Role of eosinophils in protective immunity against secondary nematode infections

Abstract Infections with parasites, especially those involving nematodes that invade tissues, induce a strong Th2-type immune response, which increases immunoglobulin E and eosinophil levels in the blood and tissues. Eosinophils are not effective against all possible helminth infections, but are known to be effective against nematode larvae. In particular, when a host is re-infected by a species of nematode that it previously encountered, the activation of acquired immunity causes robust accumulation and activation of eosinophils that damages the nematode larvae. Eosinophil production and activation processes are mainly induced by interleukin (IL)-5, which is produced by Th2 cells and group 2 innate lymphoid cells, and eosinophils have been shown to generally participate in host defense, inflammation, and immunomodulation. Recently, several papers have reported host defense by non-antigen-specific immune activation, in which group 2 innate lymphoid cells and Th2 cells produce interleukin (IL)-5 and IL-13 in response to IL-33 stimulation. This immune activation is produced by migrating larvae of a species that differs from the species previously encountered. Eosinophils also play an important role in the eradication of migrating larvae. Thus, eosinophils contribute to host defense in both antigen-specific and non-antigen-specific manners.


Parasitic infections
Parasitic infections are not common in developed countries, but a World Health Organization survey revealed that intestinal parasites continue to infect billions of people in tropical and subtropical areas [1]. Thus, intestinal parasitic infections are regarded as a medically important "Neglected tropical diseases". Depending on the type and number of parasites, parasitic infection often causes severe anemia and malnutrition, and becomes chronic. Infections in children cause developmental disabilities, delays in cognitive development, and potential mortality. Moreover, parasitic infections have economic consequences due to treatment cost and loss of work time [2].
Parasites can be classified into small protozoa, which constitute unicellular eukaryotes (e.g., malaria, toxoplasma, and amoeba) and large multicellular helminths (e.g., trematodes, cestodes, and nematodes). Parasites infect via various routes and in various sites, and the corresponding host immune responses vary depending on the properties of the particular parasites, as the amounts of circulating IgE and eosinophils increase due to helminthic infections. Nevertheless, parasitic infections often become chronic, as many worms acquire the ability to evade host immune responses. Here, we discuss the role of eosinophils in host defense against helminth infections.

Immune responses to nematodes
Immune responses to parasites exhibit some general trends. First, T cells are essential, as demonstrated by the inability of nude mice to successfully eliminate parasitic infections [3][4][5]. Typically, a Th1-type immune response is induced by protozoal infection [6], whereas a Th2-type immune response is induced by helminthic infection [7]. For example, studies using Leishmania have shown the importance of the activation of phagocytes (e.g., macrophages) by interferon (IFN)-c produced by Th1 cells, to control protozoal infections [6]. In contrast, to restrict the growth and activity of large helminths that cannot be phagocytosed by macrophages, various effector factors are activated by cytokines produced by Th2 cells. In particular, the activation of effector antibodies and granulocytes (e.g., eosinophils, basophils, and mast cells), as well as mucus secretion and contraction of smooth muscle in mucosal tissues, such as gastrointestinal tract and trachea, promote the expulsion of helminths from the body [8][9][10]. Mice with a predominately Th2type immune response are resistant to infection by Trichuris muris, whereas mice with a predominately Th1-type immune response are susceptible to infection by T. muris [11]. While it remains unclear why  naïve T cells differentiate into Th2 cells in hosts  infected with helminths, there are at least two possible reasons. First, helminths have few Toll-like receptor ligands that induce dendritic cells to produce interleukin (IL)-12, which is important for Th1 cell differentiation. Second, parasites produce excretory/ secretion molecules that suppress IL-12 production while enhancing the production of Th2 cell-inducing cytokines (thymic stromal lymphopoietin [TSLP], IL-25, and IL-33) from non-hematopoietic epithelial cells [12][13][14][15].
The role of innate immunity against helminth infection is also a focus of active investigations. In particular, helminths damage epithelial and endothelial cells in various tissues when they infect or move in the host; these damaged cells release cytokines such as TSLP, IL-25, and IL-33, which activate group 2 innate lymphocyte cells (ILC2s) and stimulate the production of large amounts of Th2 cytokines [16,17]. ILC2s are regarded as important sources of Th2 cytokines because the production and activation of Th2 cells requires a considerable amount of time; it is not sufficiently rapid to control primary or early stages of infection [18].
Th2 cells and ILC2s produce Th2 cytokines such as IL-4, IL-13, IL-9, and IL-5. IL-4 is mainly produced by Th2 cells and is important for Th2 cell differentiation, as well as for the activation of the class switching mechanism in B cells that enables IgE production. The receptor for IL-4 consists of IL-4Ra and the common c-chain, expressed on hematopoietic cells; it uses Signal Transducer and Activator of Transcription 6 (STAT6) and insulin receptor substrate-1 for intracellular signal transduction [19]. Notably, IL-13 signal transduction also involves IL-4Ra and STAT6; thus, its actions partially overlap with those of IL-4. However, the receptor for IL-13 uses IL-13Ra, largely expressed on non-hematopoietic cells, instead of the common c-chain; therefore, the actions of IL-13 differ from those of IL-4 [20,21]. IL-4 efficiently stimulates hematopoietic cells, whereas IL-13 mainly contributes to the activation of non-hematopoietic cells [20]. IL-4 and IL-13 induce (1) the differentiation of mucosal epithelial cells into goblet cells for mucus secretion [22]; (2) the proliferation of epithelial cells, thereby promoting epithelial turnover [23]; (3) the production of antimicrobial peptides from epithelial cells [24] and Paneth cells [25], as well as the production of IL-25 from tuft cells [26][27][28]; (4) the contraction of smooth muscle in mucosal tissue, thereby enhancing peristaltic movement [29]; (5) the production of chemokines such as C-C Motif Chemokine Ligand (CCL) 11, CCL17, CCL22, and CCL24 from various cells [30,31]; (6) the differentiation of M2 macrophages [32]; and (7) the activation and class switching of B cells [33]. The actions of IL-4 and IL-13 are important for the elimination of the rodent intestinal parasitic nematode, Nippostrongylus brasiliensis [8]. In addition, IL-9 enhances the proliferation, survival, and activation of mast cells, T cells, and ILC2s [34]; IL-5 is a growth and survival factor for eosinophils, which is essential for eosinophil-mediated host defense [35].

Proliferation and activation of eosinophils
Eosinophils are multinucleated leukocytes with granules that are rich in basic proteins; the numbers of these cells increase in blood and sites of inflammation during allergic reactions and helminth infections. IL-5 secretion from Th2 cells and ILC2s is important for eosinophil proliferation and survival. Th2 cells and ILC2s also produce IL-13, which facilitates recruitment of eosinophils to the site of infection or the site of allergen entry by stimulating the production of chemokines (e.g., CCL11 and CCL24) from various cells [30,36]. IL-33 is produced by various cells, such as epithelial cells, endothelial cells, and fibroblasts; this cytokine strongly stimulates the production of Th2 cytokines from Th2 cells and ILC2s [37][38][39][40]. IL-33 also has the capacity to directly activate recruited eosinophils at sites of infection and allergen entry [41].
The function of eosinophils as a defense against parasites is well-known; however, eosinophils have been reported to have additional roles in allergic inflammation and tissue repair, as well as immunomodulation [42][43][44]. Regarding the eosinophilmediated mechanism of defense against parasites, binding to the worm larvae through antibody or complement, followed by release of intracellular granules, is presumed to cause injury to invading worms [45][46][47][48][49]. The numbers of eosinophils increase with helminth infection. L€ offler found a link between parasite infection and pulmonary eosinophilia (L€ offler's syndrome) [50]. Although the mechanism underlying this eosinophil accumulation has been unknown for many years, we elucidated the mechanism using a Strongyloides venezuelensis infection model [38]. Some parasitic enteric nematode larvae, including S. venezuelensis and N. brasiliensis, do not migrate directly to the gut upon percutaneous or oral infection. Instead, they migrate to the lungs via the bloodstream, then penetrate the alveolar cavity and travel to the throat, where they are swallowed. Finally, they reach the small intestine and mature [51,52]. As the larvae pass through the lungs, they cause damage to lung tissue, stimulating the release of IL-33 from type II alveolar epithelial cells. IL-33-deficient mice showed a marked reduction in eosinophil accumulation upon S. venezuelensis infection. Conversely, nasal administration of IL-33 can induce pulmonary eosinophilia even in Rag2-deficient mice that lack both T cells and B cells [38]. IL-33 stimulates ILC2s to proliferate and accumulate in the lungs, and induces production of IL-5 and IL-13 by ILC2s. Excretory/secretory products from N. brasiliensis stimulate neuronal production of neuromedin U, which stimulates ILC2s to produce Th2 cytokines. These combined actions result in the onset of lung eosinophilic inflammation [53].

Eosinophils in host defense
Because the functions of IL-5 and eosinophils appeared to be closely related, their in vivo functions in eosinophil-mediated host defense has been clarified in studies using IL-5-deficient mice [54,55] and neutralizing antibodies against IL-5 [56]. Neutralization or deficiency of IL-5 strongly suppresses eosinophil accumulation during helminth infection; however, some eosinophils remain in mouse tissues. Therefore, eosinophil-deficient mice have been developed and used for experimental analyses [42,57]. Eosinophils were first shown to have anti-parasitic activity through in vitro experiments on schistosome larvae [46]. When eosinophils and schistosome larvae were cultured in the presence of IgE, IgG or complement, the eosinophils exhibited damaging activity against the larvae [46,48,58]. However, the role of IgE-mediated eosinophil activation in vivo remains unclear, as high-affinity FceRIa is often not expressed on the surface of eosinophils in mice, which are the most common experimental animal model [59]. In addition, even eosinophil-deficient mice infected with schistosomes exhibit phenotypes similar to wild-type mice. Following scenario account for the differences in vitro and in vivo: At the initial stage of infection, infecting Schistosoma mansoni larvae induce a Th1type immune response in mice without increasing the number of eosinophils, subsequently the host immune response changes to Th2-type after the larvae mature to adults and begin to lay eggs [60]. The adult worms, different from larvae, are resistant to attack from eosinophils even though the number of eosinophils increases by the action of Th2 cytokines.
In addition, the absence of eosinophils had no particular effect in the mouse intestinal helminth T. muris infection model [61]. T. muris is transmitted by the oral intake of eggs; larvae hatch in the intestine and become adults without migration to other tissues [62]. In contrast, Heligmosomoides polygyrus infection occurs through oral ingestion of the larvae; these larvae invade the intestinal lining, where they undergo some growth before returning to the lumen and becoming adults. When eosinophil-deficient mice were infected with H. polygyrus, the intestines of these mice showed more worms than those of wild-type mice. In a model of microfilariae infection induced by intravenous administration of microfilariae of Brugia malayi, the number of microfilariae decreases immediately after infection in wild-type mice, but remains high after infection in eosinophildeficient mice. When eosinophil-deficient mice were infected with the tissue-migrating intestinal nematode N. brasiliensis, the number of adults found in the intestinal tract was similar to that of wild-type mice; however, eosinophil-deficient mice showed increased numbers of eggs in feces, compared with the numbers of eggs observed in wild-type mice. Thus, eosinophils act to protect against intestinal parasitic nematodes that invade tissues.

Immune modulation by eosinophils
However, it has also been reported that eosinophils may promote infection. New larvae of Trichinella spiralis form in the intestinal tract; they then migrate to muscles, where they invade muscle fibers and become capsular larvae [63]. At the stage of new larva infection, IL-10-producing CD4 þ T cells or dendritic cells are activated by eosinophil-derived IL-10. Subsequently, CD4 þ T/dendritic cell-derived IL-10 suppresses the production of nitric oxide (NO), such that larvae can survive [64]. In contrast, eosinophil-deficient mice exhibit lower numbers of cystic larvae due to insufficient differentiation of IL-10-producing CD4 þ T cells, as well as enhanced expression of IFN-c and enhanced production of NO [65]. Because these results were not observed in mice with neutralizing antibodies against IL-5, and were not observed in mice deficient in IL-5, the increased number of larval infections caused by eosinophils is thought to be due to the presence of resident eosinophils, those are not affected by IL-5 [66][67][68][69][70]. These results are potentially related to the ability of parasites to exploit the immunoregulatory functions of eosinophils.
Resident eosinophils are thus presumed to have an immunoregulatory function; they are present in non-infection/non-allergy (i.e., steady state) conditions [43]. Because they have the ability to enhance or suppress immunity, they may be associated with various diseases.

Eosinophil-mediated host protection in acquired immunity
When a host is infected with a parasite, the host acquires immunity to the parasite by retaining memory of specific antigens and thus becomes resistant to reinfection by the same parasite. Reinfecting wild-type mice with N. brasiliensis results in the capturing of parasite larvae in the skin and fewer larvae subsequently migrate to the lungs [71]; however, many larvae reach the lungs in eosinophildeficient mice [72] (Figure 1). Eosinophils support T. spiralis survival during primary infection as described above. However, during reinfection, eosinophils inhibit the migration of Figure 1. Primary and secondary immune responses in helminth infections. (Primary infection) Nematode infection induces differentiation of Th2 cells to induce Th2-type immune responses, such as mucus production and mastocytosis, as well as proliferation and accumulation of eosinophils and basophils, production of nematode antigen-specific antibodies, and differentiation of M2 macrophages. Some nematodes are expelled by these effectors, while others evade these effectors and develop chronic infection. Some nematodes exhibit a lung stage before they become adults in the intestines. When larvae penetrate the lung, they damage lung tissue, which leads to the release of epithelial cell-related cytokines (e.g., IL-33, TSLP, and IL-25) from epithelial and stromal cells; these cytokines induce proliferation of group 2 innate lymphoid cells and subsequent production of cytokines (e.g., IL-5 and IL-13), resulting in the accumulation of eosinophils. Thus, nematode infection induces pulmonary eosinophilia at 1-2 weeks after infection. However, eosinophils cannot damage larvae in the lungs, because nematodes have already moved to the intestines and developed into adult worms. (Secondary infection) If a host is re-infected by a species of nematode that it has previously encountered, Th2 cells and antibodies respond to the parasites robustly and promptly, thus trapping and damaging them at the sites of infection (e.g., skin) and at the lungs, through the actions of effector cells such as basophils and macrophages, prior to migration of the larvae to the intestines. If a host is infected by a species of nematode that it has not previously encountered, Th2 cells and antibodies cannot respond to the parasite antigen with effectiveness similar to that of the response to the sensitizing parasite; thus, the larvae can migrate to the lungs. Following tissue damage by migrating larva, stromal cells produce IL-33. The lungs of an infected host have large numbers of Th2 cells and/or ILC2s, which rapidly react to IL-33 by producing Th2 cytokines. Thus, infected hosts exhibit nonantigen-specific resistance by attacking migratory larvae through the actions of rapidly accumulated eosinophils. new larvae [73]. Parasite-specific antibodies are critical for eosinophil-mediated resistance to infection. Antibodies enhance the in vitro activity of eosinophils against T. spiralis larvae [74]. These antibodies have also been shown to synergize with basophils and M2 macrophages to inhibit the migration of N. brasiliensis and H. polygyrus [71,75].

Non-antigen-specific host defense
Recently, we found that innate immunity against nematode infection also involves a memory component. S. venezuelensis-experienced mice were resistant to the same nematode infection through the actions of Th2 cells [76], but were also resistant to infection by another parasite, N. brasiliensis, regardless of the presence of CD4 þ T cells [77]. This resistance was dependent on ILC2s: the ILC2s of mice that had experienced S. venezuelensis infection remained highly reactive even after expulsion of the nematode; they then responded rapidly and robustly to N. brasiliensis infection. In primary infections, the activation of ILC2s is slow, such that when ILC2s produce IL-5 and eosinophils accumulate in the lung, the nematode has already moved from the lung to the intestine; therefore, eosinophils are not very effective against parasites at this stage of infection. However, in infection-experienced mice, large amounts of IL-5 and IL-13 are promptly produced by memory-like ILC2s, which results in rapid accumulation of eosinophils at the site of infection during the early stage of infection; these eosinophils then attack the invading larvae. Importantly, eosinophils are essential for this resistance. Moreover, IL-33 is required for antigen-non-specific resistance, as IL-33-deficient mice did not exhibit protection against secondary infection. Similarly, Martinez-Gonzalez et al. reported that treatment with allergen or IL-33 induced the differentiation of memory-like ILC2s in the lung [78]. They demonstrated that IL-33 alone was sufficient for the induction of memory-like ILC2s; however, in our experiments, IL-33 alone could not induce differentiation of these cells. Thus, the role of IL-33 in the induction of memorylike cell differentiation is controversial. Such parasite species-nonspecific responses are also exhibited by Th2 cells. In response to IL-33 stimulation, Th2 cells produced IL-5 and IL-13 in an antigen-independent manner, which facilitated resistance to N. brasiliensis infection following infection by another lung migratory nematode, Ascaris suum [79] (Figure 1). In addition to nematode infection, allergens can induce non-antigen-specific host defense.
Gazzinelli-Guimaraes et al. recently reported that house dust mite sensitization significantly limit the number and size of A. suum larva in the lungs, in an eosinophil-dependent manner [80]. Furthermore, Filbey et al. demonstrated that mice could acquire CD4 þ T cells/eosinophil dependent resistance against N.brasiliensis infection following infections by H.polygyrus or T. muris, although the life cycles of these species do not include a lung stage [81]. These data are in contrast to the findings of previous reports in which helminth infections were shown to suppress the inflammatory responses in several autoimmune diseases [82][83][84]. Thus, helminths exhibit complicated immune modulation, and further studies are needed to determine the mechanisms involved in the control of inflammatory diseases and helminth infections.
Thus, eosinophils do not work effectively against every helminth; however, in certain conditions, they contribute to host defense against parasites, especially nematode larvae. In particular, during reinfection, eosinophils participate in anti-parasite immunity by accumulating at the site where larvae are present, before the larvae mature into adults; therefore, it is important that eosinophils gather at the appropriate time and place.