Helminths: Immunoregulation and Inflammatory Diseases—Which Side Are Trichinella spp. and Toxocara spp. on?

Macropathogens, such as multicellular helminths, are considered masters of immunoregulation due to their ability to escape host defense and establish chronic infections. Molecular crosstalk between the host and the parasite starts immediately after their encounter, which influences the course and development of both the innate and adaptive arms of the immune response. Helminths can modulate dendritic cells (DCs) function and induce immunosuppression which is mediated by a regulatory network that includes regulatory T (Treg) cells, regulatory B (Breg) cells, and alternatively activated macrophages (AAMs). In this way, helminths suppress and control both parasite-specific and unrelated immunopathology in the host such as Th1-mediated autoimmune and Th2-mediated allergic diseases. However, certain helminths favour the development or exacerbation of allergic responses. In this paper, the cell types that play an essential role in helminth-induced immunoregulation, the consequences for inflammatory diseases, and the contrasting effects of Toxocara and Trichinella infection on allergic manifestations are discussed.


Introduction
Immune responses induced by helminths are predominantly of the 2 type involving cytokines such as interleukin-3 (IL-3), IL-4, IL-5, IL-9, IL-10, and IL-13. ese cytokines mediate immune responses typically characterized by increased levels of circulating IgE antibodies, eosinophils, basophils, and mast cells [1]. During infection, the immune system is exposed to different helminth-derived molecules, including proteins, lipids, and glycoconjugates present either at the surface of the worms or in the excretory-secretory (ES) products [2]. Interaction of helminth-derived molecules with host cells can result in a shi of the immune response, from an in�ammatory towards an anti-in�ammatory type of response. Helminth-derived molecules can modify dendritic cells (DCs) function and downregulate adaptive immune responses, through the induction of a regulatory network that include regulatory T (Treg) cells, alternatively activated macrophages (AAMs), and regulatory B (Breg) cells. e induced immunosuppresive network, together with cytokines produced by diverse hematopoietic and nonhematopoietic cells as integral part of immunoregulatory pathways, appears to be essential for parasite survival and its effect can be extended to other in�ammatory disorders such as allergies and autoimmune diseases [3,4]. However, the association between helminth infections and allergy does not always have an unequivocal outcome. While certain helminth infections protect against allergic diseases (reviewed in [5]), other helminths can exacerbate this immunopathology (reviewed in [6]). Here, the role of DCs, Treg, and other regulatory cells in helminth-induced immunoregulation, the consequences for in�ammatory diseases, and the contrasting effects of Toxocara and Trichinella infections on allergic manifestations are discussed.

Dendritic Cells
DCs are sentinels on alert for possible danger signals to immediately activate local innate immune responses and subsequently, aer antigen presentation, initiate the proper adaptive immune responses. Interaction with DCs determines the function and cytokine production of lymphocytes. DCs play therefore an essential role in shaping the immune response and controlling the course of infection [7]. ese cells are located throughout the body forming a complex network that allows them to communicate with different populations of lymphocytes. Different DC subsets may have distinct locations, where they acquire antigens to be transported to the draining lymph nodes for T-cell priming [8]. DCs as well as other innate immune cells possess various families of pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), NOD-like receptors, RIG-like receptors, and the C-type lectin receptors (CLRs) that allow them to recognize a great variety of pathogen-associated molecular patterns (PAMPs). Aer pathogen recognition via various PRR, DCs produce molecules that induce polarization of different types of responsiveness such as 1-, 2-, 17-, or Treg-related. e response of DCs to pathogens is mediated in large part via TLR, with input from other PRR resulting in changes in gene expression that leads to DCs maturation. Maturation of these cells refers to a transition from a resting state into a more dynamic state in which the cells present antigen in the context of MHC, express costimulatory molecules such as CD40, CD80, and CD86, and secrete a broad spectrum of cytokines and chemokines [9]. TLR-mediated responses are controlled mainly by the MyD88-dependent pathway, which is used by all TLR except TLR3-and by the TIR-domain-containing adapter-inducing interferon--(TRIF)-dependent pathway, which is used by TLR3 and TLR4 [10]. TLRs have been implicated in the recognition of helminth products by DC. For instance, Lacto-N-fucopentaose III (LNFPIII) produced by trematode Schistosoma mansoni, and ES-62, a phosphorylcholine-containing protein secreted by the nematode Acanthocheilonema viteae, can condition DCs to induce 2 responses through TLR4 [11]. Likewise, monoacetylated phosphatidyl serine lipids from schistosomes speci�cally instruct DCs to preferentially induce IL-10-producing Treg in a TLR2-dependent manner [12]. is was also demonstrated in TLR2-de�cient mice that showed a reduced number of CD4+CD25+ Treg cells and immunopathology during schistosomiasis [13]. CLRs on DCs also play an important role in sensing helminth glycans. Studies using schistosomal antigens suggest that helminth glycans may be the conserved molecular pattern that instructs DCs via CLR to drive 2-polarized responses [14]. Other recent studies demonstrate that host-like glycan antigens expressed by many helminths are recognized by DCs via lectin receptors [2]. Schabussova et al. reported on blood group-like glycans from T. canis that bind the lectin DC-SIGN (Dendritic Cell-Speci�c Intercellular adhesion molecule-3-Grabbing Nonintegrin) [15], which may enable the activation of signal transduction pathways involving Raf-1 and subsequent modulation of DC maturation resulting in skewing towards a 2 responses [16]. Lewis X antigen, a host-like glycan expressed on the surface of schistosomes in all life stages which is also present in secreted products such as the soluble egg antigens (SEAs), also binds to DC-SIGN [17].
DC maturation is considered to be essential for DCs to induce T-cell responses. It has become clear that DCs responding to helminth products do not mature in the conventional way upon encountering parasitic antigens but acquire a semimatured status and are still capable of inducing T-cell polarization. Several studies support the �ndings that helminth products fail to directly activate DCs and other studies show that helminth products suppress DC maturation. For instance, SEA suppresses lipopolysaccharide-(LPS-) induced activation of immature murine DCs, as indicated by decreased MHC class II and costimulatory molecules expression in addition to IL-12 production. is resulted in increased LPS-induced production of IL-10 by DC aer incubation by SEA [18]. Pretreatment of DCs and macrophages with ES-62 also inhibits their ability to produce IL-12p70 in response to LPS [19]. In another study, a mixture of high molecular weight components from Ascaris suum was found to reduce the LPS-induced expression of MHCII, CD80, CD86, and CD40 molecules on mouse CD11c+ DCs and to hampered T-cell proliferative responses in vitro. is inhibitory effect was abolished in IL-10-de�cient mice [20]. Fasciola hepatica tegumental antigen alone did not induce cytokine production or cell surface marker expression on murine DCs� however, it signi�cantly suppressed cytokine production and cell surface marker expression in DCs matured with a range of TLR and non-TLR ligands [21]. In vitro studies on the impact of T. spiralis excretory/secretory products (TspES) on mice DCs revealed that these parasitic antigens suppress DC maturation induced by LPS derived from different bacteria [22]. In this study, different TLR agonists were used showing that the suppressive effect of TspES on DC maturation is restricted to TLR4. ese helminth products were also shown to interfere with the expression of several genes related to the TLR-mediated signal transduction pathways. For rat bonemarrow-derived DC it has been shown that aer incubation with TspES these DCs acquire a semimatured status which is re�ected in moderate upregulation of CD86, signi�cant upregulation of ICAM1 (Intercellular Adhesion Molecule 1), and no upregulation of MHC II, accompanied by impaired production of IL-12 p70 [23].

Regulatory T Cells
Treg cells control peripheral immune responses and are likely to play a central role in autoimmune, infectious, and allergic diseases. ree phenotypes of Treg have been described to date, categorized according to their origin, function, and expression of cell surface markers: natural Treg cells (CD4+CD25+Foxp3+) and inducible Treg cells that include the IL-10-producing Tr1 cells and the Foxp3+ T cells induced in the periphery [24]. In spite of the complexity of regulatory cell types, CD4+CD25+Foxp3+ Treg cells are the most prominent population of immunoregulatory cells known so far to be induced during helminth infections [4].
Early studies had already suggested regulatory T-cell activity during chronic helminth infections in humans. Doetze et al. reported that IL-10 and transforming growth factor-(TGF-) production mediated the hyporesponsiveness observed in PBMC from individuals with generalized onchocercosis caused by the �larial nematode Onchocerca volvulus [25]. In a study with �lariasis patients, lymphedema was associated with a de�ciency in the expression of Foxp3, GITR (glucocorticoid-induced tumournecrosis-factor-receptor-related protein), TGF-, and CTLA-4 (cytotoxic T-lymphocyte antigen 4), known to be expressed by Treg cells [26], while in children infected with intestinal nematodes (Ascaris lumbricoides and Trichuris trichiura) high levels of IL-10 and TGF-in addition to generalized T-cell hyporesponsiveness were found [27,28]. Likewise, Schistosome-infected individuals in Kenya and Gabon had higher CD4+CD25+ and CD4+CD25+Foxp3+ T-cell levels compared with uninfected individuals [29]. One of the studies providing evidence on the suppressive effect of Treg cells from helminth-infected individual is the one reported by Wammes et al. [30]. In this study, carried out in Indonesia, Treg cells from geohelminth-infected individuals were more effective at suppressing proliferation and IFN-production by effector T cells in response to malaria antigens and BCG than Treg cells from healthy individuals. A �larial parasite of humans, Brugia malayi, was found to secrete TGH-2 (transforming growth factor homologue-2), a homologue of host TGF- [31]. Since the recombinant TGH-2 can bind to the mammalian TGF-receptor, it has been suggested that it can promote the generation of regulatory T cells, as it has been shown for mammalian TGF-. In another study a signi�cant increased expression of Foxp3 and regulatory effector molecules such as TGF-, CTLA-4, PD-1 (programmed death 1) and ICOS (inducible costimulatory molecule) was found in �lariainfected compared to uninfected individuals in response to live infective-stage larvae or micro�lariae of Brugia malayi [32].
Various studies on the role of Treg cells in helminth infections have used animal models. In mice, CD25+ Treg cells were shown to restrain the pathology to helminth eggs during schistosome infection [13] and to Trichuris muris in the gut [33]. Moreover, depletion of CD25+ Treg cells with combined antibodies to CD25 and GITR resulted in enhanced immunity to �larial nematode Litomosoides sigmodontis in mice [34]. Generation of Treg cells with elevated expression of Foxp3 during helminth infection has also been demonstrated. For instance, infection of BALB/c mice with Brugia pahangi third-stage larvae (L3) resulted in expansion of a population of CD4+CD25+ T cells which was highly enriched in Foxp3 and IL-10 gene expression [35]. Induction of Treg cells was demonstrated to be necessary to establish a chronic L. sigmodontis infection since depletion of Treg cells in susceptible mice prior to infection enhanced parasitic killing and cleared the infection [36]. In chronic infection with the gastrointestinal helminth Heligmosomoides polygyrus, it was established that levels of Foxp3 expression within the CD4+ T-cell population of mice mesenteric lymph nodes were signi�cantly increased and that puri�ed CD4+CD25+ Treg cells possess suppressive activity in vitro [37,38].
e effect of TspES on T-cell activation in vitro was investigated using splenocytes derived from ovalbumin-(OVA)-TCR transgenic D011.10 mice that were incubated with TspES-pulsed DC+OVA. Results indicate that the presence of TspES resulted in expansion of CD4+CD25+ T cells that express high levels of Foxp3+. ese Treg cells were shown to have suppressive activity and to produce TGF-. Together these results indicate that T. spiralis secretion products can induce expansion of functional Treg cells in vitro [22]. In a rat model, the infection with T. spiralis is accompanied with the increase proportion of Foxp3+ Treg cells [23]. In vitro studies showed that DCs stimulated with TspES caused strong 2 polarization, accompanied by elevated production of the regulatory cytokines IL-10 and TGF- [23]. However, unlike the mouse model described previously, conditioned rat DCs generated no increase in the proportion of CD4+CD25+Foxp3+ T cells. In vivo T-cell priming with TspES stimulated DCs resulted in mixed 1/2 cytokine response, with the dominance of the 2 type and elevated levels of regulatory cytokines. Signi�cant increase in the proportion of CD4+CD25+Foxp3+ cells was found in spleen cells of recipients that received TspES stimulated DCs compared to the control value obtained from rats that received DCs cultivated in medium only.

Other Regulatory Cells
Helminth infections may also lead to expansion of immunoregulatory cells other than Treg cells, including alternatively activated macrophages (AAMs) and regulatory B cells. Signals encountered during migration by developing macrophages determine their function at sites of in�ammation or infection. Among these signals, cytokines are responsible for the development of highly divergent macrophage phenotypes: classically activated and AAMs [39]. Prieto-Lafuente et al. reported that the homologues of the mammalian cytokine macrophage migration inhibitory factor (MIF) expressed by Brugia malayi synergized with IL-4 to induce the development of suppressive AAMs in vitro [40]. One pathway for this effect may be through the MIFmediated induction of IL-4R expression on macrophages, amplifying in this way the potency of IL-4 itself. us, in a 2 environment, MIF may prevent the classical activation of macrophages. e suppressive effect of AAMs on the immune response is most likely dependent on the expression of arginase-1 (Arg-1) as indicated by studies in which mice macrophages lacking Arg-1 failed to suppress 2 responses (reviewed in [41]).
B cells possess a variety of immune functions, including production of antibodies, presentation of antigens, and production of cytokines. IL-10-producing regulatory B cells have great potential to regulate T-cell-mediated in�ammatory responses [5] and to downmodulate experimental autoimmune encephalomyelitis, collagen-induced arthritis, and in�ammatory bowel disease [42]. In addition, in mouse models of chronic parasitic in�ammation, such as chronic schistosomiasis, IL-10-producing B cells were also reported to be associated with protection against anaphylaxis [43]. Moreover, H. polygyrus-infected mice induced regulatory B cells that can downmodulate both allergy and autoimmunity in an IL-10 independent manner [44].

Helminth Infections and
In�ammator� �iseases e helminth-induced immunosuppresive network may not only be bene�cial for the parasite, but it can also have bene�cial outcomes for the host, reducing allergic and autoimmune diseases [41,45]. Epidemiological, cross-sectional studies support an inverse correlation between allergic diseases and helminth infection [46,47] including infections by nematode species like A. lumbricoides and Necator americanus [48]. An increased skin reactivity to house dust mites was found aer antihelminthic treatment against infection with A. lumbricoides and T. trichiura [49]. Studies performed in animal model system have con�rmed that helminth infection can protect against allergic disease and in particular lung-associated in�ammation. For instance, S. mansoniinfected BALB/c mice were protected against OVA-induced experimental allergic airway in�ammation (EAAI) as indicated by reduction of eosinophils in BAL, 2 cytokine production, OVA-speci�c IgE levels and reduction of the number of in�ammatory cells in lungs. �ere, induction of CD4+CD25+Foxp3+ regulatory T cells was independent of IL-10 [50]. Dittrich et al. found that chronic infection with the �larial parasite Litomosoides sigmodontis suppressed all pathological features of the OVA-induced EAAI model [51]. Additionally, these authors observed signi�cantly increased numbers of Treg cells in spleen and mediastinal lymph nodes in infected OVA-treated mice compared to OVAcontrols animals. Suppression of EAAI during the course of H. polygyrus infection was shown to involve the induction of Treg cells [52]. Infection with the same parasite resulted also in the inhibition of allergic response to peanut extract [53]. Several epidemiological studies have investigated the protective effect of parasitic infections in different autoimmune diseases like multiple sclerosis and type 1 diabetes [54]. Studies indicate that persons infected with chronic parasitic worm infections have lower rates of in�ammatory bowel disease (IBD) than persons without these infections [55]. Experiments carried out using animal models of human autoimmune diseases have shown that parasites can interfere with autoimmunity. Schistosoma mansoni infection has been shown to protect from type 1 diabetes [56] and reduces the severity of EAE [57] while infection with H. polygyrus suppresses the experimental colitis [58]. Infection with L. sigmodontis prevented diabetes in NOD mice. In this study, protection was associated with increased 2 responses and Treg cell numbers [59].
e immunomodulatory effect of helminth-derived products has been extensively studied. Table 1 provides an overview of different helminthic antigens with immunoregulatory properties. Findings regarding the use of parasite antigens to suppress experimental in�ammatory diseases are summarized in Table 2.
Although the majority of data suggest that infection with helminths is associated with a suppression of allergic and autoimmune responses, some examples provide the opposite view. Epidemiological studies indicate that infection with Ascaris spp, Toxocara spp, Fasciola hepatica, hookworms, or Enterobius vermicularis has no protective effects or even enhanced allergic responses (reviewed in [95]).
ere are also experimental studies that show that infection with some helminths have a positive association with allergy. A study using a murine model has shown that T. canis infection results in exacerbation of EAAI [96]. Other animal experiments provided evidence that parasites like Nippostrongylus brasiliensis [97] and B. malayi [98] could also induce or exacerbate allergic responses. Exacerbation of anaphylaxis has been shown to occur during T. spiralis infection [99]. e links between infections and autoimmunity are complex and there is scarce evidence on the induction or exacerbation of autoimmune responses by helminths [100].

Contrasting Effect of Toxocara and Trichinella Infections on In�ammator� �iseases
Toxocara canis and Toxocara cati are roundworms of dogs and cats, respectively, that can also infect humans worldwide. Aer ingestion of the infectious Toxocara eggs, the larvae migrate to the intestine, liver, and lungs. While in dogs and cats under the age of 6 months, the larva migrate back to the intestine; in humans migration continues to other organs where they can persist for many years [101]. Toxocara infection results in the induction of 2 cells that make cytokines such as IL-4, IL-5, and IL-13, which induce responses to the parasite such as increased IgE levels and eosinophilia (reviewed in [6]). Trichinella spiralis is also a roundworm that infects different mammals including humans and mice. Aer ingestion of Trichinella infected meat, the larvae migrate to the intestine and matures to the adult stage, the parasites mate, and �nally the newborn larvae (NBL) migrate to striated muscle cells where they become encysted. Infection with T. spiralis is characterized by the induction of a 1 type of response at the beginning of the intestinal phase. When the NBL disseminate, a dominant 2 type of response develops which is essential for parasite expulsion [102]. Ingestion of both Toxocara spp. and Trichinella spp. commonly results in chronic infections. Interestingly these helminths have a contrasting effect on in�ammatory diseases, while infections with Trichinella spp. can suppress (reviewed in [103]) Toxocara spp. exacerbate in�ammatory diseases [6]. Studies using animal models for human autoimmune and allergic diseases indicate that Trichinella infection ameliorates these immune disorders (  [78]. T. spiralis infection also ameliorated autoimmune diabetes in NOD mice [89] and modulated severity of the disease in the experimental model of multiple sclerosis (MS), namely, experimental autoimmune encephalomyelitis (EAE) in Dark Agouti rats in a dosedependent manner [90]. In this study severity of EAE as Block neutrophil protease and promote 1 type of response [69] Brugia malayi L3 larvae ALT-1/2 proteins Inhibit macrophage resistance and present good �larial vaccine candidate [70] Toxocara canis TES32-C type lectin (CTL) Inhibit TLR responses on DC and compete with host lectins for ligands, thereby blocking host immunity [71,72] Heligmosomoides polygyrus Excretory-secretory antigen (HES)

Trichuris suis
Soluble antigens of adult worm EAE [77] Ancylostoma ceylanicum Soluble and excretory-secretory antigens of adult worm DSS-induced colitis [  judged by cumulative disease index, maximal clinical score, duration of illness, and the number of mononuclear cells in�ltrating the spinal cord in T. spiralis infected animals were all reduced in comparison to the uninfected EAE-induced group. In a following study, these authors reported that alleviation of the disease in infected-EAE rats coincided with reduced IFN-and IL-17 production and increased IL-4, IL-10, and TGF-production. ey suggested that mechanisms underlying the observed bene�cial effect include 2 and regulatory responses provoked by the parasite. Transfer of T-cell-enriched spleen cells from T. spiralis-infected rats that contained a higher proportion of CD4+CD25+Foxp3+ regulatory T cells into rats in which EAE was induced caused amelioration of EAE, which indirectly points to the role of Treg in restraining in�ammatory conditions [91]. Boles [94]. Trichinella pseudospiralis is markedly different from T. spiralis in that it is smaller in size and that the muscle stage larvae are not surrounded by a capsule [104]. Whether the mechanisms involved in immunosuppression varies depending on Trichinella species remains to be investigated. Infection with T. spiralis can also ameliorate EAAI [92]. In this study, the concentrations of IL-10 and TGF-were signi�cantly increased and the recruitment of Treg into draining lymph nodes was elevated as the result of T. spiralis infection. is protective effect has been recently shown to occur during acute as well as chronic phases of Trichinella infection [105]. Protection against EAAI to OVA was stronger during the chronic phase of infection and associated with increased numbers of splenic CD4+CD25+Foxp3+ Treg cells with suppressive activity. Adoptive transfer of CD4+ T cells from chronically infected mice with elevated numbers of Treg cells in the spleen induced partial protection against EAAI [105]. e possible mechanisms by which helminths or their products could inhibit allergic responses are depicted in Figure 1.
In contrast to the suppressive effect of Trichinella infections on allergic diseases experimental as well as epidemiological studies indicate that Toxocara infections are risk factors for allergies, including allergic asthma [6].
Studies using murine models for toxocariosis indicate that infection with T. canis leads to persistent pulmonary in�ammation, eosinophilia, increase levels of circulating IgE, airway hyperreactivity, and production of -2 type cytokines. Pulmonary in�ammation has been shown to develop as soon as 48 hours aer infection, it occurs in a dosedependent manner, and it can persist up to 2 or 3 months. Eosinophil counts also increase in the bronchoalveolar lavage (BAL) of Toxocara-infected mice [106,107]. Relative quan-ti�cation of cytokine expression in lungs of mice infected with different T. canis doses showed that while a proportional increased expression of the IL-4, IL-5, and IL-10 transcripts was observed, the expression of IFN-was not different from that of uninfected controls [107]. Results from this study indicate that infection of mice with T. canis results in a dominant 2 type of immune response, independent of the inoculum size [108]. In addition, infection of BALB/c mice with 1,000 T. canis embryonated eggs results in hyperreactivity of the airways that persisted up to 30 days p.i. Evaluation of parasite burden revealed that few T. canis larvae were still present in the lungs of infected mice at 60 days p.i. which could explain the persistent pulmonary in�ammation observed in these mice [107].
Common features between allergic asthma and toxocariosis are the induction of a 2-cell mediated immune response including the production of high levels of IgE, and eosinophilia. In addition, infection with Toxocara spp. shares common clinical features with allergic asthma such as in�ammation of the airways accompanied with wheezing, coughs, mucus hypersecretion, and bronchial hyperreactivity. In order to study the effect of Toxocara infection on allergic manifestations two murine models were combined, namely, the murine model for toxocariosis described above and a murine model for allergic airway in�ammation. For this study BALB/c mice were infected with 500 embryonated T. canis eggs and exposed to OVA sensitization followed by OVA-challenge. Results indicate that infection with T. canis in combination with OVA treatment led to exacerbation of pulmonary in�ammation; eosinophilia; airway hyperresponsiveness; increase of OVA speci�c and total IgE; increased expression of IL-4 compared to mice that were only T. canis infected or OVA treated. e observed exacerbation of EAAI was independent of the timing of infection in relation to allergen exposure. In conclusion, infection with T. canis leads to exacerbation of EAAI [96]. Several factors may in�uence the differential effect of helminth infections on allergic diseases [6]. One of these factors is whether the host is de�nitive or accidental. e normal or de�nitive hosts for T. canis are dogs whereas humans are accidental hosts for this parasite. In an accidental host the parasite does not usually develop to the adult stage and in case of Toxocara spp. the continuous migration of the larvae through different organs including the lungs can cause more damage comparing to what happens in a de�nitive host where migration is transitory. T. spiralis can infect many different mammals including mice and humans in which the parasite completes its life cycle. e infected mammal is therefore a de�nitive host for this helminth. And although T. spiralis pass through the lung microvascular system on its way to the skeletal muscle, it is a rapid process in which the larvae are usually not trapped in the lungs [109]. It is likely that there are differences between parasites of humans such as T. spiralis that have evolved with their host and have developed strategies to survive without causing much damage compared to parasites such as Toxocara spp. for which humans are accidental host [6,110].
In conclusion, helminths induce an anti-in�ammatory response, which could ameliorate in�ammatory diseases� however, this is not a universal property of all helminths and different factors such as the helminth species, and whether the host is de�nitive or accidental, the parasite load and acute versus chronic infections may all in�uence the overall effect of helminth infections on in�ammatory diseases. Identi�cation of the helminth molecules that induce immunosuppression and elucidation of the mechanisms involved is essential for the development of alternative strategies for prevention and�or treatment of in�ammatory diseases.