Introduction

Multiple sclerosis and environmental factors

Multiple sclerosis (MS) is an inflammatory disorder causing central nervous system (CNS) demyelination and axonal injury. Its etiology remains elusive; however, several lines of evidence support the concept that autoimmunity plays a major role in disease pathogenesis [1]. Autoimmune diseases are currently considered to arise from the interplay between genetic susceptibility and environmental factors [2, 3]. Based on findings of several genome-wide association studies, the major genetic component of MS is believed to result from common allelic variants in many genes, perhaps acting as cooperative networks [4]. In addition, the substantial discordance in prevalence of MS between monozygotic twins suggests that additional factors, such as environmental modulators, are also be involved [5].

One of the most striking illustrations of the importance of the environment in MS pathogenesis results from the particular geographical distribution of the disease. Prevalence rates are increased in high latitude regions, and are reduced near the equator [6]. Population migration studies indicate that individuals moving from areas of low risk to areas of high risk, particularly before the age of 15 years, show a similar incidence of MS as host country populations, suggesting the presence of either a protective factor in the region of origin or, alternatively, a harmful factor in the adopted region [7]. Identifying these environmental factors and elucidating how they increase autoimmune disease risk would help develop new treatment strategies for the disease. Although to date no specific environmental factor has been firmly linked to disease pathogenesis, putative environmental risk factors under analysis include: low exposure to ultraviolet (UV) radiation, vitamin D deficiency, cigarette smoking, and Epstein-Barr virus (EBV) infection [8].

Although a role of systemic infections in precipitating MS flares is well-accepted, there is an ongoing debate as to whether infections enhance or ameliorate the risk of developing autoimmune diseases [9, 10]. Several studies implicate infectious environmental factors present during childhood and young adulthood as strong determinants of MS risk. Microbial infections have also been identified as triggers inducing autoimmunity, resulting in clinical disease manifestations in genetically predisposed individuals. Alternatively, infections might accelerate sub-clinical autoimmune processes.

Conversely, however, some epidemiological and experimental studies support the hygiene hypothesis, which posits that infections protect rather than induce or accelerate autoimmune diseases like MS [11]. In line with this concept, Leibowitz and coworkers suggested in 1966 that greater MS prevalence correlated with high levels of sanitation in childhood environments [12]. Support for this hypothesis comes from epidemiological data demonstrating an inverse relation between infections, and allergic as well as autoimmune diseases in the developed world during the last five decades, even after adjusting for improvements in access to medical attention and diagnostic capabilities [11]. The rise observed in autoimmune disease prevalence is also too rapid to be considered secondary to genomic alterations, therefore, implying that some critical environmental change must have taken place. Progressive industrial development has pushed human migration from rural areas to cities, exposing the immune system to new environments, and a decreased incidence of many infectious diseases has resulted from better medical treatments. Epidemiological investigations demonstrating an inverse correlation between the global distribution of MS and that of the parasite Trichuris trichiura, a common human pathogen, further strengthen this hypothesis. MS prevalence appears to fall steeply once a critical threshold of T. trichiura prevalence (about 10 %) is exceeded in any given population [13]. Thus, dichotomous distribution of MS and T. trichiura infection hint at a helminth infection protecting against MS development. Indeed, regions of the world where poor sanitary conditions generate endemic areas of parasitoses show lower prevalence of allergic and autoimmune diseases. Additionally, evidence for a causal effect of parasites on reducing allergies and autoimmune diseases stems from reports that clearance of infection using antihelminth treatment increases reactivity to skin tests against different allergens, as well as disease activity in MS patients [14, 15•].

Immunoregulatory mechanisms induced by helminths

In endemic areas, many if not most helminth-infected individuals are relatively asymptomatic. Manifest disease occurs often in individuals with reduced immunity, rendering them more susceptible to infection and presenting with a very high worm burden. Maintaining a disease-tolerant or asymptomatic state requires adequate balance between immune regulatory mechanisms present both in the host and the helminth. Chronic helminth infections have continuous and profound effects on immune system function [16••, 17•]; a finely tuned immune regulatory network governing susceptibility and resistance to helminths exists which is both redundant and parallel, in order to exclude parasites while minimizing collateral pathology. Current investigations have shown that peripheral T cells from infected patients are unresponsive to stimulation with parasite antigens, and responses to other antigens are also reduced [18]. Innate immune cells have been recognized as major contributors of interleukin-4 (IL-4), IL-5, and IL-13, setting the stage for a more potent adaptive anti-helminth type 2 T-cell helper response (Th-2) [19]. This response is initially induced during helminth infections by IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) produced by epithelial cells in the gut or lung [17•, 20]. In addition, innate type 2 mechanisms promote transformation of the intestinal environment through mast cell, epithelial cell, and goblet cell activation and differentiation, helping maintain the mucosal barrier and limiting aberrant responses triggered by the gut infection [21]. Induced CD4+ Th-2 cells then drive different anti-parasite mechanisms, such as class-switched antibody production, cytokine secretion, and activation of innate defense molecules [17•]. However, the fact that typical Th-2 associated diseases (e.g. allergies) can benefit from helminth infections, challenges the notion that Th-2 polarized responses are the primary mechanism behind the protection induced by helminth infections [22]. Moreover, although Th-2 responses play a crucial role in resistance to helminths, one should not forget that they also cause detrimental effects [23]. It has been suggested that in parasite-infected patients with stronger regulatory responses, production of key Th-2 effector cytokines (IL-5 and IL-13) and IFN-γ are down regulated, while Th-2 regulatory cytokines (IL-4 and IL-10) continue to be produced [24, 25]. Likewise, IgG4 titers rise, and IgE levels fall [26]. Interestingly, IgG4/IgE ratio is modulated by IL-10 and TGF-β [27]. In addition to CD4+ Th-2 responses, regulatory FoxP3+ regulatory T cells, IL-10 producing regulatory B cells, IFN-γ and IL-17 inhibition, IL-10 and TGF-β release, and induction of alternatively activated (or M2) macrophages have also been identified as key components of the immune regulatory network functioning during helminth infections [16••, 24]. Induction of these different cell populations can account for the ability of helminth infections to dampen allergies and autoimmune diseases, as has been observed in several study cohorts.

Helminth therapy in experimental autoimmune encephalomyelitis

Evidence in favor of a causal relation between helminth infections and reduced severity of autoimmune disease derive from animal models. Many examples exist in both spontaneous and induced models of human autoimmune diseases where helminth infection, or products thereof, influence the course of autoimmune pathology [28–36]. Experimental autoimmune encephalomyelitis (EAE), which mimics some of the clinical and pathological characteristics of MS, has been used in several studies investigating the impact of helminth infections or their products on disease severity and immunological response (Table 1). In most EAE models, prior infection with helminths, or exposure to nonviable ova or parasite-secreted products, reduced both incidence and severity of the disease. These observations would indicate the presence of a systemic anti-inflammatory milieu generated by multiple cell types and molecular mediators influencing autoimmune response [28–32, 43]. Heterogeneity of these immunological responses can be attributed to helminth species, helminth-derived products, age at which infection was acquired, and infection intensity. Helminths are often considered a homogeneous group, but significant differences exist between species. Importantly, there is evidence from mouse models that helminth infections, under certain conditions, can also exacerbate disease [44]. Therefore, caution is recommended when interpreting data from animal models.

Table 1 Overview of studies that have investigated the effects of helminth therapy in experimental autoimmune encephalomyelitis
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Treatment

Epidemiological and experimental data support the hypothesis that helminth administration in a controlled clinical setting (“helminth therapy”) could relieve or attenuate immune responses, providing a possible starting point for the development of a treatment against autoimmune diseases . In this regard, in an observational, prospective, double-cohort study, our group demonstrated that relapsing-remitting MS (RRMS) patients infected with different parasites (Hymenolepis nana, Trichuris trichiura, Ascaris lumbricoides, Strongyloides stercolaris and Enterobius vermicularis) showed significantly lower number of relapses, minimal changes in disability scores, as well as significantly lower MRI activity, compared to uninfected MS individuals. Parasite-driven protection led to the development of IL-10 and TGF-β secreting cells, as well as CD4+CD25+FoxP3+ regulatory T cells, while simultaneously inhibiting T-cell proliferation and suppressing IFN-γ and IL-12 production [45]. In addition, helminth infections in MS patients induced regulatory B cells capable of dampening the immune response through IL-10 production [46]. Interestingly, when some of these patients received antihelminthic drug treatment for worsening of parasite-associated symptoms, a major reduction in parasite egg numbers per gram faeces was observed, as well as significant increase in clinical and radiological MS activity. Flares were accompanied by substantial increase in IFN-γ and IL-12 producing cell numbers and a decline in IL-10, TGF-β and regulatory T cells, providing evidence of direct suppression of the autoimmune response as a result of the helminth infection [15•]. These observations indicate that helminth therapy can induce protection not only through prevention, since helminths can be present before an autoimmune disease develops, but also after autoimmune response is established. Moreover, it is important to note that the immunosuppressive effects mediated by parasite infections end once the parasite has been eliminated. Helminth therapy has already been used in clinical trials associated with allergy [47] and autoimmune diseases including inflammatory bowel diseases (IBD) [48, 49] and MS [50•]; several more clinical trials are currently underway in other diseases (NCT01040221; NCT01070498) [51]. It must be pointed out that not all helminth infections can be deemed therapeutically equal, and some might worsen a disease [44, 52]. Therefore, parasite species selection is crucial. In this respect, most current studies use either Trichura suis (pig whipworm) or Necator americanus (human hookworm). Use of parasites that do not permanently colonize humans, or use of organisms that can be administered at low infection intensity and eliminated with antihelminthic drugs, decrease the potential for accidental disease transmission to healthy subjects. Indeed, T. suis ova use has recently been approved by the US Food and Drug Administration and the European Medicines Agency as an investigational medicinal product (IMP), while N. americanus has been granted an IMP license by the Medicines and Healthcare Regulatory Authority in the UK.

Nevertheless, possible caveats should be considered when assessing these trials. Data from animal models demonstrating a favourable influence on EAE outcome have preferentially been observed during pre-immunization and inductive phases [28, 30–32, 53••], suggesting that it may be more difficult to suppress an ongoing reaction than prevent its development. Moreover, many trials use asymptomatic infection doses, which are significantly lower than those present during natural infections, and, therefore, possibly insufficient to suppress pathology [54].

Clinical trials using helminth therapy in MS are summarized in Table 2. Initially encouraging results on effects of helminth infections in IBD [48, 49] have led to trials being initiated to establish whether T. suis has any effect on MS [53••]. The first clinical trial on helminth therapy in MS was the HINT (Helminth-induced immunomodulation therapy) study [50•]. In this small scale, safety oriented trial, five newly diagnosed RRMS patients received 2,500 T. suis ova (TSO) each, administered orally every 2 weeks for 3 months. The mean number of gadolinium (Gd)-enhancing MRI lesions fell from 6.6, at baseline, to 2.0 at the end of treatment, and rose again to a mean of 5.8 lesions 2 months after TSO was discontinued. Treatment was associated with relative increases in IL-4 and IL-10 levels in serum, as well as elevation of C-reactive protein and antibody to T. suis excretory/secretory products (IgG1 and IgA), indicating robust systemic immunity response to T. suis colonization. Peripheral CD4+CD5+FoxP3+ cells modestly increased in two of the five study subjects, and TSO was well-tolerated. Minor gastrointestinal symptoms (FDA grade1), observed in three of five subjects, were transient. Although MRI study results seem promising, they should be interpreted with caution due to the small sample size and the short follow up duration. After reviewing the HINT 1 study results, regulatory authorities approved a follow up clinical trial (HINT 2; NCT00645749) with 18 relapsing remitting MS patients, studied for 20 months using a baseline versus treatment design. Final results of this trial are expected to be reported in 2014.

Table 2 Clinical trials using helminth therapy in MS
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In another study (Trichuris Suis Ova Therapy for Relapsing Multiple Sclerosis - A Safety Study, TRIMS A; NCT01006941) conducted at the Danish Multiple Sclerosis Center of Copenhagen University Hospital, 10 RRMS patients were treated with 2,500 TSO every 2 weeks for 3 months. The primary outcome measure was MRI activity established based on the number of new or enlarging T2 lesions, number of Gd-enhancing lesions and volume of T2 lesions. Brain MRI testing was performed every 3 weeks. The investigators concluded that TSO seemed to be safe and well-tolerated. However, no clinical, MRI or immunological signals indicating benefit were observed [55].

Investigators at Charite University in Berlin, Germany, conducted the first exploratory study in secondary progressive MS patients [56]. Four patients were surveyed during 6 months of therapy with 2,500 TSO, given orally every 2 weeks. The study was focused on T cell modulation as well as on innate immune response. Stimulated peripheral blood mononuclear cells showed slight down-regulation of Th-1 associated cytokine patterns, with temporal increase of Th-2 associated cytokines such as IL-4. A double blind placebo controlled phase II trial (Trichuris Suis Ova in Recurrent Remittent Multiple Sclerosis and Clinically Isolated Syndrome, TRIOMS; NCT01413243) has been initiated by the same investigators [58]. The study will recruit 50 patients with RRMS or clinically isolated syndrome with clinical activity, not undergoing any standard therapy. Patients will receive either 2,500 TSO every 2 weeks, or placebo, for a 12-month treatment period and will be followed up for an additional period of 6 months.

Another phase II double blind, placebo controlled study (Worms for Immune Regulation of Multiple Sclerosis, WIRMS; NCT01470521) has begun at the University of Nottingham. The study will enroll 72 RRMS and secondary progressive MS patients with superimposing relapse, which will be treated with dermally administered hookworms, N. americanus (25 live larvae) or placebo. Worms will be allowed to remain in residence for a full 9 months. Investigators speculate that this period of residence will establish and maintain immune regulatory mechanisms of sufficient magnitude to translate into the anti-inflammatory effect, and consequently generate therapeutic benefit. The cumulative number of new and active lesions on T2 weighted MRI will be the primary outcome measure. Regulatory network induction (Treg induction, regulatory B cells, Tr1 cells and NK cells) will be the secondary outcome measure.

Clearly, at this time a number of critical issues need to be addressed in further investigations. Questions remain regarding which helminth is most effective, and at what dose, which is the best route of administration or the optimal timing of infection in relation to disease onset, whether helminth-derived molecules have the same efficacy as live parasites, and what the optimal treatment schedule should be.

Standard dosage

For T. suis, 2,500 TSO given orally every 2 weeks has been used in most clinical trials with good tolerance.

For N. americanus, patients have received 10 -100 live larvae administered dermally [59]. The lack of effect of this infection in different clinical settings has been attributed to insufficient parasite inoculum dosing [54]. However, it must be pointed out that due to the requisite extra-intestinal phase of its life cycle and the fact that humans are natural hosts, there is probably a much narrower therapeutic window between effective immune modulation and unacceptable side effects in the case of this helminth.

Main drug interactions

Although the effects of helminth therapy on vaccine efficacy have not been evaluated, several studies have shown that helminths can influence vaccine efficacy by modulating host immune response, in particular when Th1-like and cell-dependent responses are required. S. mansoni infection was shown reduce BCG-induced protective response against Mycobacterium tuberculosis in mice [60]. Likewise, helminth infections dramatically reduced malaria DNA vaccine immunogenicity [61]. Moreover, epidemiological studies have demonstrated that Schistosoma spp. infections decrease the efficacy of vaccines against tetanus [62] and hepatitis B virus [63]. Overall effects of helminth therapy on vaccine efficacy need to be further investigated.

Main side effects

TSO has been extensively study in IBD patients, even while on concomitant immunosuppressive drugs, without observing any significant side effects. In the HINT 1 study some patients presented transient diarrhea and upper abdominal pain (lasting 3 – 5 days). The symptoms peaked 30 – 50 days after TSO treatment initiation and could be related to initial T. suis larvae colonization, which induced an innate inflammatory immune response in the gut [50•]. They were not observed in earlier TSO studies in IBD patients, perhaps because they were occurring in the context of moderate gastrointestinal pathology in the study population [59]. Nor did these symptoms interfere with patients’ daily life activities.

An additional concern in helminth therapy is whether helminth colonization may worsen other pathogenic bacterial, parasite or viral infections, especially in immunocompromised hosts. Enhancement of disease and pathology by co-infection of T. suis and Campylobacter jejuni or T. trichiura has been described. However, this has never been observed in TSO-treated patients [64, 65].

Another helminth studied in clinical trials thus far is the hookworm N. americanus. In previous studies of helminth therapy the most common hookworm-related side effect was localized maculopapular rash at skin entry site, which began within a day or so of infection and typically lasted 2 – 5 days. In some patients, rash recurred approximately 2 – 3 weeks after infection for up to 10 days before disappearing. The most troublesome adverse effects were gastrointestinal symptoms, such as diarrhea and abdominal pain. The other most commonly reported symptoms were malaise and fatigue, which occurred between week six to seven of treatment and have been associated with systemic eosinophilia, rather than with a direct effect caused by parasites. Dose-ranging studies of therapeutic N. americanus infection have shown side effects to be dose dependent. Doses higher than 10 larvae correlated with more frequent and severe adverse events than low-dose inocula. All symptoms disappeared completely after subjects were treated with mebendazole [59, 66].

Special points

Monitoring

It is recommended that careful monitoring for gastrointestinal adverse events be part of any study on helminth therapy for MS [67]. In addition, patients considered for helminth therapy may require screening for carriage of other potential pathogens prior to initiating treatment, to avoid coinfections which might result in more serious complications, including septicemia [53••].

Further perspectives

To promote the host immune regulatory network reactions described above, helminths have evolved to secrete a wide range of molecules able to target various host cells, altering immune response. During recent decades, a number of helminth-derived immunomodulatory molecules have been characterized, both in terms of structure and bioactivity [68, 69]. Although this approach might overcome a number of safety concerns regarding the use of live helminths as therapeutic agents, controversies still exist on whether live infection is a prerequisite for suppression of inflammatory responses in different disease models of autoimmunity.

ES-62 a glycoprotein from the rodent nematode Acanthonema vitae has been widely investigated as an immunomodulatory molecule. Its administration after collagen-induced arthritis induction in mice resulted in significant reduction of disease severity and progression [70]. Likewise, when rDiAg, a product from the filarial parasite Dirofilaria immitis, was administered to NOD mice it prevented insulinitis and diabetes onset [71]. Furthermore, treatment with soluble products from T.suis, S. mansoni, and Trichinella spiralis caused strong reduction in EAE severity, and significant suppression of pro-inflammatory phenotype in human dendritic cells, and consequently in the generating human Th-1 and Th-17 effector cells [37]. These few examples of helminth-derived immunomodulatory products illustrate the potential of these molecules to serve as drugs or templates for drug design. However, much yet remains to be explored to move the field from observations in animal models to clinical practice; issues relating to in vivo stability and pharmacodynamics of helminth-derived molecules, delivery methods, as well as immunogenicity need to be overcome if new therapeutic modalities are to be developed.