IFN-γ are mediating colitis inamousemodel of intestinal inflammation (Buonocore et al., 2010). Fur-thermore, ILCs regulate adaptive immune responses and limit effectorCD4+ T cell responses to commensal bacteria (Hepworth et al., 2013).Research on how helminths influence ILCs in animal models of IBD islacking but the fact that these cells intervene high in the inflammatorycascade and that they have proven regulatory functions makes themhighly interesting new targets for treatment of IBD.

5.2. Effect of helminths and their products on the adaptive immune systemin inflammatory bowel disease models

5.2.1. T cellsIn Crohn's disease, gastrointestinal inflammation is mediated

through Th1 and Th17 cells (Moreels & Pelckmans, 2005; Wallaceet al., 2014). During helminth infection, thehost evokes a strongTh2 im-mune response (Fig. 3) (Maizels et al., 2004). The release of Th2 cyto-kines such as IL-4, IL-5 and IL-13 is important in gastrointestinalworm expulsion from the gut. Both IL-4 and IL-13 promote mucus se-cretion, enhance intestinal smooth muscle contractility and stimulatefluid secretion into the lumen. IL-5 is important for the destruction ofsome larval forms (Weinstock et al., 2005). This Th2 response is broadlycharacterized by the activation of eosinophils, basophils and mast cellsand the production of IgE (Diaz & Allen, 2007). The cytokines producedby Th1 and Th2 cells cross-regulate each other's development and activ-ity (Hunter &McKay, 2004; Khan & Fallon, 2013). Furthermore, Th1 andTh2 cytokines can inhibit Th17 development (Bi et al., 2007). In addi-tion, helminths induce immunosuppressive Treg cells through a varietyof different mechanisms (Fig. 3) (McGuirk & Mills, 2002; Cools et al.,2007). In this way, the helminth evoked immune response can counter-act the Th1/Th17 response found in patients with Crohn's disease(Fig. 2B). The discovery of the IL-17 pathwaywas a major breakthroughin the immunopathogenesis of several inflammatory conditions (Zhanget al., 2007) as there seems to be an important imbalance in Th17/Tregcells in IBD and other inflammatory diseases. Investigation of the effectof helminths and their products on the Th17/Treg pathway in animalmodels for IBD is extensive (Tables 1 and 3). For example, we have pre-viously shown that treatment with S. mansoni soluble worm proteins(SmSWP) caused a decrease in IL-17 and an increase in the relative ex-pression of IL-10 and TGF-β in T cells isolated from the colon of micewith TNBS-induced colitis (Ruyssers et al., 2009). A. ceylanicum adultworm extracts (AcAw) attenuated DSS colitis in mice by downregulat-ing Th1 and Th17 cytokines (Cancado et al., 2011). In accordance,A. caninum excretory/secretory products (AcES) suppress murineTNBS and DSS colitis (Ruyssers et al., 2009; Cancado et al., 2011;Ferreira et al., 2013) and induce IL4+/IL10+ CD4+ T cells in mesenteric




lymph nodes and colon (Ferreira et al., 2013). An earlier study by Hunt-er and colleagues (2005) showed the importance of IL-10 in the protec-tion of Hymenolepsis diminuta against DNBS-induced colitis. A role forTreg cells has also been proposed by the group ofWeinstock inmultiplestudies e.g. showing that infection of mice with H. polygyrus bakeri pro-motes the production of IL-10 and TGF-β by lamina propria T cells andinduces CD8+ Treg cells (Metwali et al., 2006; Setiawan et al., 2007)and showing that the adoptive transfer of colonic forkhead box p3(Foxp3)+/IL-10+ Treg cells isolated from H. polygyrus bakeri-infectedmice was sufficient to prevent colitis in a mouse model of IBD (Hanget al., 2013). The majority of experimental studies listed in Tables 1and 3 indicate a downregulation of Th1 and Th17 responses with anessential role of Treg cells in controlling these immune responses. How-ever, the important question of ‘who is regulating the regulators’ cur-rently remains, hence the continued attention to the innate arm of theimmune system.

5.2.2. B cellsWithin the adaptive immune system, B cells are known as positive

regulators of the humoral immune response by the production of anti-bodies (Fujimoto, 2010). Regulatory B cells suppress immune responsesand are important in the regulation of intestinal homeostasis and inhi-bition of inflammatory cascades (Kayama & Takeda, 2012). Helminthictherapy in multiple sclerosis patients led to the production of IL-10from regulatory B cells resulting in a reduction of inflammation(Correale et al., 2008; Ben-Ami Shor et al., 2013). To our knowledge,the role of B cells has not been investigated yet in the field of helminthictherapy in IBD which leaves another caveat for more research.

6. Conclusions

Helminths and their products are able to attenuate chronic in-flammation in IBD and in other disorders that result from an overlyaggressive immune response. Today the use of helminthic therapyfor autoimmune disorders is only available in clinical trial settings.More research, both in animalmodels andwell designed and controlledrandomized clinical trials, is necessary to validate the treatment andeventually commercialize helminthic or helminth-derived therapyfor IBD.

This review provided a detailed overview of the experimental andclinical studies investigating the therapeutic potential of helminth-based therapy in IBD and focused on the underlying immunologicalmechanisms bywhich helminths and their products provide protectionagainst intestinal inflammation. There is solid evidence that helminthsand their products induce multiple immunomodulatory pathways indifferent animal models of IBD, including downregulation of proinflam-matory cytokines and induction of regulatory cytokines and regulatoryT cells. Although many studies have looked at the role of T cells in thisfield, the focus is now shifting towards the effect of helminths andtheir products on cells of the innate immune system. The role of macro-phages and dendritic cells is currently being investigated bymultiple re-search groups showing a more regulatory phenotype after helminthictreatment. There is a major gap in our understanding of how helminthsinfluence intestinal lymphoid cells andwhether these cells subsequent-ly contribute to the attenuation of colitis. Therefore, ILCs are an interest-ing cell population that deserves more attention in future research.Furthermore, knowledge on the role of B cells is limited, leaving inter-esting opportunities for finding additional mechanisms by which hel-minths provide protection against IBD.

Although our understanding on the beneficial effect of helminthsand their products on IBD is continuously growing, it is clear thatmore in-depth research is necessary to further unravel all mechanismsinvolved. Furthermore, the identification and characterization ofhelminth-derived immunomodulatory molecules that mimic the


protective effect of living helminths remains important as it opensnew perspectives for the treatment of IBD.

Conflict of interest statement

The authors declare that there are no conflicts of interest.The manuscript has not been published elsewhere and is not under

consideration elsewhere.

Acknowledgment

This work was supported by a grant from the Belgian IBD Researchand Development (BIRD) group (BIRD Research Grant 2012A) and thelegacy Deceunynck (4692).

This work was also supported by the FWO through a Research Grant(1515314N) to Nathalie Ruyssers.

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