Parasitic worms known as helminths represent an important health problem in large parts of Africa, South America and Asia. Once their larvae enter the body, they head to the gut where they mature into adults and start laying eggs. In areas with poor sanitation, these may then get passed on to other individuals. To defend the body, the immune system sends large numbers of immune cells to the gut, but it usually struggles to eliminate the parasites. Without deworming medication, the infection can last for many years.

Scientists study helminth infections in the laboratory by using worms that naturally infect mice. Understanding exactly how the immune system responds to the infection is essential to grasp why it fails to clear the worms. However, it is difficult to extract immune cells from an infected gut, as the infection creates strong local responses – such as an intense ‘slime’ production to try to flush out the worms.

The standard procedure to obtain immune cells from the gut consists of three steps: collecting a gut segment and washing it, stripping away the surface layers with chemicals, and finally using enzymes to digest the tissues, which are then filtered to obtain individual cells. However, this protocol is not able to extract cells during infection. Ferrer-Font et al. therefore methodically refined every step of this method, and finally succeeded in obtaining millions of immune cells from infected guts.

For the first time, these cells could then be studied and identified using a new technology called spectral flow cytometry. Over 40 immune cell types were followed throughout the course of infection, revealing that many ‘first responders’ immune cells were recruited to the gut early on, when the worms were still larvae. However, these cells disappeared once the worms developed into adults. These findings were confirmed by microscopy, which also showed that the first responder cells were found around the developing larvae, likely attacking them. When the adult worms developed, these cells were replaced by other immune cells, which also decreased the longer the worms were present in the gut.

This new extraction process established by Ferrer-Font et al. can also be paired with other technologies that can, for example, reveal which genes are turned on in individual cells. This could help map out exactly how the body fights helminth infections, and how to improve this response. The method could also be useful to extract immune cells from the gut in other challenging scenarios, such food allergies or inflammatory bowel disorders.

Recent advances in single cell analysis have significantly increased our understanding of multiple diseases and cell types in different tissues (Svensson et al., 2018; Hwang et al., 2018). However, many of these technologies require single cell suspensions as an input, which limits our assessment of difficult-to-process tissues (Hwang et al., 2018; Nguyen et al., 2018; Chen et al., 2018). One prominent example is the intestine, which is at the center of many research questions that focus on nutrient uptake (Kiela and Ghishan, 2016), host-microbiome interactions (Belkaid and Hand, 2014; Tilg et al., 2019; Sekirov et al., 2010), local and systemic immune tolerance (Worbs et al., 2006; Harrison and Powrie, 2013; Whibley et al., 2019) and gastrointestinal diseases and infections (Mowat and Agace, 2014; Sell and Dolan, 2018; Fletcher et al., 2013; Hendrickson et al., 2002; Saleh and Elson, 2011), but represents a challenging tissue to digest (Weigmann et al., 2007; Reißig et al., 2014).

The standard digestion procedure to isolate intestinal immune cells located in the small intestinal lamina propria consists of three steps (Weigmann et al., 2007; Reißig et al., 2014; Scott et al., 2016; Esterházy et al., 2019) (Figure 1a). First the intestinal segment of interest is collected, opened longitudinally to remove its luminal content, washed and cut into small pieces. These pieces then undergo several wash steps with EDTA containing wash buffers at 37°C to remove the epithelial layer and make the lamina propria accessible for enzymatic digestion. Lastly, the tissue is enzymatically digested with collagenases (Collagenase VIII from Clostridium histolyticum being among the most popular) and later filtered to obtain a single cell suspension.

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While this method results in a high cell yield from steady state intestines, the isolation of cells from severely infected segments remains challenging (Reißig et al., 2014; Scott et al., 2016; Webster et al., 2020) (Figure 1b). One of the most prominent examples are infections with intestinal helminths, which represent over 50% of all parasitic infections in human and livestock populations (McSorley and Maizels, 2012; Hotez et al., 2008; Jourdan et al., 2018; Jackson et al., 2009). This has limited our analysis of anti-parasite immune responses to imaging approaches, phenotypical observations in transgenic mouse strains or the assessment of secondary locations like the draining lymph nodes, blood or spleen (McSorley and Maizels, 2012; Maizels and McSorley, 2016; Mishra et al., 2014), which might only partially reflect local immunity. As helminth infections are strongly linked to chronic impairments that affect nutrition availability (Koski and Scott, 2001; Crompton and Nesheim, 2002); memory, cognition and physical development (Ezeamama et al., 2005; Pabalan et al., 2018; Nokes et al., 1992); changes in the microbiota (Gause and Maizels, 2016; Ramanan et al., 2016) and modulation of local and systemic immunity (Mishra et al., 2014; Maizels et al., 2009), an optimized digestion protocol is needed to further investigate the infected intestinal tissue.

Difficulties with intestinal digests during helminth infection have been associated with a strong anti-parasite type 2 immune response that drives mucus production (Hashimoto et al., 2009; von Moltke et al., 2016), alters the epithelium (Gerbe et al., 2016; Howitt et al., 2016), induces immune cell infiltration (Inclan-Rico and Siracusa, 2018) and causes tissue remodelling (Motran et al., 2018; Boyett and Hsieh, 2014) (Figure 1c). In order to investigate a model of both acute and chronic helminth infection, we infected C57BL/6 mice with Heligmosomoides polygyrus bakeri (also known as Heligmosomoides bakeri; Behnke and Harris, 2010), a naturally occurring rodent parasite with an exclusive intestinal life cycle (Monroy and Enriquez, 1992; Reynolds et al., 2012). Infective L3 larvae penetrate the intestinal tissue of the duodenum within 24 hr of ingestion, undergo larval development in the muscularis externa and return to the lumen within 10 days post infection, where the adult worms mate and develop a chronic infection in C57BL/6 mice (Reynolds et al., 2012; Smith et al., 2016). The peak of acute immunity is usually studied around day 14 post infection and we focused on this time point and the heavily infected duodenum (Filbey et al., 2014; Elliott et al., 2008), to optimize our digestion protocol.

In order to develop a digestion protocol for heavily infected intestines, we followed a systematic approach and optimized each step of the standard intestinal digestion protocol. First, we modified the EDTA wash steps to remove the increased amount of mucus but did not observe an improvement in cell yield (Figure 1d and Figure 1—figure supplement 1; digestion protocols #1, 2, 6). This was followed by testing a variety of collagenases that have been reported for intestinal digests, as we hypothesized that the intestinal remodeling that occurred during helminth infection could negatively impact the digestion procedure. Indeed, we found that Collagenase A from Clostridium histolyticum (Figure 1d and Figure 1—figure supplement 1; digestion protocols #7, 8), but not Collagenase VIII, Collagenase D, Dispase or Liberase TM (Figure 1d and Figure 1—figure supplement 1; digestion protocols #3–5), showed an increase in cell yield when used in conjunction with the standard digestion protocol.

To further optimize the protocol, we focused on Collagenase A-based digestion and increased and modified the wash steps and observed a further increase in cell yield (Figure 1d and Figure 1—figure supplement 2; digestion protocols #9–12). Importantly, strong vortexing after each wash step significantly improved the outcome of digestion (Figure 1d and Figure 1—figure supplement 2; digestion protocol #13), suggesting that the epithelium is harder to remove in helminth-infected tissues. Indeed, observations from Stat6ko mice confirmed that the physiological changes that impair the intestinal digest using the standard protocol, were all linked to type two immune responses, as intestines from infected Stat6ko mice could readily be digested (Figure 1—figure supplement 3). We also assessed intra-epithelial cells in the EDTA wash, but could not detect any CD45+ cells in preparations from infected animals, emphasizing that our protocol should only be used to isolate lamina propria cells. Several intestinal cell isolation protocols also utilize a final gradient centrifugation step to further isolate immune cells (Weigmann et al., 2007; Esterházy et al., 2019). However, in our hands this resulted in a dramatic drop in cell yield and was therefore omitted (Figure 1d and Figure 1—figure supplement 2; digestion protocol #14). Our optimized lamina propria cell isolation protocol for H. polygyrus-infected intestines thus included three 10 min 2 mM EDTA wash steps (each followed by vigorous vortexing) and a 30 min digest with 1 mg/ml Collagenase A, 20% FCS and 0.05 mg/ml DNase (see Appendix 1 for step-by-step instructions).

When we compared intestinal digests from naïve animals using the standard or optimized cell isolation protocol, we observed highly comparable outcomes (Figure 1d; digestion protocols #1 and 13). Both digestion protocols resulted in a cell yield of 3–6 million live cells per duodenum with 70–80% viability and 20–30% frequency of CD45+ cells. To assess the effectiveness of our digestion protocol during the different stages of H. polygyrus infection, we harvested the duodenum from naïve C57BL/6 mice and at day 7, day 14 and day 28 of H. polygyrus infection, which represented time points of larval development in the muscularis externa, as well as acute and chronic adult worm infection, respectively. We observed that samples from all time points could be successfully digested using our optimized digestion protocol and that duodenal digests from 14- and 28 days post infection yielded 3–6 million live cells per sample (Figure 1e,f). We furthermore observed a consistent doubling of the cell count to 8–11 million live cells per duodenum at day seven post infection and observed a similar trend when we quantified CD45+ cells in cryosections from these time points (Figure 1f,g).

To understand these differences and validate whether our protocol was suitable for subsequent single cell analysis and immunophenotyping, we characterized the isolated cells further using a 23-color spectral flow cytometry panel that incorporated many of the hallmark surface and intracellular markers for type two immune responses that have been associated with helminth infections (Maizels and McSorley, 2016; Reynolds et al., 2012) (see Supplementary file 1 for details regarding markers, fluorophores, clones and staining concentrations used). Our staining panel was designed to identify both innate and adaptive immune cell populations and allowed us to assess eosinophils, neutrophils, different subsets of monocytes, macrophages and dendritic cells, as well as the three main populations of innate lymphoid cells (ILC1, ILC2, and ILC3) and effector T cells populations (Th1, Th2, Th17), T regulatory cells and B cells as well as their proliferation through ki67 expression within the same panel.

To guarantee optimal staining conditions, we tested our optimized digestion protocol on splenocytes and compared digested to non-digested cells, as collagenase digests can negatively affect surface epitope integrity. While we observed a reduction in the MFIs of several markers (namely Ly6G, MHCII, CD45 and CD127), all positive stained cell populations could be clearly identified (Figure 1—figure supplement 4). Isolated intestinal lamina propria cells also proved a challenge for intracellular staining, as different commercial intracellular staining kits significantly affected the cellular, but not debris, scatter profiles and varied in the resolution of intracellularly antibody staining (Figure 1—figure supplement 5). In our hands, the eBioscience FoxP3/Transcription Factor Staining Buffer Set yielded the best results and was used henceforth.

We isolated immune cells from the three main stages of H. polygyrus infection (day 7, day 14 and day 28), representing larval development, as well as acute and chronic worm infection and used a combination of high-dimensional analysis tools and manual gating strategies to assess changes within each immune cell population (Figure 2a and Figure 2—figure supplement 1 and Figure 2—figure supplement 2). In line with previous findings (Inclan-Rico and Siracusa, 2018), we observed a strong infiltration of immune cells such as neutrophils and monocytes at day 7 post infection, which we verified by confocal microscopy and were primarily localized around the developing larvae explaining the increase in total cell number at this timepoint (Figure 2a–c and Figure 2—figure supplement 3). At later time points this inflammatory response receded, which was likely linked to the worms exiting the intestinal tissue and inhabiting the lumen. Peak expression of RELMα in resident macrophages, which is a hallmark for their alternative activation and wound repair responses (Esser-von Bieren et al., 2013; Krljanac et al., 2019), was observed at day 14 and was again localized within the granulomas (Figure 2b,c and Figure 2—figure supplement 3). While type two innate lymphoid cells did not increase in frequency over time, ki67 expression increased, suggesting cell proliferation and activation (Figure 2d), as previously described (von Moltke et al., 2016; Schneider et al., 2018). GATA3+ Th2 cells, important drivers of type two immunity (Reynolds et al., 2012; Mohrs et al., 2005), were detected throughout all stages of infection, increased in frequency over time and showed high ki67 expression (Figure 2b). Interestingly, ki67 expression strongly decreased at day 28 post infection for all cell types analyzed (Figure 2d and Figure 2—figure supplement 4), which could be linked to the strong immunomodulatory properties reported during chronic worm infection (Maizels and McSorley, 2016; Grainger et al., 2010) (Figure 2e).

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To validate that our cell isolation protocol resulted in an accurate representation of intestinal immune responses, we quantified B220+, Siglec F+, CD3+ CD4+, CD3+ CD4- and CD64+ cells using confocal microscopy (Figure 2—figure supplement 5a–c) and compared their frequency to our spectral flow cytometry data. We observed that the frequencies of B220+, CD3+ CD4+, and CD64+ cells were highly comparable between confocal microscopy and spectral flow cytometry, while Siglec F+ cells were overrepresented in our spectral flow cytometry data and CD3+ CD4- cells were underrepresented. However, changes within immune cell populations at the peak of H. polygyrus infection were faithfully reported by both confocal microscopy and spectral flow cytometry (Figure 2—figure supplement 5d), emphasizing that cell ratios defined by single cell analysis need to be carefully validated within the tissue before conclusions are drawn.

Another important conclusion from our analysis was that the strong inflammatory immune responses that we had observed during H. polygyrus development in the muscularis externa, were specific to the infected tissue and were not observed to the same extent in the draining lymph nodes. Furthermore, ratios of ILC populations and T helper subsets were also strikingly different between the lamina propria and the draining lymph nodes at steady state, as were their proliferation kinetics and changes in proportion throughout the course of H. polygyrus infection (Figure 2—figure supplement 6 and Figure 2—figure supplement 7).

To highlight the potential of our protocol for future studies that utilize current single-cell analysis tools, such as single cell RNA sequencing, we assessed the RNA quality of purified B cells, CD4 T cells and macrophages isolated from naïve or day 14 hr. polygyrus-infected mice (Figure 2—figure supplement 8a,b). Our RNA quality analysis using the Agilent TapeStation resulted in high RIN numbers (range 6.8–10 for naïve and 7.0–10 for day 14 hr. polygyrus samples) (Figure 2—figure supplement 8e). However, no clear separation of the 18S and 28S peaks could be observed on the gel image or the electropherograms (Figure 2—figure supplement 8c,d). While the RIN numbers might not have been correctly calculated, and a technical optimization of the TapeStation protocol might be necessary, no RNA degradation was observed in naïve and day 14 hr. polygyrus samples, suggesting that the extraction of high-quality RNA is feasible from both naïve and day 14 hr. polygyrus-infected mice using our cell isolation protocol.

Thus, our cell isolation protocol and high-dimensional analysis allowed us to characterize many known hallmarks of innate and adaptive anti-parasite immune responses throughout the entire course of helminth infection. We were able to validate these changes using confocal microscopy and while we could observe differences in the reported cell ratios, changes between naïve and infected samples were faithfully reported by both approaches.

Importantly, many of these changes were only observed locally, highlighting the requirement for good cell isolation techniques to investigate intestinal responses against helminths directly.

In addition to flow cytometric immunophenotyping, we were also able to extract high-quality RNA from cells isolated with our protocol, which could accelerate single-cell discoveries of local helminth immune responses through current single-cell analysis tools, such as single cell RNA sequencing, which has previously been unfeasible.

Lamina propria and lymph node data sets can be downloaded from flowrepository (http://flowrepository.org/id/FR-FCM-Z28B).

1. 1. Ferrer-Font L
   2. Mayer JU

   (2019) FlowRepository

   Lamina propia and MLN cells during H. polygyrus infection.

   http://flowrepository.org/id/FR-FCM-Z28B

1. 1. Behnke J
   2. Harris PD

   (2010) Heligmosomoides bakeri: a new name for an old worm?

   Trends in Parasitology 26:524–529.

   https://doi.org/10.1016/j.pt.2010.07.001

   • PubMed
   • Google Scholar
2. 1. Belkaid Y
   2. Hand TW

   (2014) Role of the Microbiota in immunity and inflammation

   Cell 157:121–141.

   https://doi.org/10.1016/j.cell.2014.03.011

   • PubMed
   • Google Scholar
3. 1. Boyett D
   2. Hsieh MH

   (2014) Wormholes in host defense: how helminths manipulate host tissues to survive and reproduce

   PLOS Pathogens 10:e1004014.

   https://doi.org/10.1371/journal.ppat.1004014

   • PubMed
   • Google Scholar
4. 1. Chen X
   2. Teichmann SA
   3. Meyer KB

   (2018) From tissues to cell types and back: single-cell gene expression analysis of tissue architecture

   Annual Review of Biomedical Data Science 1:29–51.

   https://doi.org/10.1146/annurev-biodatasci-080917-013452

   • Google Scholar
5. 1. Crompton DW
   2. Nesheim MC

   (2002) Nutritional impact of intestinal helminthiasis during the human life cycle

   Annual Review of Nutrition 22:35–59.

   https://doi.org/10.1146/annurev.nutr.22.120501.134539

   • PubMed
   • Google Scholar
6. 1. Elliott DE
   2. Metwali A
   3. Leung J
   4. Setiawan T
   5. Blum AM
   6. Ince MN
   7. Bazzone LE
   8. Stadecker MJ
   9. Urban JF
   10. Weinstock JV

   (2008) Colonization with Heligmosomoides polygyrus suppresses mucosal IL-17 production

   Journal of Immunology 181:2414–2419.

   https://doi.org/10.4049/jimmunol.181.4.2414

   • PubMed
   • Google Scholar
7. 1. Esser-von Bieren J
   2. Mosconi I
   3. Guiet R
   4. Piersgilli A
   5. Volpe B
   6. Chen F
   7. Gause WC
   8. Seitz A
   9. Verbeek JS
   10. Harris NL

   (2013) Antibodies trap tissue migrating helminth larvae and prevent tissue damage by driving IL-4Rα-independent alternative differentiation of macrophages

   PLOS Pathogens 9:e1003771.

   https://doi.org/10.1371/journal.ppat.1003771

   • PubMed
   • Google Scholar
8. 1. Esterházy D
   2. Canesso MCC
   3. Mesin L
   4. Muller PA
   5. de Castro TBR
   6. Lockhart A
   7. ElJalby M
   8. Faria AMC
   9. Mucida D

   (2019) Compartmentalized gut lymph node drainage dictates adaptive immune responses

   Nature 569:126–130.

   https://doi.org/10.1038/s41586-019-1125-3

   • Google Scholar
9. 1. Ezeamama AE
   2. Friedman JF
   3. Acosta LP
   4. Bellinger DC
   5. Langdon GC
   6. Manalo DL
   7. Olveda RM
   8. Kurtis JD
   9. McGarvey ST

   (2005) Helminth infection and cognitive impairment among filipino children

   The American Journal of Tropical Medicine and Hygiene 72:540–548.

   https://doi.org/10.4269/ajtmh.2005.72.540

   • PubMed
   • Google Scholar
10. 1. Filbey KJ
    2. Grainger JR
    3. Smith KA
    4. Boon L
    5. van Rooijen N
    6. Harcus Y
    7. Jenkins S
    8. Hewitson JP
    9. Maizels RM

    (2014) Innate and adaptive type 2 immune cell responses in genetically controlled resistance to intestinal helminth infection

    Immunology and Cell Biology 92:436–448.

    https://doi.org/10.1038/icb.2013.109

    • PubMed
    • Google Scholar
11. 1. Fletcher SM
    2. McLaws M-L
    3. Ellis JT

    (2013) Prevalence of gastrointestinal pathogens in developed and developing countries: systematic review and meta-analysis

    Journal of Public Health Research 2:9–53.

    https://doi.org/10.4081/jphr.2013.e9

    • Google Scholar
12. 1. Gause WC
    2. Maizels RM

    (2016) Macrobiota - helminths as active participants and partners of the Microbiota in host intestinal homeostasis

    Current Opinion in Microbiology 32:14–18.

    https://doi.org/10.1016/j.mib.2016.04.004

    • PubMed
    • Google Scholar
13. 1. Gerbe F
    2. Sidot E
    3. Smyth DJ
    4. Ohmoto M
    5. Matsumoto I
    6. Dardalhon V
    7. Cesses P
    8. Garnier L
    9. Pouzolles M
    10. Brulin B
    11. Bruschi M
    12. Harcus Y
    13. Zimmermann VS
    14. Taylor N
    15. Maizels RM
    16. Jay P

    (2016) Intestinal epithelial tuft cells initiate type 2 mucosal immunity to helminth parasites

    Nature 529:226–230.

    https://doi.org/10.1038/nature16527

    • PubMed
    • Google Scholar
14. 1. Grainger JR
    2. Smith KA
    3. Hewitson JP
    4. McSorley HJ
    5. Harcus Y
    6. Filbey KJ
    7. Finney CA
    8. Greenwood EJ
    9. Knox DP
    10. Wilson MS
    11. Belkaid Y
    12. Rudensky AY
    13. Maizels RM

    (2010) Helminth secretions induce de novo T cell Foxp3 expression and regulatory function through the TGF-β pathway

    The Journal of Experimental Medicine 207:2331–2341.

    https://doi.org/10.1084/jem.20101074

    • PubMed
    • Google Scholar
15. 1. Harrison OJ
    2. Powrie FM

    (2013) Regulatory T cells and immune tolerance in the intestine

    Cold Spring Harbor Perspectives in Biology 5:a018341.

    https://doi.org/10.1101/cshperspect.a018341

    • PubMed
    • Google Scholar
16. 1. Hashimoto K
    2. Uchikawa R
    3. Tegoshi T
    4. Takeda K
    5. Yamada M
    6. Arizono N

    (2009) Depleted intestinal goblet cells and severe pathological changes in SCID mice infected with Heligmosomoides polygyrus

    Parasite Immunology 31:457–465.

    https://doi.org/10.1111/j.1365-3024.2009.01123.x

    • PubMed
    • Google Scholar
17. 1. Hendrickson BA
    2. Gokhale R
    3. Cho JH

    (2002) Clinical aspects and pathophysiology of inflammatory bowel disease

    Clinical Microbiology Reviews 15:79–94.

    https://doi.org/10.1128/CMR.15.1.79-94.2002

    • PubMed
    • Google Scholar
18. 1. Hotez PJ
    2. Brindley PJ
    3. Bethony JM
    4. King CH
    5. Pearce EJ
    6. Jacobson J

    (2008) Helminth infections: the great neglected tropical diseases

    Journal of Clinical Investigation 118:1311–1321.

    https://doi.org/10.1172/JCI34261

    • PubMed
    • Google Scholar
19. 1. Howitt MR
    2. Lavoie S
    3. Michaud M
    4. Blum AM
    5. Tran SV
    6. Weinstock JV
    7. Gallini CA
    8. Redding K
    9. Margolskee RF
    10. Osborne LC
    11. Artis D
    12. Garrett WS

    (2016) Tuft cells, taste-chemosensory cells, orchestrate parasite type 2 immunity in the gut

    Science 351:1329–1333.

    https://doi.org/10.1126/science.aaf1648

    • PubMed
    • Google Scholar
20. 1. Hwang B
    2. Lee JH
    3. Bang D

    (2018) Single-cell RNA sequencing technologies and bioinformatics pipelines

    Experimental & Molecular Medicine 50:96.

    https://doi.org/10.1038/s12276-018-0071-8

    • PubMed
    • Google Scholar
21. 1. Inclan-Rico JM
    2. Siracusa MC

    (2018) First responders: innate immunity to helminths

    Trends in Parasitology 34:861–880.

    https://doi.org/10.1016/j.pt.2018.08.007

    • PubMed
    • Google Scholar
22. 1. Jackson JA
    2. Friberg IM
    3. Little S
    4. Bradley JE

    (2009) Review series on helminths, immune modulation and the hygiene hypothesis: immunity against helminths and immunological phenomena in modern human populations: coevolutionary legacies?

    Immunology 126:18–27.

    https://doi.org/10.1111/j.1365-2567.2008.03010.x

    • PubMed
    • Google Scholar
23. 1. Jacobson KA
    2. Rose J
    3. Zeller R

    (2008) Hematoxylin and eosin staining of tissue and cell sections

    Cold Spring Harbor Protocols 2008:pdb.prot4986.

    https://doi.org/10.1101/pdb.prot4986

    • Google Scholar
24. 1. Johnston CJC
    2. Robertson E
    3. Harcus Y
    4. Grainger JR
    5. Coakley G
    6. Smyth DJ
    7. McSorley HJ
    8. Maizels R

    (2015) Cultivation of Heligmosomoides polygyrus: an immunomodulatory nematode parasite and its secreted products

    Journal of Visualized Experiments 52412.

    https://doi.org/10.3791/52412

    • Google Scholar
25. 1. Jourdan PM
    2. Lamberton PHL
    3. Fenwick A
    4. Addiss DG

    (2018) Soil-transmitted helminth infections

    The Lancet 391:252–265.

    https://doi.org/10.1016/S0140-6736(17)31930-X

    • Google Scholar
26. 1. Kiela PR
    2. Ghishan FK

    (2016) Physiology of intestinal absorption and secretion

    Best Practice & Research Clinical Gastroenterology 30:145–159.

    https://doi.org/10.1016/j.bpg.2016.02.007

    • PubMed
    • Google Scholar
27. 1. Koski KG
    2. Scott ME

    (2001) Gastrointestinal Nematodes, nutrition and immunity: breaking the negative spiral

    Annual Review of Nutrition 21:297–321.

    https://doi.org/10.1146/annurev.nutr.21.1.297

    • PubMed
    • Google Scholar
28. 1. Krljanac B
    2. Schubart C
    3. Naumann R
    4. Wirtz S
    5. Culemann S
    6. Krönke G
    7. Voehringer D

    (2019) RELMα-expressing macrophages protect against fatal lung damage and reduce parasite burden during helminth infection

    Science Immunology 4:eaau3814.

    https://doi.org/10.1126/sciimmunol.aau3814

    • PubMed
    • Google Scholar
29. 1. Lamprecht MR
    2. Sabatini DM
    3. Carpenter AE

    (2007) CellProfiler: free, versatile software for automated biological image analysis

    BioTechniques 42:71–75.

    https://doi.org/10.2144/000112257

    • PubMed
    • Google Scholar
30. 1. Maizels RM
    2. Pearce EJ
    3. Artis D
    4. Yazdanbakhsh M
    5. Wynn TA

    (2009) Regulation of pathogenesis and immunity in helminth infections

    The Journal of Experimental Medicine 206:2059–2066.

    https://doi.org/10.1084/jem.20091903

    • PubMed
    • Google Scholar
31. 1. Maizels RM
    2. McSorley HJ

    (2016) Regulation of the host immune system by helminth parasites

    Journal of Allergy and Clinical Immunology 138:666–675.

    https://doi.org/10.1016/j.jaci.2016.07.007

    • PubMed
    • Google Scholar
32. 1. Mayer JU
    2. Demiri M
    3. Agace WW
    4. MacDonald AS
    5. Svensson-Frej M
    6. Milling SW

    (2017) Different populations of CD11b⁺ dendritic cells drive Th2 responses in the small intestine and Colon

    Nature Communications 8:15820.

    https://doi.org/10.1038/ncomms15820

    • PubMed
    • Google Scholar
33. 1. McSorley HJ
    2. Maizels RM

    (2012) Helminth infections and host immune regulation

    Clinical Microbiology Reviews 25:585–608.

    https://doi.org/10.1128/CMR.05040-11

    • PubMed
    • Google Scholar
34. 1. Mishra PK
    2. Palma M
    3. Bleich D
    4. Loke P
    5. Gause WC

    (2014) Systemic impact of intestinal helminth infections

    Mucosal Immunology 7:753–762.

    https://doi.org/10.1038/mi.2014.23

    • PubMed
    • Google Scholar
35. 1. Mohrs K
    2. Harris DP
    3. Lund FE
    4. Mohrs M

    (2005) Systemic dissemination and persistence of Th2 and type 2 cells in response to infection with a strictly enteric nematode parasite

    The Journal of Immunology 175:5306–5313.

    https://doi.org/10.4049/jimmunol.175.8.5306

    • PubMed
    • Google Scholar
36. 1. Monroy FG
    2. Enriquez FJ

    (1992) Heligmosomoides polygyrus: a model for chronic gastrointestinal helminthiasis

    Parasitology Today 8:49–54.

    https://doi.org/10.1016/0169-4758(92)90084-F

    • PubMed
    • Google Scholar
37. 1. Motran CC
    2. Silvane L
    3. Chiapello LS
    4. Theumer MG
    5. Ambrosio LF
    6. Volpini X
    7. Celias DP
    8. Cervi L

    (2018) Helminth infections: recognition and modulation of the immune response by innate immune cells

    Frontiers in Immunology 9:664.

    https://doi.org/10.3389/fimmu.2018.00664

    • PubMed
    • Google Scholar
38. 1. Mowat AM
    2. Agace WW

    (2014) Regional specialization within the intestinal immune system

    Nature Reviews Immunology 14:667–685.

    https://doi.org/10.1038/nri3738

    • PubMed
    • Google Scholar
39. 1. Nguyen QH
    2. Pervolarakis N
    3. Nee K
    4. Kessenbrock K

    (2018) Experimental considerations for Single-Cell RNA sequencing approaches

    Frontiers in Cell and Developmental Biology 6:108.

    https://doi.org/10.3389/fcell.2018.00108

    • PubMed
    • Google Scholar
40. 1. Nokes C
    2. Grantham-McGregor SM
    3. Sawyer AW
    4. Cooper ES
    5. Bundy DA

    (1992) Parasitic helminth infection and cognitive function in school children

    Proceedings. Biological Sciences 247:77–81.

    https://doi.org/10.1098/rspb.1992.0011

    • PubMed
    • Google Scholar
41. 1. Pabalan N
    2. Singian E
    3. Tabangay L
    4. Jarjanazi H
    5. Boivin MJ
    6. Ezeamama AE

    (2018) Soil-transmitted helminth infection, loss of education and cognitive impairment in school-aged children: a systematic review and meta-analysis

    PLOS Neglected Tropical Diseases 12:e0005523.

    https://doi.org/10.1371/journal.pntd.0005523

    • PubMed
    • Google Scholar
42. 1. Ramanan D
    2. Bowcutt R
    3. Lee SC
    4. Tang MS
    5. Kurtz ZD
    6. Ding Y
    7. Honda K
    8. Gause WC
    9. Blaser MJ
    10. Bonneau RA
    11. Lim YA
    12. Loke P
    13. Cadwell K

    (2016) Helminth infection promotes colonization resistance via type 2 immunity

    Science 352:608–612.

    https://doi.org/10.1126/science.aaf3229

    • PubMed
    • Google Scholar
43. 1. Reißig S
    2. Hackenbruch C
    3. Hövelmeyer N

    (2014) Isolation of T cells from the gut

    Methods in Molecular Biology 1193:21–25.

    https://doi.org/10.1007/978-1-4939-1212-4_3

    • PubMed
    • Google Scholar
44. 1. Reynolds LA
    2. Filbey KJ
    3. Maizels RM

    (2012) Immunity to the model intestinal helminth parasite Heligmosomoides polygyrus

    Seminars in Immunopathology 34:829–846.

    https://doi.org/10.1007/s00281-012-0347-3

    • PubMed
    • Google Scholar
45. 1. Saleh M
    2. Elson CO

    (2011) Experimental inflammatory bowel disease: insights into the host-microbiota dialog

    Immunity 34:293–302.

    https://doi.org/10.1016/j.immuni.2011.03.008

    • PubMed
    • Google Scholar
46. 1. Schindelin J
    2. Arganda-Carreras I
    3. Frise E
    4. Kaynig V
    5. Longair M
    6. Pietzsch T
    7. Preibisch S
    8. Rueden C
    9. Saalfeld S
    10. Schmid B
    11. Tinevez JY
    12. White DJ
    13. Hartenstein V
    14. Eliceiri K
    15. Tomancak P
    16. Cardona A

    (2012) Fiji: an open-source platform for biological-image analysis

    Nature Methods 9:676–682.

    https://doi.org/10.1038/nmeth.2019

    • PubMed
    • Google Scholar
47. 1. Schmidt AJ
    2. Mayer JU
    3. Wallace PK
    4. Ronchese F
    5. Price KM

    (2019) Simultaneous polychromatic immunofluorescent staining of tissue sections and consecutive imaging of up to seven parameters by standard confocal microscopy

    Current Protocols in Cytometry 91:633.

    https://doi.org/10.1002/cpcy.64

    • Google Scholar
48. 1. Schneider C
    2. O'Leary CE
    3. von Moltke J
    4. Liang HE
    5. Ang QY
    6. Turnbaugh PJ
    7. Radhakrishnan S
    8. Pellizzon M
    9. Ma A
    10. Locksley RM

    (2018) A Metabolite-Triggered tuft Cell-ILC2 circuit drives small intestinal remodeling

    Cell 174:271–284.

    https://doi.org/10.1016/j.cell.2018.05.014

    • PubMed
    • Google Scholar
49. 1. Scott CL
    2. Wright PB
    3. Milling SW
    4. Mowat AM

    (2016) Isolation and identification of conventional dendritic cell subsets from the intestine of mice and men

    Methods in Molecular Biology 1423:101–118.

    https://doi.org/10.1007/978-1-4939-3606-9_7

    • PubMed
    • Google Scholar
50. 1. Sekirov I
    2. Russell SL
    3. Antunes LC
    4. Finlay BB

    (2010) Gut Microbiota in health and disease

    Physiological Reviews 90:859–904.

    https://doi.org/10.1152/physrev.00045.2009

    • PubMed
    • Google Scholar
51. 1. Sell J
    2. Dolan B

    (2018) Common gastrointestinal infections

    Primary Care: Clinics in Office Practice 45:519–532.

    https://doi.org/10.1016/j.pop.2018.05.008

    • PubMed
    • Google Scholar
52. 1. Smith KA
    2. Filbey KJ
    3. Reynolds LA
    4. Hewitson JP
    5. Harcus Y
    6. Boon L
    7. Sparwasser T
    8. Hämmerling G
    9. Maizels RM

    (2016) Low-level regulatory T-cell activity is essential for functional type-2 effector immunity to expel gastrointestinal helminths

    Mucosal Immunology 9:428–443.

    https://doi.org/10.1038/mi.2015.73

    • PubMed
    • Google Scholar
53. 1. Svensson V
    2. Vento-Tormo R
    3. Teichmann SA

    (2018) Exponential scaling of single-cell RNA-seq in the past decade

    Nature Protocols 13:599–604.

    https://doi.org/10.1038/nprot.2017.149

    • PubMed
    • Google Scholar
54. 1. Tilg H
    2. Zmora N
    3. Adolph TE
    4. Elinav E

    (2019) The intestinal Microbiota fuelling metabolic inflammation

    Nature Reviews. Immunology 336:973.

    https://doi.org/10.1038/s41577-019-0198-4

    • Google Scholar
55. 1. von Moltke J
    2. Ji M
    3. Liang HE
    4. Locksley RM

    (2016) Tuft-cell-derived IL-25 regulates an intestinal ILC2-epithelial response circuit

    Nature 529:221–225.

    https://doi.org/10.1038/nature16161

    • PubMed
    • Google Scholar
56. 1. Webster HC
    2. Andrusaite AT
    3. Shergold AL
    4. Milling SWF
    5. Perona-Wright G

    (2020) Isolation and functional characterisation of Lamina propria leukocytes from helminth-infected, murine small intestine

    Journal of Immunological Methods 477:112702.

    https://doi.org/10.1016/j.jim.2019.112702

    • PubMed
    • Google Scholar
57. 1. Weigmann B
    2. Tubbe I
    3. Seidel D
    4. Nicolaev A
    5. Becker C
    6. Neurath MF

    (2007) Isolation and subsequent analysis of murine Lamina propria mononuclear cells from colonic tissue

    Nature Protocols 2:2307–2311.

    https://doi.org/10.1038/nprot.2007.315

    • PubMed
    • Google Scholar
58. 1. Whibley N
    2. Tucci A
    3. Powrie F

    (2019) Regulatory T cell adaptation in the intestine and skin

    Nature Immunology 20:386–396.

    https://doi.org/10.1038/s41590-019-0351-z

    • PubMed
    • Google Scholar
59. 1. Worbs T
    2. Bode U
    3. Yan S
    4. Hoffmann MW
    5. Hintzen G
    6. Bernhardt G
    7. Förster R
    8. Pabst O

    (2006) Oral tolerance originates in the intestinal immune system and relies on antigen carriage by dendritic cells

    The Journal of Experimental Medicine 203:519–527.

    https://doi.org/10.1084/jem.20052016

    • PubMed
    • Google Scholar

Author details

1. Laura Ferrer-Font

   Malaghan Institute of Medical Research, Wellington, New Zealand

   Contribution

   Conceptualization; Formal analysis; Investigation; Visualization; Methodology; Writing - review and editing

   Competing interests

   No competing interests declared

2. Palak Mehta

   Malaghan Institute of Medical Research, Wellington, New Zealand

   Contribution

   Formal analysis, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

3. Phoebe Harmos

   Malaghan Institute of Medical Research, Wellington, New Zealand

   Contribution

   Formal analysis, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

4. Alfonso J Schmidt

   Malaghan Institute of Medical Research, Wellington, New Zealand

   Contribution

   Investigation, Visualization, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

5. Sally Chappell

   Malaghan Institute of Medical Research, Wellington, New Zealand

   Contribution

   Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

6. Kylie M Price

   Malaghan Institute of Medical Research, Wellington, New Zealand

   Contribution

   Supervision, Funding acquisition, Writing - review and editing

   Competing interests

   No competing interests declared

7. Ian F Hermans

   Malaghan Institute of Medical Research, Wellington, New Zealand

   Contribution

   Supervision, Funding acquisition, Writing - review and editing

   Competing interests

   No competing interests declared

8. Franca Ronchese

   Malaghan Institute of Medical Research, Wellington, New Zealand

   Contribution

   Supervision, Funding acquisition, Writing - review and editing

   Competing interests

   No competing interests declared

9. Graham le Gros

   Malaghan Institute of Medical Research, Wellington, New Zealand

   Contribution

   Supervision, Funding acquisition, Writing - review and editing

   Competing interests

   No competing interests declared

10. Johannes U Mayer

    Malaghan Institute of Medical Research, Wellington, New Zealand

    Contribution

    Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

    For correspondence

    jmayer@malaghan.org.nz

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-6225-7803

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