Equine enteroid-derived monolayers recapitulate key features of parasitic intestinal nematode infection

doi:10.21203/rs.3.rs-3342735/v1

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Equine enteroid-derived monolayers recapitulate key features of parasitic intestinal nematode infection

https://doi.org/10.21203/rs.3.rs-3342735/v1

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published 27 Feb, 2024

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Stem cell-derived organoid cultures have emerged as attractive experimental models for infection biology research regarding various types of gastro-intestinal pathogens and host species. However, the large size of infectious nematode larvae and the closed structure of 3-dimensional organoids often hinder studies of the natural route of infection. To enable easy administration to the apical surface of the epithelium, organoids from the equine small intestine, i.e. enteroids, were in the present study used to establish epithelial monolayer cultures. These monolayers were functionally tested by stimulation with IL-4 and IL-13, and/or exposure to infectious stage larvae of the equine nematodes Parascaris univalens, cyathostominae and/or Strongylus vulgaris. Effects were recorded using transcriptional analysis combined with histochemistry, immunofluorescence-, live-cell- and scanning electron microscopy. These analyses revealed heterogeneous monolayers containing both immature and differentiated cells including tuft cells and mucus-producing goblet cells. Stimulation with IL-4/IL-13 increased tuft- and goblet cell differentiation as demonstrated by the expression of DCLK1 and MUC2. In these cytokine-primed monolayers, the expression of MUC2 was further promoted by co-culture with P. univalens. Moreover, live-cell imaging revealed morphological alterations of the epithelial cells following exposure to larvae even in the absence of cytokine stimulation. Thus, the present work describes the design, characterization and usability of an experimental model representing the equine nematode-infected small intestinal epithelium. The presence of tuft cells and goblet cells whose mucus production is affected by Th2 cytokines and/or the presence of larvae opens up for mechanistic studies of the casual interactions between nematodes and the equine intestinal mucosa.

Equine

organoid

enteroid

enteroid-derived

monolayer

nematode

larvae

intestine

in vitro

Stem cell-derived intestinal organoid cultures provide unique opportunities for detailed studies of the host-pathogen interplay at the intestinal epithelium of various animal species [1, 2]. To date, most published work on organoid-pathogen interactions involves viruses, bacteria and protozoa [3–5], while the organoid technology has only just begun to be explored for studies of gastrointestinal (GI) nematodes [6–9].

Intestinal epithelial cells sensing parasite antigens will alarm immune cells and regulate the ensuing expulsion response [10]. Initiation of this response is mainly orchestrated by enterocytes and tuft cells that react to nematodes and/or their products by releasing the alarmins IL-25, IL-33 and TSLP, which in turn activate T helper type 2 (Th2) immune cell polarization with production of IL-4 and IL-13. Together, these cytokines activate a series of effector functions collectively termed the “weep and sweep” response. The main characteristics of this response is expansion of tuft- and goblet cell populations, increased mucus production and increased intestinal peristalsis to trap and expel worms [10–11]. Thus, to reflect these early events of nematode intestinal infection in vitro, a relevant organoid model system is dependent on the presence and flexible differentiation of appropriate cell lineages, such as tuft- and goblet cells.

Both the large size of GI nematode larvae and the natural inwards-facing polarity of 3-dimensional (3D) organoids pose challenges in using traditional basal-out organoids to model host-parasite interactions at the natural site of infection. However, nematodes of both sheep, Teladorsagia circumcincta, and cattle, Ostertagia ostertagi, were shown to burrow into the 3D organoid lumen from the basolateral side although this is not occurring in vivo [8, 9]. Moreover, the protozoan parasite Trypanosoma cruzi could invade murine colon organoids (colonoids) from both the apical and basolateral surface [12]. These organoid models also illustrated interesting physical effects such as swelling when exposed to O. ostertagi and intracellular replication by T. cruzi. However, this may be difficult to reproduce for parasite species that exclusively infect apically, especially when focusing on epithelial invasion and how it can be hindered.

To expose the apical surface of the epithelium, 3D organoids can be used to generate 2D monolayers [13], which facilitates delivery of infectious agents and/or their products to the apical compartment. This approach was recently used for infecting monolayers of murine ceacal organoids with Trichuris muris larvae, providing novel insights into the early stages of intestinal whipworm invasion [7]. Furthermore, growing organoid monolayers in transwell culture systems allows for separate apical and basolateral manipulation of the epithelium making it possible to mimic the environmental conditions encountered by epithelial cells in vivo [reviewed in 14]. It should however be noted that the different conformations in which intestinal organoids can be grown vary greatly in dimensions and cell differentiation patterns. For example, growing organoids as monolayers tend to promote an immature state rather than full differentiation, which can potentially affect the ability of pathogens to interact with the epithelial cells [3]. Thus, the presence and functional activity of defined epithelial cell populations is essential to determine for each type of organoid set-up.

The present study was undertaken to explore the utility of previously established equine small intestinal 3D enteroids and enteroid-derived 2D monolayers [15], focusing on key epithelial functions in the response to nematode infection. To model the relevant infection conditions, the equine enteroid monolayers were basolaterally stimulated with the Th2 polarizing cytokines IL-4 and IL-13 and/or exposed to the infectious larval stage (L3) of three equine GI nematodes, namely cyathostomins, Parascaris univalens and Strongylus vulgaris. Effects, including the presence of tuft cells and mucus-producing goblet cells, were studied using transcriptional analysis combined with histochemistry, immunofluorescence imaging and scanning electron microscopy (SEM). Finally, a recently developed method for live-cell imaging of enteroid monolayers [16] was adapted for the present experimental set-up, enabling differential interference contrast (DIC) microscopy of the apical monolayer surface during exposure to nematode larvae.

Culture of equine enteroids

Equine enteroids were cultured from cryopreserved jejunal enteroids using a previously published protocol [15]. Briefly, frozen enteroids from two individual horses were thawed, washed, fragmented and suspended in ice-cold Matrigel (Corning™) supplemented with the recombinant human growth factors Noggin (100 ng/mL; Peprotech), R-spondin (500 ng/mL; Peprotech), EGF (50 ng/mL; Corning™), Wnt3a (100 ng/mL; Peprotech), Y-27632 (10 µM; Peprotech), SB202190 (10 µM; Tocris Bioscience™), LY2157299 (500 nM; A ChemBlock) and CHIR99021 (2.5 µM; Tocris Bioscience™). Approximately 20–40 crypts in 50 µL Matrigel were plated as domes in 24-well plates (Nunc) and covered by 0.5 mL enteroid growth medium (EGM), i.e. DMEM/F12 containing 1x GlutaMAX (Gibco™), 200 IU/mL penicillin, 100 µg/mL streptomycin and 10 mM HEPES (Invitrogen, CA, USA), 1×N-2 and 1×B-27 (Gibco™). Growth factors as specified above were added every second day and the enteroids were propagated by passage every 4–5 days.

Establishment of equine enteroid monolayers

Equine enteroid monolayers were generated as previously described [15]. Briefly, enteroids at day 4–5 after passage were disrupted to a single cell suspension by 10 min incubation in 1x TrypLE Express Enzyme (Gibco™) at 37 ⁰C and mechanically dissociated by pipetting. The TrypLE dissociation was stopped by addition of four volumes ice-cold EGM containing 5% fetal calf serum (FCS; Invitrogen) and 10 µM Y-27632 and the fragmented enteroids thereafter resuspended in EGM supplemented with the growth factors as specified above.

Monolayers were cultured on semi-permeable transwell polycarbonate inserts with a 0.4 µm pore size for 12-well plates (Thermo Fisher; Fig. 1). The transwell membranes were prepared by 1–2 h incubation in a 1:30 mix of Matrigel and EGM and then air-dried for 10 min prior to use. The enteroid cells were seeded at a concentration of 40–50 000 cells/cm² and cultured in 0.5 mL growth factor-supplemented EGM in the upper chamber and 1.5 mL in the lower chamber of the well. Half of the medium volume (0.25 and 0.75 mL, respectively) was replaced every second day. The monolayer integrity was monitored every 2–3 day by measuring the trans-epithelial electrical resistance (TEER) using an Epithelial Volt-Ohm Meter (Millicell ERS-2, Millipore). Monolayers reaching a TEER of > 800 ohm*cm² were characterized by histological stainings and used for larval exposure experiments. In the present experimental set-up, this condition was achieved after 5–6 days of culture. The enteroids and/or enteroid monolayers were exposed to various stimuli and characterized as outlined in Fig. 1 and specified below.

Preparation of cyathostomin, S. vulgarisandP. univalensthird stage larvae

Third stage cyathostomin, S. vulgaris and P. univalens larvae (L3) were prepared from fecal samples collected from naturally infected privately owned horses, using previous published protocols [17, 18]. Before use, L3s of all three species were decontaminated by 24 h incubation in PBS containing 400 IU/mL penicillin, 200 IU/mL streptomycin, 1 µg/mL amphotericin and 30 µg/mL polymyxin B (Sigma-Aldrich, USA). Strongylus vulgaris and cyathostomin L3s were thereafter exsheated by 4–5 min incubation in 0.2% sodium hypochlorite pre-warmed to 37 ⁰C, then repeatedly washed in PBS at RT. All centrifugation steps were performed for 5 min at 50 x g. For all preparations, the endotoxin content was below 0.23 IU/mL as determined by the LAL assay (Pierce™).

Combined apical and basolateral stimulation with Th2-polarizing cytokines and nematode larvae

Transwell-grown equine enteroid monolayers were primed basolaterally with a combination of 25 ng/mL equine yeast-derived recombinant IL-4 and 25 ng/mL IL-13 (eqIL-4/IL-13; Kingfisher Biotech, Inc) at the second day of culture and kept in parallel with unstimulated monolayers. After a total culture time of 5–6 days, both types of cultures were apically subjected to ~ 20 living L3 of P. univalens, S. vulgaris or cyathostomins, or maintained as untreated controls. After 20 or 48 h incubation, the monolayers were harvested and compared by qPCR analysis or immunofluorescence imaging, respectively (Fig. 1B).

RNA isolation and cDNA synthesis

Transwell-grown equine enteroid monolayers intended for qPCR were set up in duplicates that were pooled at harvest to ensure enough material. RNA was extracted by combining Trizol (Invitrogen, USA) with the E.Z.N.A total RNA kit (Omega Biotek, USA), as previously described [19]. To make cDNA, 1.2 µg of RNA was treated with RQ1 RNAse-free DNAse (Promega) followed by cDNA synthesis using the GoScript Reverse Transcription System (Promega). To ensure that all genomic DNA had been eliminated, -RT controls were run in parallel. The samples were diluted 1:5 in nuclease-free H₂O and stored at -20 ⁰ C until use.

qPCR analysis

The expression of cell lineage markers EPCAM (epithelial cells), PCNA (proliferative cells), SOX9 (proliferative- and stem cells), LYZ (Paneth cells), CGA (enteroendocrine cells), MUC2 (goblet cells) and DCLK1 (tuft cells) was examined by qPCR using previously published primers [15]. Specific cytokine and chemokine transcript responses was quantified using primers for equine IL-5, IL-8, IL-18 and TGF-β [15, 18]. In addition, sequences for CXCL10 (ENSECAT00000013951) and MIF (ENSECAG00000012792) were identified in the equine genome (GCA_002863925.1) in ENSEMBL [20]. Primers were designed to flank intronic sequences using Primer3web [21] and ordered from Eurofins Genomics (Galten, Denmark). Primers were optimized for qPCR regarding annealing temperature and concentration to a 95–100% efficiency (Additional file 1). In addition, PCR products were confirmed by gel electrophoresis and by Sanger sequencing. Duplicate reactions of 2 µL cDNA in 23 µL qPCR mix (i.e. Qiagen Quantitect SYBR Green PCR mix + nuclease free H₂O + primers) were run on a CFX96 Touch PCR machine (Bio-Rad), starting at 95°C for 15 s followed by 40 cycles of 95°C for 15 s, the assay specific annealing temperature for 30 s and 72°C for 30 s. Based on a previous evaluation of equine enteroid samples [15], the three reference genes GAPDH, HPRT and SDHA were selected for normalization of data. The fold change value for the gene of interest was calculated by normalization to the geometric mean for the reference genes followed by calibration to the untreated control [22]. Differences in gene expression between treatments were calculated on ΔΔCt values using the one-way ANOVA followed by Dunnett’s multiple comparisons test or the paired Student’s T-test using the Graph Pad software (Prism 7.0). P-values < 0.05 were regarded as significant. When indicated, gene expression data is reported as mean ± SD.

Histological sectioning and staining of enteroid monolayers

Transwell-grown equine enteroid monolayers cultured in EGM for 5–6 days were fixed with Methanol-Carnoy’s solution (60% methanol, 30% chloroform and 10% glacial acetic acid) to ensure preservation of mucus. The membranes were cut from the plastic frame and divided into three strips, approximately 4 mm wide. The membrane strips were placed between foam pads in embedding cassettes, dehydrated overnight (routine program, 13–14 h) in a tissue processor (ThermoFisher Excelsior) and transferred to an embedding station. To prepare monolayer cross sections, the strips were oriented perpendicularly and embedded in paraffin in an embedding base mold. After cooling, the samples were sectioned in a rotary microtome (Microm/ThermoFisher) into 4 µm sections and placed on slides (SuperFrost Plus). To visualize acidic and neutral mucins, the slides were stained with the Alcian Blue (AB) and Periodic acid-Schiff´s (PAS) technique, respectively, using slightly modified standard protocols. Briefly, the slides were dried overnight at 37 ⁰ C followed by incubation at 60 ⁰ C, deparaffinized, rehydrated and stained in AB, pH 2.5. The slides were then oxidized in 0.5% periodic acid and stained in Schiff´s reagent using Mayer´s haematoxylin as a nuclear counterstain. Finally, the samples were dehydrated, cleared and mounted with coverslips.

Immunofluorescence staining of enteroids and enteroid monolayers

To visualize tuft cells and mucus-producing goblet cells, rabbit anti-human DCLK1 (ab31704; Abcam), diluted 1:1000, previously referred to as DCAMKL1 [23] or rabbit anti-MUC2 (PA5-79702; Thermo Fisher), diluted 1:100, were used as primary antibodies. As secondary antibody, goat anti-rabbit IgG labelled with Alexa Fluor™ 488 (1:600; ab150077; Abcam) was used. The labelling conditions for the anti-DCLK1 and anti-MUC2 antibodies were set on enteroids harvested at day 4 after passage, either cultured in plain EGM or in EGM supplemented with eqIL-4/IL-13 (25 ng/mL) for the last 48h of culture. The enteroids were harvested in cell recovery solution, washed three times in PBS + 0.1% BSA and fixed in 4% paraformaldehyde for 30 min at RT. The fixed enteroids were permeabilized and blocked with staining buffer, i.e. 1x BD Cytoperm™ permeabilization buffer plus (BD Biosciences) in PBS + BSA, for 30 min at RT. The enteroids were incubated with primary and secondary antibodies diluted in staining buffer for 24 h each at RT. In between incubations, the enteroids were washed three times in staining buffer. Finally, the stained enteroids were resuspended in a small volume (< 1 mL) of PBS and placed on a microscope slide immediately before imaging.

Transwell-grown equine enteroid monolayers were fixed in Methanol-Carnoy’s solution for 30 min at RT followed by a gentle rinse in PBS + BSA. The monolayers were incubated with anti-MUC2 diluted in PBS + BSA for 4 hours at RT, gently washed and thereafter incubated with the secondary antibody for another 4 hours. Monolayers were also stained with Alexa Fluor® 488 Phallodin (Sigma Aldrich) for detection of actin filaments and counterstained with DAPI (Bio-Rad Laboratories, Inc) according to the manufacturer’s protocols. Stained membranes were cut out from the inserts and mounted on glass microscope slides using ProLong Diamond Antifade Mountant (Invitrogen).

Confocal laser scanning microscopy

Fluorescence images and z-stacks were captured using an inverted LSM800 laser scanning confocal microscope equipped with 405, 488, 560 and 633 nm lasers and the Zen acquisition software (Carl Zeiss). 20x/1.4 NA, or 40x/1.4 NA water emulsion objectives were used and Z-stacks were acquired in 25 z-sections at 0.66 µm intervals. The images were processed in the Zen blue (Zeiss) or Fiji [24] softwares. All images belonging to the same group were acquired and processed using the same settings.

Scanning electron microscopy

SEM analysis was performed on transwell-grown equine enteroid monolayers cultured for 8 days and after 48h exposure to a cocktail of 15–20 each of P. univalens, S. vulgaris and cyathostomin L3s and compared to untreated control cultures. The monolayers were washed in PBS and fixed by 24 h incubation at 4 ⁰C in 2.5% glutaraldehyde (Sigma) in 0.1 M PHEM buffer (60 mM piperazine-N,N9-bis(2-ethanesulfonic acid), 25 mM HEPES, 10 mM EGTA and 4 mM MgSO4) at pH 6.9. The samples were prepared for SEM by repeated dehydration in graded ethanol and critical point drying (Leica EM CPD300) and then coated with 5-nm platinum (Quorum Q150T-ES sputter coater). Images were captured by a field emission scanning electron microscope (Carl Zeiss Merlin) using in-lens and in-chamber secondary electron detectors at accelerating voltage of 4 kV and probe current of 100 pA.

Live-cell imaging of equine enteroid monolayers upon exposure to nematode larvae

A method to improve the optical conditions for live-cell imaging, described in detail for human enteroid monolayers [16] was applied. In brief, equine enteroid monolayers were grown on alumina Whatman Anodisc membranes (13-mm-diameter with 0.2 µm pores) placed within custom-designed 3D-printed holders denoted “Apical Imaging Chambers” (AICs). To prepare the surface for Matrigel coating, the alumina membranes were pre-treated by 1 h soaking in 20% H₂O₂ at RT followed by a rinse in sterile distilled H₂O (dH₂O) and 5 min incubation in 0.1 mg/mL poly-L-lysine (Sigma-Aldrich). The poly-L-lysine coated membranes were air-dried o.n., soaked in Matrigel diluted 1:30 in dH₂O for 1 h and thereafter air-dried o.n. again. After coating, the membranes were mounted within the AICs and placed in 12-well plates (Nunc, Thermo Fisher Sci). Approximately 25–35 000 enteroid cells in 150 µL growth factor supplemented EGM were seeded into the top compartment of the AICs and 600 µL growth factor supplemented EGM was added to the bottom of the well. After the cells had adhered to the membrane, an additional 250 µL medium was added to cover the AIC. Thereafter, half the medium volume in the well (500 µL) was changed every 2–3 days. The AIC monolayers were kept in culture for 5–6 days before exposure to nematode larvae and microscopy. Live-cell imaging was performed using a custom-built upright microscope described in detail in van Rijn & Eriksson et al, [16]. Briefly, the microscope was equipped with a heated 60x/1.0 NA objective (Nikon CFI APO NIR, 2.8 mm WD), a differential interference contrast (DIC) oil condenser (Nikon d-CUO, 1.4 NA), and placed in a microscope chamber maintaining 37°C, 5% CO₂. The AIC-monolayers were placed in 35-mm glass-bottom dishes (Cellvis, Mountain View, CA, USA) containing 3 mL growth factor supplemented EGM and exposed to a cocktail of 15–20 each of P. univalens, exsheated S. vulgaris and exsheated cyathostomin L3s. Nematode-exposed and non-exposed control cultures were monitored for 3 days and DIC images were acquired at 0, 24, 48 and 72 hours. To obtain an even field of illumination, the images were processed by subtracting a Gaussian blurred projection (30-pixel sigma) from the original images using the Fiji software [24].

Characterization of equine enteroid monolayers

Equine enteroids were disrupted to single cells and cultured on semipermeable transwell membranes (Fig. 1). At the present culture conditions, confluent monolayers were achieved after 2–3 days of culture and TEER values of at least 800 ohm*cm² were recorded after a total culture time of 5–6 days. At this time, the equine enteroid monolayers expressed the cell-lineage marker genes SOX9, LYZ, PCNA EPCAM, CGA, MUC2 and DCLK1 indicating the presence of stem cells, immature proliferative cells, paneth cells, absorptive epithelial cells, enteroendocrine cells, goblet cells and tuft cells, respectively (Fig. 2A). Actin staining of the monolayers (Fig. 2B) and HE staining of cross-sections (Fig. 2C) demonstrated two-dimensional growth with no gaps or cellular overlap. Staining with AB and AB-PAS indicated cells containing acidic (Fig. 2D) and neutral (Fig. 2E) mucins as well as a 0.5–0.8 µm thick mucin lining at the apical brush border (Fig. 2E). Thus, the gene expression data indicated the establishment of a heterogenous enteroid monolayer containing also secretory cell types, which was further supported by mucin stainings.

Th2 cytokines promote tuft- and goblet cell differentiation in equine enteroids and enteroid monolayers

Transwell-grown equine enteroid monolayers were next basolaterally stimulated with eqIL-4/IL-13 and evaluated for alterations in epithelial cell differentiation. This stimulation resulted in a significantly higher gene expression of the goblet cell marker MUC2 (P = 0.0015; Fig. 3A) and the tuft cell marker DCLK1 (p = 0.004; Fig. 3B) compared to the unstimulated controls. In contrast, the gene encoding LYZ was downregulated after stimulation (Table 1), indicating a reduced differentiation of paneth cells or reduced paneth cell activity in response to eqIL-4/IL-13-treatment. The expression of CGA, EPCAM, SOX9 and PCNA was unaffected by this cytokine stimulation (Table 1).

Table 1

Alterations in gene expression after basolateral cytokine stimulation and/or apical exposure to nematode larvae
Gene name	IL-4 + IL-13	P. univalens	P. univalens + IL-4 + IL-13	Cyathostomins	Cyathostomins + IL-4 + IL-13	S. vulgaris	S. vulgaris + IL-4 + IL-13
SOX9	0.54 ± 0.08	1.06 ± 0.08	0.51 ± 0.09	1.05 ± 0.16	0.57 ± 0.07	0.98 ± 0.10	0.52 ± 0.22
LYZ	0.23 ± 0.23	0.74 ± 0.18	0.13 ± 0.05	0.93 ± 0.18	0.17 ± 0.06	0.86 ± 0.24	0.24 ± 0.12
PCNA	0.61 ± 0.14	0.96 ± 0.12	0.44 ± 0.08	0.94 ± 0.03	0.54 ± 0.10	1.10 ± 0.09	0.69 ± 0.18
CGA	0.54 ± 0.11	1.07 ± 0.15	0.67 ± 0.13	1.06 ± 0.32	0.58 ± 0.16	1.18 ± 0.31	0.52 ± 0.12
EPCAM	0.89 ± 0.17	1.13 ± 0.04	0.96 ± 0.22	1.05 ± 0.12	0.86 ± 0.16	1.02 ± 0.09	0.72 ± 0.06
IL-5	1.02 ± 0.49	1.01 ± 0.31	1.15 ± 0.27	1.04 ± 0.20	1.21 ± 0.42	0.71 ± 0.32	0.59 ± 0.28
IL-8	2.11 ± 1.24	1.11 ± 0.35	1.82 ± 1.12	1.02 ± 0.16	2.82 ± 2.50	0.90 ± 0.19	1.92 ± 1.32
TGF-β	0.55 ± 0.13	1.24 ± 0.12	0.78 ± 0.27	1.10 ± 0.16	0.62 ± 0.26	0.84 ± 0.32	0.49 ± 0.17
IL-18	0.61 ± 0.20	1.19 ± 0.14	0.82 ± 0.14	1.02 ± 0.10	0.72 ± 0.12	0.78 ± 0.28	0.54 ± 0.25
CXCL10	1.21 ± 1.04	1.27 ± 0.27	1.52 ± 1.41	0.81 ± 0.43	1.39 ± 1.26	1.05 ± 0.69	0.85 ± 0.43
MIF	0.76 ± 0.18	1.14 ± 0.27	1.05 ± 0.15	1.21 ± 0.45	0.88 ± 0.22	0.78 ± 0.33	0.65 ± 0.21
Transwell-grown equine enteroid monolayers, generated from two horses, performed at two independent experiments were primed basolaterally with IL-4 and IL-13, apically subjected to ~ 20 living L3 of P. univalens, S. vulgaris or cyathostomins, combination of basolaterally IL-4 and IL-13 and apically to L3 or kept as untreated controls. After incubation, the monolayers were harvested and compared by qPCR analysis. Relative expression (fold change ± SD) of cell lineage markers, cytokine- and chemokine genes was normalized to the geometrical mean for the reference genes (GAPDH, HPRT and SDHA) and calibrated to that in the controls.

To confirm the cytokine-induced mRNA expression of MUC2 and DCLK1 on protein level, immunofluorescence labeling conditions were elaborated using 3D enteroids grown in EGM supplemented with eqIL-4/IL-13 for 48h and compared to unstimulated enteroids. Confocal laser scanning microscopy (Fig. 3C) showed MUC2-positive staining in the enteroid lumen of both untreated and IL-4/IL-13 stimulated enteroids, illustrating mucus production by goblet cells. The presence of tuft cells was confirmed by DCLK1 staining, which appeared primarily localized to the cytoplasmic area of the cells. Compared to unstimulated enteroids, eqIL-4/IL-13-stimulated enteroids featured a higher number of DCLK1-positive cells (Fig. 3C). Taken together, stimulation of both equine enteroids and enteroid monolayers with eqIL-4/IL-13 induced a secretory lineage differentiation pattern, which recapitulates findings from studies of nematode infections in vivo [25, 26].

Equine enteroid monolayer differentiation in response to P. univalens, cyathostomin orS. vulgarisinfection in the absence and presence of Th2 cytokine stimulation

To further model the conditions of nematode infection, transwell-grown equine enteroid monolayers were basolaterally primed with eqIL-4/IL-13 and thereafter apically exposed to the infective stage of P. univalens, cyathostomin, or S. vulgaris larvae. For comparison, parallel monolayer cultures were either apically exposed to larvae, basolaterally exposed to cytokines, or kept as untreated controls. After 20 h of larval exposure, expression of cell-lineage marker genes and a selected panel of cytokine and chemokine genes was analyzed by qPCR (schematic in Fig. 1B).

Exposure of unprimed monolayer cultures to P. univalens, cyathostomin or S. vulgaris did not induce differential expression of any of the cytokine, chemokine or cell-lineage marker genes tested (Fig. 4A-B, Table 1). Interestingly however, the expression of MUC2 was significantly higher in eqIL-4/IL-13-primed cultures exposed to P. univalens compared to cultures only stimulated with eqIL-4/IL-13 (p = 0.02; Fig. 4A). A similar trend was also indicated in eqIL-4/IL-13-primed cultures subjected to cyathostomins, but this did not reach statistical significance (p = 0.08; Fig. 4A). DCLK1 gene expression was upregulated at similar levels in all eqIL-4/IL-13-primed cultures, but seemed to remain unaffected by larval exposure (Fig. 4B). Transcripts encoding IL-25, IL-33, TNF-α and TSLP were either below the detection limit of the qPCR used, or showed a low expression in only one of the technical replicates. Together, this data suggests that nematode exposure on its own has a marginal, or only a transient, effect on enteroid monolayer transcription as measured in bulk, but that larvae may boost IL-4/IL-13 induction of goblet cell MUC2 transcript levels.

The capability of the monolayers to respond with mucus production to nematode larvae and/or Th2 cytokines was further assessed by immunofluorescence staining and confocal microscopy. After 48h exposure to P. univalens or S. vulgaris L3s, the enteroid monolayers were fixed in Carnoy’s solution and stained for MUC2. As illustrated in Fig. 4C, Z-stack imaging indicated an elevated production of MUC2 in response to basolateral stimulation with eqIL-4/IL-13, whereas almost no MUC2-positive staining was detected in the untreated cultures (Fig. 4C). Compared to the untreated control, a slight increase of MUC2-positive staining was also noted in unprimed cultures exposed to P. univalens or S. vulgaris. The confocal images thus support the gene expression data (Fig. 4A), further implying that the equine enteroid monolayers contain mucus-producing cells and that their frequency and secretion is affected by apical or basolateral stimulation(s) relevant to nematode infection.

Scanning electron microscopy of equine enteroid monolayers reveals epithelial heterogeneity and nematode – epithelial cell interactions

Larvae-exposed enteroid monolayers and untreated control cultures were next fixed and imaged by SEM. In both types of cultures, a heterogenous epithelial cell layer with different degrees of microvilliated surfaces was observed (Fig. 5A-B). Structures resembling goblet cell orifices (Fig. 5C) and apical microvillus tufts (Fig. 5D) were found in both larval-exposed and untreated monolayers. Thus, the formation of heterogenous equine enteroid monolayers encompassing also secretory cell lineages is supported by the SEM analysis. Despite repeated washing during preparation of the enteroid monolayers, L3 larvae were still found unevenly distributed across the monolayer surface in the infected samples (Fig. 5E-F). Notably, these larvae frequently displayed accumulations of enteroid cells/cell debris attached at their anterior end.

Live-cell imaging of equine enteroid monolayers during exposure to P. univalens, cyathostomin andS. vulgarislarvae highlights morphological alterations to the apical epithelial surface

To visualize the nematode – epithelium interaction dynamics at the interface of infection, conditions for live-cell microscopy were elaborated for the equine enteroid monolayers in co-culture with infective stage nematode larvae (Fig. 6). The cells successfully attached to the alumina membranes placed in AICs and generated a confluent polygonal cell layer within 2–3 days of culture (Fig. 6A). After a total culture time of 5–6 days, the monolayers were subjected to co-infection of P. univalens, cyathostomin and S. vulgaris L3s, and monitored by DIC microscopy. High-resolution images of both larvae (Fig. 6B) and the apical surface of the monolayers (Fig. 6C) were recorded over a period of 0–72 h. During this time, larvae of all three species remained active and motile, appearing to probe the monolayer surface (Additional file 2). There were no signs of larval penetration or stable attachment to the epithelium. However, in agreement with the SEM analysis, pronounced epithelial cell/cell debris clustering was again noticed on the parasite´s anterior end (Fig. 6B).

Of further interest, morphological changes appeared on the monolayers apical surface after 48 h of larval exposure and remained at 72 h post-challenge. These changes included the emergence of protruding cells with a “ruffled” surface structure clearly distinct from the typical enterocyte morphology, that were unevenly distributed over several patches of the monolayer. These patches were often found underlying one or several strongyle larvae and were completely absent in the control cultures (Fig. 6C). This suggests that although we found no dramatic effects on enteroid monolayer transcription assessed in bulk at 20h post-challenge (Fig. 4), nematode exposure nevertheless impacts epithelial cell characteristics in affected regions.

Equine GI nematode infections are an increasing problem worldwide due to the rapid development of anthelmintic resistance in P. univalens and cyathostomins [27]. To find alternative treatments, a better understanding of the parasite interactions with the host intestinal barrier is needed. In that context, equine enteroids are attractive experimental models that can partially recapitulate the structure and function of the small intestinal epithelium [15, 28]. However, the large size of nematode larvae and the closed structure of traditional basal-out 3D enteroids complicate studies of the natural route of infection. To address this issue, equine enteroids were in the present study adapted into 2D monolayer cultures allowing easy administration of nematode larvae to the apical surface of the epithelium. These enteroid monolayers were functionally perturbed by basolateral stimulation with Th2 polarizing cytokines and/or apical exposure to the equine GI nematodes P. univalens, cyathostomins and S. vulgaris, and thereafter characterized for gene expression and morphology.

It was recently demonstrated that equine enteroids can be cultured in 3D conformation, as well as in open conformation as a 2D monolayer [15, 28–30]. One consequence of plating out enteroid cells on a flat surface is that the organizational hierarchy with crypt-like domains rich in stem cells and villus-like regions containing differentiated cells is lost. Although there seem to be some degree of crypt-like spatial organization in enteroid monolayers of murine origin under some conditions [13, 31, 32], such cultures primarily contain immature cells with high proliferative activity that do not differentiate without modification of the culture medium [3, 33]. By removing or reducing the growth factors that stimulate the Wnt pathway, enteroids and enteroid monolayers of man and mice can be transformed from a stem-cell like to a more differentiated state [33]. In such cultures, dual Wnt and notch inhibition is generally needed for expansion of goblet cells [33, 34]. With this background, it was unexpected to find that the equine enteroid monolayers expressed the goblet cell marker MUC2 after 5–6 days of culture despite being kept in the presence of Wnt-stimulating factors. This is in consistence with our previous study that showed similar gene expression levels of MUC2 after 2–3 days of monolayer culture [15]. Further in support of this finding, presumed goblet cell orifices appearing as ring-shaped (crater)-like features containing secretory vesicles were in the present study observed by SEM. The combined expression of CGA, DCLK1, EPCAM, MUC2, SOX9 and PCNA further suggests that the established culture conditions upholds a population of proliferative cells alongside the differentiation of secretory cell lineages. A similar heterogenous gene expression profile was recently demonstrated for bovine enteroid monolayers using an in-house composed medium [35], emphasizing the need to optimize the culture conditions for each animal species and experimental setup.

To verify the presence of goblet cells, transwell-grown equine enteroid monolayers were carefully recovered, sectioned and stained for acidic and neutral mucins. This procedure verified a single cell layer interspersed with occasional mucin-containing cells. In addition, AB-PAS staining revealed a thin layer of mucins situated at the apical brush border, likely representing the membrane-bound mucins that build up the intestinal glycocalyx [36]. Similar staining procedures have illustrated changes in goblet cell distribution and mucin content in various equine intestinal disorders [37] and following the inflammatory response to equine cyathostomins [38]. Therefore, it seemed vital to assess if functional goblet cells are present and can be flexibly induced in equine enteroid monolayers aimed for GI-nematode research.

Even though intestinal mucus production is essential for the “weep and sweep” response occurring at expulsion of worms from the intestinal lumen [10, 25, 26, 39], the mucin components and/or associated proteins are likely also important for initial protection against invading larvae [10, 11]. Since the differentiation of goblet cells and their mucus production is promoted by IL-4 and IL-13, these type 2 cytokines were added into the growth medium of 3D enteroids or to the lower chambers of transwell-grown enteroid monolayers during the last 48 hours of culture. Z-stack imaging of enteroid monolayers illustrated a marked increase in MUC2-positive staining after stimulation with eqIL-4/IL-13, compared to the weakly stained untreated control monolayers. In the 3D enteroids, intense staining of MUC2 was found in the lumen of both untreated and eqIL-4/IL-13-stimulated enteroids, likely reflecting the accumulation of mucus in these closed enteroid structures over time. Thus, mimicking the Th2 cytokine response typically evoked by GI nematode infection dramatically boosts mucin production by equine enteroid monolayers.

The production of IL-4 and IL-13 during nematode infection in vivo is mainly initiated by the alarmins IL-25, IL-33 and TSLP released by epithelial and stromal cells [40]. An important producer of IL-25 is the rather recently described chemosensory tuft cell [reviewed in 41] that respond to GI nematodes and other intestinal insults [42]. In this context, mouse intestinal organoids have been indispensable in improving our understanding of the role of epithelial tuft cells in the initiation and regulation of type 2 immune responses against nematodes [6, 25, 39, 43, 44]. In accordance, the expression of DCLK1 was increased in both equine enteroids and enteroid monolayers by basolateral eqIL-4/IL-13 stimulation, as shown by immunofluorescence microscopy and gene expression analysis, respectively. Furthermore, cells with a tuft cell-resembling morphology, as described for other species [6, 45], were observed by SEM. Taken together, the gene expression data, immunohistochemical staining, confocal and scanning electron microscopy imply that the equine enteroid monolayers contain tuft cells and mucus-producing goblet cells whose frequency and expression is affected by basolateral stimulation with Th2 cytokines linked to nematode infection.

We have previously shown that equine enteroid monolayers respond to apical stimulation with viral and bacterial pathogen-associated molecular patterns (PAMPs) by inducing gene expression for anti- and pro-inflammatory cytokines [15]. However, no differential expression of these cytokines was observed in transwell cultures of equine enteroid monolayers after 20 h exposure to GI nematode larvae, regardless of whether the monolayers had been primed with eqIL-4/IL-13 or not. The only significant effect of nematode larvae was on the expression of MUC2 in monolayers that had been eqIL-4/IL-13-primed before exposure to P. univalens larvae. Effects on MUC2 production was also indicated by z-stack confocal imaging after 48 h exposure to P. univalens or S. vulgaris larvae.

A current major limitation of the transwell culture system is its poor compatibility with live-cell imaging. To overcome this, a novel method for imaging pathogen interactions with human enteroid monolayers was recently demonstrated using Salmonella enterica Typhimurium and Giardia intestinalis as models for bacterial and protozoan infections, respectively [16]. This technology is built on custom imaging chambers that support monolayer growth while optimizing conditions for DIC microscopy to give sufficient optical contrast and resolution for tracing individual microbes atop the epithelium. To test if these AICs are also compatible to study the infection dynamics of equine nematodes, conditions for co-culturing equine enteroid monolayers with cyathostomins, P. univalens and S. vulgaris L3s on AICs were established. During the entire co-incubation time of 72 h, the larvae remained motile across the monolayer surface. Despite this, no signs of stable larval attachment or invasion of the monolayer was observed. Notably, however, the larvae frequently accumulated epithelial cell debris at their anterior end while probing the monolayers. If this behaviour is relevant to nematode foraging, attempts at damaging the epithelial cell layer integrity, or some other aspect of the nematode infection cycle remains an intriguing question for future studies.

Moreover, the live-cell imaging revealed that epithelial cells with an altered apical morphology reproducibly appeared after 48 h of larval exposure, suggesting that either transient larval attachments or excretory/secretory (ES) products released at sites of contact affect the single cell characteristics of the epithelium. While the important role of nematode ES products in establishing and maintaining infections has been known for decades [46, 47], the secretome of equine nematodes and the effects of released ES products on the equine intestinal mucosa remain to be resolved. Although additional experiments are needed to evaluate the putative role of ES products in the present study, the results indicate that equine enteroid monolayers could serve as useful model for studying direct effects of ES products on the equine intestinal epithelium. Future studies should also explore if conditions can be optimized to visualize successful nematode traversal of the epithelial cell layer. This may include testing different states of cellular differentiation and/or increasing the pliability of the infection model, e.g. by culturing the enteroid monolayers atop loose hydrogel scaffolds [48], or introducing an air-liquid interface [6, 49]. Regardless, the imaging technologies elaborated on here will provide a meaningful basis for future studies of nematode infection dynamics at the intestinal epithelial barrier.

In conclusion, an experimental model representative of the nematode-infected equine small intestine that can be analyzed by various imaging techniques was established. These equine enteroid monolayers contain tuft cells and mucus-producing goblet cells whose differentiation and relative abundance can be controlled by addition of Th2 polarizing cytokines. Co-incubation with nematode larvae enables detailed studies of parasite-induced effects on the intestinal epithelium, demonstrating the potential for using enteroid monolayers as an in vitro tool to study host-nematode interactions in the equine gut.

ACKNOWLEDGEMENTS

We gratefully acknowledge Ana Lopes and Jens Eriksson at the Dept of Medical Biochemistry and Microbiology, Uppsala University, for introducing us to your live-cell imaging facility and sharing your expertise with us. The authors also acknowledge the facilities and technical assistance of the Umeå Core Facility Electron Microscopy (UCEM) at the Chemical Biological Centre (KBC), Umeå University, a part of the National Microscopy Infrastructure NMI (VR-RFI 2019-00217).

AUTHOR’S CONTRIBUTIONS

Conceptualization and study design, SH, ET, FM, CF and MS; laboratory work, SH, ET, FM, CF, AN and BH; acquisition of parasite material, ET, ET and PS; analysis and interpretation of data, SH, ET, FM, CF, and MS; writing original draft, SH; reviewing and editing, CF, ET, FM and MS. All authors red and approved the final manuscript.

FUNDING

The project was funded by the Swedish-Norwegian Foundation for Equine Research (H-20-47-568) and by Formas (2019-00809)

AVAILIBILITY OF DATA AND MATERIALS

The data that support the findings of this study are available on request from the corresponding author

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

The experiments were approved by the regional committee for animal experimentation in Uppsala, Sweden (ID: 5.8.18-08784/2020).

COMPETING INTERESTS

The authors declare that they have no competing interests

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Additionalfile1.docx
Additional file 1. Table of novel primers for qPCR.
Additionalfile2.avi
Additional file 2. Live-cell imaging of a P. univalens L3 atop equine intestinal epithelial cells. Equine enteroid monolayers were grown in custom imaging chambers and monitored by live-cell microscopy during exposure to infective stage P. univalens, S. vulgaris and cyathostomin larvae. The movie shows a P. univalens L3 above the monolayer surface after two days co-incubation. Scale bar = 50 µm.
Additionalfile3.avi
Additional file 3. Live-cell imaging of a S. vulgaris L3 atop equine intestinal epithelial cells. Equine enteroid monolayers were grown in custom imaging chambers and monitored by live-cell microscopy during exposure to infective stage P. univalens, S. vulgaris and cyathostomin larvae. The movie shows a S. vulgaris L3 above the monolayer surface after two days co-incubation. Scale bar = 50 µm.

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Equine enteroid-derived monolayers recapitulate key features of parasitic intestinal nematode infection

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Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Culture of equine enteroids

Establishment of equine enteroid monolayers

Combined apical and basolateral stimulation with Th2-polarizing cytokines and nematode larvae

RNA isolation and cDNA synthesis

qPCR analysis

Histological sectioning and staining of enteroid monolayers

Immunofluorescence staining of enteroids and enteroid monolayers

Confocal laser scanning microscopy

Scanning electron microscopy

Live-cell imaging of equine enteroid monolayers upon exposure to nematode larvae

RESULTS

Characterization of equine enteroid monolayers

Th2 cytokines promote tuft- and goblet cell differentiation in equine enteroids and enteroid monolayers

DISCUSSION

Declarations

References

Supplementary Files

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