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Antibodies Trap Tissue Migrating Helminth Larvae and Prevent Tissue Damage by Driving IL-4Rα-Independent Alternative Differentiation of Macrophages

  • Julia Esser-von Bieren,

    Affiliation Swiss Vaccine Research Institute and Global Health Institute, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

  • Ilaria Mosconi,

    Affiliation Swiss Vaccine Research Institute and Global Health Institute, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

  • Romain Guiet,

    Affiliation Bioimaging and Optics Core Facility, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

  • Alessandra Piersgilli,

    Affiliation Institute of Animal Pathology, University of Bern, Bern, Switzerland

  • Beatrice Volpe,

    Affiliation Swiss Vaccine Research Institute and Global Health Institute, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

  • Fei Chen,

    Affiliation Center for Immunity and Inflammation, New Jersey Medical School, Newark, New Jersey, United States of America

  • William C. Gause,

    Affiliation Center for Immunity and Inflammation, New Jersey Medical School, Newark, New Jersey, United States of America

  • Arne Seitz,

    Affiliation Bioimaging and Optics Core Facility, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

  • J. Sjef Verbeek,

    Affiliation Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands

  • Nicola L. Harris

    nicola.harris@epfl.ch

    Affiliation Swiss Vaccine Research Institute and Global Health Institute, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

Antibodies Trap Tissue Migrating Helminth Larvae and Prevent Tissue Damage by Driving IL-4Rα-Independent Alternative Differentiation of Macrophages

  • Julia Esser-von Bieren, 
  • Ilaria Mosconi, 
  • Romain Guiet, 
  • Alessandra Piersgilli, 
  • Beatrice Volpe, 
  • Fei Chen, 
  • William C. Gause, 
  • Arne Seitz, 
  • J. Sjef Verbeek, 
  • Nicola L. Harris
PLOS
x

Abstract

Approximately one-third of the world's population suffers from chronic helminth infections with no effective vaccines currently available. Antibodies and alternatively activated macrophages (AAM) form crucial components of protective immunity against challenge infections with intestinal helminths. However, the mechanisms by which antibodies target these large multi-cellular parasites remain obscure. Alternative activation of macrophages during helminth infection has been linked to signaling through the IL-4 receptor alpha chain (IL-4Rα), but the potential effects of antibodies on macrophage differentiation have not been explored. We demonstrate that helminth-specific antibodies induce the rapid trapping of tissue migrating helminth larvae and prevent tissue necrosis following challenge infection with the natural murine parasite Heligmosomoides polygyrus bakeri (Hp). Mice lacking antibodies (JH−/−) or activating Fc receptors (FcRγ−/−) harbored highly motile larvae, developed extensive tissue damage and accumulated less Arginase-1 expressing macrophages around the larvae. Moreover, Hp-specific antibodies induced FcRγ- and complement-dependent adherence of macrophages to larvae in vitro, resulting in complete larval immobilization. Antibodies together with helminth larvae reprogrammed macrophages to express wound-healing associated genes, including Arginase-1, and the Arginase-1 product L-ornithine directly impaired larval motility. Antibody-induced expression of Arginase-1 in vitro and in vivo occurred independently of IL-4Rα signaling. In summary, we present a novel IL-4Rα-independent mechanism of alternative macrophage activation that is antibody-dependent and which both mediates anti-helminth immunity and prevents tissue disruption caused by migrating larvae.

Author Summary

Intestinal helminths present a pressing problem in developing countries with approximately 2 billion people suffering from chronic infection. To date no successful vaccines are available and a detailed mechanistic understanding of anti-helminth immunity is urgently needed to improve strategies for prevention and therapy. Antibodies form a crucial component of protective immunity against challenge infections with intestinal helminths. However, the exact mechanisms by which antibodies target these large multi-cellular parasites have remained obscure. We now demonstrate that helminth-specific antibodies induce the rapid trapping of tissue migrating helminth larvae by activating phagocytes. In the absence of antibodies or their receptors, helminth-infected mice developed extensive tissue damage, revealing a novel role for antibodies in limiting parasite-caused tissue disruption. Furthermore, helminth-specific antibodies reprogrammed macrophages to express wound-healing factors such as the arginine-metabolizing enzyme Arginase-1. Interestingly, the Arginase-1 product L-ornithine directly impaired the motility of helminth larvae. In summary, our study provides detailed mechanistic insights into how antibodies can modulate phagocyte function to provide protection against a large multi-cellular parasite. Our findings suggest that novel anti-helminth vaccines should target the larval surface and activate wound-healing macrophages to provide rapid protection against tissue-disruptive larvae.

Introduction

Intestinal helminths present a major global health burden, particularly in developing countries. Patients that are infected with nematodes such as Ascaris lumbricoides, Trichuris trichuria or Necator americanus often develop severe pathology and impaired responsiveness to vaccines [1] [2] [3] [4]. Approximately 2 billion people are infected with intestinal nematodes, with the most severe infections often found within school children [5] [6]. Although such infections can be treated by chemotherapy, worm burdens typically reach pretreatment levels within 6 months [7] [5]. Moreover, drug resistant helminths present a pressing problem for livestock [8], raising concerns about the long-term validity of chemotherapy amongst human populations [9] [10] [11] [6] [12] [13]. Unfortunately, no efficacious vaccines against intestinal nematodes are available to date, making an improved understanding of host immunity imperative.

Macrophages are highly plastic immune cells that can fulfill diverse tasks in immunity, metabolism and wound-healing depending on their tissue location and inflammatory context [14]. In the context of bacterial infection, the activation of classically activated macrophages by serum components such as antibodies and complement can enhance the phagocytosis and killing of bacterial or fungal pathogens [15]. By contrast, type 2 immune responses associated with helminth infection and allergies are characterized by a predominance of alternatively activated macrophages (AAM) that appear to play a role in anti-helminth immunity and wound repair through ill-defined mechanisms [16] [17].

Infection with the natural murine parasite Heligmosomoides polygyrus bakeri (Hp) is a common model used to study immunity against helminth infection [18]. Following primary (1°) infection with Hp, C57BL/6 mice develop a chronic infection [19]. In contrast, Hp fails to establish chronicity after challenge infection, largely due to the rapid development of a protective TH2 type granuloma around the tissue invasive larvae [20] [21] [22]. The highly concentrated accumulation of Arginase-1 (Arg1) expressing alternatively activated macrophages in inflammatory lesions is a hallmark of type 2 responses associated with allergy or helminth infection [23] [24] [25]. Recent reports have demonstrated an important role for type 2 cytokine driven alternative activation of macrophages in protective immunity against intestinal helminth infection [16] [26] [27]. Previous work [28] [29], including a study from our own group [30], additionally identified helminth-specific antibodies as essential components of immunity against Hp. Passive transfer of IgG or immune serum could also confer resistance to Hp, Ascaris suum and Strongyloides ratti [31] [32] [33], and antibody production has been found to correlate with protection in human helminth infection [34] [35] [36]. Whilst antibodies contribute to protective immunity against Hp and other nematodes such as Trichuris muris or whipworms such as Trichinella spiralis, which cause chronic infections [37] [38], they are not required for rapid expulsion of the hookworm Nippostrongylus braziliensis [29]. However, the mechanism of antibody-mediated immunity against intestinal helminths has remained obscure.

In the current study we investigated the mechanisms of antibody-mediated immunity following challenge Hp infection and demonstrate that antibodies function to trap helminth larvae and to prevent parasite-induced tissue damage. Using newly developed tools for image analysis, we show that antibody-activated macrophages upregulate Arg1 and immobilize infective and tissue dwelling larvae. Intriguingly, the reprogramming of macrophages by helminth-specific antibodies did not require IL-4Rα signaling, indicating that antibody activation of macrophages during helminth infection represents a novel pathway of alternative macrophage differentiation. In summary we have shown that antibodies in the presence of helminth antigens can elicit a novel subtype of IL-4Rα independent alternatively activated macrophages, which we refer to as helminth-antibody activated macrophages (HAAM).

Results

Mice deficient in antibodies or activating antibody receptors exhibit impaired early immunity to H. polygyrus bakeri challenge infections

Our previous work on antibody mediated protective immunity against Hp has largely been focused on late time points (day 14–20) after challenge infection, when adult worms can be found in the lumen of the small intestine [30]. However, protective immunity is initiated as early as day four of challenge Hp infection [16] [29], when larvae have first invaded the intestinal mucosa. Thus, in order to investigate a potential early effect of antibodies in the memory response to Hp, we analysed the numbers of larvae that had invaded the small intestine of infected antibody-deficient JH−/− mice (for accession numbers of gene name abbreviations used in this manuscript see Table 1). JH−/− mice carry a deletion of the joining fragment of the immunoglobulin heavy chain locus and are devoid of mature B-cells and antibodies [39]. As shown in Fig. 1A, and in line with previous work [29], JH−/− harbored significantly higher numbers of L4 on day 4 after challenge infection. As previous work indicated a small but significant contribution of cellular activation via activating Fc receptors on the numbers of adult worms [30], we additionally assessed numbers of larvae in challenge infected mice lacking activating Fc receptors (FcRγ−/−). Interestingly, these mice also exhibited clearly increased larval burdens (Fig. 1A).

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Figure 1. Antibody and FcRgamma-chain deficient mice show increased larval burdens, larger granulomas, a stronger tendency to develop necrosis and higher in-tissue motility as compared to wildtype mice.

Mice were challenge infected with 200 infective L3 larvae after drug cured 1° infection and sacrificed on day 4 p.i.; (A) Numbers of tissue invasive larvae were analysed with a modified Baermann apparatus. (B) Paraffin sections of the duodenum of infected mice were hematoxylin and eosin (H&E) stained, (C/D) granuloma areas were quantified in light microscopy images of the largest cross-section of serial sections with larvae (C) or without larvae (D). (E) Necrosis in H&E-stained sections was scored by a blinded pathologist. (F–H) Absolute (F) and relative (H) in-tissue motility or larval length (G) was assessed by light microscopy combined with our Fiji macro. Pooled data of two independent experiments with 5 mice per group are shown as mean + SEM (*p<0.05, **p<0.01, ***p<0.001, Mann-Whitney test).

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Table 1. Accession numbers for abbreviations of gene name used in the text.

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Antibody and FcRgamma-deficient mice exhibit larger granulomas, a tendency to develop necrosis and greater larval motility

In addition to the increased numbers of larvae, we observed that JH−/− and FcRγ−/− mice exhibited larger granulomas at day 4 post challenge Hp infection. Assessment of hematoxylin and eosin (H&E) stained tissue sections revealed that granulomas in all sets of mice were characterized by epitheloid macrophages and eosinophils and often contained cuticle remains or intact larvae (Fig. 1B). Quantification of the inflamed area for lesions containing intact larvae (Fig. 1B, left column), or for lesions without intact larvae (Fig. 1B, right column), showed that JH−/− and FcRγ−/− mice had indeed developed more extensive granulomas around intact larvae (Fig. 1C) and larger “cysts” without visible larvae (Fig. 1D). Finally, pathological scoring of H&E stained granuloma sections showed an increased tendency to develop necrosis in both JH−/− and FcRγ−/− mice (Fig. 1E).

We rationalized that a failure to efficiently trap larvae in the granuloma might contribute to the observed increase in tissue necrosis. As the assessment of larval numbers by a modified Baermann apparatus only gives an indirect measure of larval motility [16], we quantified in-tissue motility directly by microscopy. Our analysis of time-lapse movies (Text S1, Fig. S1) showed that in-tissue motility was increased in the absence of antibodies or activating antibody receptors (Movie S1, Fig. 1F). During our analysis, we also noted pronounced differences in the size of larvae between wildtype, FcRγ−/− and JH−/− mice (Fig. 1G). We therefore calculated the difference in the larval position after 60s relative to the total larval area. In FcRγ−/− and JH−/− mice, the movement of the larvae was still observed to be greater than that seen for wildtype mice even when the movement was normalized to larval size (Fig. 1H).

Cellular compositions of granulomas are similar in wildtype and antibody-deficient mice

Due to the apparent involvement of cellular activation via Fc receptors in antibody-mediated protection against Hp (Fig. 1) we performed a flow cytometric analysis of granuloma cell populations, with a focus on macrophages and granulocytes as major FcR expressing cells. As the absence of antibodies resulted in a defective immune response already at day 4 post challenge infection, we compared granuloma cell populations from challenge infected JH−/− and wildtype mice by flow cytometry using the gating strategy described in Fig. S2A. No significant differences in the cellular infiltrate were noted between the two strains, with the exception of basophils that were absent from the granuloma of antibody-deficient mice (Fig. S2B). The absence of basophils observed in JH−/− mice was in keeping with our previous work, showing an important role for antibodies in basophil expansion during Hp infection [40]. However, basophils represent a minor cell population in the granuloma and basophil depletion during challenge infection only had a minor impact on protective immunity [40]. Thus, we concluded that antibody-FcRγ-chain-mediated protection is likely to involve other cell types.

Granuloma macrophages express high levels of antibody receptors and efficiently bind IgG1 and IgG3

The similarities in the cell recruitment between wildtype and antibody deficient mice suggested that antibodies might differentially activate resident or recruited cells at the site of infection. As our previous data indicated a predominant role of IgG [30] we analysed surface IgG on macrophages (F4/80high, Ly6G), eosinophils (SSChigh, SiglecF+) and neutrophils (Ly6G+ Gr1+) by flow cytometry. Our analysis showed that macrophages, which were the most abundant cell type in day 4 granulomas, displayed the highest surface levels of IgG, with eosinophils exhibiting moderate binding and neutrophils very little binding (Fig. 2A, left panel). We then confirmed the different IgG binding capacities of macrophages and eosinophils in vitro with bone marrow derived cells incubated with Hp immune serum (Fig. 2A, right panel). Further characterization of granuloma macrophages by flow cytometry showed high surface expression of activating FcγRs (CD64 and CD16) as well as CD11b, which is involved in complement-mediated immune complex binding [41] (Figs. 2B, S2C). In addition, macrophages displayed low levels of IgE but considerable levels of IgG1 and IgG3 on their surface (Fig. 2C).

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Figure 2. Macrophages are the major IgGhigh population in the granuloma following challenge Hp infection and express high levels of antibody and complement receptors.

(A) 25–30 granuloma were excised from the small intestine of challenge Hp infected mice on day 4 p.i. and isolated granuloma cells (left panels) or immune serum activated bone marrow derived cells (right panels) were stained for cell surface markers and IgG. (B) Expression of CD64, CD16/32 and CD11b on granuloma macrophages from C57BL/6 or FcRγ−/− mice; (C) Levels of IgE, IgG1 and IgG3 on granuloma macrophages from C57BL/6 or FcRγ−/− mice; Representative FACS plots from two independent experiments with 4–5 mice per group are shown.

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When comparing granuloma macrophages in challenge Hp infected wildtype and FcRγ−/− mice, we observed that FcRγ−/− mice displayed reduced levels of surface IgG1 and IgG3 (Figs. 2C, S2C). Interestingly CD11b levels were also reduced on granuloma macrophages of FcRγ−/− mice (Fig. S2C). Thus, in addition to the engagement of activating Fc receptors, helminth-antigen-antibody immune complexes might bind to CD11b in a complement dependent manner [42] [43] [44].

Antibodies act to upregulate Arginase-1 expression by macrophages in vitro and in vivo

We next investigated potential differences in the activation of macrophages in the presence or absence of specific antibodies following challenge infection with Hp. As RNA samples from granulomas were not of sufficient quality for microarray analysis, we performed in vitro experiments with bone marrow-derived macrophages (BMMac) that we incubated with Hp immune serum (1∶50, v∶v) and infective Hp L3 larvae (500 larvae/106 cells). Gene expression was compared using microarray analysis of macrophages incubated with larvae alone or with larvae in combination with immune serum. The microarray analysis identified a total of 216 genes that were differentially expressed (up- or down- regulated more than 1.5 fold). Among the ten most downregulated genes, we identified several TH1 associated genes such as interleukin 12b (IL12b), interferon regulatory factor 1 (Irf1) and interferon inducible GTPase 1 (Iigp1). However for our further experiments, we focused on the ten genes that were found to be upregulated more than two-fold by the combination of immune serum and larvae. Of note, these included several factors involved in tissue repair processes such as angiogenesis, cell proliferation, and remodeling as well as granulocyte recruitment and activation, or type 2 immunity (Table 2). Subsequent qPCR analysis confirmed the significant up-regulation of CXCL3, CXCL2, IL-33, Jag1 and Arg1 (Figs. S3, 3A). However, Arg1 was the only gene that was upregulated to a lesser extent in macrophages from FcRγ−/− mice. Moreover, Arg1 was moderately upregulated when BMMac were stimulated with larvae and purified 2° IgG (100 µg/ml), which could be greatly enhanced, when naïve serum was added together with 2° IgG and larvae (Fig. S3J). Hence, helminth-specific IgG can reprogram macrophages and this effect is amplified by the presence of complement components.

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Figure 3. Arg1 is induced by immune serum and Hp larvae in vitro and Arg1 expressing macrophages are less abundant in the granulomas from antibody and FcRgamma-chain deficient mice.

(A) Arg1 expression in BMMac from C57BL/6 or FcRγ−/− mice cultured in the presence or absence of larvae and/or immune serum was quantified by qPCR. (B) Paraffin sections of the upper duodenum of challenge Hp (day 4 p.i.) infected C57BL/6, FcRγ−/− or JH−/− mice were immune-fluorescently stained for Arg1 (green), F4/80 (red) and nuclei (blue), representative pictures are shown. (C–F) Intensities of Arg1 and F4/80 were quantified using Fiji and Cell Profiler software. (C) Scatter plots of intensities for F4/80 and Arg1 for all detected cells in the region of interest (ROI) (D) Frequency of Arg1+ F4/80+ (intensity >0.035) cells in the ROI, (E) Mean intensity of Arg1 and F4/80 staining in all detected cells in the ROI (F) Total intensity of Arg1 and F4/80 staining per ROI; Pooled data from two independent experiments with 3–6 mice per group are shown as mean + SEM (*p<0.05, **p<0.01); (G) Arg1+ macrophages are absent from the granuloma of 1° Hp infected mice (day 14 p.i.), representative picture.

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Table 2. Fold induction and function of the 10 most upregulated genes in macrophages co-cultured with immune serum in combination with larvae as compared to larvae alone.

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We next investigated the impact of antibodies on Arg1 protein expression by macrophages in vivo. Arg1 expression in granuloma sections from wildtype, JH−/− or FcRγ−/− mice was analysed by immunofluorescence and confocal microscopy (Fig. 3B). In keeping with our gene expression data, granulomas of antibody-deficient mice harbored significantly lower numbers of Arg1high macrophages (Fig. 3B). Quantification of the intensities of the Arg1 and F4/80 staining for each cell resulted in scatter plots, which clearly showed that most F4/80+ cells expressed Arg1 in the granuloma of C57BL/6 but not of FcRγ−/− or JH−/− mice (Fig. 3C). Furthermore, significant differences were noted not only for the frequency of Arg1high macrophages (Fig. 3D) but also for the mean Arg1 intensity in all detected cells (Fig. 3E) and the total Arg1 intensities in the region of interest (Fig. 3F). Of note, we could not detect any Arg1 expressing cells in the granuloma of 1° Hp infected C57BL/6 mice (Fig. 3G), indicating that effector mechanisms that arise only following challenge infection are required for Arg1 upregulation.

Immune serum from challenge-infected mice induces the adherence of macrophages to Hp larvae in vitro

In our in vitro co-cultures of bone marrow derived macrophages (BMMac) and larvae, we observed that immune serum from challenge Hp infected C57BL/6 mice (collected at day 4 p.i.) induced the adherence of macrophages to larvae (Fig. 4A). By contrast, serum from 1° Hp infected C57BL/6 or challenge Hp infected JH−/− mice showed an attenuated capacity to induce macrophage adherence (Fig. 4A). Immune serum-induced adherence could be confirmed using macrophages from the peritoneal cavity of naïve mice (Fig. S4A), or using BMMac of a different genetic background (Balbc) (Fig. S4B). As IgG+ eosinophils were also present in the granuloma, we additionally tested the ability of these cells to adhere to larvae. Bone marrow-derived eosinophils activated with immune serum hardly adhered to larvae, and addition of these cells to macrophage cultures decreased rather than increased macrophage adherence (Fig. S4B). These data indicate that Hp-specific antibodies predominantly act on macrophages to promote cellular adherence to larvae.

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Figure 4. Antibodies from challenge Hp infected wildtype mice induce adherence of bone marrow derived macrophages to Hp larvae in vitro, causing larval immobilization dependent on FcRgamma-chain and complement component 3; Larval infectivity and viability is not affected by adhering macrophages.

(A) BMMac from C57BL/6 mice were co-cultured with larvae in the presence or absence of serum from 1° or challenge Hp infected C57BL/6 or JH−/− mice, adherent macrophages per larva were counted in light microscopy images. (B) Correlation of the number of adhering BMMac and larval motility, (C/D) C57BL/6 or FcRγ−/− BMMac were cultured with larvae in the presence or absence of immune serum from C57BL/6 or FcRγ*C3−/− mice. Adherent macrophages per larva were counted in light microscopy images (C) and larval motility was quantified by Fiji (D). (E) Representative time lapse pictures, (F) Larval infectivity was analysed by infecting C57BL/6 wildtype mice with 100 larvae recovered from BMMac co-cultures in the presence or absence of immune serum. Adult worms in the three parts of the small intestine were counted on day 10 p.i.. (G) The larval viability relative to control (cultured without BMMac) after co-culture with or without immune serum was quantified by ATP-assay. Pooled data from three independent experiments with bone marrow from 2–3 mice or with 5 mice per group are shown as mean + SEM (*p<0.05, **p<0.01, ***p<0.001, Mann-Whitney test).

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To rule out a possible contribution of IL-4 from immune serum we determined the role of this cytokine in macrophage adherence. Addition of IL-4 to macrophage larvae co-cultures did not alter immune serum-induced macrophage adherence and larval immobilization (Fig. S5A) or larval fitness (Fig. S5B), and levels of IL-4 or IL-13 present in the immune serum or macrophage-larvae co-culture supernatants were all below the ELISA detection limit (Figs. S5C, D).

Macrophages immobilize Hp larvae in an antibody-, FcγR- and complement-dependent manner

We also noted that larvae covered in helminth-antibody activated macrophages exhibited a striking reduction of larval motility. To analyze this in a quantitative manner we developed a method to analyze larval motility in time-lapse movies. This analysis showed a clear correlation between the number of adherent macrophages per larvae and the reduction in larval motility, measured as the difference in the larval shape between consecutive frames of time-lapse movies (Fig. 4B) (detailed description Fig. S1B–D). We next explored the role of activating FcRs and complement in immune serum-induced macrophage adherence to Hp larvae. When co-cultured with larvae in the presence of immune serum, FcRγ−/− macrophages adhered less efficiently to larvae as compared to wildtype macrophages (Fig. 4C). Using BMMac from mice lacking all IgG receptors (FcγRI/II/III/IV−/−), we confirmed the important role of IgG for efficient trapping of larvae (Fig. S4E). As expected, FcγRI/II/III/IV−/− displayed no detectable surface IgG, CD16/32 (FcγRII/III) or CD64 (FcγRI) and both FcγRI/II/III/IV−/− and C57BL/6 BMMac did not show IgM binding (Fig. S4F). To rule out a possible role of IgE for larval trapping, we studied macrophage adherence and larval immobilization, using immune serum from challenge infected IgE−/− mice. IgE-deficient immune serum potently induced BMMac adherence as well as larval immobilization (Figs. S4G, H).

As for FcRγ−/− mice, our previous work had shown a small but significant contribution of complement to reducing adult worm numbers following challenge Hp infection [30]. Thus to determine a possible contribution of complement in larval immobilization we used complement component 3 (C3)-deficient immune serum in our macrophage assays. C3-deficient immune serum had a clearly reduced ability to promote macrophage adherence to wildtype macrophages and adherence of macrophages from FcRγ−/− mice was completely abolished (Fig. 4C). Moreover, FcRγ−/− macrophages had a lower capacity to trap larvae in the presence of wildtype immune serum, and failed completely to immobilize larvae when incubated with C3-deficient immune serum (Figs. 4D/E) (Movie S2). These data indicate that immune serum acts to promote macrophage adherence and larval immobilization in a manner involving the contribution of activating FcRs and complement.

Of note, immune serum also triggered adherence of BMMac to tissue dwelling L4 stage larvae, which we recovered from the small intestine of infected mice. Although L4 larvae were less motile than infective L3 larvae, immune-serum induced macrophage adherence also had a negative effect on their motility in vitro (Movie S3).

In order to study a potential effect of immune serum-induced macrophage adherence on larval fitness, we next infected mice with larvae recovered from immune serum-supplemented macrophage co-cultures. Adult worm counts on day 10 p.i. indicated that larvae cultured with BMMac and immune serum for 24h were still as infective as larvae cultured with macrophages alone (Fig. 4F). As another measure of larval fitness, we quantified ATP levels in larval homogenates following their recovery from macrophage co-cultures. Again we could not detect any significant difference in the viability after culture with BMMac alone, or with a combination of immune serum and BMMac (Fig. 4G). Taken together these data suggest that helminth-antibody-dependent macrophage activation promotes their adherence to result in an impact on larval motility but not on larval viability.

Absence of Arginase-1 reduces immune serum-induced larval trapping

Our previous data demonstrated that antibody-FcγR interactions upregulated Arg1 gene expression in macrophages co-cultured with larvae in vitro, and were necessary for Arg1 protein expression by granuloma macrophages in vivo. Moreover Arg1 has previously been reported to contribute to protective immunity against Hp [16]. We therefore investigated a possible contribution of this enzyme to macrophage adherence and larval immobilization. We performed in vitro co-cultures of bone marrow-derived macrophages from Arg1f/fTie2-Cre [45] [46] or Tie2-Cre [47] mice with Hp larvae in the presence of immune serum. The Tie2-Cre deleter was originally reported to result in Cre recombinase mediated gene ablation in endothelial cells [47], but was later discovered to show robust (80–90%) recombination also in hematopoietic cells [48]. We confirmed Arg1f/f and Tie2-Cre transgene expression and partial deletion of Arg1 in Arg1f/fTie2-Cre mice (Figs. S5H–J). We could not observe any difference in the capacity of Arg1f/fTie2-Cre and wildtype Arg1Tie2-Cre macrophages to adhere to larvae (Fig. 5A). However, Arg1f/fTie2-Cre macrophages had a reduced ability to immobilize larvae after incubation with immune serum (Figs. 5B, C) (Movie S4). To further confirm the role of Arg1 in antibody- mediated larval trapping, we added the Arg1 inhibitor S-(2-boronoethyl)-L-cysteine (BEC) to co-cultures of BMMac and larvae in the presence of immune serum. Addition of BEC in vitro resulted in reduced macrophage adherence and clearly impaired larval immobilization (Figs. 5, E).

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Figure 5. Arg1 is needed for to efficient larval trapping in vitro and in vivo and L-ornithine or polyamines but not urea can reduce larval motility in vitro.

(A/B) BMMac from Tie2-Cre or Argf/fTie2-Cre mice were co-cultured with larvae in the presence or absence of immune serum from C57BL/6 mice and adherence (A) and motility (B) were assessed by light microscopy. Dashed line in panel B depicts motility of larvae in the presence of BMMac without addition of immune serum. (C) Representative time lapse pictures; (D/E) Adherence and motility of C57BL/6 BMMac in the presence of the Arg1 inhibitor BEC (1 µM); (F/G) C57BL/6 mice were treated with BEC (0.2%) during challenge infection and larval motility (F) and numbers (G) were analysed; (H/I) Larvae were cultured in L-arginine free medium with or without supplementation with L-arginine (570 µM) +/− L-ornithine, putrescine, spermidine or spermine (each 100 µM) (H) or urea (I) and motility was quantified. Pooled data from two independent experiments (3–6 mice per group) are shown as mean + SEM (*p<0.05, **p<0.01, ***p<0.001, Mann-Whitney test).

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Finally, we tested the relevance of Arg1 in larval trapping in vivo, by treating challenge-infected C57BL/6 mice with BEC (0.2%, oral gavage, once daily). In keeping with previous findings [16], BEC treatment resulted in higher larval burdens (Fig. 5F). Moreover, larvae in BEC treated mice showed higher in-tissue motility (Fig. 5G, Movie S5).

Arg1 catalyzes the breakdown of L-Arginine into urea and L-ornithine, and BMMac produced considerable amounts of urea when activated with immune serum and Hp larvae (Fig. S5F). We first investigated a potential effect of L-arginine depletion by culturing larvae in L-arginine-free medium. However, we did not observe significant changes in the larval motility in the absence of L-arginine (Fig. 5H). As larvae are surrounded by large numbers of Arg1 expressing macrophages in challenge Hp infected mice, trapped larvae are likely to be exposed to high levels of Arg1 products. To replicate this process in vitro we incubated larvae with L-ornithine or polyamines (putrescine, spermidine and spermine) or urea. While urea did not significantly affect larval motility (even at 100 µM) (Fig. 5I), L-ornithine and polyamines clearly impaired larval movement (Fig. 5H, Movie S6). These data implicate L-arginine metabolism to polyamines as a possible mechanism by which Arg1 contributes to protective immunity against Hp.

Antibodies can induce Arg1 independently of IL-4Rα signaling

IL-4 has long been recognized as a major factor responsible for the upregulation of Arg1 [49]. Thus, we analysed Arg1 expression in BMMac after culture with larvae and immune serum in the presence of IL-4. IL-4 alone clearly induced Arg1 to similar levels as observed for immune serum (Fig. 6A), and the combination of IL-4 plus immune serum resulted in a slightly stronger Arg1 induction, both on the mRNA and enzymatic activity level (Figs. 6A, B).

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Figure 6. Antibodies can induce Arg1 and larval trapping by macrophages independently of IL-4Ralpha signaling.

(A) Arg1 expression in BMMac treated with larvae and immune serum and/or IL-4 or was quantified by qPCR and calculated relative to untreated control (B) Urea production in macrophages (treated as in (A)) was quantified after performing an Arg1 activity assay. (C) Sections of the small intestine from challenge-infected C57BL/6 or IL-4Ralpha−/− mice, treated with immune or naïve serum were stained for Arg1 (green) and F4/80 (red) and counterstained with DAPI (blue). Representative pictures from two independent experiments with 3–4 mice per group are shown. (D) Frequencies of Arg1+F4/80+ cells, mean intensities or total intensities of Arg1 and F4/80 were quantified using Fiji and Cell Profiler software. Dashed line depicts background fluorescence (E). Arg1 expression in wildtype or IL-4Ralpha−/− BMMac treated with larvae and immune serum was quantified by qPCR. (F/G) BMMac adherence (F) and larval motility (G) were analysed after co-culture with larvae in the presence or absence of immune serum. (H/I) Adherence (H) and motility (I) after treatment with larvae and purified 2° IgG −/+ naïve serum (NS). Dashed line depicts motility of larvae with BMMac alone. Pooled data from two independent experiments with 3–4 mice per group are shown as mean + SEM (*p<0.05, **p<0.01, ***p<0.001, Mann-Whitney test).

http://dx.doi.org/10.1371/journal.ppat.1003771.g006

Arg1 expression during helminth infection is normally associated with an AAM phenotype dependent upon IL-4Rα signaling [49] [16] [50]. Hence, we further investigated the role of IL-4Rα signaling on antibody-induced upregulation of Arg1 following challenge Hp infection. As IL-4Rα−/− mice exhibit a strongly impaired antibody response during primary and challenge infections with Hp [40], we treated challenge-infected mice with immune serum (collected at day 4 p.i.) from immune C57BL/6 mice. As shown in Fig. 6C, IL-4Rα−/− mice showed reduced numbers of F4/80+ macrophages surrounding tissue dwelling larvae. However, treatment with immune serum did result in an increase in Arg1+ macrophages within the granuloma of IL-4Rα−/− mice, indicating that Hp-specific antibodies can induce Arg1 expression in vivo independently of IL-4Rα signaling (Figs. 6C, D). To confirm that the ability of antibodies to trigger Arg1 expression in macrophages occurs independently of IL-4Rα signaling, we performed co-cultures of IL-4Rα−/− BMMac with Hp larvae. As shown in Fig. 6E, Arg1 was potently upregulated in IL-4Rα−/− BMMac in response to immune serum and larvae. Taken together these data indicate that IL-4 and antibodies can upregulate expression of Arg1 independently, but when present together will act in an additive manner.

Lastly, we investigated a potential role of IL-4Rα and other effector mechanisms that may contribute to immune serum-triggered larval trapping by macrophages. We observed that IL-4Rα−/− BMMac displayed a significantly reduced capacity to adhere (Fig. 6F), but were still able to immobilize larvae (Fig. 6G). Furthermore, IgG antibodies purified from challenge immune serum promoted weak adherence and moderate larval immobilization, which could be greatly enhanced by naïve serum (Figs. 6H, I). These data suggest that helminth specific antibodies together with complement components can trigger larval trapping even in the absence of other effector molecules that may be present in challenge immune serum.

Discussion

Numerous studies have documented a role for antibodies in providing protective immunity against helminths [34] [35] [36] [51] [37] [28] [30] [29], yet the mechanisms by which antibodies act against these large multicellular parasites have remained elusive. Our current work demonstrates a novel effector function of antibodies in activating macrophages to modulate the expression of genes normally associated with the alternatively activated phenotype. Antibody-mediated macrophage activation also triggered macrophage adherence to helminth larvae and resulted in a potent suppression of larval motility.

Interestingly, activating Fc receptors and complement C3 component acted together to activate macrophages and to limit larval motility in vitro. Crosstalk between the complement cascade and activating Fc receptors is well known to occur in autoimmunity [52]. We have previously shown that genetic ablation of either FcRγ-chain or the complement component C3 led to a significant but minor defect in the emergence of adult worms following challenge Hp infection between days 14–28. Our current data suggest that these two pathways act in a redundant and additive manner to promote antibody-induced larval trapping and to provide immunity against challenge Hp infections. The rapid activation of cells via Fc receptors is likely to be of particular importance during the early response (day 4) against the tissue dwelling larvae, as mice genetically deficient for FcRγ or B cells exhibited similar increases in larval motility and tissue necrosis within intestinal granulomas. The tissue dwelling stage of helminth larvae has been suggested to be a primary target of antibody-mediated protective immunity, which may be advantageous due to the immune-suppressive capacities of adult worms [53]. Our data further suggest that the antibody-mediated activation of granuloma macrophages mainly involves IgG1 and/or IgG3 isotypes, which may form immune complexes with helminth antigens or directly bind to the larval surface. As essential components of the larval cuticle are shared between different helminths, future work should delineate the specificities of protective antibody isotypes in order to identify potential vaccine targets.

Interestingly, although immune serum efficiently activated macrophages to immobilize larvae in vitro, it did not impact on larval viability. Given that the destruction of a large extracellular parasite such as Hp is likely to result in severe inflammation, the trapping and starvation of invasive larvae might be more beneficial for the host. In line with this view we observed that type 2-associated antibody production functioned to limit tissue necrosis and to induce tissue repair genes. Thus, type 2 immunity may act to limit larval migration through tissues, leading to a halt in the parasitic lifecycle and additionally serving to prevent excessive tissue damage. However we cannot rule out the possibility that other immune-mediated mechanisms also contribute to the killing of helminth larvae in vivo.

Of particular interest, arginase was required for the ability of helminth-antibody activated macrophages to inhibit larval motility as well as for efficient larval trapping in vivo. Arg1 expressing AAM are strongly associated with helminth infection [16] [54] [46] [55], and are typically regarded as controlling inflammation and tissue repair [46] [55] rather than as protective immune cells [56]. However, in keeping with our data Anthony et al. [16], previously reported a protective role for Arg1 expressing AAM in Hp infection. Our work expands on these findings to indicate that antibodies are essential for the robust Arg1 expression by macrophages following Hp infection. We also show that the Arg1 product L-ornithine and the polyamine metabolites putrescine, spermidine and spermine have a direct negative effect on larval motility, suggesting that excessive polyamines can impact on larval metabolism in a manner that interferes with motility. The finding that L-ornithine immobilizes larvae at concentrations that are below usual serum levels of this metabolite (around 50 µM) is surprising especially as granuloma may be expected to be a “leaky” environment. However, under homeostatic conditions, cell-free L-ornithine levels in the intestine might be relatively low due to the co-expression of Arginase 2 and L-ornithine metabolizing enzymes in enterocytes [57]. Thus, Arg1 activity might serve not only to control aberrant inflammation and fibrosis during parasite infection [46], but to also create a metabolically unfavorable environment for parasites. Future studies will be required to delineate the exact mechanism(s) by which polyamines affect larval motility.

Of note, IL-4Rα−/− mice failed to accumulate the large numbers of macrophages that were observed in the granuloma of wildtype mice, indicating that IL-4Rα signaling is necessary for the recruitment and/or expansion of macrophages at the site of infection [16] [58]. Thus, even if macrophages could immobilize larvae in the absence of IL-4Rα signaling in vitro, IL-4Rα signaling might be essential for the accumulation of macrophages in vivo, which can then be activated by antibodies to immobilize tissue invasive larvae. This suggests that in vivo, antibodies and IL-4 may work together to elicit a potent activation of Arg1 expressing macrophages, which mediate protection. This might be due to the convergence of IL-4Rα and FcRγ-chain signaling at the level of downstream signaling events such as Spleen tyrosine (Syk) or PI3 kinases [59] [60] [61]. In a lung model of Schistosoma infection, Arg1 expression in the granuloma was found to be tightly concentrated around the egg despite abundant AAM and type 2 cytokines (IL-4 and IL-13) in the overall granuloma environment [25]. Thus, it is tempting to speculate that antibodies directed against helminth eggs or larvae can promote local Arg1 expression in areas of high antigen availability.

Previous publications have indicated that macrophages activated by type 2 cytokines express high levels of Arg1 [16] [49] [27], whilst macrophages activated by immune complexes and TLR ligands in vitro express high levels of IL-10 and low levels of Arg1 [62]. In our hands, antibodies together with helminth larvae elicited potent Arg1 expression even in the absence of IL-4Rα signaling. Interestingly, immune complexes were previously shown to induce a robust FcR- and complement-mediated activation of C/EBPbeta, and C/EBPbeta has previously been associated with Arg1 induction in response to bacterial infection [63] [45], indicating it may also play a role in our model. Importantly the prominent expression of Arg1 by helminth-antibody activated macrophages did not correlate with other markers typical of AAM, such as Relmα. Thus the helminth-antibody activated macrophages described in this study seem to represent a novel “regulatory” macrophage type, which expresses Arg1 and wound healing genes in the absence of Relmα. The exact role of these helminth-antibody activated macrophages in tissue repair during the resolution phase of intestinal helminth infection would be of great interest for future studies.

In addition to the “canonical” pathway of Arg1 induction via the IL-4/IL-13/Stat6 pathway [64], Arg1 expression can be elicited via MyD88/IL-6/Stat3 and adenosine dependent mechanisms [65] [66]. Here, we have identified a novel pathway, which leads to the induction of Arg1 in the context of an adaptive type 2 response and which involves antibody-helminth interactions. The existence of multiple pathways for the induction of Arg1 may serve to achieve redundancy in an essential anti-inflammatory effector mechanism functional in many settings of infection, inflammation and wound healing.

In summary, we have demonstrated that antibodies mediate the activation of macrophages resulting in Arg1 expression and immobilization of tissue invasive helminth larvae. Macrophages often form a large component of the inflammatory infiltrate following allergen challenge or helminth infection, and our data indicate that antibodies form a previously unrecognized component of macrophage regulation during type 2 immune responses.

Materials and Methods

All animal experiments were approved by the office Affaires vétérinaires (1066 Epalinges, Canton Vaud, Sitzerland) with the authorization Number 2238 according to the guidelines set by the service de la consummation et des affaires vétérinaires federal (Canton Vaud, Switzerland).

Mice

C57BL/6, BALB/c, JH−/−, FcRγ-chain−/− [67], C3−/− [68], IgE−/− [69] and IL-4Rα−/− [70] were bred and maintained under specific pathogen free conditions at the Ecole Polytechnique Fédérale (EPFL) de Lausanne, Switzerland. FcγRI/II/III/IV−/− mice were bred and maintained at Leiden University, Netherlands and Arg1fl/flTie2-Cre [45] and Tie2-Cre [47] mice were bred and maintained at New Jersey Medical School, USA. The following primers were used for genotyping of Arg1fl/flTie2-Cre mice: wildtype Arg1/Arg1fl/fl: 5′-TGCGAGTTCATGACTAAGGTT-3′ (forward), 5′-AAAGCTCAGGTGAATCGG-3′(reverse); Tie2-Cre: 5′-CGCATAACCAGTGAAACAGCATTGC-3′ (forward) 5′-CCCTGTGCTCAGACAGAAATGAGA-3′ (reverse).

Infection, parasitology and collection of immune serum

C57BL/6 mice were infected with 200 Hp L3 larvae by oral gavage. Worms were cleared by treatment with two courses of Cobantril (Interdelta - Givisiez, Fribourg, Switzerland) 28 days after primary infection. 14 days later mice were challenge-infected with 200 larvae. BEC (Cayman Chemicals, Ann Arbor, MI) was administered (0.1 ml, 0.2%) once daily from the first day of infection by oral gavage. Mice were sacrificed on day 4 post infection and immune serum was collected from the inferior vena cava. Adult worms were counted under a stereomicroscope on longitudinally opened small intestines of 1° or challenge-infected mice. Tissue dwelling L4 larvae were counted after recovery with a modified Baermann apparatus [16].

Histology, immunofluorescence stainings and confocal microscopy

Serial paraffin sections were stained with hematoxylin and eosin. Granulomas were identified by light microscopy and serial sections were used for pathological scoring (details see Text S1) or immunofluorescence staining for Arginase-1 and F4/80. Stained tissue sections were imaged with an inverted point scanning confocal microscope (Zeiss LSM 710) with a Plan-Apochromat (63×/1.4 NA or 40×/1.3 NA) objective and pixel intensities were analysed using a custom built CellProfiler pipeline (for details see Text S1).

Flow cytometry

In vitro cultured cells or cells isolated from granuloma were stained with fluorescently labeled monoclonal antibodies (see Text S1) and acquired on a BD LSRII flow cytometer (BD, Franklin Lakes, NJ).

Luminescent Cell Viability Assay for assessment of larval viability

Larvae were recovered from co-cultures with macrophages with or without immune serum, washed thoroughly with enzyme free cell dissociation solution (Milipore, Billerica, MA) to remove all adherent cells, counted and homogenized in a cell/tissue homogenizer in RPMI1640 by using 0.1 mm Zirconia/Silica beads (BioSpec Products, Inc.). ATP levels in larval homogenates were analysed by CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI).

Culture and treatment of murine bone marrow cells and peritoneal macrophages

Peritoneal macrophages were isolated by plating peritoneal wash cells on petri dishes overnight and by removing non-adherent cells. Bone marrow was flushed from the femur and tibia of wildtype or transgenic mice and passed through cell strainers (70 µm). Cells (106/ml) were cultured in M-CSF (L929) supplemented medium (RPMI, 10% FCS, penicillin/streptomycin, β-mercaptoethanol) for 7 days as described previously [71]. On day 7, macrophages were harvested and stimulated for 24 h as indicated with L3 larvae (500 larvae/106 cells),(10 ng/ml) (Peprotech, Rocky Hill, NJ), or immune sera (1∶50 v/v). For differentiation into eosinophils, bone marrow cells were cultured as described [72] in the presence of Flt3 and SCF (Peprotech, Rocky Hill, NJ) for 4 days and in the presence of IL-5 (Peprotech, Rocky Hill, NJ) for subsequent 8 days. For RNA extraction, cells were re-suspended in Trizol and kept at −80°C.

Adherence of macrophages to larvae was determined by manual counting of bright field microscopy pictures taken with an Olympus AX70 microscope (UPLAN 10×/0.3 NA objective).

Measurement of larval motility

Time-lapse series (60 s: 20 frames, 3 s intervals) of larvae in the small intestine ex vivo or in vitro were acquired in order to measure their motility with an Olympus AX70 microscope (UPLAN 10×/0.3 NA objective). Larval motility was quantified by using custom-made Fiji macros (for details see Text S1).

qPCR and microarray analysis

RNA was extracted with a Direct-zol RNA MiniPrep kit (Zymo Research, Irvine, CA) and reverse transcribed using RevertAid cDNA synthesis reagents (Thermo Scientific, Waltham, MA) for qPCR analysis. QPCR was performed using SYBR Green I Master Mix (Eurogentec, Liege, Belgium) on an Applied Biosystems 7900HT System (for qPCR primer sequences see Table S1). Microarray analysis was performed using Affymetrix mouse arrays (Affymetrix, Santa Clara, CA, USA) (for details see Text S1).

ELISA

Concentrations of IL-4 or IL-13 in cell culture supernatants or mouse serum were quantified by using ELISA Ready-SET-Go! Kits (eBioscience, San Diego, CA).

Arginase-1 activity assay

Macrophage arginase-1 activity was determined according to previously published methods [73]. Briefly, adherent macrophages were lysed and conversion of L-arginine was quantified indirectly by measuring urea production with a QuantiChrom Urea Assay (BioAssay Systems, Hayward, CA).

Supporting Information

Figure S1.

Image processing for motility analysis ex vivo and in vitro and quantification of immune-fluorescent staining for Arg1 and F4/80. (A) First (a) and last (a′) frame of an ex vivo time-lapse acquisition. Mask obtained for the first (b) and last (b′) frame based on a manual drawing. XOR operator on first and last frame masks (c). Common parts to both masks become null (black) due to the XOR operation. (B) Montage of time-lapses series of a larva co-cultured with macrophages (Arg1f/fTie2-Cre) and immune serum in vitro (10 frames, 3 s interval). (C) Steps of the image processing. (a) Raw images, (b) “find edges” filtered images, (c) “median filter” filtered image, (d) “threshold manually selected” segmented image. (D) Temporal color code of the z-projection of mask series using the “Spectrum” look-up table (a). Z-Projections of mask (b, b′), contour (c, c′) and convex hull (d, d′) obtained for the time-lapses series from an in vitro experiment using knock-out (KO) (Arg1f/fTie2-Cre) (b, c, d) or wildtype (WT) (C57BL/6) macrophages (b′, c′, d′). (E) Original DAPI staining image (a), nuclei identified (b) and their outlines on the DAPI staining image (c); expanded nuclei mask used to measure in F4/80 and Arg1 channels (d).

doi:10.1371/journal.ppat.1003771.s001

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Figure S2.

Except for basophils, granuloma cell populations are largely overlapping in challenge Hp infected wildtype and antibody deficient mice. (A) Gating strategy for the flow cytometry analysis of granuloma cell populations; (B) Characterisation of granuloma cell populations in C57BL/6 and JH−/− mice on day 4 post challenge infection according to the gating strategy in (A). (C) MFI of CD11b, IgG1 and IgG3 on granuloma macrophages from challenge infected C57BL/6 and FcRγ−/− mice. Pooled data from two independent experiments with 4–6 mice per group are shown as mean + SEM (*p<0.05, **p<0.01).

doi:10.1371/journal.ppat.1003771.s002

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Figure S3.

Immune serum in combination with Hp larvae induces the expression of genes involved in granulocyte recruitment and activation, TH2 responses and wound healing and purified 2° IgG in combination with naïve serum upregulates Arg1 expression. mRNA levels normalized to GAPDH expression and relative to untreated cells for Cxcl3 (A), Cxcl2 (B), Il33 (C), Jag1 (D), S100A8 (E), Emp2 (F), Tpbpa (G), Trem1 (H), Inhba (I) or Arg1 (J). (A–I) Expression of the ten most-upregulated genes in BMMac treated with immune serum and larvae versus larvae alone identified by microarray was analysed by qPCR using cDNA from BMMac from C57BL/6 or FcRγ−/− mice cultured in the presence or absence of larvae and/or immune serum. (J) Expression of Arg1 after treatment with purified 2° IgG −/+ naïve serum. Pooled data from three independent experiments with bone marrow from 2–3 mice per group are shown as mean + SEM (*p<0.05, **p<0.01, ***p<0.001, Mann-Whitney test).

doi:10.1371/journal.ppat.1003771.s003

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Figure S4.

Antibody-induced adherence to Hp larvae occurs in different types of macrophages but not eosinophils and is independent of IgE but dependent on Fc receptors and IgG. (A) Adherence of peritoneal macrophages to larvae in response to immune serum from 1° and challenge Hp infected C57BL/6 mice. (B) Immune serum does not induce adherence of bone marrow derived eosinophils to Hp larvae. (C/D) Eosinophils fail to immobilize larvae. (E) FcγRI/II/III/IV−/− macrophages show reduced larval trapping. (F) Surface levels of IgG, IgM, CD16/32 (FcγRIII/II) and CD64 (FcγRI) on C57BL/6 and FcγRI/II/III/IV−/− macrophages was analysed by flow cytometry; (G/H) Immune serum from IgE−/− mice efficiently induces adherence (G) and immobilization (H); CD16 deficient macrophages show normal immune serum induced adherence (G) and a minor defect in larval immobilization (H); Peritoneal macrophages from C57BL/6 mice or BM-derived macrophages or eosinophils from C57BL/6, BalbC, or FcγRI/II/III/IV−/− mice were co-cultured with larvae in the presence or absence of immune serum from Hp infected C57BL/6, BalbC or IgE−/− mice for 24 h. Adherent macrophages per larva were counted in light microscopy images. Larval motility was quantified by Fiji (as described in Experimental Procedures). Immune serum activated BMMac were stained for surface IgG, IgM and antibody receptors. Pooled data and representative histograms from two independent experiments with peritoneal wash or bone marrow from 2–4 mice per group are shown as mean + SEM (*p<0.05, **p<0.01, ***p<0.001, Mann-Whitney test).

doi:10.1371/journal.ppat.1003771.s004

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Figure S5.

Presence of IL-4 does not change adherence or larval viability in co-cultures with macrophages and immune serum and co-culture supernatants contain negligible levels of IL-4 and IL-13; Genotyping and deletion efficiency for Arg1f/fTie2-Cre mice. (A-C) BMMac were cultured with larvae or larvae and immune serum (IS) in the presence or absence of IL-4 (10 ng/ml) for 24 h. (A/B) Macrophage adherence and larval motility were determined by light microscopy. (C) Larval viability was assessed by CellTiter-Glo Assay and normalized to the larval viability after culture for 24 h in BMMac medium without BMMac or IS. (D-G) IL-4 or IL-13 in serum (D/E) or cell culture supernatants (F/G) was quantified by ELISA. (H/I) Arg1f/f, WT Arg1 or Tie2-Cre transgene expression was determined in tissue biopsies from Arg1f/fTie2-Cre or Tie2-Cre mice. (J) Relative expression of Arg1 mRNA was determined in BMMac from the same mice as in H/I. Data from two independent experiments are shown as mean + SEM (***p<0.001, Mann-Whitney test).

doi:10.1371/journal.ppat.1003771.s005

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Movie S1.

In vivo motility of larvae in granuloma of antibody and FcRγ deficient versus wildtype mice. A piece of the upper duodenum of challenge Hp infected mice was removed at day 4 p.i. and carefully flattened between two glass slides for imaging with an Olympus AX70 microscope (10× objective). Movies of granulomas, containing clearly visible larvae were recorded over a time frame of 60 s (20 frames). Movies were processed using iMovie software. Representative examples are shown.

doi:10.1371/journal.ppat.1003771.s006

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Movie S2.

FcRγ and C3 dependent pathways contribute to immune serum induced trapping of Hp larvae by BMMac. Larvae were incubated with BMMac from C57BL/6 or FcRγ−/− mice in the presence of immune serum from C57BL/6 or FcRγ*C3−/− mice for 24 h. Suspensions from co-cultures were transferred to glass slides, carefully covered with cover slips and time lapse-movies were immediately recorded with an Olympus AX70 microscope. Movies were processed using iMovie software. Representative examples are shown.

doi:10.1371/journal.ppat.1003771.s007

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Movie S3.

Immune serum promotes adherence of BMMac to L4 larvae in vitro. Larvae recovered from the small intestine of challenge-infected mice were incubated with BMMac in the presence of or absence of immune serum for 24 h. Suspensions from co-cultures were transferred to glass slides, carefully covered with cover slips and time lapse-movies were immediately recorded with an Olympus AX70 microscope. Movies were processed using iMovie software. Representative examples are shown.

doi:10.1371/journal.ppat.1003771.s008

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Movie S4.

Arg1 deficiency impairs immune serum induced trapping of larvae by BMMac in vitro. Larvae were incubated with BMMac from Arg1f/fTie2-Cre or Tie2-Cre mice in the presence of immune serum from challenge Hp infected C57BL/6 mice for 24 h. Suspensions from co-cultures were transferred to glass slides, carefully covered with cover slips and time lapse-movies were immediately recorded with an Olympus AX70 microscope. Movies were processed using iMovie software. Representative examples are shown.

doi:10.1371/journal.ppat.1003771.s009

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Movie S5.

The arginase inhibitor BEC prevents larval immobilization during challenge infection in vivo. C57BL/6 mice were treated with S-(2-boronoethyl)-L-cysteine (BEC) (0.2%, 100 µl by oral gavage) during challenge Hp infection. Mice were sacrificed on day 4 p.i. and small intestines were removed. Pieces of the upper duodenum were carefully covered with cover slips and time lapse-movies were immediately recorded with an Olympus AX70 microscope. Movies were processed using iMovie software. Representative examples are shown.

doi:10.1371/journal.ppat.1003771.s010

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Movie S6.

L-ornithine and polyamines reduce larval motility in vitro. Larvae were incubated in BMMac medium supplemented with L-ornithine or polyamines (100 µM) for 24 h, aliquots were transferred to glass slides and time lapse movies were recorded immediately with an Olympus AX70 microscope. Movies were processed using iMovie software. Representative examples are shown.

doi:10.1371/journal.ppat.1003771.s011

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Text S1.

Supporting Materials and Methods and qPCR primer sequences. Detailed descriptions of experimental procedures, materials and data analysis methods including Fiji macros and Cell profiler pipelines can be found in the Supporting Information (Text S1).

doi:10.1371/journal.ppat.1003771.s012

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Acknowledgments

The authors would like to thank the staff of the Bioimaging and optics facility (EPFL), particularly Dr. Olivier Burri for help with microscopy and image analysis; the Genomic Technologies facility (University of Lausanne) for help with microarray analysis and the staff of the animal facility at EPFL for providing high-quality husbandry.

Author Contributions

Conceived and designed the experiments: JEvB NLH. Performed the experiments: JEvB IM AP FC BV. Analyzed the data: JEvB NLH. Contributed reagents/materials/analysis tools: RG WCG JSV AS. Wrote the paper: JEvB NLH.

References

  1. 1. Beaver PC (1975) Biology of soil-transmitted helminths: the massive infection. Health Lab Sci 12: 116–125.
  2. 2. Crompton DW (1988) The prevalence of Ascariasis. Parasitol Today (Regul Ed) 4: 162–169.
  3. 3. Stephenson LS, Holland CV, Cooper ES (2000) The public health significance of Trichuris trichiura. Parasitology 121 Suppl: S73–95.
  4. 4. Cooper PJ, Chico ME, Losonsky G, Sandoval C, Espinel I, et al. (2000) Albendazole treatment of children with ascariasis enhances the vibriocidal antibody response to the live attenuated oral cholera vaccine CVD 103-HgR. J Infect Dis 182: 1199–1206 doi:10.1086/315837.
  5. 5. Bethony J, Brooker S, Albonico M, Geiger SM, Loukas A, et al. (2006) Soil-transmitted helminth infections: ascariasis, trichuriasis, and hookworm. Lancet 367: 1521–1532 doi:10.1016/S0140-6736(06)68653-4.
  6. 6. Albonico M, Allen H, Chitsulo L, Engels D, Gabrielli A-F, et al. (2008) Controlling soil-transmitted helminthiasis in pre-school-age children through preventive chemotherapy. PLoS Negl Trop Dis 2: e126 doi:10.1371/journal.pntd.0000126.
  7. 7. Appleton CC, Mosala TI, Levin J, Olsen A (2009) Geohelminth infection and re-infection after chemotherapy among slum-dwelling children in Durban, South Africa. Ann Trop Med Parasitol 103: 249–261 doi:10.1179/136485909X398212.
  8. 8. Sangster NC (1999) Anthelmintic resistance: past, present and future. Int J Parasitol 29: 115–124 discussion 137–138.
  9. 9. Geerts S, Gryseels B (2000) Drug resistance in human helminths: current situation and lessons from livestock. Clin Microbiol Rev 13: 207–222.
  10. 10. Geerts S, Gryseels B (2001) Anthelmintic resistance in human helminths: a review. Trop Med Int Health 6: 915–921.
  11. 11. Galvani AP (2005) Age-dependent epidemiological patterns and strain diversity in helminth parasites. J Parasitol 91: 24–30 doi:10.1645/GE-191R1.
  12. 12. Taylor-Robinson DC, Maayan N, Soares-Weiser K, Donegan S, Garner P (2012) Deworming drugs for soil-transmitted intestinal worms in children: effects on nutritional indicators, haemoglobin and school performance. Cochrane Database Syst Rev 11: CD000371 doi:10.1002/14651858.CD000371.pub5.
  13. 13. Khieu V, Schär F, Marti H, Sayasone S, Duong S, et al. (2013) Diagnosis, treatment and risk factors of Strongyloides stercoralis in schoolchildren in Cambodia. PLoS Negl Trop Dis 7: e2035 doi:10.1371/journal.pntd.0002035.
  14. 14. Wynn TA, Chawla A, Pollard JW (2013) Macrophage biology in development, homeostasis and disease. Nature 496: 445–455 doi:10.1038/nature12034.
  15. 15. Aderem A, Underhill DM (1999) Mechanisms of phagocytosis in macrophages. Annu Rev Immunol 17: 593–623 doi:10.1146/annurev.immunol.17.1.593.
  16. 16. Anthony RM, Urban JF Jr, Alem F, Hamed HA, Rozo CT, et al. (2006) Memory T(H)2 cells induce alternatively activated macrophages to mediate protection against nematode parasites. Nat Med 12: 955–960 doi:10.1038/nm1451.
  17. 17. Gordon S (2003) Alternative activation of macrophages. Nat Rev Immunol 3: 23–35 doi:10.1038/nri978.
  18. 18. Camberis M, Le Gros G, Urban J Jr (2003) Animal model of Nippostrongylus brasiliensis and Heligmosomoides polygyrus. Curr Protoc Immunol Chapter 19: Unit 19.12 doi:10.1002/0471142735.im1912s55.
  19. 19. Robinson M, Wahid F, Behnke JM, Gilbert FS (1989) Immunological relationships during primary infection with Heligmosomoides polygyrus (Nematospiroides dubius): dose-dependent expulsion of adult worms. Parasitology 98(Pt 1): 115–124.
  20. 20. Urban JF Jr, Katona IM, Finkelman FD (1991) Heligmosomoides polygyrus: CD4+ but not CD8+ T cells regulate the IgE response and protective immunity in mice. Exp Parasitol 73: 500–511.
  21. 21. Urban JF Jr, Katona IM, Paul WE, Finkelman FD (1991) Interleukin 4 is important in protective immunity to a gastrointestinal nematode infection in mice. Proc Natl Acad Sci USA 88: 5513–5517.
  22. 22. Anthony RM, Rutitzky LI, Urban JF Jr, Stadecker MJ, Gause WC (2007) Protective immune mechanisms in helminth infection. Nat Rev Immunol 7: 975–987 doi:10.1038/nri2199.
  23. 23. Zimmermann N, King NE, Laporte J, Yang M, Mishra A, et al. (2003) Dissection of experimental asthma with DNA microarray analysis identifies arginase in asthma pathogenesis. J Clin Invest 111: 1863–1874 doi:10.1172/JCI17912.
  24. 24. Hesse M, Modolell M, La Flamme AC, Schito M, Fuentes JM, et al. (2001) Differential regulation of nitric oxide synthase-2 and arginase-1 by type 1/type 2 cytokines in vivo: granulomatous pathology is shaped by the pattern of L-arginine metabolism. J Immunol 167: 6533–6544.
  25. 25. Barron L, Smith AM, El Kasmi KC, Qualls JE, Huang X, et al. (2013) Role of arginase 1 from myeloid cells in th2-dominated lung inflammation. PLoS ONE 8: e61961 doi:10.1371/journal.pone.0061961.
  26. 26. Zhao A, Urban JF Jr, Anthony RM, Sun R, Stiltz J, et al. (2008) Th2 cytokine-induced alterations in intestinal smooth muscle function depend on alternatively activated macrophages. Gastroenterology 135: 217–225.e1 doi:10.1053/j.gastro.2008.03.077.
  27. 27. Yang Z, Grinchuk V, Urban JF Jr, Bohl J, Sun R, et al. (2013) Macrophages as IL-25/IL-33-Responsive Cells Play an Important Role in the Induction of Type 2 Immunity. PLoS ONE 8: e59441 doi:10.1371/journal.pone.0059441.
  28. 28. Wojciechowski W, Harris DP, Sprague F, Mousseau B, Makris M, et al. (2009) Cytokine-producing effector B cells regulate type 2 immunity to H. polygyrus. Immunity 30: 421–433 doi:10.1016/j.immuni.2009.01.006.
  29. 29. Liu Q, Kreider T, Bowdridge S, Liu Z, Song Y, et al. (2010) B cells have distinct roles in host protection against different nematode parasites. J Immunol 184: 5213–5223 doi:10.4049/jimmunol.0902879.
  30. 30. McCoy KD, Stoel M, Stettler R, Merky P, Fink K, et al. (2008) Polyclonal and specific antibodies mediate protective immunity against enteric helminth infection. Cell Host Microbe 4: 362–373 doi:10.1016/j.chom.2008.08.014.
  31. 31. Khoury PB, Stromberg BE, Soulsby EJ (1977) Immune mechanisms to Ascaris suum in inbred guinea-pigs. I. Passive transfer of immunity by cells or serum. Immunology 32: 405–411.
  32. 32. Behnke JM, Parish HA (1979) Expulsion of Nematospiroides dubius from the intestine of mice treated with immune serum. Parasite Immunol 1: 13–26.
  33. 33. Murrell KD (1981) Protective role of immunoglobulin G in immunity to Strongyloides ratti. J Parasitol 67: 167–173.
  34. 34. Woolhouse ME, Taylor P, Matanhire D, Chandiwana SK (1991) Acquired immunity and epidemiology of Schistosoma haematobium. Nature 351: 757–759 doi:10.1038/351757a0.
  35. 35. Hagel I, Lynch NR, Di Prisco MC, Rojas E, Pérez M, et al. (1993) Ascaris reinfection of slum children: relation with the IgE response. Clin Exp Immunol 94: 80–83.
  36. 36. Atmadja AK, Atkinson R, Sartono E, Partono F, Yazdanbakhsh M, et al. (1995) Differential decline in filaria-specific IgG1, IgG4, and IgE antibodies in Brugia malayi-infected patients after diethylcarbamazine chemotherapy. J Infect Dis 172: 1567–1572.
  37. 37. Blackwell NM, Else KJ (2001) B cells and antibodies are required for resistance to the parasitic gastrointestinal nematode Trichuris muris. Infect Immun 69: 3860–3868 doi:10.1128/IAI.69.6.3860-3868.2001.
  38. 38. Gurish MF, Bryce PJ, Tao H, Kisselgof AB, Thornton EM, et al. (2004) IgE enhances parasite clearance and regulates mast cell responses in mice infected with Trichinella spiralis. J Immunol 172: 1139–1145.
  39. 39. Chen J, Trounstine M, Alt FW, Young F, Kurahara C, et al. (1993) Immunoglobulin gene rearrangement in B cell deficient mice generated by targeted deletion of the JH locus. Int Immunol 5: 647–656.
  40. 40. Herbst T, Esser J, Prati M, Kulagin M, Stettler R, et al. (2012) Antibodies and IL-3 support helminth-induced basophil expansion. Proc Natl Acad Sci USA 109: 14954–14959 doi:10.1073/pnas.1117584109.
  41. 41. Sanchez-Madrid F, Nagy JA, Robbins E, Simon P, Springer TA (1983) A human leukocyte differentiation antigen family with distinct alpha-subunits and a common beta-subunit: the lymphocyte function-associated antigen (LFA-1), the C3bi complement receptor (OKM1/Mac-1), and the p150,95 molecule. J Exp Med 158: 1785–1803.
  42. 42. Díaz de Ståhl T, Dahlstrom J, Carroll MC, Heyman B (2003) A role for complement in feedback enhancement of antibody responses by IgG3. J Exp Med 197: 1183–1190 doi:10.1084/jem.20022232.
  43. 43. Hazenbos WL, Heijnen IA, Meyer D, Hofhuis FM, Renardel de Lavalette CR, et al. (1998) Murine IgG1 complexes trigger immune effector functions predominantly via Fc gamma RIII (CD16). J Immunol 161: 3026–3032.
  44. 44. Ding JW, Zhou T, Zeng H, Ma L, Verbeek JS, et al. (2008) Hyperacute rejection by anti-Gal IgG1, IgG2a, and IgG2b is dependent on complement and Fc-gamma receptors. J Immunol 180: 261–268.
  45. 45. El Kasmi KC, Qualls JE, Pesce JT, Smith AM, Thompson RW, et al. (2008) Toll-like receptor-induced arginase 1 in macrophages thwarts effective immunity against intracellular pathogens. Nat Immunol 9: 1399–1406 doi:10.1038/ni.1671.
  46. 46. Pesce JT, Ramalingam TR, Mentink-Kane MM, Wilson MS, El Kasmi KC, et al. (2009) Arginase-1-expressing macrophages suppress Th2 cytokine-driven inflammation and fibrosis. PLoS Pathog 5: e1000371 doi:10.1371/journal.ppat.1000371.
  47. 47. Kisanuki YY, Hammer RE, Miyazaki J, Williams SC, Richardson JA, et al. (2001) Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo. Dev Biol 230: 230–242 doi:10.1006/dbio.2000.0106.
  48. 48. Constien R, Forde A, Liliensiek B, Gröne HJ, Nawroth P, et al. (2001) Characterization of a novel EGFP reporter mouse to monitor Cre recombination as demonstrated by a Tie2 Cre mouse line. Genesis 30: 36–44.
  49. 49. Munder M, Eichmann K, Modolell M (1998) Alternative metabolic states in murine macrophages reflected by the nitric oxide synthase/arginase balance: competitive regulation by CD4+ T cells correlates with Th1/Th2 phenotype. J Immunol 160: 5347–5354.
  50. 50. Herbert DR, Orekov T, Perkins C, Rothenberg ME, Finkelman FD (2008) IL-4R alpha expression by bone marrow-derived cells is necessary and sufficient for host protection against acute schistosomiasis. J Immunol 180: 4948–4955.
  51. 51. Nyindo M, Kariuki TM, Mola PW, Farah IO, Elson L, et al. (1999) Role of adult worm antigen-specific immunoglobulin E in acquired immunity to Schistosoma mansoni infection in baboons. Infect Immun 67: 636–642.
  52. 52. Schmidt RE, Gessner JE (2005) Fc receptors and their interaction with complement in autoimmunity. Immunol Lett 100: 56–67 doi:10.1016/j.imlet.2005.06.022.
  53. 53. Harris NL, Pleass R, Behnke JM (2013) Understanding the role of antibodies in murine infections with Heligmosomoides (polygyrus) bakeri: 35 years ago, now and 35 years ahead. Parasite Immunol doi:10.1111/pim.12057.
  54. 54. Marsland BJ, Kurrer M, Reissmann R, Harris NL, Kopf M (2008) Nippostrongylus brasiliensis infection leads to the development of emphysema associated with the induction of alternatively activated macrophages. Eur J Immunol 38: 479–488 doi:10.1002/eji.200737827.
  55. 55. Chen F, Liu Z, Wu W, Rozo C, Bowdridge S, et al. (2012) An essential role for T(H)2-type responses in limiting acute tissue damage during experimental helminth infection. Nature Medicine 18: 260–6 Available: http://www.ncbi.nlm.nih.gov/pubmed/22245779. Accessed 19 January 2012.
  56. 56. Murray PJ, Wynn TA (2011) Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol 11: 723–737 doi:10.1038/nri3073.
  57. 57. Ozaki M, Gotoh T, Nagasaki A, Miyanaka K, Takeya M, et al. (1999) Expression of arginase II and related enzymes in the rat small intestine and kidney. J Biochem 125: 586–593.
  58. 58. Jenkins SJ, Ruckerl D, Cook PC, Jones LH, Finkelman FD, et al. (2011) Local macrophage proliferation, rather than recruitment from the blood, is a signature of TH2 inflammation. Science 332: 1284–1288 doi:10.1126/science.1204351.
  59. 59. Indik ZK, Park JG, Hunter S, Schreiber AD (1995) The molecular dissection of Fc gamma receptor mediated phagocytosis. Blood 86: 4389–4399.
  60. 60. Ennaciri J, Girard D (2009) IL-4R(alpha), a new member that associates with Syk kinase: implication in IL-4-induced human neutrophil functions. J Immunol 183: 5261–5269 doi:10.4049/jimmunol.0900109.
  61. 61. Weisser SB, McLarren KW, Voglmaier N, van Netten-Thomas CJ, Antov A, et al. (2011) Alternative activation of macrophages by IL-4 requires SHIP degradation. Eur J Immunol 41: 1742–1753 doi:10.1002/eji.201041105.
  62. 62. Edwards JP, Zhang X, Frauwirth KA, Mosser DM (2006) Biochemical and functional characterization of three activated macrophage populations. J Leukoc Biol 80: 1298–1307 doi:10.1189/jlb.0406249.
  63. 63. Yan C, Zhu M, Staiger J, Johnson PF, Gao H (2012) C5a-regulated CCAAT/enhancer-binding proteins β and δ are essential in Fcγ receptor-mediated inflammatory cytokine and chemokine production in macrophages. J Biol Chem 287: 3217–3230 doi:10.1074/jbc.M111.280834.
  64. 64. Rutschman R, Lang R, Hesse M, Ihle JN, Wynn TA, et al. (2001) Cutting edge: Stat6-dependent substrate depletion regulates nitric oxide production. J Immunol 166: 2173–2177.
  65. 65. Qualls JE, Neale G, Smith AM, Koo M-S, DeFreitas AA, et al. (2010) Arginine usage in mycobacteria-infected macrophages depends on autocrine-paracrine cytokine signaling. Sci Signal 3: ra62 doi:10.1126/scisignal.2000955.
  66. 66. Csóka B, Selmeczy Z, Koscsó B, Németh ZH, Pacher P, et al. (2012) Adenosine promotes alternative macrophage activation via A2A and A2B receptors. FASEB J 26: 376–386 doi:10.1096/fj.11-190934.
  67. 67. Takai T, Li M, Sylvestre D, Clynes R, Ravetch JV (1994) FcR gamma chain deletion results in pleiotrophic effector cell defects. Cell 76: 519–529.
  68. 68. Wessels MR, Butko P, Ma M, Warren HB, Lage AL, et al. (1995) Studies of group B streptococcal infection in mice deficient in complement component C3 or C4 demonstrate an essential role for complement in both innate and acquired immunity. Proc Natl Acad Sci USA 92: 11490–11494.
  69. 69. Oettgen HC, Martin TR, Wynshaw-Boris A, Deng C, Drazen JM, et al. (1994) Active anaphylaxis in IgE-deficient mice. Nature 370: 367–370 doi:10.1038/370367a0.
  70. 70. Mohrs M, Ledermann B, Köhler G, Dorfmüller A, Gessner A, et al. (1999) Differences between IL-4- and IL-4 receptor alpha-deficient mice in chronic leishmaniasis reveal a protective role for IL-13 receptor signaling. J Immunol 162: 7302–7308.
  71. 71. Hoss A, Zwarthoff EC, Zawatzky R (1989) Differential expression of interferon alpha and beta induced with Newcastle disease virus in mouse macrophage cultures. J Gen Virol 70(Pt 3): 575–589.
  72. 72. Dyer KD, Moser JM, Czapiga M, Siegel SJ, Percopo CM, et al. (2008) Functionally competent eosinophils differentiated ex vivo in high purity from normal mouse bone marrow. J Immunol 181: 4004–4009.
  73. 73. Allen JE, Wynn TA (2011) Evolution of Th2 immunity: a rapid repair response to tissue destructive pathogens. PLoS Pathog 7: e1002003 doi:10.1371/journal.ppat.1002003.
  74. 74. Geiser T, Dewald B, Ehrengruber MU, Clark-Lewis I, Baggiolini M (1993) The interleukin-8-related chemotactic cytokines GRO alpha, GRO beta, and GRO gamma activate human neutrophil and basophil leukocytes. J Biol Chem 268: 15419–15424.
  75. 75. Wente MN, Keane MP, Burdick MD, Friess H, Büchler MW, et al. (2006) Blockade of the chemokine receptor CXCR2 inhibits pancreatic cancer cell-induced angiogenesis. Cancer Lett 241: 221–227 doi:10.1016/j.canlet.2005.10.041.
  76. 76. Benedito R, Roca C, Sörensen I, Adams S, Gossler A, et al. (2009) The notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis. Cell 137: 1124–1135 doi:10.1016/j.cell.2009.03.025.
  77. 77. Bailis W, Yashiro-Ohtani Y, Fang TC, Hatton RD, Weaver CT, et al. (2013) Notch simultaneously orchestrates multiple helper T cell programs independently of cytokine signals. Immunity 39: 148–159 doi:10.1016/j.immuni.2013.07.006.
  78. 78. Wadehra M, Iyer R, Goodglick L, Braun J (2002) The tetraspan protein epithelial membrane protein-2 interacts with beta1 integrins and regulates adhesion. J Biol Chem 277: 41094–41100 doi:10.1074/jbc.M206868200.
  79. 79. Gordon LK, Kiyohara M, Fu M, Braun J, Dhawan P, et al. (2013) EMP2 regulates angiogenesis in endometrial cancer cells through induction of VEGF. Oncogene doi:10.1038/onc.2012.622.
  80. 80. Bouchon A, Dietrich J, Colonna M (2000) Cutting edge: inflammatory responses can be triggered by TREM-1, a novel receptor expressed on neutrophils and monocytes. J Immunol 164: 4991–4995.
  81. 81. Bleharski JR, Kiessler V, Buonsanti C, Sieling PA, Stenger S, et al. (2003) A role for triggering receptor expressed on myeloid cells-1 in host defense during the early-induced and adaptive phases of the immune response. J Immunol 170: 3812–3818.
  82. 82. Cheng P-C, Lin C-N, Chen Y-J, Chang F-S, Tsaihong JC, et al. (2011) Triggering receptor expressed on myeloid cells (TREM)-1 participates in Schistosoma mansoni inflammatory responses. Parasite Immunol 33: 276–286 doi:10.1111/j.1365-3024.2011.01284.x.
  83. 83. Hu D, Cross JC (2011) Ablation of Tpbpa-positive trophoblast precursors leads to defects in maternal spiral artery remodeling in the mouse placenta. Dev Biol 358: 231–239 doi:10.1016/j.ydbio.2011.07.036.
  84. 84. Ehrchen JM, Sunderkötter C, Foell D, Vogl T, Roth J (2009) The endogenous Toll-like receptor 4 agonist S100A8/S100A9 (calprotectin) as innate amplifier of infection, autoimmunity, and cancer. J Leukoc Biol 86: 557–566 doi:10.1189/jlb.1008647.
  85. 85. Passey RJ, Xu K, Hume DA, Geczy CL (1999) S100A8: emerging functions and regulation. J Leukoc Biol 66: 549–556.
  86. 86. Wills-Karp M, Rani R, Dienger K, Lewkowich I, Fox JG, et al. (2012) Trefoil factor 2 rapidly induces interleukin 33 to promote type 2 immunity during allergic asthma and hookworm infection. J Exp Med 209: 607–622 doi:10.1084/jem.20110079.
  87. 87. Phillips DJ, Woodruff TK (2004) Inhibin: actions and signalling. Growth Factors 22: 13–18.
  88. 88. Ogawa K, Funaba M, Chen Y, Tsujimoto M (2006) Activin A functions as a Th2 cytokine in the promotion of the alternative activation of macrophages. J Immunol 177: 6787–6794.