Innate immunity

Innate immunity in mice to larval S. stercoralis is highly efficient, with parasite elimination occurring within 5–7 days p.i. (Abraham et al. Reference Abraham, Rotman, Haberstroh, Yutanawiboonchai, Brigandi, Leon, Nolan and Schad1995). Analysis of the parasite microenvironment, within the implanted diffusion chamber, revealed the presence of infiltrated eosinophils, neutrophils and macrophages. If parasites were implanted in mice in diffusion chambers that prevented cell ingress, parasite killing did not occur. This suggested that contact between cells and the worms was required for parasite killing and that soluble factors from the host were insufficient to kill the worms (Abraham et al. Reference Abraham, Rotman, Haberstroh, Yutanawiboonchai, Brigandi, Leon, Nolan and Schad1995). Elimination of neutrophils and eosinophils from naïve mice by monoclonal antibody treatment resulted in an increase in parasite survival (O'Connell et al. Reference O'Connell, Hess, Santiago, Nolan, Lok, Lee and Abraham2011a ), suggesting that these cells are active participants in the protective innate immune response. Similarly, S. venezuelensis induced an increase of eosinophils and mononuclear cells in the blood, peritoneal cavity fluid and bronchoalveolar lavage fluid (Machado et al. Reference Machado, Ueta, Lourenco, Anibal, Roque-Barreira and Faccioli2007, Reference Machado, Carlos, Lourenco, Souza, Sorgi, Silva, Ueta, Ramos, Aronoff and Faccioli2010). Studies with S. ratti have revealed that cellular infiltrates, consisting of neutrophils, eosinophils and macrophages, were observed in the skin in response to the invading larvae in rats and mice during both primary and secondary responses (Dawkins et al. Reference Dawkins, Muir and Grove1981; McHugh et al. Reference McHugh, Jenkins and McLaren1989) and that granulocytes are crucial in the early defence against migrating larvae of S. ratti in mice (Watanabe et al. Reference Watanabe, Noda, Hamano, Koga, Kishihara, Nomoto and Tada2000).

Innate immunity – summary

The proposed sequence of events leading to protective innate immunity to larval S. stercoralis starts with neutrophils coming into contact with live worms as the worms migrate through the tissues. This contact induces the neutrophils to die through the process of etosis thereby releasing DNA that traps the larvae and prevents their movement. Cells, including neutrophils and eosinophils, undergo chemotaxis to the trapped worms and upon contact release toxic molecules that kill the parasite in collaboration with complement component C3b. Finally, eosinophils have the added capability of acting as APC to initiate the adaptive immune response to the parasite (Fig. 1).

Fig. 1. Innate immunity to Strongyloides stercoralis L3 in mice. Neutrophil recruitment to the L3 microenvironment is parasite specific and results in neutrophil production of additional chemokines MIP-2 and KC. Eosinophils are also preferentially recruited by parasite components and are required for the innate but not adaptive immune response. Eosinophil granular proteins, human and mouse MBP and human ECP are toxic to L3. Eosinophils also act as APC, presenting parasitic antigens, resulting in the induction of Th2 cells. Neutrophils die by etosis and release NETs to trap larvae. Neutrophils utilize MPO to kill L3 during the innate and adaptive immune responses. L3 killing by granulocytes is C3 dependent. C3b potentiates adherence of effector cells to L3. Abbreviations: APC, antigen presenting cells; ECP, eosinophil cationic protein; MBP, major basic protein; MPO, myeloperoxidase.

Adaptive immunity

The adaptive immune response in mice, induced by immunization with live larvae, is highly effective with greater than 90% of the challenge larvae killed within 24 h (Abraham et al. Reference Abraham, Rotman, Haberstroh, Yutanawiboonchai, Brigandi, Leon, Nolan and Schad1995). Several components have been identified that are integral to the protective adaptive immune response.

Adaptive immunity – summary

In summary, the adaptive immune response is initiated by eosinophils functioning as APC stimulating CD4⁺ T cells to produce the Th2 cytokines, IL-4 and IL-5. B cells produce IgM, which collaborates with macrophages, and IgG, which collaborates with neutrophils, to kill the larvae. Eosinophils do not participate in killing the parasites in the adaptive immune response, but are required for inducing the production of IgM. Complement activation, specifically C3b, is required for the accelerated killing in the adaptive immune response. Neutrophils kill through a process dependent on TLR4 and MPO. In addition to the antibody dependent killing, macrophages that have been alternatively activated by IL-4 also kill the parasites as part of a triad composed of macrophages, neutrophils and complement (Fig. 2).

Fig. 2. Adaptive immunity to Strongyloides stercoralis L3 in mice. Immunization of mice with L3i results in Th2 cell derived secretion of IL-5 and IL-4. IL-5 is required for eosinophils, which collaborate with B cells to produce parasite specific IgM. IL-4 is required for the development of alternatively activated macrophages that interact with neutrophils, complement and IgM to kill during the adaptive immune response. Accelerated L3 killing by neutrophils during the adaptive response is mediated via MPO and complement component C3 and is IgG dependent. Abbreviation: MPO, myeloperoxidase.

Innate intestinal effector cells

Several lines of evidence suggest that mucosal mast cells are the central effector cells mediating expulsion of S. ratti and S. venezuelensis from the intestine. Mast cells were induced in the small intestine during S. ratti and S. venezuelensis infection in mice (Khan et al. Reference Khan, Horii, Tiuria, Sato and Nawa1993) and rats (Shintoku et al. Reference Shintoku, Kadosaka, Kimura, Takagi, Kondo and Itoh2013). Mouse mast cell protease 1 (mMCP1), a protease that is specific for mucosal mast cells (Reynolds et al. Reference Reynolds, Stevens, Lane, Carr, Austen and Serafin1990), was elevated in the serum of S. ratti and S. venezuelensis infected mice (Sasaki et al. Reference Sasaki, Yoshimoto, Maruyama, Tegoshi, Ohta, Arizono and Nakanishi2005; Eschbach et al. Reference Eschbach, Klemm, Kolbaum, Blankenhaus, Brattig and Breloer2010). Similarly rat mast cell protease II was elevated in intestinal tissue of S. ratti infected rats (Wilkes et al. Reference Wilkes, Bleay, Paterson and Viney2007).

WBB6F1-Kit^W/W−v (W/W^v) mice carry a mutation in the stem cell factor (SCF) receptor Kit that results in mast cell deficiency. Release of S. ratti (Nawa et al. Reference Nawa, Kiyota, Korenaga and Kotani1985) and S. venezuelensis (Khan et al. Reference Khan, Horii, Tiuria, Sato and Nawa1993) eggs and L1 in the feces was prolonged in W/W^v mice. Reconstitution with wild type bone marrow cells partially reverted the phenotype suggesting a contribution of mast cells to intestinal immunity. Nawa et al. also described increased numbers of L3 in the head of S. ratti infected W/W^v mice during first infection (Nawa et al. Reference Nawa, Kiyota, Korenaga and Kotani1985) and numbers of migrating S. venezuelensis L3 in W/W^v mice were not recorded (Khan et al. Reference Khan, Horii, Tiuria, Sato and Nawa1993). Therefore a formal differentiation between mast cell function in immunity to migrating larvae and parasitic adults in W/W^v mice is missing. Since W/W^v mice suffer from several other haematological and non-haematological deficiencies including a drastic reduction in the number of basophils, it is difficult to definitively attribute their phenotype to mast cell deficiency alone (Reber et al. Reference Reber, Marichal and Galli2012).

Support for a role of intestinal mast cells in expulsion of parasitic adults originates from two studies using Notch 2 and phosphatidylinositol-3 kinase (PI3K) subunit p85α deficient mice. Conditional Notch 2 deficient mice show a changed intestinal mast cell distribution (Sakata-Yanagimoto and Chiba, Reference Sakata-Yanagimoto and Chiba2015). Accumulation of mast cells predominantly in the lamina propria instead of the intestinal epithelial layer was associated with increased fecal egg release during S. venezuelensis infection (Sakata-Yanagimoto et al. Reference Sakata-Yanagimoto, Sakai, Miyake, Saito, Maruyama, Morishita, Nakagami-Yamaguchi, Kumano, Yagita, Fukayama, Ogawa, Kurokawa, Yasutomo and Chiba2011). PI3Kp85α^/− mice lack gastrointestinal and peritoneal mast cells while still containing dermal mast cells. These mice displayed prolonged fecal release of S. venezuelensis eggs and prolonged presence of parasitic adults in the intestine (Fukao et al. Reference Fukao, Yamada, Tanabe, Terauchi, Ota, Takayama, Asano, Takeuchi, Kadowaki, Hata Ji and Koyasu2002). Still, as PI3Kp85α deficiency results in impaired Kit signalling that was most likely causing their mast cell deficiency, these mice do not represent a Kit independent mouse model for mast cell deficiency either.

Kit independent mast cell deficient mice were generated recently by heterozygous transgenic expression of Cre recombinase under the control of the mast cell specific carboxypeptidase A3 promoter (Cpa3^Cre/wt) that led to deletion of mast cells in mucosal and connective tissues via genotoxic mechanisms (Feyerabend et al. Reference Feyerabend, Weiser, Tietz, Stassen, Harris, Kopf, Radermacher, Moller, Benoist, Mathis, Fehling and Rodewald2011). Cpa3^Cre mice have reduced basophil numbers while other features of the immune system are normal. In line with the former studies using the mixed background WBB6F1-Kit ^W/W−v mice, mast cell deficient S. ratti infected Cpa3^Cre/wt mice displayed higher parasite burden and prolonged fecal release of eggs and L1 compared with their mast cell competent Cpa3^wt/wt littermates (Blankenhaus et al. Reference Blankenhaus, Reitz, Brenz, Eschbach, Hartmann, Haben, Sparwasser, Huehn, Kuhl, Feyerabend, Rodewald and Breloer2014). Using mice that lack mucosal and connective tissue mast cells (Feyerabend et al. Reference Feyerabend, Weiser, Tietz, Stassen, Harris, Kopf, Radermacher, Moller, Benoist, Mathis, Fehling and Rodewald2011) or selectively connective tissue mast cells (Dudeck et al. Reference Dudeck, Dudeck, Scholten, Petzold, Surianarayanan, Kohler, Peschke, Vohringer, Waskow, Krieg, Muller, Waisman, Hartmann, Gunzer and Roers2011) or basophils (Ohnmacht et al. Reference Ohnmacht, Schwartz, Panzer, Schiedewitz, Naumann and Voehringer2010), we are currently re-evaluating overlapping and separate roles of mast cells and basophils during S. ratti infection in mice. Mice lacking either connective tissue mast cells or basophils displayed an initial elevation in intestinal parasite burden but eventually terminated infection with wild type kinetics whereas mice that lacked both connective tissue mast cells and mucosal mast cells were unable to terminate S. ratti infection for more than 150 days. Here, immunity to tissue migrating L3 was not affected since differences in parasite burden were restricted to the intestinal life stage of S. ratti. These results show that basophils and connective tissue mast cells contribute to early intestinal immunity but are dispensable for final expulsion of parasitic adults whereas mucosal mast cells represent essential terminal effector cells that mediate the final expulsion of S. ratti from the intestine (Reitz and Breloer unpublished results).

Eosinophils and neutrophils predominantly mediate immunity to tissue migrating L3 as reviewed in the section “Control and eradication of migrating L3 in the tissue- S. stercoralis” and are dispensable for intestinal immunity to S. ratti and S. venezuelensis. Although eosinophils expanded in the intestinal tissue of S. ratti and S. venezuelensis infected mice, abrogation of this eosinophilia by anti-IL-5 treatment or in IL-5-deficient mice did not abrogate intestinal immunity (Korenaga et al. Reference Korenaga, Hitoshi, Yamaguchi, Sato, Takatsu and Tada1991a ; Ovington et al. Reference Ovington, McKie, Matthaei, Young and Behm1998; Watanabe et al. Reference Watanabe, Sasaki, Hamano, Kishihara, Nomoto, Tada and Aoki2003). Increased eosinophilia in IL-5 transgenic mice did not accelerate termination of infection but reduced numbers of parasitic adults after oral infection (El-Malky et al. Reference El-Malky, Maruyama, Hirabayashi, Shimada, Yoshida, Amano, Tominaga, Takatsu and Ohta2003). However, it should be noted that analysis of intestinal immunity to S. ratti and S. venezuelensis infection in eosinophil deficient mice has not been performed. Depletion of Ly6G⁺ neutrophils and Ly6C⁺ monocytes by anti-Gr1 (RB6–85C) treatment increased numbers of S. ratti L3 in the tissue and subsequent intestinal parasite burden (Watanabe et al. Reference Watanabe, Noda, Hamano, Koga, Kishihara, Nomoto and Tada2000). If Gr1⁺ cells were depleted after the completion of the tissue migrating phase i.e. at day 3 p.i., the intestinal parasite burden did not change but fecal egg release increased. Thus, granulocytes are more important in controlling migrating L3 in the tissue as demonstrated for S. stercoralis (Galioto et al. Reference Galioto, Hess, Nolan, Schad, Lee and Abraham2006) than for eradication of adults from the intestine although they may contribute to parasite control by affecting fecundity.

Adaptive immune response

RAG1^−/− mice that lack T and B cells displayed comparable numbers of parasitic adults in the intestine at day 6 p.i. (Breloer et al. Reference Breloer, Hartmann, Blankenhaus, Eschbach, Pfeffer and Jacobs2015) demonstrating that initial control of S. ratti can be maintained in the absence of adaptive immunity. However, termination of infection clearly depends on a concerted action of innate effectors and the adaptive immune system, as infection was drastically prolonged for up to 1 year in T cell deficient nude mice (Dawkins et al. Reference Dawkins, Mitchell and Grove1982a ). CD4⁺ T cells were the dominant cells contributing to timely termination of infection, since MHC-I^−/− mice which lack CD8⁺ T cells displayed an unchanged course of S. venezuelensis infection whereas MHC-II^−/− mice, which lack CD4⁺ T cells, displayed a delay in clearance of infection by 1 week (Goncalves et al. Reference Goncalves, Rodrigues, Silva, Goncalves, Cardoso, Beletti, Ueta, Silva and Costa-Cruz2008; Rodrigues et al. Reference Rodrigues, Silva, Goncalves, Cardoso, Alves, Goncalves, Beletti, Ueta, Silva and Costa-Cruz2009, Reference Rodrigues, Cardoso, Goncalves, Silva, Massa, Alves, Ueta, Silva and Costa-Cruz2013). As the authors did not quantify tissue migrating larvae, the net effect of absent CD4⁺ T cells could reflect specifically a less efficient eradication of migrating L3 as shown before in CD4⁺ T cell depleted mice for S. stercoralis (Rotman et al. Reference Rotman, Schnyder-Candrian, Scott, Nolan, Schad and Abraham1997) or a combination of less efficient eradication of tissue migrating L3 and expulsion of parasites from the intestine.

The canonical T cell response to helminth infection is the Th2 response that is characterized by production of the cytokines IL-3, IL-4, IL-5, IL-9 IL-10 and IL-13 and the subsequent induction of antibody isotype switch to IgG1 and IgE (Allen and Maizels, Reference Allen and Maizels2011). Infection of rats (Wilkes et al. Reference Wilkes, Bleay, Paterson and Viney2007; Chiuso-Minicucci et al. Reference Chiuso-Minicucci, Marra, Zorzella-Pezavento, Franca, Ishikawa, Amarante, Amarante and Sartori2010) or mice (Machado et al. Reference Machado, Ueta, Lourenco, Anibal, Roque-Barreira and Faccioli2007; Eschbach et al. Reference Eschbach, Klemm, Kolbaum, Blankenhaus, Brattig and Breloer2010; Blankenhaus et al. Reference Blankenhaus, Reitz, Brenz, Eschbach, Hartmann, Haben, Sparwasser, Huehn, Kuhl, Feyerabend, Rodewald and Breloer2014) with S. ratti and S. venezuelensis provoked all features of this canonical Th2 response. Thereby the magnitude of Th2 cytokine production was positively correlated to the infection dose in S. venezuelensis and S. ratti infected mice (Eschbach et al. Reference Eschbach, Klemm, Kolbaum, Blankenhaus, Brattig and Breloer2010; Schilter et al. Reference Schilter, Pereira, Eschenazi, Fernandes, Shim, Sousa, Teixeira and Negrao-Correa2010) and rats (Bleay et al. Reference Bleay, Wilkes, Paterson and Viney2007) while single or repeated infection doses both elicited comparable Th2 responses (Paterson et al. Reference Paterson, Wilkes, Bleay and Viney2008).

Evidence for a contribution of the Th2 immune response to anti-Strongyloides immunity is supported by several studies using mice with defective Th2 polarization. Signal transducer and activator of transcription (STAT) 6^−/− mice showed prolonged fecal egg release by 11 days compared with wild type mice (Sasaki et al. Reference Sasaki, Yoshimoto, Maruyama, Tegoshi, Ohta, Arizono and Nakanishi2005). Injection of IL-27 or transgenic overexpression of IL-27 in S. venezuelensis infected mice resulted in reduced IgE and IL-4 production and a 10 day prolongation of egg release in the feces (Yoshimoto et al. Reference Yoshimoto, Yasuda, Mizuguchi and Nakanishi2007).

Transgenic overexpression of IL-12 was shown to abrogate immunity against S. stercoralis L3 in the tissue (Rotman et al. Reference Rotman, Schnyder-Candrian, Scott, Nolan, Schad and Abraham1997). The reciprocal promotion of Th2 polarization in IL-12^−/− C57BL/6 mice elevated several aspects of S. venezuelensis specific Th2 response such as eosinophilia, the production of IL-13, IL-10, IL-3, IL-5 as well as IgG1 and IgE response but resulted only in a slight decrease in parasitic adult numbers in the intestine day 7 p.i. in one study (Machado et al. Reference Machado, Carlos, Lourenco, Sorgi, Silva, Ramos, Ueta, Aronoff and Faccioli2009). Unchanged parasite burden in the intestine on days 7, 10 and 14 p.i. and unchanged fecal egg release were reported in another study where IL-12^−/− mice were infected with a lower dose of S. venezuelensis L3i (Negrao-Correa et al. Reference Negrao-Correa, Pinho, Souza, Pereira, Fernandes, Scheuermann, Souza and Teixeira2006).

Interestingly, the induction of a Th1 driven anti-S.ratti HSP60 response induced by vaccination with SrHSP60 and characterized by antigen-specific production of IFNγ and elevation of S. ratti specific IgG2b and IgG2c with simultaneous absence of IL-13 production, did not protect mice from subsequent infections (Nouir et al. Reference Nouir, Eschbach, Piedavent, Osterloh, Kingsley, Erttmann, Brattig, Liebau, Fleischer and Breloer2012). By contrast, susceptibility of vaccinated mice was increased leading to elevated numbers of adults in the intestine and increased fecal release of S. ratti DNA. However, if the anti-SrHSP60 response was biased towards Th2 by vaccination with alum precipitated SrHSP60, antigen-specific IgG1 and IL-13 were induced, IFNγ was absent and mice were protected from subsequent infection thus highlighting the importance of intact Th2 response for control of S. ratti infection.

Type 2 innate lymphoid cells (ILC2) that are activated in response to tissue derived alarmins play an important role in the initiation of Th2 immune responses (Licona-Limon et al. Reference Licona-Limon, Kim, Palm and Flavell2013b ). Strongyloides venezuelensis infection triggered production of the alarmin IL-33 by alveolar epithelial type II cells (ATII) in the lung day 5 and 7 p.i. (Yasuda et al. Reference Yasuda, Muto, Kawagoe, Matsumoto, Sasaki, Matsushita, Taki, Futatsugi-Yumikura, Tsutsui, Ishii, Yoshimoto, Akira and Nakanishi2012). The authors provide evidence that IL-33 induced expansion of IL-5 and IL-13 producing ILC2 in the lungs of infected mice days 7–15 p.i. IL-33^−/− mice showed no ILC2 induction, reduced eosinophilia and reduced local induction of IL-5 and IL-13 mRNA upon S. venezuelensis infection. Parasite burden recorded as fecal release of eggs was increased, clearance of infection was delayed by 1 day and intestinal mMCP1 concentrations at days 7–14 were reduced. Increased S. venezuelensis fecal egg release in IL-33^−/− mice was partially restored by intranasal treatment with IL-33. Interestingly, nasal application of chitin also induced IL-33 in ATII cells, suggesting that presence of nematode parasites such as S. venezuelensis can be sensed by conserved “pathogen associated molecular patterns”.

As parasite burden was quantified by fecal egg release and the number of migrating L3 in the lung was not assessed, discrimination between IL-33 mediated eradication of L3 and adults remains difficult. On the one hand IL-33 mediated eosinophilia occurred in the lung, suggesting that immunity to L3 in the lung was improved. On the other hand IL-33 was not detected during the tissue migration phase of S. venezuelensis day 1–3 but was elevated after day 4, when S. venezuelensis has already reached the intestine. Thus IL-33 induced ILC2 that promote type 2 immunity may affect the intestinal life stage as well.

Cytokines

Mice lacking the IL-4Rα chain, part of the IL-4 and IL-13 receptor, showed delayed eradication of S. venezuelensis, although initial parasite burden were alike (Negrao-Correa et al. Reference Negrao-Correa, Pinho, Souza, Pereira, Fernandes, Scheuermann, Souza and Teixeira2006). Using bone marrow chimeras, the authors provide evidence that IL-4Rα expressed on lymphocytes was dispensable for timely termination of S. venezuelensis infection while mice that were deficient for IL-4Rα selectively on non-hematopoietic cells displayed the same delay in S. venezuelensis expulsion as complete IL-4Rα^−/− mice. A comparable impact of IL-4Rα signalling on non-hematopoietic cells was shown for control of Nippostrongylus brasiliensis infection in the intestine, possibly via activation of intestinal smooth muscle cells or intestinal epithelial cells that express IL-4Rα (Finkelman et al. Reference Finkelman, Shea-Donohue, Morris, Gildea, Strait, Madden, Schopf and Urban2004). It is conceivable that IL-4 and IL-13 trigger IL-4Rα mediated signalling redundantly since both cytokines are induced with comparable kinetics during S. ratti infection (Eschbach et al. Reference Eschbach, Klemm, Kolbaum, Blankenhaus, Brattig and Breloer2010) and neither IL-13 neutralization nor IL-4 deficiency delayed parasite expulsion from the intestine (Watanabe et al. Reference Watanabe, Hamano, Yada, Noda, Kishihara, Nomoto and Tada2001; Blankenhaus et al. Reference Blankenhaus, Reitz, Brenz, Eschbach, Hartmann, Haben, Sparwasser, Huehn, Kuhl, Feyerabend, Rodewald and Breloer2014). A significant elevation of S. ratti eggs passed with feces in IL-4^−/− mice was recorded at day 6 p.i. only, while fecal egg release was unchanged at day 5 and at days 7–13.

IL-5 neutralization or deficiency predominantly compromised immunity to migrating L3 in tissues as reviewed in the section “Control and eradication of migrating L3 in the tissue- S. stercoralis”, but neither changed kinetics of parasite expulsion during first infection nor interfered with immunity to the intestinal life stage in immune mice during second infection (Korenaga et al. Reference Korenaga, Hitoshi, Yamaguchi, Sato, Takatsu and Tada1991a ; Ovington et al. Reference Ovington, McKie, Matthaei, Young and Behm1998; Watanabe et al. Reference Watanabe, Sasaki, Hamano, Kishihara, Nomoto, Tada and Aoki2003). Since overexpression of IL-5 promoted expulsion of adults 1 day or 3 days after implantation (El-Malky et al. Reference El-Malky, Maruyama, Hirabayashi, Shimada, Yoshida, Amano, Tominaga, Takatsu and Ohta2003), IL-5 may contribute to intestinal immunity but apparently is not essential.

Repeated injection of IL-3 reduced parasite burden and reciprocally increased the number of intestinal mast cells and mMCP1 concentration in serum and intestinal tissue of nude mice and immune competent C57BL/6 mice (Abe and Nawa, Reference Abe and Nawa1988; Abe et al. Reference Abe, Sugaya, Ishida, Khan, Tasdemir and Yoshimura1993). As IL-3 injection did not reduce parasite burden in W/W^v mice, IL-3 apparently acted via activation of mast cells or other effector cells that are absent or dysfunctional in W/W^v mice. The numbers of L3 recovered from heads of infected mice were unchanged in the presence or absence of IL-3 while numbers of parasitic adults were reduced in IL-3 treated mice even after oral infection with adult worms. Thus IL-3 activated effector cells attacked the adult parasites in the intestine rather than the migrating L3 in the tissues. The accelerated expulsion of adults in orally infected mice was observed as early as 6 h p.i., suggesting that initial establishment and not survival of adults in the intestine was compromised by IL-3 treatment (Abe et al. Reference Abe, Sugaya, Ishida, Khan, Tasdemir and Yoshimura1993). Supporting these findings, IL-3 deficient mice displayed delayed expulsion of S. venezuelensis that was correlated to reduction in mast cell and basophil expansion during infection (Lantz et al. Reference Lantz, Boesiger, Song, Mach, Kobayashi, Mulligan, Nawa, Dranoff and Galli1998, Reference Lantz, Min, Tsai, Chatterjea, Dranoff and Galli2008). In summary, these studies provide direct evidence that IL-3 supports expulsion of S. ratti and S. venezuelensis from the intestine. Interestingly, W/W^v mice and IL-3^−/− mice terminated infection with a delay of maximal 8 days whereas W/W^v mice, that additionally lacked IL-3, displayed prolongation of egg release for more than 50 days, suggesting a certain level of redundancy in IL-3 mediated effects.

Several lines of evidence suggest that IL-9 acts in concert with IL-3 to induce mast cell activation and S. ratti expulsion from the intestine. IL-9 is a cytokine with pleiotropic function that was shown to promote mast cell expansion and recruitment (Noelle and Nowak, Reference Noelle and Nowak2010). Injection of recombinant IL-9 reduced numbers of S. ratti adults in the small intestine of BALB/c and C57BL/6 mice whereas neutralization of endogenous IL-9 increased intestinal parasite burden (Blankenhaus et al. Reference Blankenhaus, Reitz, Brenz, Eschbach, Hartmann, Haben, Sparwasser, Huehn, Kuhl, Feyerabend, Rodewald and Breloer2014). Increased parasite burden in anti-IL-9 treated mice was correlated to reduced mMCP1 concentrations in the serum, indicating reduced activation of mucosal mast cells in the absence of endogenous IL-9. Injection of recombinant IL-9 alone did not trigger mMCP1 release as shown for IL-3 (Abe et al. Reference Abe, Sugaya, Ishida, Khan, Tasdemir and Yoshimura1993) but synergistically increased the mMCP1 release induced by recombinant IL-3 if both cytokines were injected together (Sasaki et al. Reference Sasaki, Yoshimoto, Maruyama, Tegoshi, Ohta, Arizono and Nakanishi2005).

IL-9 production was further elevated in S. ratti infected mice that were depleted of Foxp3⁺ regulatory T cells (T_reg) (Blankenhaus et al. Reference Blankenhaus, Reitz, Brenz, Eschbach, Hartmann, Haben, Sparwasser, Huehn, Kuhl, Feyerabend, Rodewald and Breloer2014) or were deficient for a negative co-regulatory receptor (B and T lymphocyte attenuator, BTLA) or its ligand (herpes virus entry mediator, HVEM) (Breloer et al. Reference Breloer, Hartmann, Blankenhaus, Eschbach, Pfeffer and Jacobs2015). Increased IL-9 production was correlated to reduced parasite burden in the intestine and reduced release of S. ratti L1 in the feces, while numbers of migrating L3 in head and lung were not reduced. Thus elevated IL-9 in T_reg depleted or BTLA deficient mice improved immunity to the intestinal life stage of S. ratti. As only mast cell competent Cpa3^wt/wt but not mast cell deficient Cpa3^Cre/wt littermates benefited from elevated IL-9 in T_reg depleted mice, this study provides direct evidence that IL-9 functions as additional activator of mast cells during S. ratti infection (Blankenhaus et al. Reference Blankenhaus, Reitz, Brenz, Eschbach, Hartmann, Haben, Sparwasser, Huehn, Kuhl, Feyerabend, Rodewald and Breloer2014). It should be noted that an additional putative function for IL-9 as enhancer of smooth muscle contractility that was shown to promote expulsion of Trichuris muris (Klementowicz et al. Reference Klementowicz, Travis and Grencis2012) has not been investigated so far.

IL-9 is part of the canonical Th2 response (Allen and Maizels, Reference Allen and Maizels2011) but the existence of a specialized Th9 cell is currently debated (Schmitt et al. Reference Schmitt, Klein and Bopp2014) and specifically Th9 and not Th2 cells were shown to protect mice against N. brasiliensis infection (Licona-Limon et al. Reference Licona-Limon, Henao-Mejia, Temann, Gagliani, Licona-Limon, Ishigame, Hao, Herbert and Flavell2013a ). T cells derived from S. ratti-infected mice clearly secreted IL-9 upon antigen-specific stimulation ex vivo, but flow cytometric analysis revealed that IL-9 was produced by a low frequency of both CD4⁺ T cells and CD4⁻ cells in vivo (Blankenhaus et al. Reference Blankenhaus, Reitz, Brenz, Eschbach, Hartmann, Haben, Sparwasser, Huehn, Kuhl, Feyerabend, Rodewald and Breloer2014). As several non-T cells, including ILC2 and mast cells can principally secrete IL-9, the possible sources of IL-9 during S. ratti infection still need to be elucidated.

An unexpected role in promoting intestinal immunity was demonstrated for IL-18 in concert with IL-2. Daily injections with a combination of IL-18 and IL-2 induced intestinal mastocytosis and elevation of serum concentrations of mMCP1, IL-3, IL-4, IL-9 and IL-13 (Sasaki et al. Reference Sasaki, Yoshimoto, Maruyama, Tegoshi, Ohta, Arizono and Nakanishi2005). As mMCP1 induction by IL-18/IL-2 treatment was CD4⁺ T cell dependent and could be replaced by injection of IL-3 and IL-9, the authors suggested that IL-3 and IL-9 producing CD4⁺ T cells were induced and subsequently caused the elevation of mMCP1. IL-2/IL-18 treated mice rapidly expulsed S. venezuelensis adults that were implanted in the intestine showing that specifically intestinal immunity was increased. Accelerated expulsion in IL-2/IL-18 treated mice was correlated with increased mastocytosis and not observed in W/W^v mice. Supporting these findings, IL-18^−/− mice and mice deficient for IL-18 receptor displayed a transient reduction of mMCP1 release at days 7 and 10 p.i. and a delay in termination of S. venezuelensis by 4 days. Induction of mMCP1 by IL-18 and IL-2 treatment was independent of functional STAT6, i.e. independent of a classic Th2 polarization. STAT6^−/− mice also displayed a delayed termination of S. venezuelensis infection by 11 days but additional neutralization of IL-18 and IL-2 in STAT6^−/− mice prolonged infection even further and reduced mMCP1 in serum beyond day 14. Thus it was hypothesized that two synergistic pathways for mast cell activation exist, one that depends on STAT6 mediated classic Th2 polarization and one STAT6 independent that functions via IL-18/IL-2.

Antibodies

A central function of S. ratti-specific IgM and IgG is promoting the eradication of migrating L3 in the tissue during first and second infection and in vaccinated mice, as reviewed in the section “Control and eradication of migrating L3 in the tissue- S. stercoralis”. Still, evidence for additional contribution of B cells to intestinal immunity was provided using B cell deficient JHD mice (El-Malky et al. Reference El-Malky, Maruyama, Al-Harthi, El-Beshbishi and Ohta2013). Strongyloides venezuelensis adults that were directly implanted in the intestine, thus bypassing the tissue migration phase, established better within B cell deficient mice compared with wild type mice. The number of remaining adults 5 days after implantation was increased and egg release was prolonged. Numbers of intestinal eosinophils and mucosal mast cells were comparable, suggesting that activation and not recruitment of effector cells was compromised in B cell deficient mice. However, since indicators of mast cell degranulation such as serum concentration of mMCP1 were not recorded, this hypothesis was not formally tested.

The FcRγ subunit chain is associated with the function of several Fc receptors such as the high affinity receptor for IgG, FcγRI and the high affinity receptor for IgE, FcεRI. Mice lacking the FcRγ chain displayed no IgE mediated anaphylaxis in vivo and cross-linking surface associated IgE did not induce mast cell degranulation in vitro (Takai et al. Reference Takai, Li, Sylvestre, Clynes and Ravetch1994). Strongyloides venezuelensis expulsion from the intestine was delayed in FcRγ^−/− mice upon s.c infection. (Onah et al. Reference Onah, Uchiyama, Nagakui, Ono, Takai and Nawa2000). Since oral infection also led to prolonged egg release in FcRγ^−/− mice compared with wild type mice during first and second infection, intestinal immunity was impaired (Onah and Nawa, Reference Onah and Nawa2004). Nevertheless mast cell numbers increased in the intestine at day 13 p.i. and mMCP1 concentrations were even elevated in FcRγ^−/− mice (Onah et al. Reference Onah, Uchiyama, Nagakui, Ono, Takai and Nawa2000). The authors concluded that protective immunity during first infection depended on IgE/FcεR mediated activation of intestinal effector cells, while recruitment of mast cells to the intestine of S. venezuelensis infected mice was independent of functional FcεR. However, it should be noted that the FcRγ chain is involved in signal transduction via several Fc receptors as well as C-type lectin receptors (Takai et al. Reference Takai, Li, Sylvestre, Clynes and Ravetch1994), thus the phenotype of the FcRγ^−/− mice may not be caused by defective FcεR alone.

Direct evidence for contribution of both FcεR and FcγRIII in expulsion of S. venezuelensis from the small intestine was provided using mice deficient in activation-induced cytidine deaminase (AID) (Matsumoto et al. Reference Matsumoto, Sasaki, Yasuda, Takai, Muramatsu, Yoshimoto and Nakanishi2013). B cells cannot undergo affinity maturation and subsequent class switch in AID^−/− mice resulting in intact S. venezuelensis specific IgM but defective IgG1 and IgE responses. Initial parasite burdens were comparable in AID^−/− and wild type mice but final expulsion of S. venezuelensis was 8 days delayed. Transfer of wild type immune serum to AID^−/− mice accelerated expulsion of S. venezuelensis. Thereby the serum was transferred after completion of the tissue migration phase at day 6/7 p.i., showing that antibodies targeted the parasitic adult in the intestine. IgG and IgE acted redundantly since both isotypes, purified from immune serum, promoted expulsion. Depletion of either isotype reduced the protective effect of the serum, while only simultaneous depletion of IgE and IgG abrogated protective efficiency. Furthermore, transfer of immune serum in mice lacking either FcγRIII or FcεR1α still promoted expulsion while purified S. venezuelensis specific IgG or IgE did not accelerate parasite expulsion in mice lacking the respective Fc receptor. Supporting these findings, timing of S. venezuelensis infections was unchanged in mice lacking either FcγRIII or FcεR1α (Matsumoto et al. Reference Matsumoto, Sasaki, Yasuda, Takai, Muramatsu, Yoshimoto and Nakanishi2013). Also neutralization of IgE by application of anti-IgE mAb did not prolong larval output during S. ratti infection although IgE depletion from the serum of infected mice was controlled to be complete from day 0 to day 28 p.i. (Korenaga et al. Reference Korenaga, Watanabe and Tada1991b ), thus emphasizing the redundant functions of IgE and IgG.

As transfer of S. venezuelensis specific IgG and IgE did not promote expulsion of S. venezuelensis adults in W/W^v mice Ig function was dependent on mast cells or other effector cells absent in W/W^v mice (Matsumoto et al. Reference Matsumoto, Sasaki, Yasuda, Takai, Muramatsu, Yoshimoto and Nakanishi2013). It should be noted that the authors did not compare the effect of S. venezuelensis specific Ig transfer in WBB6F1-W/W^v mice to WBB6F1^+/+ mice but to C57BL/6 mice as WBB6F1^+/+ mice displayed ten times lower parasite burden without Ig treatment highlighting the importance of the genetic background on susceptibility.

Mechanism of S. ratti and S. venezuelensis expulsion from the intestine

The data accumulated so far strongly suggest that mast cells, probably alongside with additional yet poorly characterized intestinal effector cells, promote final expulsion of S. ratti and S. venezuelensis from the intestine. Thereby IL-3, IL-9 and Strongyloides spp. specific antibodies of IgG and IgE isotype trigger mast cell activation. Mast cells contain a plethora of preformed effector molecules including proteases, lysosomal enzymes, cytokines, biogenic amines and proteoglycans that are released upon activation. Additionally the de novo synthesis of effector molecules such as lipid mediator prostaglandin and leukotriene and platelet activation factor is induced (Wernersson and Pejler, Reference Wernersson and Pejler2014).

Prostaglandin E2 (PGE₂) (Machado et al. Reference Machado, Carlos, Lourenco, Souza, Sorgi, Silva, Ueta, Ramos, Aronoff and Faccioli2010) and Leukotriene B4 (LTB₄) (Machado et al. Reference Machado, Ueta, Lourenco, Anibal, Sorgi, Soares, Roque-Barreira, Medeiros and Faccioli2005) were elevated in the lung and in the intestine of S. venezuelensis infected mice. Inhibition of LTB₄ synthesis using either an inhibitor (MK886) or mice lacking an enzyme involved in LTB synthesis (5-lipoxygenase) increased parasite burden in small intestines and fecal egg release suggesting a contribution of LTB₄ to parasite control. The phospholipid mediator platelet activation factor (PAF) has also been implicated in intestinal immunity since PAF receptor deficient mice displayed delayed parasite expulsion leading to increased adult numbers at day 12 p.i. (Negrao-Correa et al. Reference Negrao-Correa, Souza, Pinho, Barsante, Souza and Teixeira2004).

mMCP1 is a protease specifically expressed by mucosal mast cells (Reynolds et al. Reference Reynolds, Stevens, Lane, Carr, Austen and Serafin1990) and elevation of mMCP1 concentration in the serum indicates mast cell activation. Elevated mMCP1 concentrations in the serum of S. ratti and S. venezuelensis infected mice were associated with reduced intestinal parasite burden in several studies (Abe et al. Reference Abe, Sugaya, Ishida, Khan, Tasdemir and Yoshimura1993; Sasaki et al. Reference Sasaki, Yoshimoto, Maruyama, Tegoshi, Ohta, Arizono and Nakanishi2005; Blankenhaus et al. Reference Blankenhaus, Klemm, Eschbach, Sparwasser, Huehn, Kuhl, Loddenkemper, Jacobs and Breloer2011, Reference Blankenhaus, Reitz, Brenz, Eschbach, Hartmann, Haben, Sparwasser, Huehn, Kuhl, Feyerabend, Rodewald and Breloer2014; Breloer et al. Reference Breloer, Hartmann, Blankenhaus, Eschbach, Pfeffer and Jacobs2015). Reciprocal reduction in systemic mMCP1 concentration upon IL-9 neutralization was correlated with increased parasite burden (Blankenhaus et al. Reference Blankenhaus, Reitz, Brenz, Eschbach, Hartmann, Haben, Sparwasser, Huehn, Kuhl, Feyerabend, Rodewald and Breloer2014), suggesting a causative link between mMCP1 and parasite expulsion. FcRγ deficient mice, by contrast, displayed a clear delay in S. venezuelensis expulsion while both mastocytosis and mMCP1 release were comparable or even increased in the absence of the FcRγ chain at later time points (Onah et al. Reference Onah, Uchiyama, Nagakui, Ono, Takai and Nawa2000). Since mast cells can be partially activated, leading to secretion of some effector molecules without complete degranulation (Wernersson and Pejler, Reference Wernersson and Pejler2014), mMCP1 may be an indicator of mast cell activation that does not necessarily indicate complete degranulation and may not contribute to protection in all settings. It should be noted that the course of S. ratti or S. venezuelensis infection in mMCP1 deficient mice that display delayed expulsion of another gastrointestinal parasite, Trichinella spiralis (Knight et al. Reference Knight, Wright, Lawrence, Paterson and Miller2000), has not been investigated so far, thus direct evidence regarding the contribution of mMCP1 to expulsion of Strongyloides spp. is missing.

Strongyloides ratti does not invade the intestinal wall but rather embeds itself in the mucosa of the intestine tightly attached to the layer of intestinal epithelial cells (Dawkins et al. Reference Dawkins, Robertson, Papadimitriou and Grove1983). Oral implantation resulted in quick and stable adherence to the intestine after 4 h in naïve but not in immune mice. This intestinal immunity was inversely correlated to mast cell numbers and was compromised in W/W^v mice (Maruyama et al. Reference Maruyama, Yabu, Yoshida, Nawa and Ohta2000). In vitro incubation of S. venezuelensis adults with the mast cell products chondroitin sulphate (CHS) A and E or heparin reduced their ability to invade the mucosa after subsequent intestinal implantation. It is conceivable that CHS contributes to expulsion of parasitic adults by preventing adhesion to the intestinal mucosa. In support of this hypothesis, the increased intestinal parasite burden in FcRγ^−/− mice upon oral infection was correlated with a reduction in free CHS present in intestinal washings (Onah and Nawa, Reference Onah and Nawa2004). Thereby, the number of mast cells in the intestine and release of mMCP1 were not changed (Onah et al. Reference Onah, Uchiyama, Nagakui, Ono, Takai and Nawa2000) and total content of CHS in gut homogenates was actually increased, suggesting that mast cells were recruited but not fully activated in the absence of FcRγ chain. Differences in CHS release were also implicated in the reduced intestinal susceptibility of 129/SvJ mice to S. venezuelensis infection compared with C57BL/6 mice (Nakamura-Uchiyama et al. Reference Nakamura-Uchiyama, Nagao, Obara, Ishiwata and Nawa2001). Therefore, the release of CHS by fully activated mast cells apparently promotes expulsion of parasitic adults from the intestine by preventing their adhesion to the intestinal epithelial cell layer.

Nitric oxide (NO) an inflammatory mediator that is produced by macrophages and granulocytes was induced in macrophages exposed to antigens derived from S. venezuelensis L3 and parasitic adults in vitro and during the tissue migration phase of S. venezuelensis infection in vivo (Ruano et al. Reference Ruano, Lopez-Aban, Gajate, Mollinedo, De Melo and Muro2012). Treatment of BALB/c mice with NO donors reduced numbers of L3 in the lung and in the intestine in normal mice whereas treatment with an inhibitor of the inducible NO synthase reciprocally increased parasite burden (Ruano et al. Reference Ruano, Lopez-Aban, Fernandez-Soto, de Melo and Muro2015). Of note, treatment with NO donors also reduced the drastically increased parasite burden and fecal egg release in immune suppressed mice. Since mice were infected by s.c. injection of S. venezuelensis L3i, the reduced number of parasites in the intestine could reflect the already reduced number of migrating L3. Thus the target of NO, L3 or adult, remains to be elucidated.

In light of the many effector molecules, including cytokines that are produced by intestinal effector cells or are present in mast cell granules (Wernersson and Pejler, Reference Wernersson and Pejler2014), the analysis of potential effector molecules that contribute to expulsion of S. ratti and S. venezuelensis from the intestine is rather fragmented to date. The data accumulated so far regarding the pathways contributing to parasite expulsion from the intestine are summarized in Fig. 3.

Fig. 3. Control and eradication of parasitic S. venezuelensis and S. ratti adults from the small intestine. Migrating S. venezuelensis L3 induce tissue-derived IL-33 in the lung. IL-33 triggers the expansion of IL-5 and IL-13 producing ILC2 that subsequently promote parasite control and initiation of Th2 response. Classical STAT6 dependent Th2 response results in production of IL-13, IL-4, IL-5, IL-3 and IL-9. Additional IL-9 may be produced by IL-18/IL-2 activated CD4⁺ T cells and other undefined sources. IL-27 and IL-12 antagonize initiation of Th2. Foxp3⁺ T_reg and BTLA-HVEM mediated signalling dampens IL-9 production during S. ratti infection. IL-4 triggers B cell differentiation to IgG1 and IgE producing plasma cells. IgG and IgE as well as IL-3 and IL-9 activate mast cells and probably other intestinal effector cells. Mast cell activation results in mMCP1 release with unknown contribution to parasite expulsion. The mast cell product CHS interferes with attachment of S. venezuelensis to IEC. Other potential mast cell products LTB₄, PGE₂ and PAF as well as IL-4 and IL-13 receptor engagement on somatic cells promotes parasite expulsion via unknown mechanisms. Abbreviations: BTLA, B and T lymphocyte attenuator; CHS, chondroitin sulphate; HVEM, herpes virus entry mediator; IEC, intestinal epithelial cells; LTB4, Leukotriene B4; mMCP1, mouse mast cell protease 1;PAF, platelet activation factor; PGE₂, Prostaglandin E2; STAT, signal transducer and activator of transcription.

Immune evasion

Helminth parasites manage to establish chronic infections by actively dampening the immune response of their hosts (McSorley and Maizels, Reference McSorley and Maizels2012). Rodent specific S. ratti and S. venezuelensis, by contrast, cause transient infections that are terminated after 3–4 weeks (Dawkins et al. Reference Dawkins, Grove, Dunsmore and Mitchell1980; Sato and Toma, Reference Sato and Toma1990). Since parasitic females have the capacity to live for more than 1 year in the absence of adaptive immunity (Gardner et al. Reference Gardner, Gems and Viney2006) the timely termination of S. ratti and S. venezuelensis infection in wild type mice and rats apparently reflects efficient and unsuppressed function of the immune system. In spite of this, recent evidence suggests that active suppression of the host's immune response is required for S. ratti to survive even the first week within the intestine of immune competent mice (Blankenhaus et al. Reference Blankenhaus, Klemm, Eschbach, Sparwasser, Huehn, Kuhl, Loddenkemper, Jacobs and Breloer2011, Reference Blankenhaus, Reitz, Brenz, Eschbach, Hartmann, Haben, Sparwasser, Huehn, Kuhl, Feyerabend, Rodewald and Breloer2014; Breloer et al. Reference Breloer, Hartmann, Blankenhaus, Eschbach, Pfeffer and Jacobs2015).

Infection of BALB/c and C57BL/6 mice induced transient expansion of Foxp3⁺ T_reg numbers first at day 2 p.i. in popliteal and inguinal lymph nodes and later on day 7–14 p.i. in MLN. The Foxp3⁺ T_reg population contracted to naïve levels by day 36 p.i., thus kinetics of Foxp3⁺ T_reg expansion and contraction resembled the parasites migration path through the host, with 2 days tissue migration and prolonged presence in the intestine until complete clearance after 1 month (Blankenhaus et al. Reference Blankenhaus, Reitz, Brenz, Eschbach, Hartmann, Haben, Sparwasser, Huehn, Kuhl, Feyerabend, Rodewald and Breloer2014). The transient depletion of Foxp3⁺ T_reg in BALB/c mice before s.c. infection with S. ratti L3i reduced parasite burden in the intestine and fecal release of eggs and L1 at any time point of infection until clearance. As T_reg depletion did not affect eradication of migrating L3 in lung and head tissue, specifically intestinal immunity was improved (Blankenhaus et al. Reference Blankenhaus, Klemm, Eschbach, Sparwasser, Huehn, Kuhl, Loddenkemper, Jacobs and Breloer2011). T_reg depletion led to a generalized increase in Th2 associated IL-3, IL-5, IL-13 and IL-10 production by MLN cells and elevated IgE and mMCP1 concentration in the serum. However, the improved intestinal immunity in T_reg depleted mice was not due to increased Th2 response in general but was caused by increased IL-9 production and subsequent IL-9 driven accelerated activation of mucosal mast cells. Improved resistance and accelerated mMCP1 release in T_reg depleted BALB/c mice were abrogated by IL-9 neutralization while IL-13 neutralization had no effect (Blankenhaus et al. Reference Blankenhaus, Reitz, Brenz, Eschbach, Hartmann, Haben, Sparwasser, Huehn, Kuhl, Feyerabend, Rodewald and Breloer2014). Additional deficiency of mast cells in T_reg depleted Cpa3^Cre/wt mice increased parasite burden to wild type levels while IL-9 production was still elevated. Thus mast cells apparently represent one effector cell population that was first activated by IL-9 and subsequently contributed to improved S. ratti expulsion in T_reg depleted mice.

The depletion of T_reg (DEREG) mouse model used for these studies causes only transient T_reg depletion (Lahl et al. Reference Lahl, Loddenkemper, Drouin, Freyer, Arnason, Eberl, Hamann, Wagner, Huehn and Sparwasser2007). T_reg numbers returned to normal levels day 6 p.i. and T_reg depletion at later time points after infection i.e. at day 4 p.i. did not reduce parasite burden (Blankenhaus et al. Reference Blankenhaus, Klemm, Eschbach, Sparwasser, Huehn, Kuhl, Loddenkemper, Jacobs and Breloer2011) suggesting that suppression of IL-9 mediated mast cell activation by Foxp3⁺ T_reg was achieved during the first days of infection.

Comparison of BALB/c and C57BL/6 mice showed that T_reg expansion and phenotype during S. ratti infection was similar in both mouse strains. T_reg function was apparent in both strains as T_reg depletion elevated Th2 associated cytokine production and IgE concentration in C57BL/6 mice and BALB/c mice to the same extent (Blankenhaus et al. Reference Blankenhaus, Reitz, Brenz, Eschbach, Hartmann, Haben, Sparwasser, Huehn, Kuhl, Feyerabend, Rodewald and Breloer2014). Also efficiency of T_reg depletion and repopulation kinetics was comparable in BALB/c and C57BL/6 DEREG mice. Strikingly, T_reg depletion in C57BL/6 mice did not reduce parasite burden in high dose or low dose infections and did not significantly increase mMCP1 release and IL-9 production. Thus, expanding Foxp3⁺ T_reg mediate control of IL-9 production and subsequent mast cell activation in S. ratti infected BALB/c mice in a non-redundant manner while this is not the case in C57BL/6 mice. As immunity to S. ratti in C57BL/6 mice also depends on IL-9 (Blankenhaus et al. Reference Blankenhaus, Reitz, Brenz, Eschbach, Hartmann, Haben, Sparwasser, Huehn, Kuhl, Feyerabend, Rodewald and Breloer2014) and C57BL/6 are even more susceptible to S. ratti infection than BALB/c mice, it was hypothesized that additional regulatory elements of the immune system maintain control of IL-9 production and subsequent mast cell activation in S. ratti infected C57BL/6 mice in the absence of T_reg.

Expression of the regulatory receptor BTLA (Murphy and Murphy, Reference Murphy and Murphy2010) increased predominantly on CD4⁺ T lymphocytes in MLN of day 7 S. ratti infected C57BL/6 mice (Breloer et al. Reference Breloer, Hartmann, Blankenhaus, Eschbach, Pfeffer and Jacobs2015). Absence of either BTLA or its ligand HVEM reduced parasite burden in the intestine and fecal release of eggs and L1 throughout infection. Numbers of migrating L3 in lungs and heads of BTLA^−/− and HVEM^−/− mice were not reduced suggesting that intestinal immunity improved in the absence of BTLA-HVEM mediated signalling. Thereby specifically IL-9 production by MLN increased and mMCP1 release in the serum was accelerated in BTLA^−/− and HVEM^−/− mice. Thus BTLA-HVEM mediated signalling interfered with IL-9 driven mast cell activation and promoted S. ratti survival in wild type C57BL/6 mice.

Although expression of the regulatory receptor cytotoxic T lymphocyte attenuator 4 (CTLA4) increased on Foxp3⁻ effector T cells in MLN of S. ratti infected BALB/c and C57BL/6 mice (Blankenhaus et al. Reference Blankenhaus, Klemm, Eschbach, Sparwasser, Huehn, Kuhl, Loddenkemper, Jacobs and Breloer2011; and Blankenhaus unpublished results) it played only a minor role during immune evasion. Blockade of CTLA4 mediated signalling elevated S. ratti specific and polyclonal Th2 response in both mouse strains, but did not reduce fecal release of S. ratti eggs and L1. Intestinal parasite burden was significantly reduced in anti-CTLA4 treated BALB/c mice at day 7 p.i but not at day 6 or day 8 p.i., thus providing no consistent increase in resistance.

In summary these studies show that suppression of IL-9 driven mast cell activation via induction of Foxp3⁺ T_reg predominantly in BALB/c mice or BTLA expression predominantly in C57BL/6 mice was central for establishment of even transient intestinal infection by S. ratti in immune competent mice.

Bystander suppression

Helminth-induced immune suppression spills over to bystander antigens and may change the outcome of allergy, vaccination or co-infections (McSorley and Maizels, Reference McSorley and Maizels2012). In this context, the suppression of some aspects of Ovalbumin (OVA) induced allergy were reported in S. stercoralis infected mice (Wang et al. Reference Wang, Nolan, Schad and Abraham2001). Implantation of L3 prior to immunization with OVA decreased eotaxin concentration in the lung as well as OVA-specific IgE production to intranasal challenge OVA exposure, although S. stercoralis did not induce a patent infection in BALB/c mice and L3 were never present in the lung. S. venezuelensis infection induced lung airway hyporesponsiveness (AHR) in the absence of other allergic stimuli (Silveira et al. Reference Silveira, Nunes, Cara, Souza, Correa, Teixeira and Negrao-Correa2002) but the more dramatic increase in AHR elicited by experimental OVA induced asthma was ameliorated by S. venezuelensis infection during OVA challenge in sensitized mice (Negrao-Correa et al. Reference Negrao-Correa, Silveira, Borges, Souza and Teixeira2003).

Still the direct comparison of the immune modulatory effects induced by transient S. ratti infection to long lasting Litomosoides sigmodontis infection showed that chronic nematode infection caused a stable suppression of immune responses to bystander antigens, whereas transient S. ratti infection predominantly polarized bystander immune responses towards a Th2 phenotype. Mice that carried a chronic L. sigmodontis infection displayed drastically reduced IgG responses to model antigen vaccination affecting both Th1 associated IgG2 and Th2 associated IgG1 responses (Hartmann et al. Reference Hartmann, Haben, Fleischer and Breloer2011; Haben et al. Reference Haben, Hartmann and Breloer2014). By contrast, transient S. ratti infection suppressed Th1 associated IgG2 responses to model antigen vaccination more pronounced than Th2 associated IgG1 responses (Hartmann et al. Reference Hartmann, Eschbach and Breloer2012). Modulation of vaccination response was only observed if vaccination was performed during maximal S. ratti parasite burden. Also production of Th1 associated IFNγ by anti-CD3 stimulated spleen cells and MLN was only transiently suppressed during S. ratti infection while anti-CD3 engagement induced production of Th2 associated cytokines at the same time (Eschbach et al. Reference Eschbach, Klemm, Kolbaum, Blankenhaus, Brattig and Breloer2010). Furthermore, concurrent infection with L. sigmodontis interfered with the induction of Plasmodium berghei specific cytotoxic lymphocytes by experimental vaccination whereas concurrent S. ratti infection did not abrogate vaccination efficacy under similar conditions (Kolbaum et al. Reference Kolbaum, Tartz, Hartmann, Helm, Nagel, Heussler, Sebo, Fleischer, Jacobs and Breloer2012b ).

In line with these findings, multiple infections with S. venezuelensis did not change the course of experimental autoimmune encephalomyelitis in Lewis rats (Chiuso-Minicucci et al. Reference Chiuso-Minicucci, Van, Zorzella-Pezavento, Peres, Ishikawa, Rosa, Franca, Turato, Amarante and Sartori2011) and the pathology induced in a model of type I diabetes resulting in weight loss, hyperglycemia and islet infiltration was only mildly ameliorated by a regimen of previous S. venezuelensis immunization and infection (Peres et al. Reference Peres, Chiuso-Minicucci, da Rosa, Domingues, Zorzella-Pezavento, Franca, Ishikawa, do Amarante and Sartori2013). The beneficial effect of S. venezuelensis infection was correlated to transient induction of Th2 associated IL-5 and IL-10 suggesting that Th1 associated islet inflammation was attenuated by the pre-existing Th2 polarization rather than by active suppression. Chronic L. sigmodontis infection, by contrast, clearly prevented autoimmune diabetes in non-obese mice independent of a Th2 polarization by TGFβ mediated immune suppression (Hubner et al. Reference Hubner, Shi, Torrero, Mueller, Larson, Soloviova, Gondorf, Hoerauf, Killoran, Stocker, Davies, Tarbell and Mitre2012).

Co-infection

It is conceivable that the transient Th2 polarization observed in Strongyloides spp. infected mice would interfere with the initiation of protective immune responses to infection with parasites that are predominantly controlled by Th1 associated mechanism. However, Plasmodium yoelii and Leishmania major were efficiently controlled in S. ratti co-infected mice (Kolbaum et al. Reference Kolbaum, Ritter, Zimara, Brewig, Eschbach and Breloer2011, Reference Kolbaum, Eschbach, Steeg, Jacobs, Fleischer and Breloer2012a ). Thereby the S. ratti induced Th2 response was outcompeted by the P. yoelii and L. major induced Th1 polarization although mice were first infected with S. ratti for 6 days to establish Th2 polarization before co-infection was performed. Despite the suppressed Th2 polarization in co-infected mice, the S. ratti infection itself was also efficiently controlled and terminated, even if L. major infections were performed 14 days before S. ratti infection to allow full establishment of Th1 polarization (Kolbaum et al. Reference Kolbaum, Ritter, Zimara, Brewig, Eschbach and Breloer2011). These studies suggest that the immune system is sufficiently elastic to cope with two infections requiring opposing types of immune response. Still, pre-existing S. venezuelensis infection interfered with an efficient response to intravenous Mycobacterium bovis co-infection 3 days later. Thereby bacterial load in lung increased at day 28 of M. bovis infection and lung concentration of IL-17 but not of IFNγ decreased (Dias et al. Reference Dias, de Castro, Alves, Rezende, Rodrigues, Machado, Fernandes, Negrao-Correa, Teixeira and Ferreira2011). Also numbers of S. venezuelensis adults increased in co-infected mice and Th2 response recorded as concentration of IL-4 and IL-13 in lung and intestinal tissues decreased (Carmo et al. Reference Carmo, Vicentini, Dias, Alves, Alves, Brandi, De Paula, Fernandes, Barsante, Souza, Teixeira, Negrao-Correa and Ferreira2009). It should be noted that the course of Mycobacterium tuberculosis aerosol infection was not changed by pre-existing S. ratti infection (Stubbe, Breloer and Hölscher, unpublished results).

In summary these findings suggest that transient S. ratti and S. venezuelensis infections in mice predominantly polarize the immune system towards a Th2 phenotype and suppress IL-9 driven mast cell activation to delay their own expulsion from the intestine but do not display the long lasting and systemic suppression of bystander immune responses observed in chronic helminth infections. Still, some bystander suppression may occur in selected settings and modulation of IL-9 driven pathology by pre-existing Strongyloides spp. infections has not been investigated.