TSLP-dependent basophils promote T_H2 cytokine responses following intestinal helminth infection

Abstract

CD4⁺ T helper type 2 (T_H2) cytokine responses promote the development of allergic inflammation and are critical for immunity to parasitic helminth infection. Recent studies highlighted that basophils can promote T_H2 cytokine-mediated inflammation and that phenotypic and functional heterogeneity exists between classical IL-3-elicited basophils versus TSLP-elicited basophils. However, whether distinct basophil populations develop following helminth infection, and their relative contributions to anti-helminth immune responses remain to be defined. Following Trichinella spiralis infection of mice, we show that basophil responses are rapidly induced in multiple tissue compartments, including intestinal-draining lymph nodes. Trichinella-induced basophil responses were IL-3-IL-3R-independent but critically dependent on TSLP-TSLPR interactions. Selective depletion of basophils following Trichinella infection impaired infection-induced CD4⁺ T_H2 cytokine responses, suggesting that TSLP-dependent basophils augment T_H2 cytokine responses following helminth infection. The identification and functional classification of TSLP-dependent basophils in a helminth infection model, coupled with their recently-described role in promoting atopic dermatitis, suggests these cells may be a critical population in promoting T_H2 cytokine-associated inflammation in a variety of inflammatory or infectious settings. Collectively, these data suggest that the TSLP-basophil pathway may represent a new target in the design of therapeutic intervention strategies to promote or limit T_H2 cytokine-dependent immunity and inflammation.

INTRODUCTION

CD4⁺ T helper type 2 (T_H2) cells, characterized by expression of IL-4, IL-5, IL-9 and IL-13, are required for pathogen clearance and tissue repair following exposure to helminth parasites (1–5). However, T_H2 cytokine responses can also promote the pathological changes associated with asthma and allergic diseases at multiple barrier surfaces (6, 7). Recent studies have identified that in addition to their well-established role as late-phase effector cells, basophils can express MHC class II, secrete IL-4, migrate into lymph nodes (LNs) and promote optimal T_H2 cytokine-mediated immune responses following exposure to some, but not all, allergens or helminth parasites (8–17).

The predominately T cell-derived cytokine IL-3 is a primary factor involved in basophil maturation, activation and trafficking (9, 18–20). However, since IL-3 is not required for basophil development (18), it was hypothesized that other factors could regulate basophil development and/or activation. Consistent with this, recent studies indicate that basophils are a heterogeneous population of cells, whose differentiation can be promoted by the epithelial cell-derived cytokine thymic stromal lymphopoietin (TSLP) cooperatively or independently of IL-3-IL-3R interactions (21). Further, TSLP-elicited basophils exhibit a distinct pattern of gene expression compared to classical IL-3-elicited basophils, respond more robustly to stimulation by IL-1 family cytokines IL-18 and IL-33 and produce higher levels of IL-4 and IL-6 (21). The identification of functional heterogeneity within the basophil lineage may, in part, explain the differential requirements for basophils in promoting optimal T_H2 cytokine responses depending on the pathogen or allergen examined. Although TSLP-dependent basophils promote T_H2 cytokine-associated inflammation in a mouse model of atopic dermatitis (21), the functional potential of IL-3-dependent versus TSLP-dependent basophils to helminth-induced T_H2 cytokine-mediated inflammation is unknown.

In the present study we demonstrate that following infection with the intestinal helminth parasite Trichinella spiralis, IL-3-IL-3R-independent, TSLP-dependent basophils are rapidly recruited into multiple tissue compartments, including intestinal-draining lymph nodes (LNs). Critically, depletion of basophils diminished the magnitude of infection-induced CD4⁺ T_H2 cytokine responses, suggesting that the rapid generation of “early-responder” TSLP-dependent basophil populations contributes to an environment permissive for optimal T_H2 cell differentiation that is required for immunity to invading helminths.

MATERIALS AND METHODS

Animals, parasitological techniques and cell isolations

WT C57BL/6 mice were ordered from Jackson ImmunoResearchLaboratories and TSLPR^−/− mice were obtained from Amgen. IL-3R^−/− mice (Csf2rb2^tm1Cgb Csf2rb^tm1Clsc) and BaS-TRECK mice were bred at the University of Pennsylvania and maintained in a specific-pathogen free environment. All experiments were performed with age, gender and genetic background-matched mice, to minimize variations in infection-induced immune responses. All experiments were performed according to guidelines from University of Pennsylvania Institutional Animal Care and Use Committee-approved protocols. Methods for maintenance, recovery, infection and isolation of Trichinella Ag were previously described (22, 23). Mice were infected with 300 Trichinella muscle larvae by oral gavage and were sacrificed at d 2, 4, 7 or 12 p.i. for assessment of basophil responses, or were sacrificed at d 12 p.i. for analysis of peak infection-induced T_H2 cytokine responses, worm burdens or humoral responses. At necropsy, single cell suspensions of mLN were prepared by passing through 70 μm nylon mesh filter. Splenocytes were isolated by homogenization followed by RBC lysis. Blood was collected by cardiac puncture, serum was isolated and peritoneal exudate cells were recovered by lavage with 10mL cold PBS. Bone marrow was isolated from femurs and single cell suspensions made by filtration through 70 μm nylon mesh filters and RBC lysis.

Neutralizing Ab treatments and cell depletions

Mice were treated with neutralizing mAb against mouse TSLP (obtained from Amgen) or mouse IL-3 (34D.11) by i.p. injection with 0.25 mg of Ab 4 h prior to infection and every 3 d after infection. Control mice received equivalent amounts of rat Ig (Control Ig). Mice were depleted of basophils by i.p. injection of 10 μg anti-FcεRI Ab (MAR-1, eBioscience) or were given control hamster Ig (eBioscience) on d 0, 1, 2, 7, 8 and 9 after infection. BaS-TRECK mice were given i.p. injections of Diphtheria toxin (500 ng) on d -1, 4 and 9 after Trichinella infection.

Flow cytometry

Cell preparations were surface stained with anti-mouse fluorochrome-conjugated mAbs against CD3ε (145-2C11), CD4 (GK1.5), CD11c (N418), CD19 (1D3), CD49b (DX5), CD123 (5B11), MHC class II (AF6-120.1), FcεRIα (MAR-1), c-kit (2B8), IgE (R35-72) and TSLPR (obtained from Amgen). For staining with intracellular cytokines, cells were stimulated for 4 h with 50 ng/mL PMA, 500 ng/mL ionomycin and 10 μg/mL brefeldin A (Sigma Aldrich), stained with cell surface Abs, fixed with paraformaldehyde, permeabilized in saponin and then stained with fluorochrome-labeled anti-IL-4 (11B11) and anti-IL-13 (eBio13A) antibodies. All Abs were from eBioscience unless specified otherwise. Cells were analyzed by flow cytometry using a FACSCanto or LSRII (BD Biosciences) and further analysis was performed using FlowJo software (Tree Star, Inc.).

Trichinella Ag-specific cell stimulations and ELISAs

Single cell suspensions of mLN from naive or infected mice were plated at 6 million cells/ml in complete medium (DMEM; Life Technologies) supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and 50 μM β-mercaptoethanol and stimulated for 48 h with 50 μg/mL Trichinella Ag. Supernatants or serum samples were assayed for IL-3, IL-4, IL-5, IL-13 and IgE using standard sandwich ELISA protocols (eBioscience).

RNA isolations and real time quantitative PCR

RNA from 1 cm sections of small intestine was isolated by homogenization in TRIzol using a TissueLyzer (Qiagen) followed by phenol-chloroform extraction and isopropanol precipitation. cDNA was generated per standard protocol with Superscript reverse transcriptase (Invitrogen) and used as input for real-time PCR. Real time data were analyzed using the ΔΔCT method whereby actin served as the endogenous gene. All reactions were run on ABI 7500 Fast Real-Time PCR System (Applied Biosystems). Samples are normalized to naïve controls

Statistics

Groups of animals were compared using Mann Whitney U tests or Student’s t-tests where applicable. P values ≤0.05 were considered significant.

RESULTS

Trichinella infection elicits rapid basophil responses in multiple compartments

Basophils have been implicated as important regulators and effectors of T_H2 cytokine responses and the recent identification of heterogeneity in the basophil lineage has provoked renewed interest in the factors that regulate basophil development, activation and function. In the present study we employed infection with the helminth parasite Trichinella spiralis to investigate regulation of basophil responses. Trichinella is a self-limiting natural intestinal nematode parasite of mice that undergoes a transient intestinal phase where the parasite occupies a partially intracellular niche within intestinal epithelial cells (24). Trichinella infection elicits peripheral basophilia (25) and CD4⁺ T_H2 cytokine responses coincident with expulsion of the intestinal stage of the parasite (26), however the temporal and spatial kinetics of Trichinella-induced basophilia have not been reported. To address this, basophil responses in the blood, spleen and mesenteric LN (mLN) of WT mice were examined in the first 12 d post-infection (p.i.). Trichinella infection resulted in significant increases in frequencies of basophils (identified as CD3⁻, CD4⁻, CD19⁻, c-kit⁻, CD49b⁺, IgE⁺ cells) in the blood and spleen between d 2 and d 12 p.i. compared to naïve mice (Fig. 1A). Most strikingly, while basophils were found in very low frequencies within mLN of naïve mice, Trichinella infection resulted in a 10-fold increase in frequencies of basophils in the mLN by d 4 p.i., correlating with significant increases in total numbers of basophils in the mLN, which persisted until d 12 p.i. (Fig. 1A, 1B). Phenotypic analysis of basophils in the mLN demonstrated expression of subunits of the receptors for both TSLP and IL-3 (TSLPR and CD123) (Fig. 1C). Together, these data demonstrate that Trichinella infection elicits the rapid population expansion of IL-3 and/or TSLP-responsive basophil populations.

Figure 1. Trichinella infection elicits rapid basophil responses in multiple compartments.

WT mice were infected with Trichinella. (A) Representative plots displaying mean frequencies ± SEM of blood, spleen and mLN basophils in naïve mice and on d 2, 4 or 7 or 12 p.i. (B) Total numbers of basophils in mLN ± SEM. (C) Expression of TSLPR and CD123 on basophils isolated from the mLN of d 4 infected mice (open black histograms). Shaded histograms for TSLPR staining represent basophils from TSLPR^−/− mice and for CD123 staining represent CD4⁺ T cells. Data are representative of 2 independent experiments, Naïve (n=2), d 2, 4, 7, 12 INF (n=3). *P<0.05 compared to Naïve (A), or day 0 (B).

Trichinella-induced basophil and CD4⁺ T_H2 cytokine responses are significantly diminished following anti-TSLP mAb treatment but not anti-IL-3 mAb treatment

Given that both IL-3 and TSLP can regulate basophil responses (18, 21), we sought to test the relative contributions of each of these cytokines to Trichinella-induced basophilia and CD4⁺ T_H2 cytokine responses. First, WT mice were infected with Trichinella and the expression of TSLP mRNA and IL-3 were assessed in the small intestine or serum at d 2, 4, 7 and d 12 p.i.. Trichinella infection elicited a 4-fold increase in TSLP mRNA expression by d 2 p.i. and TSLP expression returned to basal levels by day 7 p.i. (Fig. 2A). In contrast, serum IL-3 levels were not elevated above those seen in naïve animals until d 12 p.i. (Fig. 2B). Collectively, these data demonstrate that Trichinella infection induces rapid and transient TSLP expression but more delayed increases in IL-3 levels. Next, WT mice were infected with Trichinella and received either isotype control or neutralizing anti-IL-3 or anti-TSLP mAbs and basophil responses were assessed at d 4 p.i., a time point at which significant splenic and mLN basophilia were observed (Fig. 1A). Trichinella-infected mice treated with control Ig did not exhibit increases in the frequencies of basophils in the blood, consistent with data presented in Figure 1A, and these responses were not affected by ablation of TSLP or IL-3 (Fig. 2C, 2D). However, frequencies and absolute numbers of splenic basophils were increased in Trichinella-infected mice treated with control Ig or anti-IL-3 mAb (Fig. 2E, 2F). In contrast, treatment of mice with anti-TSLP mAb completely abolished infection-induced splenic basophil population expansion (Fig. 2E, 2F). While anti-IL-3 mAb treatment did partially reduce frequencies and total numbers basophils in the mLN (Fig. 2G, 2H), consistent with a role for IL-3 in mediating basophil homing into LNs during infection with other helminth species (9), anti-TSLP mAb treatment had a greater effect and reduced mLN basophil numbers to those seen in naive animals (Fig. 2G, 2H). Collectively, these data suggest that Trichinella-induced basophil responses are highly dependent on TSLP-TSLPR signaling.

Figure 2. Trichinella-induced basophil responses are abolished by anti-TSLP mAb treatment but not by anti-IL-3 mAb treatment.

WT mice were infected with Trichinella and (A) Tslp mRNA expression was assessed in small intestinal tissue homogenates by RT-PCR and (B) IL-3 protein levels were measured in serum by ELISA. (C–I) WT mice were treated with either control Ig or mAb to IL-3 or TSLP and infected with Trichinella and basophil responses were assessed at d 4 p.i. and CD4⁺ T_H2 cytokine responses were assessed at d 12 p.i.. Representative plots displaying (C–D) frequencies of blood basophils, (E) frequencies and (F) total numbers of splenic basophils and (G) frequencies and (H) total numbers of mLN basophils ± SEM at d 4 p.i. (I) Ex vivo intracellular IL-4 and IL-13 staining in mLN CD4⁺ cells at d 12 p.i., numbers ± SEM indicate frequency of cells in each quadrant. Data are representative of 2–3 independent experiments, Naïve (n=1–2), Control Ig (n=4–5), anti-IL-3 (n=3–4), anti-TSLP (n=4–6). *P<0.05 compared to Control Ig. ^#P<0.05 compared to anti-IL-3 Ab.

Critically, when infection-induced CD4⁺ T_H2 cytokine responses were assessed at d 12 p.i., mice treated with either control Ig or anti-IL-3 mAb displayed increases in frequencies of mLN CD4⁺ T cells that co-express the T_H2 cytokines IL-4 and IL-13, while mice treated with anti-TSLP mAb exhibited a significantly diminished response (Fig. 2I). Together, these data suggest that while both TSLP and IL-3 may be required for optimal basophil responses, TSLP appears to play a dominant role in regulating basophil responses and the magnitude of CD4⁺ T_H2 cytokine responses following Trichinella infection.

Infection-induced basophil responses are primarily IL-3-IL-3R independent but critically dependent on TSLP

In a mouse model of atopic dermatitis, TSLP-elicited basophil responses were independent of IL-3-IL-3R interactions (21). We sought to test whether TSLP-dependent basophil responses following Trichinella infection were dependent or independent of the IL-3-IL-3R pathway. To test this, WT or mice deficient in both beta chains of the IL-3R (Csf2rb2^−/− Csf2rb^−/−) were infected with Trichinella and basophil responses examined. Critically, both WT and Csf2rb2^−/− Csf2rb^−/− mice exhibited similar Trichinella-induced increases in frequencies of basophils in the blood (Fig. 3A, 3D), spleen (Fig. 3B) and mLN (Fig. 3C). In addition, both WT and Csf2rb2^−/− Csf2rb^−/− mice exhibited increases in total numbers of spleen and mLN basophils at d 4 p.i. (Fig. 3E, 3F), indicating that IL-3-IL-3R interactions are not required for Trichinella-induced basophil responses.

Figure 3. Infection-induced basophil responses are primarily IL-3-IL-3R independent but critically dependent on TSLP.

WT or IL-3R^−/− mice (Csf2rb2^−/−/Csf2rb^−/−) were infected with Trichinella. Representative plots displaying frequencies of (A, D) blood (B) spleen and (C) mLN basophils ± SEM at d 4 p.i. Total numbers of (E) spleen and (F) mLN basophils ± SEM. Csf2rb2^−/−/Csf2rb^−/− mice were treated with either control Ig or anti-TSLP mAb and infected with Trichinella and (G) frequencies of blood basophils and total numbers of (H) spleen and (I) mLN basophils were enumerated at d 4 p.i. Data shown for A–F and G–I are representative of 3 independent experiments, Naïve A–F (n=1–2), WT and Csf2rb2^−/−/Csf2rb^−/− INF A–F (n=4), Naïve G–I (n=2), Control Ig, anti-TSLP INF G–I (n=3). *P<0.05 compared to Control Ig.

To directly test whether the IL-3R-independent basophil responses that develop following Trichinella infection were dependent on TSLP-TSLPR interactions, Trichinella-infected Csf2rb2^−/− Csf2rb^−/− mice were treated with either control Ig or neutralizing anti-TSLP mAbs. While infection of Csf2rb2^−/− Csf2rb^−/−mice treated with control Ab resulted in pronounced basophil population expansion in the blood (Fig. 3G), spleen (Fig. 3H) and mLN (Fig. 3I), treatment with anti-TSLP mAb significantly diminished these responses, suggesting that TSLP directly promotes basophilia independently of IL-3-IL-3R interactions. Taken together, these data indicate that Trichinella infection is a potent stimulus for the rapid development of TSLP-dependent, IL-3-independent basophil responses.

Basophil and CD4⁺ T_H2 cytokine responses following Trichinella infection are dependent on TSLP-TSLPR interactions

To examine the influence of TSLP-TSLPR interactions on the induction of Trichinella-induced basophilia and T_H2 cytokine responses, we employed mice genetically deficient in TSLPR. WT or TSLPR^−/− mice were infected with Trichinella and basophil responses and T_H2 cytokine responses were examined at d 4 or d 12 p.i., respectively. While blood basophil responses in WT and TSLPR^−/− mice were comparable (Fig. 4A), infected TSLPR^−/− mice exhibited reduced frequencies of basophils in the bone marrow (Fig. 4B), spleen (Fig. 4C) and mLN (Fig. 4E), and total numbers of basophils in the spleen (Fig. 4D) and mLN (Fig. 4F) compared to WT mice, consistent with results observed following antibody-mediated TSLP ablation (see Figure 2). Further, TSLPR^−/− mice exhibited significantly diminished CD4⁺ T_H2 cytokine responses in the mLN compared to WT mice as measured by ex vivo intracellular cytokine staining (Fig. 4G, 4H) and production of IL-4, IL-5 and IL-13 by mLN cells following restimulation with Trichinella antigen (Fig 4I, 4J, 4K). These data indicate that TSLP-TSLPR interactions are critically important for both Trichinella-induced basophil responses and T_H2 cytokine responses, provoking the hypothesis that TSLP may also regulate T_H2 cytokine responses by eliciting basophil populations.

Figure 4. Basophil and CD4⁺ T_H2 cytokine responses following Trichinella infection are dependent on TSLP-TSLPR interactions.

WT or TSLPR^−/− mice were infected with Trichinella. Representative plots displaying (A) frequency of blood basophils, (B) frequency of bone marrow basophils, (C) frequencies and (D) total spleen basophils and (E) frequencies and (F) total numbers of mLN basophils ± SEM at d 4 p.i. (G) Ex vivo intracellular IL-4 and IL-13 staining in mLN CD4⁺ cells at d 12 p.i., numbers ± SEM indicated frequency of cells in each quadrant. (H) Total IL-4 and IL-13 double-positive CD4⁺ cells ± SEM. (I) IL-4, (J) IL-5 and (K) IL-13 concentrations in cell-free supernatants following 48 h culture of mLN cells with Trichinella Ag, measured by ELISA. Data are representative of 4 independent experiments, Naïve (n=1), WT A–F (n=4), TSLPR^−/− A–F (n=3), WT, TSLPR^−/− G–K (n=5). *P<0.05 compared to WT INF.

Basophils contribute to optimal T_H2 cytokine responses following Trichinella infection

Basophils can promote T_H2 cytokine-mediated inflammation and act as late-stage effector cells in some models of helminth infection or allergy (10, 11, 13, 27), but not in others (8, 12, 14). Paradoxical reports on the requirement of basophils for promoting optimal T_H2 cell responses may be explained by previously unrecognized functional heterogeneity between IL-3 and TSLP-elicited basophils (21). Because early Trichinella-induced basophil responses are TSLP-dependent, we tested the contribution of basophils to Trichinella-induced T_H2 cytokine responses. We utilized two commonly used methods for depleting basophils, anti-FcεRI mAb treatment or Diptheria toxin-(DT) mediated depletion in BaS-TRECK mice. Treatment of mice with anti-FcεRI mAb depleted splenic and mLN basophils in infected mice (Fig. 5A). Analysis of Trichinella-induced CD4⁺ T_H2 cytokine responses at d 12 p.i. revealed that anti-FcεRI mAb-treated mice displayed significantly reduced frequencies and total numbers of CD4⁺ T cells that co-express IL-4 and IL-13 in the mLN compared to control Ig-treated mice (Fig. 5B, 5C). It has been reported that anti-FcεRI mAb treatment can target mast cell or DC populations in some settings (8, 27, 28). While no alteration of peritoneal cavity mast cell or mLN FcεRI⁺ DC responses was observed following anti-FcεRI mAb treatment (Fig. S1A, S1C, S1D), anti-FcεRI mAb-treated mice did exhibit reduced frequencies of tissue-resident mast cells in the small intestine following Trichinella infection (Fig. S1B). Therefore as an alternative approach to selectively deplete basophils, BaS-TRECK (BaS-DTR⁺) mice were employed that allow lineage-specific basophil deletion via expression of the human DTR under the control of the proximal 3’UTR element in the mouse il4 locus (21, 29). These studies were critical to avoid the potential off-target effects of anti-FcεRI Ab treatment on mast cell populations (Fig. S1B). Treatment of Trichinella-infected BaS-DTR⁺ and littermate BaS-DTR⁻ mice with DT resulted in complete ablation of basophils in the spleen and mLN (Fig. 5D). Critically, basophil-depleted mice exhibited significantly reduced frequencies and total numbers of CD4⁺ T cells that co-express IL-4 and IL-13 in the mLN (Fig. 5E, 5F), indicating impaired induction of T_H2 cytokine responses. Basophil-depleted mice also exhibited significantly reduced IL-4, IL-5 and IL-13 production by mLN cells following restimulation with Trichinella antigen compared to control mice (Fig. 5G, 5H, 5I) and reduced serum IgE titers (Fig. 5J). Consistent with previous studies demonstrating that the intestinal phase of Trichinella infection is self-limiting and that mice naturally expel the parasite even in the absence of lymphocytes (30), basophil depletion by either anti-FcεRI Ab-treatment or DT-mediated ablation did not significantly affect the rate of intestinal worm expulsion: (d 12 p.i: Control Ig: 20±5 worms vs anti-FcεRI mAb: 39±13), or DT-mediated basophil depletion (BaS-DTR⁻ worms: 17±5 vs BaS-DTR⁺ 23±6). Together, these data indicate that TSLP-dependent basophil responses are a key contributor to the promotion of optimal CD4⁺ T_H2 cytokine responses following Trichinella infection.

Figure 5. Basophils contribute to optimal T_H2 cytokine responses following Trichinella infection.

WT mice were treated with either control Ig or anti-FcεRI Ab during Trichinella infection. (A) Representative plots displaying depletion of spleen and mLN basophils at d 12 p.i. in anti-FcεRI Ab treated mice. (B) Ex vivo intracellular IL-4 and IL-13 staining in mLN CD4⁺ T cells, numbers ± SEM indicates frequency of cells in each quadrant. (C) Total IL-4 and IL-13 positive CD4⁺ cells ± SEM from naïve and infected mice. BaS-TRECK diptheria toxin receptor (BaS-DTR)⁻ and BaS-DTR⁺ mice were treated with DT i.p and infected with Trichinella. (D) Representative plots displaying DT-mediated depletion of spleen and mLN basophils in BaS-DTR⁺ mice at d 12 p.i. (E) Ex vivo intracellular IL-4 and IL-13 staining in mLN CD4⁺ cells, numbers ± SEM indicates frequency of cells in each quadrant. (F) Total IL-4 and IL-13 double-positive CD4⁺ cells ± SEM from naïve and infected mice. (G) IL-4, (H) IL-5 and (I) IL-13 concentrations in cell-free supernatants following 48 h culture of mLN cells with Trichinella Ag, measured by ELISA. (J) Total serum IgE levels in infected mice. Data are representative of at least 2 independent experiments, Naïve A–C (n=2), Control Ig and anti-FcεRI Ab INF (n=4). Naïve D–J (n=1), BaS-DTR⁻ INF (n=5), BaS-DTR⁺ INF (n=3). *P<0.05 compared to Control Ig. **P<0.05 compared to BaS-DTR INF.

DISCUSSION

The generation of CD4⁺ T_H2 cytokine responses is critical for immunity to parasitic helminths and is also responsible for the chronic inflammation associated with many allergic diseases (1, 6, 7, 31), however the early events that drive T_H2 cytokine production remain incompletely understood. Recent evidence that basophils can contribute to optimal T_H2 cytokine-mediated immune responses following infection with some, but not all, parasitic helminth species and allergens (8–17), and that functional heterogeneity exists in the basophil lineage (21), provoked the question as to whether differences in the phenotype of responding basophil populations may explain the paradoxical roles for these cells in regulating T_H2 cytokine responses. Data from the present study demonstrate that following gastrointestinal helminth infection, TSLP rapidly elicits the population expansion of TSLP-dependent, IL-3-independent basophils. TSLP-dependent basophils are rapidly recruited into LNs and depletion of basophils results in impaired CD4⁺ T_H2 cytokine responses following infection. These data indicate that TSLP-dependent basophil populations are critical for promoting optimal CD4⁺ T_H2 cytokine responses following infection with a gastrointestinal helminth.

TSLP is a primarily epithelial-derived cytokine that, along with other epithelial-derived cytokines IL-25 and IL-33 (32–35), has been implicated in regulating T_H2 cytokine responses following exposure to infectious or allergic stimuli (36–38). For example, TSLP-TSLPR signaling is important for T_H2 cytokine responses following infections with some helminth species (37, 39). However, the mechanisms by which TSLP promotes T_H2 cytokine responses are not fully understood and TSLP can have effects on diverse cell types, including DCs via limitation of IL-12 p40 expression and upregulation of OX40L, which creates a more T_H2-permissive environment (38, 40). Data from the present study implicate TSLP-TSLPR interactions as critically important for Trichinella-induced basophil responses in the bone marrow, spleen and intestinal draining lymph nodes and also the magnitude of the infection-induced T_H2 cytokine response. This provokes the hypothesis that TSLP regulates T_H2 cytokine responses in part by eliciting basophil populations that can be rapidly recruited into intestinal-draining LNs to influence developing T_H2 cell responses. Since Trichinella infection elicits rapid TSLP expression at the site of infection, which precedes any increase in circulating IL-3 levels, this may explain how TSLP is more important in regulating the acute basophil response elicited following infection. Given that TSLP-elicited basophils are more potent IL-4 producers than classical IL-3-elicited basophils and more responsive to stimulation with the epithelial-derived cytokine IL-33 (21), these studies suggest that tissue-resident epithelial cells may be central for rapidly regulating the differentiation, mobilization and activation of TSLP-dependent basophils.

T-cell derived IL-3 is an important factor involved in basophil maturation, activation and trafficking (9, 18, 19, 41–43). While basophil responses following Trichinella infection and in a model of atopic dermatitis are largely IL-3-IL-3R-independent (21), IL-3 ablation did result in reduced recruitment of basophils into intestinal-draining lymph nodes following Trichinella infection. Collectively, these data provoke the hypothesis that TSLP and IL-3 may cooperate to regulate optimal basophil responses. However, additional studies are needed to further interrogate the contributions of TSLP and/or IL-3 to basophil responses in the context of health and disease.

In addition to TSLP and IL-3, IL-18 (44), IL-33 (45, 46), GM-CSF (45), IgE (47, 48), IgD (49), C5a (50) and immune complexes (17, 51) have also been demonstrated to regulate basophil activation, development or homing. Consistent with this, TSLPR^−/−IL-3R^−/− mice still exhibit circulating basophil populations (21) and in the present study, TSLP ablation in IL-3R^−/− mice failed to completely inhibit basophil homing to the mLN. The mechanisms by which basophil responses can be regulated are complex and are most likely regulated by multiple factors. Further research is required to classify the molecular and cellular factors involved in regulation of distinct IL-3 or TSLP-elicited basophil populations during different modes of inflammation. The development of genetically modified mice with cell-specific deletions in either IL-3R or TSLPR will provide new tools to interrogate the relative contribution of these distinct granulocyte populations in regulation of inflammation and immunity.

In conclusion, data from the present study provokes a model of the initial cellular immune response to a helminth infection, whereby epithelial cells at the site of infection produce TSLP which elicits an “early responder” population of basophils that are immediately mobilized prior to T cell activation. Later during infection, when the effector CD4⁺ T cell response has been established, levels of IL-3 are elevated and a “late responder” population of classical basophils can be elicited and maintained by IL-3 (9, 18–20). The identification and functional classification of TSLP-dependent basophils in a natural helminth infection model, coupled with their role in promoting atopic dermatitis (21), suggests these cells may be a critical population in promoting T_H2 cytokine-associated inflammation in a variety of inflammatory or infectious settings. As such, this cell population could represent a new target in the design of therapeutic intervention strategies to promote or limit T_H2 cytokine-dependent immunity and inflammation.

Abbreviations

DTR

Diptheria toxin receptor

mLN

mesenteric lymph node

T_H2

T helper type 2

TSLP

Thymic stromal lymphopoietin