Alisha Chetty,Matthew G. Darby,Jamie Pillaye,A'ishah Taliep,Adam F. Cunningham,Matthew K. O’Shea,Gnatoulma Katawa,Laura E. Layland,Manuel Ritter,William G. C. Horsnell
Induction of Siglec-FhiCD101hi eosinophils in the lungs following murine hookworm Nippostrongylus brasiliensis infection
helminths,Nippostrongylus brasilienis,eosinophils,Siglec-F,CD101,ILC2s,lung
Helminth-induced eosinophils accumulate around the parasite at the site of infection, or in parasite-damaged tissues well after the helminth has left the site. The role of helminth-elicited eosinophils in mediating parasite control is complex. While they may contribute to direct parasite-killing and tissue repair, their involvement in long-term immunopathogenesis is a concern. In allergic Siglec-FhiCD101hi, eosinophils are associated with pathology. Research has not shown if equivalent subpopulations of eosinophils are a feature of helminth infection. In this study, we demonstrate that lung migration of rodent hookworm Nippostrongylus brasiliensis (Nb) results in a long-term expansion of distinct Siglec-FhiCD101hi eosinophil subpopulations. Nb-elevated eosinophil populations in the bone marrow and circulation did not present this phenotype. Siglec-FhiCD101hi lung eosinophils exhibited an activated morphology including nuclei hyper-segmentation and cytoplasm degranulation. Recruitment of ST2+ ILC2s and not CD4+ T cells to the lungs was associated with the expansion of Siglec-FhiCD101hi eosinophils. This data identifies a morphologically distinct and persistent subset of Siglec-FhiCD101hi lung eosinophils induced following Nb infection. These eosinophils may contribute to long-term pathology following helminth infection.
Induction of Siglec-FhiCD101hi eosinophils in the lungs following murine hookworm Nippostrongylus brasiliensis infection

Helminth-induced eosinophils accumulate around the parasite at the site of infection, or in parasite-damaged tissues well after the helminth has left the site. The role of helminth-elicited eosinophils in mediating parasite control is complex. While they may contribute to direct parasite-killing and tissue repair, their involvement in long-term immunopathogenesis is a concern. In allergic Siglec-FhiCD101hi, eosinophils are associated with pathology. Research has not shown if equivalent subpopulations of eosinophils are a feature of helminth infection. In this study, we demonstrate that lung migration of rodent hookworm Nippostrongylus brasiliensis (Nb) results in a long-term expansion of distinct Siglec-FhiCD101hi eosinophil subpopulations. Nb-elevated eosinophil populations in the bone marrow and circulation did not present this phenotype. Siglec-FhiCD101hi lung eosinophils exhibited an activated morphology including nuclei hyper-segmentation and cytoplasm degranulation. Recruitment of ST2+ ILC2s and not CD4+ T cells to the lungs was associated with the expansion of Siglec-FhiCD101hi eosinophils. This data identifies a morphologically distinct and persistent subset of Siglec-FhiCD101hi lung eosinophils induced following Nb infection. These eosinophils may contribute to long-term pathology following helminth infection.

Eosinophils are a canonical feature of helminth infection. Several helminths have life cycles that involve larval migration through the human lung tissue which can be associated with significant pathology such as Loeffler’s syndrome (1). However, our understanding of how lung eosinophil responses and, in particular, their heterogeneity are influenced by helminth infection is not as comprehensive as in the case of, for example, allergic inflammation (2–4). Studies of allergic asthma have shown distinct roles of eosinophil subpopulations. For example, Siglec-Fhi ‘inflammatory’ eosinophil subsets in the allergic lungs are recruited after an allergen challenge and contribute to lung pathology (2, 5, 6). Conversely, Siglec-Fint ‘steady-state’ eosinophils associate with tissue homeostasis and protection from pathology (2, 4). The induction of such eosinophil subpopulations following helminth infection is incompletely understood. Lung involvement is a feature of the life cycle of many common human helminth infections (e.g., Ascaris sp. and hookworm), and subsequent pathology may result from parasite migration through the lungs prior to the establishment of the patent intestinal infection (7, 8). Such pathology can be striking; for example, infection with murine hookworm Nippostrongylus brasiliensis (Nb) can result in significant mechanical damage to the alveolar architecture following the larval transition from the circulatory system into the airways (9). This leads to a significant accumulation of eosinophils in the lungs and the presentation of acute allergic-like pathology (10–12). While this acute damage is transient, mice typically develop a chronic emphysema-like pathology (13). The relevance of this modeling to human pathology is supported by a recent study identifying human exposure to Ascaris lumbricoides associated with a decline in lung function later in life (14). In this study, we investigated the influence of Nb infection on the composition of Siglec-Fhi and Siglec-Fint eosinophils in the murine lungs. We also studied the longevity of these expanded populations in the lungs following parasite clearance and the contribution of Nb-elicited ILC2s in driving the accumulation of eosinophil subpopulations. We report that Nb infection leads to the accumulation of a predominant and persistent Siglec-Fhi eosinophil subset in the lungs and the expansion of this population requires the presence of non-T cell lymphocytes and is associated strongly with recruited ILC2 populations.

This study was carried out in accordance with the South African Bureau of Standards guidelines for animal work and approved by the UCT Faculty of Health Sciences Animal Ethics Committee (Project licenses 018-041, 021-012). All researchers were accredited by the South African Veterinary Council.

Wild-type BALB/c, athymic Nude, and IL-4Rα-/- (BALB/c background) mice aged 6 to 8 weeks were obtained from the specific pathogen-free breeding facility and housed in the BSL2 experimental unit at the Faculty of Health Sciences Research Animal Facility, University of Cape Town, South Africa.

Nb was maintained in Wistar rats as previously described (15). Briefly, rats were infected subcutaneously with 5000x infectious Nb L3 larvae. Feces were collected at day 6 to 8 post-infection and cultured for ~1 week to obtain hatched L3. Mice were subcutaneously infected with 500x L3 delivered in sterile water. For secondary/recall infections, mice were infected 5 weeks after the initial Nb infection.

To block lymphocyte egress from secondary lymph tissues, mice received daily intraperitoneal treatment with 0.5 mg kg-1 FTY720 (Sigma-Aldrich) as previously described (16).

Isolated lung tissue was immediately fixed in a 10% phosphate-buffered formalin solution (Sigma-Aldrich), embedded, and sectioned (Leica Biosystems). Sections were stained with Sirius red and hematoxylin. To detect eosinophil cationic protein (ECP), antigen retrieval was performed in Citrate buffer (pH 6). Sections were blocked (5% BSA in PBS) and incubated overnight at 4°C with rabbit polyclonal anti-mouse RNASE3/ECP (abcam PA5-79927). Immunohistochemical detection of ECP was performed using EnVision Detection Systems (DAKO). Sections were viewed with an Axioskop Microscope (Zeiss) and images were taken with an AxioCam HRc and AxioVision 4.7 supporting software (Zeiss).

Bone marrow-derived cells were obtained from individual mice by flushing the right femur with sterile PBS. Cells were washed with PBS and resuspended in a staining buffer (PBS + 0.5% BSA and 2mM Ethylenediaminetetraacetic acid (EDTA)) for flow cytometry staining. Whole blood was obtained from individual mice by cardiac puncture (post-mortem) in 10% vol/vol 0.5M EDTA. Red blood cells were lysed in ACK lysis buffer (Gibco™ Thermo Fisher). Cells were washed with sterile PBS and resuspended in a staining buffer until flow cytometry staining. Whole lung was isolated from individual mice, minced, and digested in Dulbecco’s Modified Eagle Medium (Gibco™ Thermo Fisher) supplemented with 100 U/ml penicillin, 100mg/ml streptomycin, 25 μg/ml Liberase™ TL (Roche), and 13 μg/ml DNase (Roche) for 1 hour at 37°C with gentle shaking. Digested lung tissue was passed through a 70 μm cell strainer (Corning® Sigma-Aldrich) and washed in sterile phosphate-buffered saline (PBS; Gibco™ Thermo Fisher). Red blood cells were lysed in ACK lysis buffer. Cells were washed and resuspended in a staining buffer.

Cells were stained in a staining buffer containing 2% heat-inactivated rat sera, 1 μg anti-mouse CD16/32 (clone: 93, BioLegend), and fluorochrome-conjugated antibodies in the dark for 20 minutes at 4°C. Eosinophils were identified using CD45 Alexa Fluor® 700 (clone: 30-F11, BioLegend), CD11b Brilliant violet (BV) 421™ (clone: M1/70, BioLegend), Ly6C FITC (clone: HK1.4, BioLegend), Ly6G APC Cy7 (clone: 1A8, BioLegend), and Siglec-F PE (clone: S17007L, BioLegend). ILC2s were characterised using CD45 Alexa Fluor® 700 (clone: 30-F11, BioLegend), lineage cocktail PE (CD3ϵ clone: 145-2C11, Ly-6G/Ly-6C clone: RB6-8C5, CD11b clone: M1/70, CD45R/B220 clone: RA3-6B2, TER-119 clone: Ter-119, BioLegend), IL-7Rα (CD127) PE Cy7 (clone: A7R34, BioLegend), ICOS APC (clone: C398.4A, BioLegend), ST2 (IL-33Rα) BV421™ (clone: DIH9, BioLegend), and KLRG1 BV510™ (clone: 2F1/KLRG1, BioLegend). 7-aminoactinomycin D (7-AAD) staining was used to identify non-necrotic (‘Live’) cells. Cells were acquired on an LSRFortessa (BD Biosciences) and FACS Aria I (BD Biosciences) for cell sorting. Single-stained and unstained controls were used to compensate for spectral overlap. Data were analyzed using FlowJo© V10 (Treestar, Ashland, OR).

FACS-sorted eosinophil subpopulations were centrifuged at 800rpm for 10 min in cytospin chambers (Shandon Cytospin 4, Thermo Fisher) and transferred onto glass slides (Sigma-Aldrich). Slides were air-dried, fixed in ice-cold methanol, and stained using Wright-Giemsa stain (Sigma-Aldrich) to visualize the cell nucleus and cytoplasm. Cytospins were imaged using a Zeiss Axioskop Microscope (Zeiss) and AxioCam HRc and AxioVision 4.7 supporting software.

Statistical analysis was performed using Prism 6 (GraphPad). Data is represented as mean ± s.e.m. The relevant details on the statistical tests performed can be found in the figure legends. A P value < 0.05 was considered significant.

We characterized lung eosinophil responses following Nb infection ( Figure 1A ). Siglec-Fint steady-state lung eosinophils were predominant in uninfected mice. This eosinophil population increased following Nb infection (Nb 9dpi) but was surpassed in number and proportion by Siglec-Fhi eosinophils ( Figure 1B ). This Siglec-Fhi eosinophil expansion in the lungs following Nb infection was independent of gender ( Supplementary Figure 1A ). Nb-expanded Siglec-Fint lung eosinophils expressed similar levels of cell surface markers Siglec-F, IL-5R, CCR3, CD11b, CD101, and MHCII as steady-state eosinophils from uninfected mice ( Figure 1C ). Nb-induced Siglec-Fhi eosinophils expressed higher levels of CD101, CD11b, and MHCII and lower expression of CCR3, compared to Siglec-Fint eosinophils ( Figure 1C ). Others have suggested that Siglec-Fhi eosinophils are terminally differentiated cells that are recruited to the lungs (2, 17). We did not find Siglec-FhiCD101hi eosinophils in the bone marrow ( Figure 1D ). We observed a small population of Siglec-Fhi eosinophils in the circulation which did not express high levels of CD101 ( Figure 1D ). To mitigate blood contamination in the lung tissue, we performed PBS perfusion following routine exsanguination via cardiac puncture and found equivalent proportions and numbers of Siglec-FhiCD101hi lung eosinophils with exsanguination + PBS-perfusion compared to exsanguination only ( Supplementary Figures 1B, C ). This suggests that following Nb infection, eosinophils recruited from the bone marrow may only acquire the full Siglec-FhiCD101hi phenotype once they have migrated to the lungs. We did not observe Siglec-FhiCD101hi eosinophils in the intestine-draining mesenteric lymph nodes ( Supplementary Figure 2 ) following Nb infection. Longitudinal analysis over the course of Nb infection (day 0, 1, 5, 9, and 14 PI) revealed that peak levels of total and Siglec-Fhi lung eosinophils occurred at day 9 post-infection, approximately 7 days after the parasite had left the lungs ( Figure 1E ). This time point also coincides with parasite clearance from the intestine and peak type 2 cytokine responses in the lungs (11, 18–20). The Nb-induced increase in lung eosinophils was also mirrored in the bone marrow ( Figure 1F ). While total eosinophil numbers started to decline over time in the lungs, proportionally Siglec-Fhi subsets continued to predominate ( Figure 1E ). Siglec-Fhi eosinophils in the lungs were significantly reduced in IL-4Rα knock-out mice (IL-4Rα-/-) when compared to wild-type mice, with no significant difference in Siglec-Fint eosinophil numbers between these groups ( Figure 1G ). This suggests type 2 immune signaling (i.e., IL-4 and IL-13) contributes to the expansion of Siglec-Fhi lung eosinophils following Nb infection.

We assessed the morphology of lung eosinophils following Nb infection and found CD101lo eosinophils exhibited ring-shaped nuclei while CD101hi eosinophils had hyper-segmented nuclei and less dense cytoplasm ( Figure 2A ), resembling steady-state and activated/inflammatory phenotypes, respectively (2, 3, 21). Further histological analysis of uninfected (steady state) and Nb 9dpi lung tissue showed recruited eosinophils displayed an activated morphology following Nb infection ( Figure 2B ). While both steady-state and Nb-induced eosinophils were positive for eosinophil cationic protein (ECP), Nb-induced eosinophil ECP was dispersed ( Figure 2C ), suggesting eosinophil degranulation in the tissue (22).

Innate type 2 lymphoid cells (ILC2s) are essential for the induction of type 2 immunity and mediate lung eosinophil expansion by secreting the type 2 cytokines IL-5 and IL-13 (23, 24). As expected, we observed a significant increase in total lung ILC2s at day 5 post-Nb infection ( Figure 3A ), which paralleled the expansion of Siglec-Fhi eosinophils ( Figure 1E ). Previous studies have characterized IL-33-responsive tissue-resident ILC2s (nILC2s) and recruited IL-25-responsive inflammatory ILC2s (iILC2s) by differential expression of the IL-33 receptor (ST2) and killer cell lectin-like receptor subfamily G member 1 (KLRG1) (25–27). We observed a significant expansion of ST2+KLRG1- ILC2s in the lungs following Nb infection, substantially more than ST2+KLRG1+ ILC2s ( Figure 3B ). We did not find ST2loKLRG1+ iILC2s (25, 26); however, ILC2 phenotypes are influenced by mouse strain, with lung ILC2s from BALB/c mice being reported to express lower levels of KLRG1 compared to C57BL/6 mice (28). Additionally, we found raised levels of ILC2s in the blood at day 9 post-Nb infection ( Figure 3C ), supporting the expansion of lung ILC2s to be attributed to cell trafficking from another site, as demonstrated by others (27, 29, 30). We also observed an increase in ST2+ CD4+ T cells in the lungs and blood following Nb infection ( Figure 3D ). We then tested whether ST2+ lymphoid recruitment to the lungs mediated induction of Siglec-Fhi lung eosinophils by treating Nb-infected mice with Fingolimod (FTY720) to block the trafficking of lymphoid cells to the lungs ( Figure 4A ) (27, 31). FTY720 treatment resulted in a significant reduction in CD4+ T cells and Lin-ICOS+ cells in circulation, compared to untreated mice ( Figure 4B ). This impaired lymphoid trafficking resulted in a significant reduction in ST2+ ILC2s and ST2+ CD4+ T cells in the lungs of FTY720-treated Nb 9dpi mice, compared to untreated counterparts ( Figure 4C ). Sugita et al., 2010 reported that FTY720 treatment can alter the egress of eosinophils from the bone marrow (32). We found that eosinophil development in the bone marrow and egress into circulation was not impacted by FTY720 treatment ( Figure 4D ). However, impaired lymphoid recruitment resulted in a significant reduction in the proportion and number of Siglec-Fhi eosinophils in the lungs ( Figure 4E ). Further analysis revealed that CD101 expression was also significantly lower on Siglec-Fhi lung eosinophils in FTY720-treated mice, compared to untreated mice ( Figure 4F ). The proportions and phenotypes of Siglec-Fint lung eosinophils were equivalent in FTY720-treated and untreated mice, while total and Siglec-Fint eosinophil cell numbers were reduced in the lungs of FTY720-treated mice at Nb 9dpi ( Supplementary Figure 3 ). Histological examination of the lung tissue revealed that lung eosinophils in FTY720-treated mice did not exhibit activated characteristics as in untreated Nb-infected mice ( Figure 4G ). These data support recruited lymphoid cells mediating accumulation of Siglec-FhiCD101hi activated eosinophils in the lungs following Nb infection. Lung-resident CD4+ T cells are also important contributors to immunity against Nb infection (16). To test whether lung CD4+ T cells contributed to the accumulation of Siglec-Fhi eosinophils, we treated athymic Nude mice (characterized by impaired CD4+ T cell responses) with FTY720 following Nb infection. Proportions of Siglec-Fhi lung eosinophils were equivalent in WT and Nude Nb 9dpi mice (WT 83.48 ± 1.59% vs. Nude 80.65 ± 2.18%); however, FTY720-treated Nude mice had significantly lower proportions and numbers of Siglec-FhiCD101hi lung eosinophils, compared to untreated Nude mice ( Figure 4H ). This was accompanied by a significant reduction in ST2+ ILC2s in the lungs of FTY720-treated Nude mice, compared to untreated counterparts ( Figure 4I ). These data support the findings that Nb-induced Siglec-FhiCD101hi lung eosinophil accumulation requires the recruitment of ST2+ ILC2s and is independent of TH2 CD4+ T cell responses. With a reduction in Siglec-FhiCD101hi eosinophils in the lungs, we also observed significantly increased numbers of lung neutrophils in FTY720-treated Nb-infected mice, compared to uninfected controls ( Figure 4J ).

We showed that Siglec-Fhi eosinophils expand in the lungs after parasite migration through the compartment ( Figure 1E ). In immunocompetent mice, Nb infection is naturally cleared after ~ 2 weeks (33). We observed that levels of Siglec-FhiCD101hi eosinophils remained significantly elevated in the lungs at 6 weeks post Nb infection ( Figure 5A ). We also observed a trend for elevated eosinophil proportions in the bone marrow at this time point ( Figure 5B ), which is the probable source for recruited eosinophils in the lungs (34). Significantly increased numbers of lung ST2+ ILC2s at 6 weeks post Nb infection, compared to uninfected mice ( Figure 5C ), may contribute to the long-term maintenance of Siglec-FhiCD101hi eosinophils in the lungs. Histological analysis of the lung tissue revealed that although Nb 6-week PI eosinophils retained Siglec-FhiCD101hi expression, their morphology resembled that of steady-state lung eosinophils ( Figure 5D ). We also found significantly elevated numbers of Siglec-Fhi eosinophils and a trend for increased ST2+ ILC2s in the lungs during a recall infection, when compared to primary infection ( Figures 5E, F ). These findings demonstrate the long-term maintenance of Siglec-FhiCD101hi eosinophils in the lungs long after parasite clearance, which is associated with lung ILC2s.

In this study, we have characterized the long-term induction of a Siglec-FhiCD101hi eosinophil subpopulation in the lungs following Nb infection and identified that this expansion is strongly associated with the recruitment of lung ILC2s. Previous studies have identified eosinophil subpopulations in mouse models of allergic lung inflammation (2, 3, 6). Percopo et al., 2017 identified a distinct subset of allergen-induced Siglec-F+Gr1hi lung eosinophils that produce immunomodulatory cytokines (35), while Mesnil et al., 2016 differentiated homeostatic Siglec-FintCD62L+CD101lo lung eosinophils and recruited pro-inflammatory Siglec-FhiCD62L-CD101hi eosinophils following an allergic challenge (2). CD101-expressing eosinophils have also been identified in the blood and biopsies of allergic patients and are suggested to be pathogenic (4, 5). Our identification of Nb-induced Siglec-Fhi lung eosinophils expressing high levels of CD101 and presenting an activated morphology (hyper-segmented nuclei and dispersed ECP+ cytoplasm), compared to steady-state and Nb-expanded Siglec-Fint eosinophils, may indicate the accumulation of a pathogenic eosinophil subpopulation in the lungs following Nb infection. Mesnil et al., 2016 demonstrated the induction of Siglec-FhiCD101hi eosinophils during allergic lung inflammation to be dependent on IL-5 (2). Moreover, inflammatory Siglec-FhiCD101hi eosinophils had increased the expression of type 2 receptor Il13ra1 (2). We found the expansion of Siglec-FhiCD101hi eosinophils in the lungs following Nb infection to be exclusively reduced in IL-4Rα-/- mice, suggesting an additional requirement for IL-4 and/or IL-13 signaling. Lung ILC2s are early responders to helminth migration through the lungs, producing IL-5 and IL-13 (23, 24, 36). Huang et al., 2018 showed intestinal ILC2s acquire an inflammatory phenotype (ST2-KLRG1hi) following helminth infection and migrate to peripheral sites, including the lungs (27). We did not observe this inflammatory ST2-KLRG1hi ILC2 phenotype in BALB/c Nb-infected mice, which is consistent with the findings of ILC2 studies in BALB/c mice (28). However, we found ST2+(KLRG1+/-) ILC2 accumulation in the lungs peaked at day 9 post-infection, with increased levels of ILC2s in circulation at this time point. These findings support ILC2 accumulation in the lungs to be, to a large extent, a result of the recruitment from other sites such as the intestine (27). This lung ILC2 expansion mirrored the peak accumulation of Siglec-FhiCD101hi eosinophils in the lungs. Through their production of type 2 cytokines, helminth-induced inflammatory ILC2s are known to regulate eosinophil accumulation (15, 37). Moreover, Huang et al., 2018 reported that inflammatory/recruited ILC2s produced more IL-13 compared to tissue-resident ILC2s (27). We found that expansion of Siglec-FhiCD101hi lung eosinophils following hookworm infection was reduced following FTY720-mediated inhibition of ILC2 recruitment to the lungs. This finding identifies helminth-elicited ILC2s to be strongly associated with the expansion of Siglec-FhiCD101hi eosinophils in the lungs, possibly through their production of type 2 cytokines IL-5 and IL-13. IL-33-responsive CD4+ T cells are also a source of type 2 cytokines in the lungs and are important for memory responses to limit Nb-induced lung damage and worm burden during a secondary infection (16, 38). However, studies have shown that CD4+ T cells are not essential for the expansion of helminth-induced lung eosinophils (23, 39). Likewise, we found that type 2 responses emanating from T cells were not essential for the expansion of Siglec-FhiCD101hi eosinophils in the lungs following Nb infection. The distinct role of Nb-induced Siglec-FhiCD101hi eosinophil subpopulations in the lungs is yet to be determined. Zhu et al., 2020 demonstrated that CD101+ eosinophils increase neutrophilic inflammation in the lungs, while CD101- eosinophils reduced neutrophil inflammation (40). In the context of Nb infection, we found a reduction in Siglec-FhiCD101hi eosinophils following FTY720 treatment to be associated with an increase in lung neutrophils; however, this could also be due to other effects such as impaired M2 macrophage activation in the lungs following FTY720 treatment (41). We observed the long-term maintenance of Nb-induced ECP+ Siglec-FhiCD101hi lung eosinophils at 6 weeks post-infection; however, their morphology resembled that of steady-state lung eosinophils. At this time point, mice typically develop emphysema-like pathology (13). Marsland et al., 2008 reported an equivalent long-term emphysema-like pathology in WT and IL-4Rα-/- Nb-infected mice (13). Further investigation is needed to determine the role of Nb-induced, IL-4Rα-dependent Siglec-FhiCD101hi lung eosinophils in long-term lung pathology. We also observed an increase in lung Siglec-Fhi eosinophils following a recall challenge. It is unclear whether Nb-induced Siglec-FhiCD101hi eosinophils have an enhanced ability to respond to secondary parasite infection or play a role in limiting tissue damage (16, 38). The long-term presence of Nb-induced Siglec-FhiCD101hi eosinophils in the lungs may also have bystander effects on unrelated infections or allergic inflammation (15, 40, 42). In summary, this study identifies that Nb infection drives the expansion of a morphologically distinct Siglec-FhiCD101hi eosinophil subpopulation in the lungs. Helminth-induced ILC2 recruitment to the lungs contributes to Siglec-FhiCD101hi eosinophil expansion. This eosinophil subpopulation persists after the resolution of parasite infection. This identifies these cells as an important associate with longer-term lung pathology following helminth migration through the lungs.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The animal study was reviewed and approved by University of Cape Town Faculty of Health Sciences Animal Ethics Commitee.

AC, MD, AT and JP performed experiments, maintained the parasite lifecycle, and analyzed data. WH, AC, MR, LEL, GK, AC and MO designed experiments, prepared figures, and wrote the paper. WH, AFC, LEL, GK and MR acquired funding and supervised the work. All authors contributed to the article and approved the submitted version.