Strongyloides ratti infection in mice: immune response and immune modulation

Abstract

Strongyloides ratti is a natural parasite of wild rats and most laboratory mouse strains are also fully permissive. The infection can be divided into three distinct phases: the tissue migration of the infective third stage larvae during the first two days, the early intestinal establishment of S. ratti parasites molting to adults on days three to six and the later intestinal parasitic phase until the end of infection. Immunocompetent mice terminate the S. ratti infection after one month and are semi-resistant to a second infection. Employing the powerful tools of mouse immunology has facilitated a detailed analysis of the initiation, execution and regulation of the immune response to S. ratti. Here we review the information collected to date on the protective immune response to migrating S. ratti larvae in tissues and to adult parasites in the intestine. We show that depending on the phase of infection, a site-specific portfolio of immune effector mechanisms is required for infection control. In addition, we summarize the strategies employed by S. ratti to evade the immune system and survive long enough in its host to replicate despite an effective immune response. Selected murine studies using the closely related Strongyloides venezuelensis will be discussed.

This article is part of the Theo Murphy meeting issue ‘Strongyloides: omics to worm-free populations’.

1. Introduction

Helminths are large multicellular parasites that affect a quarter of the human population [1,2]. Although helminth infections are rarely fatal, they induce prolonged morbidity, sometimes severe pathology and impair the working and learning performance of infected humans. Still, no vaccinations that confer sterile immunity exist. Effective anti-helminthic treatments are available. However, owing to frequent re-infections and the risk of resistance in helminth-endemic areas, this strategy is not sustainable. Therefore, a better understanding of the protective anti-helminth immune response and the antagonizing strategies employed by the parasite are needed.

Strongyloidiasis is one of the most neglected tropical diseases affecting approximately 600 million people worldwide, predominantly in southeast Asia, Africa and the western Pacific [3,4]. As the methodology used for diagnosis has a tremendous impact on the detection rate of infection, these numbers may still represent an underestimation. Immunological studies on Strongyloides stercoralis infected human subjects reported expansion of T helper 2 cells (Th2) [5,6], type 2 cytokines [7] and elevation of eosinophil, neutrophil and mast cell-derived effector molecules in the peripheral blood [8] (reviewed in [9]), reflecting the canonical type 2 immune response that is elicited by helminths in general [10].

Since the options for further hypothesis-driven, mechanistic studies are very limited with human subjects, mouse models have been used to elucidate the relevant immunological pathways in more detail. Different mouse models exist, that, however, only partially display the entire course of human infection. The human pathogenic S. stercoralis has the unique ability to cause auto-infections and thus develop chronic or even lifelong infections in humans. In mice S. stercoralis larvae cannot develop beyond the third larval stage (L3). Many insights regarding the elimination of L3 in the tissue were gained using S. stercoralis L3 that were implanted within migration chambers under the skin of experimental mice (reviewed in [11]). However, most laboratory mouse strains are fully susceptible for infection with the rodent pathogens Strongyloides ratti [12] and Strongyloides venezuelensis [13], thus providing the opportunity to study the host-parasite interaction during the entire life cycle and at different sites. Although the extremely chronic infections that are the hallmark of S. stercoralis infection in humans cannot be modelled by the acute, short-termed course of infections of S. ratti- or S. venezuelensis-infected mice, the powerful tools of mouse immunology enabled the precise analysis and dissection of the various pathways contributing to anti-Strongyloides immunity (table 1).

Table 1.

Immune effectors involved in the control of murine S. ratti infection. (Summary of murine studies using genetically modified mice or antibody (Ab)-mediated depletion in S. ratti (black) or S. venezuelensis (grey) infection. WT, wild-type; KO, knockout; ILC, innate lymphoid cells; IL, interleukin; TG, transgenic; DT, diphteria toxin; rec, recombinant; CTLA-4, cytotoxic T lymphocyte-associated protein 4; BTLA, B and T lymphocyte attenuator; HVEM, Herpes virus entry mediator.)

effector	intervention / comparison (genetic background of mice)	L3 numbers tissue to WT	worm numbers intestine to WT	egg or larval output to WT	immunology to WT	memory: protected from 2nd infection	reference
innate cell populations in S. ratti and S. venezuelensis infection
neutrophils	depletion: anti-Gr1 mAb injection (BALB/c)	increased (day 2)	increased (day 6)				[14,15]
	depletion: anti-Gr1 mAb injection (C57BL/6)	increased (day 2)		increased (day 5–9)			[16]
eosinophils	KO mice: ΔdblGATA mice to WT (BALB/c)	increased (day 2)	increased (day 6)				[14,15]
basophils	KO mice: Mcpt8Cre to WT littermates (BALB/c + C57BL/6)	similar	increased (day 6)	increased, infection terminated with WT kinetics	type 2 cytokines similar	intact	[17]
	depletion in TG mice: DT-treated Mcpt8 DTR to DT-treated WT littermates (C57BL76)			very slightly increased		intact	[18]
connective tissue mast cells	KO mice: Mcpt5Cre x DTR flox mice to WT littermates (C57BL/6)	similar (day 2)	increased (day 6)	increased, infection terminated with WT kinetics		intact	[19]
connective tissue and mucosal mast cells	KO mice: Cpa3Cre mice to WT littermates (BALB/c + C57BL/6	similar (day 1–4)	increased (day 6)	increased and prolonged (day 150)	type 2 cytokines similar	intact	[19]
	KO mice: W/Wv mice to WT (WBB6F1)			increased (day 7–14) and prolonged (> day 14)			[20]
	KO mice: W/Wv mice to WT mice (WBB6F1)			prolonged (6 days)			[21]
	KO mice: Kit W-s/W-sh mice to WT (C57BL/6) Cpa3Cre;Mcl-1fl/fl mice to WT littermates (C57BL/6)			prolonged (12 days)		intact	[18]
	depletion in TG mice: DT-treated MasTRECK mice to DT-treated WT littermates (BALB/c)			prolonged (7 days)		intact	[18]
ILC	KO mice: RAGγc KO mice to RAG KO mice (BALB/c)		increased (day 6)				[15]
	KO mice: RAGγc KO mice to RAG KO mice (BALB/c)		increased (day 8)	prolonged (270 days)			[18]
adaptive immunity in S. ratti infection and S. venezuelensis infection
T & B cells	KO mice: RAG2 KO mice to WT mice (C57BL/6)	similar (day 2)	similar (day 6)	prolonged (300 days)		failed	[19] & unpubdata
	KO mice: RAG2 KO mice to WT mice (C57BL/6)		increased (day 8)	increased and prolonged (20 days)		failed	[18]
CD4+ T cells	depletion: anti-CD4 mAb injection to isotype (C57BL/6)			prolonged (27 days)		failed	[18]
Ab	supplementation: transfer of immune serum (C57BL/6)			reduced (day 7 + 9)			[22]
	KO mice: Fc receptor-deficient mice (FcyR KO (lacking all FcR) and 5KO mice (lacking FcεRI, FcεRII, FcγRI, FcγRIIB, FcγRIII) to WT (C57BL/6)		similar (day 6)	slightly increased (day 9–21)			[19]
	KO mice: Ab-deficient Igh-J KO mice (BALB/c) and FcR-deficient FcyR KO mice to WT (C57BL/6)			prolonged (19 days)		intact	[18]
	KO mice: Ab-deficient JHD KO mice to WT (BALB/c)			increased (day 8–10)			[23]
	KO mice: FcR-deficient FcyR KO mice to WT (C57BL/6)		increased (day 10)	increased (day 9–13)			[24]
IgM	supplementation: transfer of S. ratti-specific mAb (IgM) (C57BL/6)	reduced (day 2)	reduced (day 6)				[25]
IgE	KO mice: IgE deficient Igh-7 KO mice to WT (BALB/c) and FcεRI/II KO mice to WT (C57BL/6)			prolonged (3 days)		intact	[18]
IgE and IgG	KO mice: class switch–defect AID KO mice to WT (C57BL/6)		increased (day 10)	prolonged (8 days)			[26]
cytokines in S. ratti infection and S. venezuelensis infection
IL-3	supplementation: Injection of rec. IL-3 (C57BL/6)	similar (36 h)	increased (44 h + 2 days post oral infection)		intestinal mastocytosis and mast cell activation increased		[27]
	supplementation of immunodeficient mice: Injection of rec. IL-3 in athymic mice (KSN (nu/nu))		reduced (day 14–15)	increased (day 11–15)			[28]
	KO mice: IL-3 KO mice to WT (C57BL6 and BALB/c)			prolonged (13–15 days)	basophil and mast cell expansion impaired		[29]
IL-9	supplementation: injection of rec. IL-9 (BALB/c and C57BL/6)		reduced (day 6)				[30]
	block: injection of anti-IL-9 mAb (BALB/c and C57BL/6)		increased (day 6)		mast cell activation reduced		[30]
	KO mice: IL-9 receptor KO mice to WT (BALB/c and C57BL/6)		similar (day 3) increased (day 6–10 BALB/c); (day 8–10 C57BL/6)	prolonged (12 days)	type 2 cytokines de-regulated, mast cell activation and intestinal IL-13 reduced	intact	[19,31]
IL-13	block: injection of anti-IL-13 mAb to isotype (BALB/c)		similar (day 6)				[30]
IL-4	KO mice: IL-4 KO mice to WT (C57BL/6)			slightly increased (day 4)			[32]
IL-4 and IL-13	KO mice: IL-4 receptor α KO (BALB/c)		similar (day 7) increased (day 10 + 14)	increased (day 7–14)	phenotype owing to IL-4Rα deficiency on somatic cells		[33]
IL-5	block: injection of anti- IL-5 mAb to isotype (C57BL/6)	increased (36 h)	increased (day 5)	increased (day 6 + 7)	reduced eosinophilia	intact	[34]
	KO mice IL-5 KO mice to WT (C57BL/6)		similar (day 2) increased (day 3–6)	increased (days 3–6)	reduced eosinophilia		[35]
	overexpression in TG mice: IL-5 transgenic mice (C3H/He)		reduced (day 8) and after implant. in intestine	reduced, infection terminated with WT kinetics	increased eosinophilia		[36]
	block: injection of anti- IL-5 mAb (BALB/c or C57BL/6 not specified)			slightly increased (day 9), infection terminated with WT kinetics	reduced eosinophilia	impaired	[37]
IL-12	KO mice: IL-12 p40 KO mice to WT (C57BL/6)		similar (day 7 + 10)	similar (day 7 + 10)			[33]
	KO mice: IL-12 KO (chain not specified) to WT (C57BL/6)		reduced (day 7)		type 2 cytokines increased		[38]
IL-33	supplementation: injection or i.n. application of rec. IL-33 (BALB/c)		reduced (day 4–6)		mast cell activation and ILC2 expansion increased and accelerated		[15]
	stabilization: i.n. application of the IL-33 stabilisator CCP1/2 (BALB/c)		reduced (day 6)		mast cell activation increased		[15]
	block: injection the IL-33-antagonist HpARI (BALB/c)		increased (day 6)		mast cell activation reduced		[15]
	KO mice: IL-33 KO mice to C57BL/6 WT (129Sv backcross to C57BL/6 for four generations)			increased (day 9)	mast cell activation, lung eosinophilia and ILC2 expansion reduced		[39]
TSLP	KO mice: TSLP receptor KO mice to WT (BALB/c)	increased (day 3)	increased (day 7)	increased (day 7–9)	mast cell and basophil activation, reduced		[40]
immunoregulation in S. ratti infection and S. venezuelensis infection
Foxp3+ Treg	depletion in TG mice: DT-treated DEREG mice to DT-treated littermates and untreated DEREG (BALB/c)	similar (day 2)	reduced day 6		type 2 cytokines IL-3, IL-5, IL-13, IL-10) increased, mast cell activation increased and accelerated	intact	[41]
	depletion in TG mice: DT-treated DEREG mice to DT-treated littermates (BALB/c)		reduced (day 6)	reduced, infection terminated with WT kinetics	type 2 cytokines (IL-3, IL-5, IL-13, IL-10) and IL-9 increased, mast cell activation increased and accelerated		[30]
	depletion in TG mice: DT-treated DEREG mice to DT-treated littermates (C57BL/6))		similar (day 6)	similar	type 2 cytokines (IL-3, IL-5, IL-13, IL-10) increased, IL-9 and mast cell activation similar		[30]
CTLA-4	block: injection of anti-CTLA-4 mAb (BALB/c)		reduced (day 6 + 7)	similar	type 2 cytokines (IL-3, IL-5, IL-13, IL-10) increased		[41]
BTLA	KO mice: BTLA KO mice to WT littermates (C57BL/6)	similar (day 2)	reduced (day 6)	reduced, infection terminated with WT kinetics	type 2 cytokines (IL-13) similar, IL-9 and mast cell activation increased		[42]
HVEM	KO mice: HVEM KO to WT littermates (C57BL/6)	similar (day 2)	reduced (day 6)	reduced, infection terminated with WT kinetics	type 2 cytokines (IL-13) similar, IL-9 and mast cell activation increased		[42]

2. Protective immune response to Strongyloides ratti

(a) . Life cycle and migration route

It is a well-known fact that establishment and execution of immunity is a site- and context-specific mechanism. Therefore, understanding the migration route of the parasite is the basis to understand the events happening in affected organs and how this shapes the general immune response. Analysis of the S. ratti migration path within mice and rats was undertaken using histology of various tissues or tracking of radioactively labelled L3 and led to the general understanding depicted in figure 1 (reviewed in [43–45]). Infective L3 dwell in the moist soil and actively penetrate the skin of their rodent host. Although percutaneous infection is possible under laboratory conditions, most studies inject defined numbers of L3 subcutaneously (s.c.). Within 2 days, the majority of L3 migrate via the tissue to the nasofrontal region of the head. In contrast to S. venezuelensis L3 that are known to exclusively migrate via the lung [46], approximately 90% of S. ratti L3 are localized in head tissue and only about 10% are found in the lung at day 2 post infection (p.i.) ([47,48] and M.L. Brunn, M. Breloer 2016, unpublished observations). Subsequently L3 are thought to enter the mouth, are swallowed, reach the small intestine by day 3 p.i. and moult to parasitic adults. Female parasitic adults dwell embedded in the mucosa of the intestine and reproduce via parthenogenesis. Eggs (S. venezuelensis) or hatched first stage larve (L1) (S. ratti) are released with the faeces already by day 5 to day 6 p.i. and may either directly moult to infective L3 or introduce a free-living generation [44] (figure 1). Infected immunocompetent mice and laboratory rats terminate S. venezuelensis and S. ratti infections within a month and remain semi-resistant to subsequent infections (reviewed in [45,49]).

Figure 1.

Strongyloides ratti life cycle. Infective S. ratti L3 live in the free world, actively penetrate the skin of rats or mice and migrate within 2 days via the tissue to the head. L3 are swallowed, molt via a 4th larval stage to parasitic adults and embed into the mucosa of the small intestine. The parasitic adults reproduce by parthenogenesis. Eggs as well as hatched L1 reach the free world with the faeces by day 5–6 p.i. and may either directly develop into infective L3 or introduce one free-living generation that displays sexual reproduction.

To interrogate the S. ratti migration pathway in more detail, our group compared the numbers of viable, emigrating L3 in different tissues and at various time points after infection in BALB/c mice. At the same time, S. ratti-derived DNA in these tissues was quantified to measure both, viable and already killed or immobilized L3 [14]. This study emphasized that S. ratti L3 do not migrate randomly through the tissues and body fluids of their host, but rather follow a defined route from the point of entry to the intestine. After s.c. injection into the footpad viable, motile L3 were first located in the foot, 10 min later in the skin and muscle tissues of the leg, 2 days later in the lung and in the head. First L3 arrived in the small intestine on day 3 p.i. All other tissues examined, such as the kidney and also the blood, remained parasite-free. Strongyloides ratti-derived DNA was detected in foot and leg tissues for a much longer period than viable L3, suggesting that L3 were immobilized and/or killed in these tissues. By contrast, the number of migrating L3 and the amount of S. ratti-derived DNA in head and lung tissue increased and decreased simultaneously, indicating that all L3 which survive the tissue passage to the head and lung tissue also migrate further on to the intestine. In mice encountering a second infection, the L3 were retained and eliminated directly at the site of infection as almost no migrating S. ratti L3 nor S. ratti-derived DNA was detectable in other tissues than the foot [14].

(b) . Immune response in the tissue

Only 10–20% of injected S. ratti L3 survive the tissue migration and are retrieved in head tissue and later in the intestine [14,49,50]. To identify the immune cells and pathways that mediate this efficient L3 eradication in the tissue already during a first infection, mice that lack certain immune effectors were used (table 1). Depletion of neutrophilic granulocytes using anti-Gr-1 monoclonal antibody (mAb) elevated the numbers of L3 in head and lung tissue of S. ratti-infected BALB/c [14] and C57BL/6 [16] mice at day 2 p.i. Similarly, the absence of eosinophils in ΔdblGATA mice elevated L3 numbers in the head tissue [14]. Neutrophils and eosinophils immobilized S. ratti L3 also in vitro, in the context of extracellular DNA trap formation (ETosis) by both cell types and myeloperoxidase production by neutrophils [14]. Here, L3-induced ETosis by neutrophils was first described to S. stercoralis L3 in vitro [51]. The in vitro killing of S. ratti L3 was enhanced by S. ratti-specific antibodies (Ab) present in the serum of previously infected mice [14]. Accordingly, transfer of immune serum [22] and also of a monoclonal S. ratti-specific Ab [25] reduced the numbers of migrating L3 in the head and lung tissues of day 2 S. ratti-infected mice.

By contrast, neither basophils nor mast cells contributed to the control of S. ratti in the tissue as L3 numbers in head and lung of either basophil-deficient Mcpt8-Cre [52] or mast cell-deficient Cpa3-Cre [53] mice and their wild-type littermates were alike at day 2 p.i. [17,19]. A closer examination of the parasite load over time in mast cell-competent and mast cell-deficient mice revealed that L3 appeared and disappeared with similar kinetics in the head and lung tissue and that similar numbers of L3 occurred in the intestine day 3 p.i. [19]; thus, indicating either no function or a redundant function for these cells in the immune defence against tissue-migrating S. ratti L3 during a first infection.

(c) . Early innate immune response in the intestine

Despite the unchanged numbers of L3 ‘arriving’ in the intestine at day 3 p.i., mice lacking either basophils or mast cells had elevated intestinal parasite burden three days later, i.e. day 6 p.i. and released more S. ratti DNA until clearance of infection [17,19]. These findings demonstrate a non-redundant function for mast cells and basophils in the eradication of intestinal S. ratti parasites. The importance of mast cells and basophils in controlling the intestinal parasite burden was subsequently reproduced for S. venezuelensis-infected mice [18]. In addition, older studies using mice with different mutations in the stem cell factor receptor Kit that result in mast cell-deficiency, but also cause several other alterations of the immune system [54], support the importance of mast cells in the control of intestinal S. ratti [20] and S. venezuelensis [21,29] infection.

The activation of mucosal mast cell can be monitored by the elevation of mouse mast cell protease 1 (mMCPT-1) in the serum, a protease that is specific for mucosal mast cells and is released once a mucosal mast cell degranulates. Elevated mMCPT-1 is detectable between days 3 and 4 of S. ratti infection, peaks between day 7 and 14 p.i. and returns to background levels subsequently after a month [41]. The classic mechanisms leading to mast cell activation and degranulation is the crosslinking of antigen-specific immunoglobulin E (IgE) that is associated with the high affinity Fcε receptors by a polyvalent antigen. However, innate mechanisms may act in parallel at these early time points. This is supported by the fact that generation of antigen-specific T and B cell responses take 1–2 weeks and the intestinal parasite burden of immunocompetent wild-type mice and T and B cell-deficient RAG KO mice are similar at day 6 p.i. [19]. Indeed, injection with recombinant interleukin 3 (IL-3), a cytokine involved in the recruitment and activation of mast cells, reduced intestinal S. ratti parasite burden in immunocompetent C57BL/6 mice [27] and also in athymic nude mice lacking T cells [28].

Several lines of evidence suggest that also the cytokine IL-9 contributes to the early, innate activation of mast cells. Injection of recombinant IL-9 reduced day 6 intestinal S. ratti parasite burden and neutralization of endogenous IL-9 via injection of a blocking mAb elevated day 6 intestinal S. ratti parasite burden and decreased mast cell degranulation [30]. Mice lacking the IL-9 receptor had higher intestinal S. ratti parasite burden and displayed a reciprocally reduced mast cell degranulation [31]. Thereby IL-9 receptor-deficient mice displayed similar numbers of ‘arriving’ L3 day 3 p.i. in the small intestine and elevated parasite burden at later time points. Taken together, these findings suggest that IL-9 does not contribute to the eradication of tissue migrating L3 but rather acts in the intestine, presumably by activating mucosal mast cells. IL-9 can be produced by several immune cells with T cells and group 2 innate lymphoid cells (ILC2) representing the dominant sources [55,56]. The comparison of T- and B cell-deficient RAG KO mice, that control the intestinal S. ratti parasite burden until day 6 p.i., to RAGγc KO mice, that additionally lack all ILC, revealed a drastic increase in S. ratti numbers in the intestine day 6 p.i in the absence of ILC. [15]. Thus, ILC and most likely ILC2-derived IL-9, contribute to the early mast cell-mediated anti-S. ratti immunity. Similar findings were obtained using ILC-deficient S. venezuelensis-infected mice [18]. Of note, this does not exclude an additional role for T cell-derived IL-9.

A contribution of eosinophils and neutrophils to the eradication of specifically intestinal Strongyloides parasites is difficult to dissect from the important role these cells play in the killing of tissue migrating L3. IL-5-deficient mice that display drastically reduced eosinophilia had more S. ratti parasites in the intestine from day 3 to day 6 p.i. [35]. As the number of tissue-migrating L3 was not recorded, the elevated numbers of S. ratti in the small intestine might reflect already impaired killing of L3 during the tissue during migration. However, in an experimental set-up circumventing the tissue migration, eosinophilic IL-5-trangenic mice harboured reduced numbers of S. venezuelensis parasites in the intestine 1–3 days after intestinal implantation [36]. Likewise, depletion of eosinophils in day 3 S. ratti-infected mice, i.e. after completion of the L3 tissue migration, elevated intestinal parasite burden day 6 p.i. (L. Linnemann, M. Breloer 2023, unpublished observation), thus suggesting an additional contribution of eosinophils to intestinal host defence. The in vivo role of monocytes and macrophages in anti-S. ratti immunity still needs to be investigated.

(d) . Late adaptive immune response in the intestine

Work performed in RAG KO mice that lack adaptive immunity showed that the innate immune response allows the control of intestinal parasite burden during the first week of infection [19]. Final clearance of S. ratti from the intestine, however, is strictly dependent on adaptive immunity. Here CD4⁺ T cells producing the type 2 cytokines IL-4, IL-5, IL-9 and IL-13 as well as B cells producing Strongyloides-specific Ab, predominantly IgG1 and IgE, are central as will be discussed below (table 1). RAG KO mice carry low but stable numbers of viable adults producing L1 for up to 1 year p.i. [19], which is approximately the life span of S. ratti [57]. In immunocompetent mice, S. ratti adult numbers decline rapidly after day 6–7 p.i. and adults are almost undetectable after day 10 p.i. while the more sensitive detection of Strongyloides DNA by quantitative polymerase chain reaction in the faeces shows ongoing S. ratti infection in C57BL/6 [50] and BALB/c [41] mice until 4 weeks p.i.

Mice lacking either basophils or specifically connective tissue mast cells (Mcpt5Cre x DTR flox mice [58]) terminated the S. ratti infection with similar kinetics as seen in wild-type controls, despite an initially increased intestinal parasite burden and increased fecal release of Strongyloides DNA [19]. By contrast, mice constitutively lacking both, mucosal and connective tissue mast cells remained infected for more than half a year despite functional T and B cell responses. These findings highlight the central role for mast cells, presumably mucosal mast cells, in the timely termination of S. ratti infection. While the early mast cell activation detected at day 6 p.i. seems to be predominantly mediated by innate mechanisms such as IL-3 and IL-9, eventually adaptive T and B cell responses contribute to the termination of infection, by initiating Ab-dependent mucosal mast cell activation. Accordingly, clearance of infection is delayed by about 14 days in mice lacking the IL-9 receptor, but full activation and clearance of infection is possible in the absence of IL-9-mediated signalling after 42 days [31]. Thus, adaptive Ab-driven mast cell activation can fully replace the activation induced by IL-9 and other innate factors [19,31]. In line with these findings, S. venezuelensis infection was prolonged in mice that could not produce Ab or where deficient for the respective Fc receptors [18,23,24,26].

The tracking and analysis of adaptive T cell responses to Strongyloides is complicated as no specific T cell antigens have been identified so far. Mesenteric lymph node cells (mLN) and splenocytes derived from infected but not from naive mice produced type 2 cytokines (IL-3, IL-4, IL-5; IL-13, IL-10) to a crude lysate of S. ratti L3 throughout the course of infection [50]. In line with this T helper 2 cytokine response, high titres of L3 lysate-specific IgG1 (an isotype that is associated with type 2 immunity) were elicited, while antigen-specific IgG2 (an isotype that is associated with type 1 immunity) was below the detection limit at all time points analysed. Although no S. ratti-specific IgE was detectable either, the infection elicited a significant rise in total serum IgE concentrations. Variations in the infection dose modified the magnitude of cytokines produced, however, the quality i.e. the type 2 cytokine pattern was not affected [50]. C57BL/6 and BALB/c mice both display this type 2 immune response to S. ratti infection [30,50]. Similarly, immunocompetent S. ratti-infected Wistar rats ceased to release S. ratti eggs and L1 3-4 weeks after infection in the context of a comparable type 2 immune response that was characterized by mast cell activation and IL-4 production by mLN cells restimulated with S. ratti antigen [59].

Recently, the generation of a transgenic S. ratti strain, named ‘the hulk’, that express a defined T cell epitope: ‘2W1S’ fused to green fluorescent protein allowed for the first time the direct identification and characterization of S. ratti-specific T cells employing MHC-II/2W1S peptide tetramers [60]. 2W1S-specific CD4⁺ T cells that were elicited in the lung during S. ratti ‘the hulk’ infection expressed type 2 signature genes such as the canonical Th2 transcription factor GATA binding protein 3 (GATA3) and the cytokines IL-4, IL-5, IL-13, and amphiregulin. This was confirmed on protein level as approximately 60% of the antigen-specific T cells were also positive for GATA3. Interestingly 30% of the ‘hulk’ 2W1S antigen-specific T cells expressed the transcription factor Foxp3 that identifies regulatory T cells, a finding that may reflect an immune evasive strategy of the parasite and will be discussed below. As expression of the transgenic T cell epitope 2W1S was lost after the tissue migration phase of the transgenic S. ratti ‘hulk’, it was not possible to follow antigen-specific T cell responses in the intestine with this model [60].

(e) . Adaptive immunity to a second infection

Immunocompetent mice that resolved a first S. ratti infection remain semi-resistant to a second infection, leading to drastically reduced or absent intestinal parasite burden. Thereby, the majority of S. ratti L3 are killed very early during tissue migration at the site of infection as only a very limited number of viable L3 and S. ratti-derived DNA was detectable beyond the foot used for infection [14]. Infection with irradiated L3, that cannot establish patent infections in the intestine, confer the same resistance to a second infection as a resolved first infection with viable L3 [19]. This resistance is strictly dependent on adaptive immunity since RAG KO mice display the same intestinal parasite burden during a first and second infection ([18,19] and M. L. Brunn, M. Breloer 2016, unpublished observation). Interestingly, neither mast cells, basophils, nor IL-9 mediated signalling were important for the establishment of this protective memory. Immune IL-9 receptor-deficient, mast cell-deficient and basophil-deficient mice that were infected for a second time showed drastically reduced numbers of migrating L3 in the tissue and subsequently reduced numbers of intestinal parasites [17–19,31]. In line with these findings, S. venezuelensis-infected mice also showed establishment of protective immunity to a second infection in mast cell- and basophil-deficient mice [18]. Dissecting the role of CD4⁺ T cells and B cell-derived Ab, Mukai et al. [18] showed that a lack of Ab could be compensated in a second infection with S. venezuelensis, while CD4⁺ T cells were more important. However, evidence for a contribution of S. ratti-specific Ab to the efficient killing of L3 in a second infection is provided by studies showing that eosinophil and neutrophil effector functions were enhanced by Ab in the serum of immune mice [14] and that transfer of immune serum to naïve mice reduced the number of tissue migrating larvae in vivo [22,25].

Owing to a lack of mouse models for ILC2-deficiency in otherwise immunocompetent mice, it was not possible to analyse the role of ILC2 in the establishment of protective immune memory so far. The recent development of neuromedin U receptor 1 based ILC2 depletion in immunocompetent mice allows this analysis that is ongoing at the moment [61,62]. The protective immune response to the different stages of S. ratti infection is summarized in figure 2.

Figure 2.

Site-specific anti-S. ratti immune responses. Tissue immunity: S. ratti L3 migrate after either experimental s.c. injection or natural percutaneous infection to the lung and head tissue where the peak of L3 numbers is observed day 2 p.i. Predominantly eosinophils (Eo) and neutrophils (Neu) intercept S. ratti L3 during tissue migration in the context of ETosis. This is enhanced by S. ratti-specific Ab. Early innate intestinal immunity: first L3 occur in the intestine day 3 p.i, the peak of adults is observed day 6 p.i. Basophils (Baso) and mast cells (MC) contribute to the control of intestinal parasite burden. ILC2-derived IL-9 promotes mast cell activation during the first week of infection. The specific contribution of eosinophils, neutrophils or macrophages to intestinal immunity has not been conclusively investigated. Late adaptive intestinal immunity: final ejection of S. ratti is strictly dependent on mucosal mast cells and T cells, whereby T cell-derived type 2 cytokines and B cell-derived IgE and IgG1 promote mast cell activation. (This figure was created with BioRender.com).

(f) . Initiation of the anti-Strongyloides ratti immune response

The presence of infecting pathogens is sensed via conserved ‘pathogen associated molecular pattern’ (PAMP) that interact with equally conserved ‘pattern recognition receptors' on innate immune cells. While a plethora of PAMPs has been described for bacterial pathogens, the recognition of helminth parasites is still under-investigated. First evidence arose from a study showing that migrating S. venezuelensis L3 or intranasal application of chitin, that is part of the nematode cuticula, induced the release of the alarmin cytokine IL-33 by lung epithelial cells [39]. IL-33 is a nuclear protein that is released from damaged and stressed cells and acts, among other cells, on ILC2 and mast cells [63]. IL-33-deficient mice displayed reduced eosinophilia in the lung day 7 p.i. and slightly elevated release of S. venezuelensis eggs at day 8 p.i. Conversely, treatment with recombinant IL-33 reduced egg release in IL-33-deficient mice [39].

Interrogating the function of endogenous IL-33 that is released during S. ratti infection, we took advantage of a helminth-derived IL-33 antagonist, the Heligmosomoides polygyrus derived Alarmin Release Inhibitor (HpARI) [64]. Blockade of IL-33 during infection elevated day 6 intestinal S. ratti numbers and reduced mucosal mast cell degranulation [15]. Stabilization of endogenous IL-33 using a truncation fragment of HpARI [65], reciprocally reduced intestinal S. ratti numbers and increased mucosal mast cell degranulation. Treatment with recombinant IL-33 phenocopied the stabilization of endogenous IL-33. Both intranasal or intraperitoneal application of IL-33 triggered mucosal mast cell degranulation within hours after application. Also, IL-33 application after the tissue migration phase, at day 4 p.i., reduced intestinal S. ratti numbers 2 days later. IL-33 expanded ILC2 and acted in the absence of adaptive immunity in RAG KO mice but not in ILC-deficient RAGγc KO mice. The IL-33-mediated reduction of intestinal parasite numbers was strictly dependent on the presence of mast cells and intact IL-9 receptor signalling. By contrast, the cell types responsible for intercepting S. ratti during tissue migration, namely neutrophils and eosinophils, were not involved [15]. Collectively, these data suggest that migrating S. ratti L3 in the (skin-) tissue trigger IL-33 release either via the putative PAMP chitin and/or via the mechanic tissue disruption. This IL-33 may expand ILC2 that produce IL-9. IL-9 further enhances the expansion of IL-9 receptor-competent ILC2 and promotes ILC2 activation thus contributing to the rapid activation of mucosal mast cells in the intestine and promoting parasite ejection (figure 3).

Figure 3.

Regulation of anti-S. ratti immune responses. Somatic tissue-derived IL-33 enhances IL-9- and mast cell-mediated parasite ejection partially via expansion and activation of IL-9 producing ILC2. S. ratti antagonizes the IL-9 mediated mast cell activation via expansion of Foxp3⁺ regulatory T cells (Treg) and induction of negative regulatory receptors such as B and T lymphocyte attenuator (BTLA) on effector T cells. (This figure was created with BioRender.com).

In addition to IL-33, other alarmin cytokines such as the tuft cell-derived IL-25 and the epithelial cell-derived thymic stromal lymphopoietin (TSLP) promote type 2 and anti-helminth immunity [66]. C57BL/6 mice that lacked the TLSP receptor had higher S. venezulensis parasite burden associated with reduced mast cell and basophil activation and impaired Th2 polarization [40]. This suggest that TSLP next to IL-33 promotes anti-Strongyloides immunity, while the role of IL-25 still remains to be determined.

3. Immune evasion

The multiple and efficient pathways leading to the ejection of S. ratti by immunocompetent mice and rats after approximately 4 weeks [50,59] seems to provide an example for a 100% functional mammalian immune response and a failing parasite. However, accumulating evidence suggest that this is not the case. Owing to its rapid reproduction that starts by days 5–6 p.i., 1–2 months survival within the host is sufficient for S. ratti to propagate its genes. Still, to ensure survival for this one month, S. ratti, like most helminths, actively suppresses the immune response that is directed towards itself as will be discussed below.

Strongyloides ratti infection induced the expansion of Foxp3⁺ regulatory T lymphocytes (Treg), first in the popliteal lymph node draining the site of infection (day 2 p.i.) and later in the mLN draining the intestine (day 7–14 p.i.). Transient depletion of these Treg using the ‘depletion of regulatory T cells’ (DEREG) mouse model [67] in BALB/c mice did not change the number of migrating L3 in the tissue but reduced intestinal parasite burden and release of S. ratti-derived DNA throughout infection, until clearance [41]. Depletion of Tregs resulted in increased production of type 2 cytokines, including IL-9, and accelerated and enhanced degranulation of mast cells. This was reversed by additional blockade of IL-9 but not IL-13, demonstrating that specifically the increased IL-9 production caused the accelerated mast cell activation in Treg-depleted BALB/c mice [30]. Treg depletion reduced intestinal parasite burden in mice lacking basophils [17] but not in mice lacking mucosal mast cells [30], indicating that specifically mast cell function was suppressed by the expanding Treg. Interestingly Treg depletion in mice that additionally lacked mast cells still resulted in increased IL-9 production. However, this IL-9 was not translated in reduced parasite burden in the absence of mast cells. Thereby, it was established that expanding Tregs dampened IL-9 production in the wild-type situation. After artificial depletion of Treg in DEREG mice, increased amounts of IL-9 were present that subsequently activated the mast cells more efficiently to degranulate, thus promoting the rapid ejection of S. ratti [30].

Since no S. ratti-specific T cell epitopes have been described so far, it was not possible to test if this expanding Treg were specific for S. ratti antigens. Therefore, it is highly interesting that 30% of the 2W1S model antigen-specific CD4⁺ T cells that were elicited in response to the 2W1S transgenic S. ratti ‘the hulk’, expressed Foxp3 and thus represent parasite antigen-specific Treg [60]. In line with the murine data, field studies reported an expansion of Foxp3⁺ CD4⁺ T cells in S. stercoralis HTLV-1 co-infected humans that was partially reverted by successful treatment of the Strongyloides infection [68,69].

C57BL/6 mice and BALB/c mice displayed a similar expansion of Treg during S. ratti infection [30]. Both mouse strains mount a comparable type 2 immune response to S. ratti infection and the relevance of basophils, mast cells and IL-9 was established using knock out and transgenic mice on both, BALB/c and C57BL/6 background [17,19,31] (table 1). In sharp contrast to BALB/c mice, C57BL/6 mice displayed unchanged intestinal parasite burden and S. ratti-derived DNA release after Treg depletion [30]. Although Treg depletion significantly elevated type 2 cytokine production in C57BL/6 mice, IL-9 production showed a non-significant trend to elevation and mast cell degranulation remained unchanged.

Next to Tregs, that remain the primary regulatory pathway for maintaining peripheral tolerance, a complex network of co-inhibitory receptors that is expressed on effector T cells acts as a second fine-tuning level of regulation. Infection with S. ratti increased expression of several co-inhibitory receptors on T cells in BALB/c and C57BL/6 mice. Namely cytotoxic T lymphocyte-associated protein 4, B and T lymphocyte attenuator (BTLA), programmed cell death 1 and immunoglobulin-like transcript 3 were upregulated on CD4⁺ T cell during S. ratti infection [70]. This induction was more pronounced in C57BL/6 mice compared to BALB/c mice with their F1 cross showing an intermediate phenotype. Likewise, the S. ratti infection-induced expansion of Foxp3 negative type 1 regulatory T cells (Tr1) was more pronounced in C57BL/6 mice compared to BALB/c mice [70]. Therefore, it is tempting to speculate that next to Foxp3⁺ Treg expansion, additional and redundant layers of regulation are induced in S. ratti-infected C57BL/6 mice. A more pronounced immune evasion in C57BL/6 mice could also contribute to the increased susceptibility of this mouse strain compared to BALB/c mice. Although both mouse strains mount a comparable type 2 immune response to S. ratti infection, C57BL/6 mice carry higher intestinal parasite burden, an observation that was reported in the first study describing S. ratti infection in mice [12] and reproduced in all direct comparisons of BALB/c and C57BL/6 mice to date [19,30,31,70] (table 1).

To support this hypothesis, we focused on the co-inhibitory receptor BTLA that is induced on CD4⁺ T cells in the mLN of S. ratti-infected C57BL/6 mice. BTLA (CD272) is a member of the immunoglobulin family and contains an intracellular immunoreceptor tyrosine-based inhibitory motif domain and delivers negative signals after engagement by its ligand, Herpes virus entry mediator (HVEM) [71]. Mice that were either deficient for BTLA or HVEM displayed reduced intestinal S. ratti parasite burden and reduced release of S. ratti-derived DNA throughout infection. This improved host defence was correlated to increased mucosal mast cell activation, increased IL-9 production and reverted by blockade of endogenous IL-9 [42]. Thus, expression of one co-inhibitory receptor interfered with the efficient immune response in C57BL/6 mice. Ongoing research will most likely expand the portfolio of these regulatory pathways that are triggered by S. ratti and contribute to the delay of efficient parasite ejection. The regulation of the immune response to S. ratti infection is summarized in figure 3.

Current research aims at identifying the immunomodulatory molecules that trigger expansion of Treg, expression of co-inhibitory receptors on effector T cells and may also antagonize other features of anti-S. ratti immunity. In this context, it was shown recently that tissue migrating S. venezuelensis L3 secrete the immunomodulatory protein venestatin. Venestatin binds to ‘receptor for advanced glycation end products' (RAGE), a multiligand danger receptor, thereby blocking its function [72]. Migrating L3 in the skin trigger the release of RAGE ligands and thus RAGE-mediated production of reactive oxygen species and proinflammatory cytokines that promote L3 eradication. This pathway was antagonized by venestatin, as knock down of venestatin in S. venezuelensis L3 reduced the number of L3 that successfully migrated to the lung and subsequently to the intestine in WT but not in RAGE-deficient mice [72]. As also S. ratti was shown to excrete a plethora of L3-specific and parasitic female-specific proteins [73], some of which have already shown immunomodulatory effects in vitro [74–76], a similar role of in vivo immune evasion of some of these proteins is very likely.

4. Concluding remarks

Strongyloides ratti and S. venezuelensis present excellent tools to study anti-helminth immunity in vivo in the mouse system. Using genetically modified mice, both protective immunity and immune evasion have been described in great detail. Thereby, accumulating evidence suggest that relevant immune effector pathways are highly site- and context-specific. Moreover, the genetic background of the experimental mice must be considered as C57BL/6 and BALB/c mice differ in the dominance and redundancy of immunoregulatory pathways. Finally, identification of the helminth-derived immunomodulatory molecules mediating this suppression may promote the development of efficient treatments and vaccinations against the human pathogenic S. stercoralis in the long run.

Data accessibility

This article has no additional data.

Authors' contributions

M.B.: conceptualization, writing—original draft, writing—review and editing; L.L.: conceptualization, writing—review and editing.

Both authors gave final approval for publication and agreed to be held accountable for the work performed therein.

Conflict of interest declaration

We declare we have no competing interests.

Funding

This work was funded by the German Research Association (grant no. BRE 3754/6-1).