Immunoregulation by Taenia crassiceps and Its Antigens

Taenia crassiceps is a cestode parasite of rodents (in its larval stage) and canids (in its adult stage) that can also parasitize immunocompromised humans. We have studied the immune response elicited by this helminth and its antigens in mice and human cells, and have discovered that they have a strong capacity to induce chronic Th2-type responses that are primarily characterized by high levels of Th2 cytokines, low proliferative responses in lymphocytes, an immature and LPS-tolerogenic profile in dendritic cells, the recruitment of myeloid-derived suppressor cells and, specially, alternatively activated macrophages. We also have utilized the immunoregulatory capabilities of this helminth to successfully modulate autoimmune responses and the outcome of other infectious diseases. In the present paper, we review the work of others and ourselves with regard to the immune response induced by T. crassiceps and its antigens, and we compare the advances in our understanding of this parasitic infection model with the knowledge that has been obtained from other selected models.


Introduction
Helminth parasites have developed complex and versatile mechanisms to evade the immune responses of their hosts, utilizing immunoregulatory strategies to avoid immune effector mechanisms. In general, these processes are necessary for the parasites to complete their long life cycles [1] and/or to favor host survival [2]. Despite their great evolutionary divergence and variety of stages, life cycles, and pathogenic and invasive mechanisms, helminths have developed similar strategies and induce strikingly similar immune responses, which have been called "stereotypical 2-type immune responses. " However, there are differences in the immune responses evoked by distinct helminths, mainly with regard to leukocyte involvement and the roles of these cells [3].
e stereotypical 2 response induced by helminth parasites is characterized by the secretion of high levels of anti-in�ammatory cytokines such as interleukin-6 (IL-6), IL-9, IL-10, IL-25, IL-33, and transforming growth factor-(TGF-), but the main cytokines are IL-4 and IL-13 [4]. As a consequence and/or origin of this cytokine secretion, there are alterations in leukocyte recruitment and activation, such as high levels of CD4+ T lymphocytes differentiated into 2 and T regulatory (Treg) subsets, the recruitment and activation of immunoglobulin G1 (IgG1)-and IgE-producing B cells, eosinophilia, basophilia, and mastocytocis [4,5]. Interestingly, an immature dendritic cell (iDC) phenotype with a 2-driving ability and huge populations of alternatively activated macrophages (AAMs) with the ability to suppress lymphocyte proliferation can also be found within this response [3,5,6]. Furthermore, another characteristic of 2 responses is the suppression of the immune response to bystander antigens, which may compromise the effectiveness of vaccination [7] and alter the immune response to several other antigens, even autoantigens.
It is commonly accepted that most of these changes in leukocyte phenotype and activation, as well as in the induction of the in�ammatory milieu, are dependent upon the ability of the parasite to excrete/secrete antigens with immunoregulatory properties [8][9][10][11][12]. Many research teams [13][14][15], including ours [16,17], have used these 2 responses elicited by helminths and their antigens to control autoimmune disease development as well as to alter the outcome of other infectious diseases [18].

The Immune Response to Experimental
T. crassiceps Infection: Th1/Th2 Balance and Susceptibility T. crassiceps is a helminth parasite (class Cestoda) that can be found in its adult form within the small intestine of canids, whereas the main larval stage (metacestode) can be found in the muscles, peritoneal, and pleural cavity of rodents. T. crassiceps metacestodes can also parasitize immunocompromised human patients with cancer [19], human immunode�ciency virus and hepatitis C virus [20]. In addition, this parasite can infect perfectly healthy patients, although only one case has been reported [21]. An interesting feature of T. crassiceps is its ability, or evolutive advantage, to reproduce asexually through budding at the larval stage. is characteristic permits the larval stage to maintain and colonize its hosts for long periods of time; thus, aer the intraperitoneal inoculation of a few parasites (10 to 20 metacestodes), hosts can harbor hundreds of parasites 6-8 weeks later. is feature has been useful for maintaining the parasite at the larval stage in the laboratory via passage from mouse to mouse through intraperitoneal injections, and these animals are also important sources of antigens that have been utilized for immunodiagnostic tests for cysticercosis [22]. Additionally, the fact that the larval stage of the parasite is innocuous for humans is important; although its macroscopic size facilitates the accumulation of an acute parasite burden, the parasite does not kill the host and is able to cause chronic infections with a minimum amount of damage in mice. Furthermore, the results are very reproducible. All of these features confer many advantages on this model for laboratory work and even for the development of vaccine strategies [23]. Early studies on the immune response against this parasite were performed in the late 1970s and early 1980s by Siebert and Good [24,25]. is work mainly focused on the humoral immune response against T. crassiceps and found that antibodies anti-T. crassiceps cannot be correlated with cytotoxic effects or tegument degradation. Later, following the de�nition of the dichotomous 1 and 2 responses, a new series of investigations were conducted by different groups. Most of these studies coincided with the general knowledge that, during the acute stage, murine infection with this parasite leads to the induction of a transient 1 proin�ammatory immune response with high serum levels of gamma interferon (IFN-), nitric oxide (NO), and IgG2a that lasts for the �rst 2-3 weeks and then is replaced by a dominant 2-type response rich in IL-4/IL-13, as well as IgG1 and IgE antibodies that last for at least two months ( Figure 1) [26]. Later �ndings demonstrated that spleen cells from T. crassiceps-infected mice were refractory to polyclonal stimuli such as Concanavalin A and anti-CD3 [27], indicating that infection has a clear modulatory effect on the hosts immune system. Our next studies, conducted in the late 1990s, sought to block the cytokines involved in immune regulation in vivo early during infection, such as IFN-, IL-4, and IL-10, or inject IFN-plus IL-2 to support our idea that a 1 response was efficacious at eliminating the larval stage of T. crassiceps. Early blockade of IFN-with speci�c antibodies in the �rst week of infection greatly favored the establishment of the parasite. In contrast, the injection of recombinant murine IFN-plus IL-2 at the time point improved resistance to the infection, whereas the blockade of IL-10 or IL-4 had little effect on parasite loads [28].
Our proposal that a 1 response was involved in eliminating a helminth infection was not widely accepted for several more years. Early in the 2000s, con�rmatory experiments were performed with knockout mice to show that susceptibility to the larval stage of T. crassiceps is dependent upon signal transducer and activator of transcription-6 (STAT6) signaling, a key transcription factor involved in 2 lymphocyte differentiation and alternative macrophage activation [29]. Conversely, resistance to this parasite was shown to be dependent on the IL-12/STAT4 signaling axis [30,31], which is the main inducer of 1 immunity. us, a 2-type response was found to be associated with susceptibility to helminth infection, whereas a 1-type response (dependent on STAT4) was shown to be clearly involved in protection against T. crassiceps ( Figure 1). Moreover, when the immune responses in susceptible (BALB/c) and resistant (C57BL/6) mouse strains were compared, it was found that the 1 immune responses mounted by C57BL/6 mice are stronger than those of susceptible mice [32]. ese data together sustain the notion that susceptibility to the T. crassiceps metacestode is dependent upon a 2 immune response, while resistance depends on the adequate and rapid development of a proin�ammatory response.
One of the most interesting �ndings from a separate series of studies was the fact that, in parallel to the shi from a 1-type towards a 2-type response, a distinct population of macrophages emerges; these macrophages display low IL-1 , IL-12, and nitric oxide (NO) secretion but express high levels of arginase-1 (Arg1), Ym1, resistin-like moleculealpha (RELM ), macrophage mannose receptor (MMR), and interleukin-4 receptor alpha (IL-4R ). is population has a poor ability to induce antigen-speci�c proliferative responses in T cells and a 2 driving ability [33] and is now recognized as AAMs ( Figure 2); importantly, AAMs have been reported to be present during most helminth infections [3]. Interestingly, the immune polarization toward a 2 pro�le and the establishment of an AAM population are accompanied by an increase in parasite burden during experimental murine cysticercosis caused by T. crassiceps [29] (Figure 1). ese �ndings suggest that the parasite itself may be the main impetus for this tolerogenic response.

Alternatively Activated Macrophages and Their Role in T. crassiceps Susceptibility and Immunoregulation
Two main macrophage phenotypes have been described according to the in�ammatory stimuli that induce their activation. Classically activated macrophages (CAMs) are activated through toll-like receptor (TLR) stimulation with bacterial-, virus-and protozoan-derived molecules such as lipopolysaccharide (LPS) and peptidoglycan as well as IFN-, tumor necrosis factor-alpha (TNF-) and IL-1 , which are secreted during in�ammatory responses. CAMs show enhanced phagocytic, microbicidal, and 1/17driving abilities and consequently have an important role in immunity to intracellular pathogens. ey typically express inducible nitric oxide synthase (iNOS), which is the main enzyme involved in NO production, and they also secrete proin�ammatory cytokines such as IL-1 , IL-12, IL-23, and TNF- [38]. In contrast, AAMs are induced mainly by IL-4 and IL-13 [39] stimulation through IL-4R [40], causing the activation and nuclear translocation of STAT6 [41]. Additionally, several helminth antigens have been proven to induce the alternative activation of macrophages independently of IL-4 stimuli [9][10][11]42]. AAMs may secrete high levels of IL-10 and TGF-but low levels of proin�ammatory cytokines and express the enzyme Arg-1, which competes with higher affinity than iNOS for the common substrate L-arginine and produces urea, polyamines, and L-ornithine. AAMs also express YM-1, RELM , programmed death-ligand 1 (PD-L1), and PD-L2 [3] and play a role in several aspects of the immune response, such as lymphocyte 2 differentiation [33,43], recruitment of IL-4-producing eosinophils [44], and, primarily, induction of low proliferative T cell responses [45,46] (Figure 2).
In recent years, it has been demonstrated that AAMs are a common cell population induced during diverse helminth infections [3,6], in which they have been shown to display diverse roles in host survival [2] as well as resistance to this type of infection [47] or as part of the wound healing machinery [2]. However, the role for AAMs in the T. crassiceps cysticercosis model is quite different.
e notion that IFN- [28] and STAT6 de�ciency [29] correlate with resistance to infection led us to investigate the role of CAMs and AAMs in the immune response and susceptibility to T. crassiceps. Interestingly, macrophages from infected resistant mice displayed a greater ability to induce T cell proliferative responses, secreted pro-in�ammatory cytokines and produced more NO, thereby displaying a classical activation phenotype, while macrophages from a susceptible mouse strain did the opposite and displayed an alternative phenotype [32]. We believe that macrophages that are recruited or polarized to become AAMs during T. crassiceps infection have been one of the most characterized during helminth infections. ese AAMs show an increased expression or production of Arg-1, Ym-1, RELM-, TREM-2, SLAM, MMR, mMGL, OX40-ligand, MHC-II, CD23, CCR5, IFN-R, IL-4R , TLR4, PD-L1, PD-L2, PGE2, IL-10, and IL-6. In contrast, these AAMs have a low production or expression of iNOS, IL-12, IL-15, IL-18, IL-23, IL-1 , TNF-, MIF, and NO [30,32,33,45]. us, a total of 28 different molecules have been identi�ed as altered in macrophages during experimental cysticercosis. Importantly, these molecules are intimately bound to the modulation of the immune response. erefore, the study of such a cell population has become essential to understand helminth immunology.
To gain insights into the role of macrophages in facilitating or clearing T. crassiceps infection, we developed new experimental strategies. e treatment of STAT6 KO mice, which develop CAMs and are highly resistant to infection, with an iNOS inhibitor in vivo rendered these mice susceptible to T. crassiceps infection [52]. Similarly, de�ciency in TLR2, which helps to induce pro-in�ammatory responses in mice that are otherwise genetically resistant, rendered them highly susceptible to helminth infection [53]. In contrast, the early depletion of AAMs with clodronate-loaded liposomes in susceptible BALB/c mice reduced parasite loads by 90% [54]. Together, these data demonstrate that AAMs, a cell population that plays a key role in immunomodulation during T. crassiceps infection, may also be implicated in susceptibility to this parasite, while CAMs appear to be related to resistance.
e mechanism by which AAMs mediate susceptibility to this cestode is not currently well known, but it may be the inhibition of NO production through the expression of Arg-1 [47,52], the release of prostaglandin E2, which also has immune-modulatory properties [55], or their suppressive capacity over lymphocyte proliferation [45]. Regardless, it is clear that the presence of CAMs is an important factor that contributes to host resistance. Several ma�or �ndings support this idea, including the discovery that strains of mice that are resistant to T. crassiceps infection do not develop AAMs [32]; for example, C57BL/6 mice challenged with a similar number of metacestodes as BALB/c mice develop CAMs, but if STAT4-KO mice on the same resistant genetic background are similarly challenged, they have huge parasite burdens and develop AAMs [30]. In contrast, mice with a susceptible genetic background, such as BALB/c, but lacking the STAT6 gene became highly resistant to infection and do not develop AAMs. Instead, they recruit CAMs that highly produce NO, TNF-, and IL-12 [29]. Moreover, as we stated above, the in vivo inhibition of iNOS was shown to induce susceptibility in STAT6-KO mice [52]. us, the activation state of macrophages plays a critical role in the outcome of helminth infection.
Additionally, both low proliferative responses and low lymphocyte counts in tissues near the parasite may be important factors in susceptibility to this infection, as it has been shown that there are many apoptotic lymphocytes surrounding viable metacestodes in T. solium-infected pigs [56] and during T. crassiceps infection can be seen a lower lymphocyte proliferative response in susceptible mice strains than in resistant ones [27]. ese �ndings are in line with our observations, in which we have found that AAMs induced by T. crassiceps can suppress the proliferative responses of naive T cells stimulated with anti-CD3/CD28 antibodies in vitro [45]. As we had previously detected high levels of PD-L1 and PD-L2 expression in AAMs recruited during T. crassiceps infection, we hypothesized that the Programmed death-1/Programmed death-Ligands (PD-1/PD-Ls) pathway may be involved in such inhibition. us, transwell assays and in vitro blockade of PD-L1 or PD-L2 were found to reverse the suppressive activity of these AAMs. Moreover, AAMs induced by T. crassiceps infection were also demonstrated to suppress the speci�c response of CD4 + DO11•10 cells to OVA peptide stimulation when unpolarized macrophages were used as antigen presenting cells. Again, in this assay, the blockade of the PD-1/PD-L's pathway reestablished the peptide-speci�c proliferative response of CD4 + DO11•10 cells [45]. erefore, AAMs can participate as a third party suppressive cell. is idea was con�rmed with a different set of experiments, in which we demonstrated that the presence of AAMs in a DC-mediated mixed lymphocyte reaction was sufficient to inhibit the response of CD4 + cells from a different genetic background. Mechanistically, AAMs recruited during chronic T. crassiceps infection are able to suppress immunological events mediated through distinct molecular pathways that may induce strong proin�ammatory responses ( Figure 2). We also demonstrated that the PD-1/PD-L pathway participates in modulating the anti-Taenia-speci�c cell proliferative response. �owever, whether these T cells exposed to AAMs undergo anergy and/or apoptosis and the in vivo signi�cance of the PD-1/PD-L pathway in susceptibility to T. crassiceps are currently unknown, and further research is needed to resolve these questions.

Immunoregulation by T. crassiceps Antigens
It is commonly accepted that the inhibition of proin�ammatory responses and the induction of 2 immunity during helminth infections are dependent upon the parasite's ability to excrete/secrete antigens with immunoregulatory properties that have important effects on myeloid-derived suppressor cells (MDSCs), eosinophil and basophil recruitment, DC maturation impairment, alternative macrophage activation, impaired lymphocyte proliferative responses, and, in some cases, Treg induction [8-12, 57, 58]. e �rst in vivo evidence for these conclusions is that the pharmacological treatment of helminth-infected patients can trigger pro-in�ammatory responses [59] and that much experimental data have been obtained indicating that the inoculation of helminth antigens alone has the ability to induce such immunoregulatory effects, as reviewed in [12,58]. us, it has been largely accepted that the in vivo injection of some helminth-derived antigens is able to mimic some of the immune features induced by these parasitic infections, but the mechanisms, putative receptors, and intracellular signaling pathways involved in these effects still have yet to be recognized [60]. Pioneering studies by the group led by Donald Harn at Harvard University have demonstrated that the main 2-inducing activity of injected soluble egg antigen (SEA) from Schistosoma mansoni is dependent on the intact structure of carbohydrates in the antigen [61]. us, it was hypothesized that glycoproteins are essential for 2 induction during schistosomiasis. is idea was rapidly adopted by several "helminth immunologists, " who corroborated many of the Harn's group �ndings using different sources of helminth antigens, such as Brugia, Echinococcus, Ascaris, Caenorhabditis, Hymenolepis [62] and, of course, Taenia. In this area, our team has also evaluated the effects of the in vivo inoculation of antigens derived from T. crassiceps metacestodes. e injection of a soluble extract of these larvae can rapidly (18 h post-inoculation) recruit a CD11b + F4/80 + Gr1 + cell population, consisting of what are now called myeloid-derived suppressor cells (MDSCs), which possess a strong capacity to inhibit proliferative responses in activated lymphocytes and may have an important role in inhibiting the initial 1 response to this parasite ( Figure  2) [63]. Interestingly, when T. crassiceps antigens are treated with sodium metaperiodate to alter glycan structures, these antigens lose the ability to recruit MDSCs, indicating a critical role for glycoproteins in modulating the immune response to this parasite. Further research has demonstrated that glycans present in T. crassiceps excreted/secreted (TcES) molecules are also important in modulating DC maturation [64]. DC maturation involves the upregulation of several costimulatory molecules that play important roles in antigen presentation and T cell activation, such as CD40, CD80, CD86 and major histocompatibility complex II (MHCII), as well as proin�ammatory cytokines such as IL-12 and IL-18. A fully mature DC is capable to induce T cell activation, proliferation, and differentiation into the 1 phenotype, whereas an iDC drives 2 differentiation and induces an impaired proliferative response in T cells [71]. Recently, we have found that the in vitro exposure of murine [64] and human [72] DCs to TcES impairs their maturation. DCs also become refractory to stimulation with different TLR ligands and thereby produce low levels of pro-in�ammatory cytokines such as IL-12, IL-15, and TNF-. Importantly, when these DCs exposed to TcES are used as antigen presenting cells, they are able to induce the 2 differentiation of naïve CD4 + T cells (Figure 2). Moreover, all these effects of TcES are glycan-dependent [64]. Interestingly, other research groups in this �eld have found many similarities in the responses of monocytederived dendritic cells following exposure to glycoproteins derived from Echinococcus granulosus, E. multilocularis [73,74], egg carbohydrate antigens from S. mansoni [75], or larval carbohydrate antigen from the nematode Trichostrongylid [76].
Moreover, in vivo assays have also demonstrated that carbohydrates in helminth-derived antigens are essential to bias 2-type responses to bystander antigens, and thus glycoproteins from SEA and from T. crassiceps coinjected with the unrelated proteins human serum albumin and ovalbumin, respectively, into mice were shown to induce strong 2 responses to these antigens. However, when glycan structures were altered, the 2 polarization effect of the helminth antigens was eliminated [8,61]. us, it is clear that host immune responses to helminth parasitic diseases or to bystander antigens are modulated by helminth-expressed glycans, and therefore most of these effects must be mediated by carbohydrate recognition receptors. It is important to keep in mind that the chemical composition of helminth antigens varies greatly among species, but the most common types are proteases, protease inhibitors, cytokine/chemokine homologs, antioxidant enzymes, lectins, and other carbohydrates [12]. Consequently, other receptors may also be involved in recognizing such diverse molecules. Likewise, it is critical to elucidate the protein, glycan, and lipid composition of helminth-derived molecules with immunomodulatory ability.

The Therapeutic Potential of T. crassiceps
e last two decades have witnessed a dramatic increase in the number of new cases of in�ammatory diseases in developed countries, while, at the same time, the hygiene conditions in these countries have greatly improved, leading to a reduction in the prevalence of different bacterial or parasitic infections, including helminth infections [77]. Taken together, these observations led to the postulation of the hygiene hypothesis, which states that in the absence of intense infections that modulate host immunity to a 2-type response (such as during helminth infections), the immune system then tends to present exaggerated 1 in�ammatory responses directed against microbial antigens or even autoantigens, thus leading to autoimmunity [57]. Although the contribution of genetic factors in the development of these diseases is evident, epidemiological [77][78][79] and experimental [80] evidence suggests that environmental factors can also be involved in the etiology of autoimmunity.
e main experimental evidence supporting the hygiene hypothesis came from studies in which helminth-infected mice were able to successfully control type 1 diabetes (T1D) [17,51], experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS) [13,16], in�ammatory bowel disease (IBD) [81], and rheumatoid arthritis (RA) [66]. More importantly, some studies conducted with parasite-derived antigens showed their ability to improve the outcomes of these diseases [14,51,82,83]. Helminth therapy and its likely bene�ts have started to be applied in humans; the treatment of patients with the eggs of the nonhuman parasite Trichuris suis, which is related to the human parasite T. trichuria, has been shown to moderately improve the outcome of MS [84], Crohn's disease [85], and, at a lower level, ulcerative colitis [85]. Because treatment with living organisms can generate adverse or side effects [84], treatment with helminth-derived immunomodulators is a very promising alternative [58].
Based on the notion that T. crassiceps induces strong anti-in�ammatory and long-lasting 2 responses, characterized by high systemic levels of IL-4, IL-10, and IL-13 as well as the recruitment of different regulatory cell populations such as AAMs, MDSCs, and iDCs accompanied by low T cell proliferative responses and the induction of low NO, IL-1 , IL-12, IL-15, IL-18, IL-23, TNF-, and IFN-levels that may block pathologic in�ammation, we investigated the role of this infection in the modulation and outcome of experimental autoimmune diseases such as EAE, rheumatoid arthritis, and T1D.
Recent �ndings in our laboratory show that the preinfection (8 weeks) of mice with T. crassiceps metacestodes can reduce the incidence of EAE by 50% and reduce the severity score of the disease (1 out of 5) in sick animals. is effect was accompanied by high systemic levels of IL-4, IL-10, IgG1, and IgE and low levels of IFN-, TNF-, IL-17, and IgG2a, as well as a reduced in�ammatory in�ltrate into the spinal cord. Importantly, we could �nd AAMs with strong suppressive activity over lymphocyte proliferation and a reduced number of CD3 + cells entering in the brain [16]. Other populations, such as CD4 + /CD25 + /FoxP3 + Tregs, have been associated with the secretion of high levels of IL-10, thereby suppressing 1, 2, and 17 responses. Tregs are likely to exert this regulatory effect on autoimmune diseases, but we have not been able to �nd Tregs in the brain, spleen, mesenteric lymph nodes, or peritoneal cavity of T. crassiceps-infected mice from susceptible or resistant strains [16,17]. Strikingly, an examination of the brains and spleens of T. crassicepsinfected EAE mice using �ow cytometry and rtPCR failed to reveal a signi�cant increase in CD4 + /CD25 + /FoxP3 + Treg cells [16].
Other research groups have shown that infection with S. mansoni [13], Fasciola hepatica [35], and Trichinella pseudospiralis [34] can regulate the incidence and/or severity of EAE, whereas other parasites such as Strongyloides venezuelensis [36] and T. spiralis [37,86] did not signi�cantly affect EAE development. Furthermore, the cytokines generally associated with the downregulation of EAE are IL-4, IL-5, and IL-10 as well as low IFN-, TNF-, and IL-17, but none of these other models are associated with AAMs as possible key players in the regulation of such diseases; instead, 2 CD4 + T cells are a common hallmark of EAE regulation (Table 1).
Importantly, Hymenolepis nana, T. trichuria, Ascaris lumbricoides, Strongyloides stercolaris, and Enterobius vermicularis-infected MS human patients showed a signi�cantly lower number of disease exacerbations, brain damage, and variation in disability scores during a 4.5-yearfollow up study. is protective effect was correlated with high eosinophilia, IgE titers and IL-10 + /TGF-+ Treg cell induction as well as low IL-12 and IFN-secreting cells [87], partially resembling the observations in the animal models.
Additionally, we have shown that the preinfection of mice with T. crassiceps can reduce the incidence of T1D induced by streptozotocin (STZ) up to 50%, mainly by lowering blood glucose levels to below 200 mg/dL, leukocyte in�ltration into the pancreas and, in consequence, the degree of insulitis. Such effects last for at least 6 weeks aer induction of T1D. ese protective effects were associated with high systemic levels of IL-4, a reduction in TNF-circulating levels, and the induction of AAM populations; however, analysis of the spleens did not show increased populations of Treg cells [16].
To our knowledge, only one work regarding STZ-induced diabetes and helminth-induced immunoregulation has been published in addition to ours; in this paper, it was shown that S. mansoni infection could reduce T1D incidence and pancreatic cell in�ltration, but the authors did not suggest which cell populations may be involved in the modulation of this disease [48] (Table 2).
Although other works examining the regulation of diabetes by helminth infections were primarily performed in less aggressive and slower models, such as nonobese diabetic (NOD) mice, there are several similarities and differences compared to our observations in the T. crassiceps model. Strikingly, it was shown that Heligmosomoides polygyrus infection during the early weeks of life can decrease the incidence of T1D in a mechanism dependent upon AAMs but not Tregs [15], but it has also been shown that infection with S. mansoni can signi�cantly reduce the incidence of diabetes and pancreatic damage in a scenario where AAMs together with Tregs play an important role in the regulation of the disease [49]. By contrast, the infection of mice with Litomosoides sigmodontis promotes protection and reduced insulitis that is dependent on increased Treg populations and 2 induction [51], while T1D incidence and blood glucose modulation in the Trichinella spiralis model are mainly regulated by 2 cells [50]. Together, these studies indicate that multiple pathways are involved in the modulation of experimental T1D by helminths, but some similarities can be found regarding regulatory leucocyte populations and cytokines ( Table 2).
Despite the strong regulatory activity of T. crassiceps in EAE and T1D, the infection with this parasite was shown to be unable to modify the outcome of experimental RA, given that 100% of infected animals developed medium clinical scores [65]. Strikingly, the pre-infection of mice with other helminths such as Syphacia oblevata [67] and Hymenolepis diminuta [70] can reduce the incidence [67] and severity [67,70] of experimental RA. Additionally, the pre-infection of mice with S. mansoni [68] and S. japonicum [66,69] can ameliorate RA in other models. In all of these models, the downregulation of IgG2a anti-collagen antibodies and the induction of high levels of IL-4 and IL-10 appear to be important in limiting RA progression and these effects were not achieved by T. crassiceps infection in this model (Table 3).
e main mechanisms involved in the abrogation of EAE and T1D with T. crassiceps infection may be IL-4 and IL-10 secretion as well as the induction of anergy in lymphocytes, as it has been shown that these diseases are dependent upon autoreactive lymphocyte proliferation [88,89] and commitment to 1 and 17 subsets [90]. We therefore hypothesize that AAMs are the main cell population involved in tolerance induction because we have shown that they have a strong suppressive ability over lymphocyte proliferation [45] while also having the capacity to drive 2 responses [33]. iDC populations may also be involved in this phenomenon due to their strong 2-driving abilities, but further in vivo investigation is needed to con�rm this hypothesis. Also, it would be important to research on the role of eosinophils [87] and B cells [91] in autoimmune disease regulation as these cells have been associated with MS T1D: type 1 diabetes; MLD-STZ: multiple low doses of streptozotocin; NOD: nonobese diabetic; N.S.: not speci�ed; ¶ incidence de�ned as mice with blood glucose levels greater than 230 mg/dL, while in the other models this was de�ned as blood glucose levels greater than 200 mg/dL. regulation in humans and, at least eosinophils, are strongly and rapidly recruited by T. crassiceps infection [54] (Figure 3). e absence of Treg induction during Taenia-induced immunomodulation of these autoimmune disease models reinforces the idea that AAMs and iDCs may play a central role in the induction of tolerance (Figure 3), but further investigation is needed to con�rm the role of these cell populations in disease regulation [12,15,78,79]. Additionally, we have not yet shown whether T. crassiceps infection can act both as a prophylactic and as a therapy option, and, more importantly, we have not yet investigated whether TcES may regulate the outcome of these diseases, which is one of the ultimate goals of our team.

T. crassiceps Immunoregulation: Fibrosis and Bystander Suppression
It is commonly accepted that 2 cytokines such as IL-4/13 and TGF-induce �brosis, which might be useful in wound healing but in other instances might be pathogenic as well [92,93]. Moreover, AAMs have been proposed to induce �brosis, mainly through the overexpression of Arg1, which may contribute to collagen deposition in the extracellular matrix [94,95]. In fact, it has been shown that T. crassiceps infection can induce liver �brosis in association with alternatively activated Kupffer cells and therefore exacerbate tetrachlorideinduced liver damage [96]. Moreover, several epidemiological studies show that parasite-parasite coinfections are common in developing countries, with children being the most susceptible group [97][98][99]. Furthermore, experimental data show that helminths can modify the host immune response and alter immunity to other parasites. For example, Litomosoides sigmodontis infection can alter the development of a secondary infection such as Leishmania major, increasing susceptibility to the second parasite [100]. Similarly, it has been shown that preinfection with T. crassiceps modi�es the immune response to Trypanosoma cruzi [101], Leishmania major, and L. mexicana [18], increasing susceptibility to these infections as well as tissue and organ damage resulting from the downmodulation of 1 immunity and classical macrophage activation, which are both associated with resistance to these protozoan parasites. T. crassiceps metacestodes F 3: Based on our information to date, it is possible that iDCs recruited by TcES can prime 2 differentiation, while AAMs may reinforce this activation and block pathogenic lymphocyte proliferation. Additionally, a shi from pathology-inducing CAMs to a protective AAM population can be seen, and all these changes together may protect mice from autoimmunity.
T. crassiceps infection can also negatively modulate the outcome of viral infections; an enhanced susceptibility to vaccinia virus via the suppression of cytotoxic T cell responses in mice infected with this helminth has been shown [102]. Moreover, the stimulation of mice with CpG, a bacteriaand virus-derived agonist of TLR9, can augment protective immunity to the cestode [103], opening the possibility for a cross regulation of susceptibility between virus and T. crassiceps when coinfection exists. is possibility can be extended to bacterial, protozoan, helminth, and fungal coinfections, given the discovery that TLR2 is involved in mediating resistance to this parasite [53].

Concluding Remarks
As with other helminths, infection with the cestode T. crassiceps induces strong and long-lasting 2-polarized immune responses, and high systemic levels of IL-4, IL-5, IL-10, IL-13, IgG1, and IgE as well as low NO, IL-1 , IL-12, IL-15, IL-18, IL-23, TNF-, and IFN-serum concentrations are achieved. ese changes in cytokine secretion are accompanied, induced, and/or regulated by AAMs, MDSC, eosinophil and iDC populations with suppressor and 2-driving abilities. us, the characteristics of the immune response to this parasite can be coopted to regulate the outcome of autoimmune diseases. In fact, we have successfully used the immune response to this parasite to regulate EAE and T1D incidence and severity. Despite these bene�ts, T. crassiceps immunoregulation has some drawbacks, such as the fact that infection with this cestode can exacerbate �brosis and protozoa infections. Moreover, we have seen that several T. crassiceps antigens can mimic the effects of parasite infection, making them promising 2 ad�uvants or anti-in�ammatory biocompounds that may be used in autoimmune or in�ammatory disease regulation while avoiding the pathogenic side effects of infection with the live parasite. Further investigation is needed to uncover the role of TcES in the regulation or amelioration of in�ammatory diseases and, in particular, the mechanisms it utilizes to modulate the immune response towards a distinct regulatory pro�le.