Skip to main content
Download PDF

Eimeria falciformis secretes extracellular vesicles to modulate proinflammatory response during interaction with mouse intestinal epithelial cells

Parasites & Vectors volume 15, Article number: 245 (2022) Cite this article

Abstract

Background

Protozoan parasite secretions can be triggered by various modified media and diverse physicochemical stressors. Equally, host-parasite interactions are known to co-opt the exchange and secretion of soluble biochemical components. Analysis of Eimeria falciformis sporozoite secretions in response to interaction with mouse intestinal epithelial cells (MIECs) may reveal parasite secretory motifs, protein composition and inflammatory activities of E. falciformis extracellular vesicles (EVs).

Methods

Eimeria falciformis sporozoites were allowed to interact with inactivated MIECs. Parasite secretions were separated into EV and vesicle-free (VF) fractions by discontinuous centrifugation and ultracentrifugation. Secreted EVs were purified in an iodixanol density gradient medium and the protein composition of both EV and VF fractions were analyzed by liquid chromatoraphy-tandem mass spectroscopy. The inflammatory activities of E. falciformis sporozoite EV on MIECs were then investigated.

Results

During the interaction of E. falciformis sporozoites with inactivated MIECs, the parasite secreted VF and vesicle-bound molecules. Eimeria falciformis vesicles are typical pathogenic protozoan EVs with a mean diameter of 264 ± 2 nm, and enclosed heat shock protein (Hsp) 70 as classical EV marker. Refractile body-associated aspartyl proteinase (or eimepsin), GAP45 and aminopeptidase were the main components of E. falciformis sporozoite EVs, while VF proteins include Hsp90, actin, Vps54 and kinases, among others. Proteomic data revealed that E. falciformis EV and VF proteins are aggregates of bioactive, antigenic and immunogenic molecules which act in concert for E. falciformis sporozoite motility, pathogenesis and survival. Moreover, in MIECs, E. falciformis EVs induced upregulation of gene expression and secretion of IL-1β, IL-6, IL-17, IL-18, MCP1 as well as pyroptosis-dependent caspase 11 and NLRP6 inflammasomes with the concomitant secretion of lactate dehydrogenase.

Conclusions

Eimeria falciformis sporozoite interaction with MIECs triggered the secretion of immunogenic and antigenic proteins. In addition, E. falciformis sporozoite EVs constitute parasite-associated molecular pattern that induced inflammatory response and cell death. This study offers additional insight in the secretion and protein composition of E. falciformis secretomes as well as the proinflammatory functions of E. falciformis sporozoite EVs.

Graphical Abstract

Background

Host-parasite interaction is a dynamic and phenomenal feature of parasitism. It co-opts parasite development and pathogenesis by means of cellular signaling, inflammation, cell death and, essentially, the excretion or secretion of molecules. In general, parasite secretions (or secretomes) are formed along several secretory pathways and released during the host-parasite interplay [1]. Previous studies have described and characterized the secretion of extracellular vesicles (EVs) and vesicle-free (VF) molecules from parasitic protozoa and helminths [2, 3] as well as from parasite-infected host cells [4]. Similarly, there have been several studies on the secretion, characterization and proteomic profiling of Eimeria sporozoite proteins triggered by thermal, chemical and mechanical stressors [5]. In addition, VF molecules have been induced and characterized from protozoan parasites in various modified mediums and by physicochemical stressors [5, 6]. Pathogenic protozoan EVs activated by chemical stressors and serum-starved media have also been characterized and  profiled [7]. A few studies have also indicated that host-parasite interactions could induce the secretion of vesicle-bound and VF molecules. In this regard, the formation of parasite secretomes could occur prior to [8] or during protozoan parasite attachment or invasion of the host cell [3, 8,9,10], but the description of distinct parasite secretions during interactions with the host cell, and characterization of such parasite-derived secretomes have not been demonstrated.

Extracellular vesicles are heterogeneous, sub-cellular, lipid-bound vesicles formed during pathophysiological processes and host-pathogen interactions [11]. Based on biogenesis and size, EVs are broadly classified as exosomes, microvesicles/microparticles and apoptotic bodies [7, 11,12,13,14,15]. EVs usually enclose soluble components of cellular origin, such as metabolic intermediates, glycoconjugates, lipids, proteins and nucleic acids [14]. Functionally, protozoan parasite EVs can deliver their cargoes to mediate parasite motility and development, differentiation, cytoadherence and pathogenesis [16,17,18,19,20]. During host–pathogen interaction, EVs are pivotal in such processes as intercellular communication, horizontal gene transfer, disease biomarker, antigen presentation, immune response and host cell death [14, 15, 21,22,23].

Typically, Eimeria spp. are obligate intracellular apicomplexan parasites [16]. More specifically, Eimeria falciformis naturally parasitizes caecal epithelial cells of wild and laboratory mice, causing catarrhal enteritis, hemorrhage and epithelial sloughing [17, 18]. The life-cycle of E. falciformis is similar to that of other coccidian parasites, with the sporozoites, after excystation from the sporocyst, actively migrate through the mice gut before invading the host intestinal epithelial cells (IECs) [17, 18, 25]. In general, parasitization of the host cells by apicomplexan parasites involves gliding motility, cellular attachment, invasion and spontaneous formation of excretory/secretory products with which apicomplexans modulate host cell responses [10, 26,27,28]. However, E. falciformis sporozoite proteins secreted during interaction with the host IECs have not been described [5]. Also, the secretion of parasite-derived EVs has not been characterized in Eimeria spp. [7], nor have the inflammatory responses of host IECs, such as the secretion of cytokines and formation of inflammsome complexes, to Eimeria-derived EVs.

In this study, E. falciformis sporozoites were allowed to interact with inactivated mouse IECs (MIECs), and E. falciformis sporozoite VF and EV fractions were separated, purified and analyzed. In addition, MIECs were stimulated with E. falciformis sporozoite EVs (EfSEVs) to determine differential regulation and expression of inflammatory cytokines (interleukin [IL]-1β, IL-6, IL-17, IL-18), the chemokine monocyte chemoattractant protein 1 (MCP1) and pyroptosis-dependent caspase 11 and nucleotide-oligomerization domain (NOD)-like receptor pyrin 6 (NLRP6) inflammasomes, and to reveal underlying synergy between MIEC inflammatory response and lethal activity of EfSEVs.

Methods

Parasite propagation

Parasite propagation was carried out as described earlier [17, 29, 30] with modification. In brief, 100 specific pathogen-free Balb/c mice aged between 6 and 8 weeks were raised in plastic cages placed in a pathogen-free room with adequate supply of food and sterile water. Each mouse was orally inoculated with 50 µl of 2 × 105 E. falciformis sporulated oocysts/ml phosphate-buffered saline (PBS). Oocysts shed in feces between 7- and 11-day post-infection were recovered from homogenized fecal solution, washed through a metal strainer and purified by salt flotation. Purified oocysts were washed and suspended as appropriate in 2.5% potassium dichromate followed by incubation at 28 °C and 120 rpm for approximately 10 days. Sporulated oocysts were stored at 5 °C and used within 30 days of storage.

Purification of E. falciformis sporozoites

Eimeria falciformis sporozoites were excystated and purified as earlier reported [31,32,33]. Briefly, E. falciformis sporulated oocysts were washed in PBS to remove potassium dichromate through discontinuous centrifugation. Purified oocysts were precipitated in 40 ml of antibiotic–antimycotic diluent (1 part dilutent per 100 ml) (Solarbio, Beijing China). Eimeria falciformis sporulated oocysts were then suspended in 5% (v/v) sodium hypochlorite for 20 min on ice with intermittent stirring, and  the oocysts were washed in PBS. The hypochlorite-treated sporulated oocysts were then vortexed with an equal volume of glass beads (diameter: 0.5 mm) for 3 min to release sporocysts and thereafter incubated with 0.75% (w/v) sodium taurodeoxycholate and 0.25% (w/v) trypsin in PBS for 1 h at 37 °C and 200 rmp for sporozoite excystation. The final purification step was vacuum filtration of the E. falciformis sporozoites [19, 32].

Cell and parasite culture

Mouse intestinal epithelial cells were cultivated in complete medium (Dulbecco’s modified Eagle’s medium [DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich) (v/v) and 1% antibiotic diluent (Solarbio, Beijing, China)[26]. After confluence was reached, approximately 1 × 106 MIECs were plated with complete medium and incubated overnight. To inhibit exosome secretions [27], MIECs were inactivated with 4% paraformaldehyde (Solarbio, Beijing, China) for 20 min at room temperature and washed 5 times with sterile PBS. Inactivated MIECs were re-cultured in Exo-clear cell growth medium (System Biosciences, Palo Alto, CA, USA) followed by inoculation of 2 × 106 freshly excysted E. falciformis sporozoites [28]. The parasites were maintained in the culture plates for 18 h [30] at 37 °C in a 5% CO2 humidified incubator. A non-treated control (NC) experiment was prepared in parallel in which inactivated MIECs were incubated as described above, but without E. falciformis sporozoites.

Separation of E. falciformis sporozoite secretomes

A 350-ml sample of culture supernatant was harvested without disrupting the MIEC monolayer and centrifuged at 4 °C sequentially at: 1000 g, 5 min; 2000 g, 10 min; 5000 g, 20 min; 10,000 g, 30 min. The supernatant was then transferred into polycarbonate ultracentrifugation tubes (Thermo Fisher Scientific, Waltham, MA, USA) and centrifuged at 120,000 g for 18 h in a Sorvall WX+ Ultra Series centrifuge (T-890 fixed angle rotor, k-factor 25.1; Thermo Fisher Scientific). The resulting supernatant from the ultracentrifugation was subjected to ultrafiltration through a 0.22-µm Corning sterile filter (Corning, Corning, NY, USA) at 4 °C under gravity, followed by 10-fold concentration using an Ultra 10 kDa cut-off membrane (Amicon Ultrafiltration System; MilliporeSigma, Burlington, MA, USA) and stored at − 80 °C as the E. falciformis sporozoite VF fraction [31]. The cell culture supernatant from the NCs was prepared in the same manner.

The resulting pellets from the ultracentrifugation process were pooled, reconstituted in 8 ml PBS (pH 7.2) and resolved on discontinuous iodixanol gradients solutions prepared by diluting OptiPrep™ density gradient (ODG) medium (Sigma-Aldrich) in 60% (v/v) aqueous iodixanol with sterilized 0.3 M sucrose/10 mM Tris, pH 7.2 [32, 33]. The gradient was formed by adding 1.6 ml each of 40, 20, 10 and 5% iodixanol solution into a 9-ml polycarbonate tube (Thermo Fisher Scientific) and 1 ml of EfSEV suspension was overlaid and  subjected to ultracentrifugation for 20 h at 120,000 g and 4 °C in a Sorvall WX+ Ultra Series centrifuge (T-890 fixed angle rotor, k-factor 25.1; Thermo Fisher Scientific). Seven gradient layers were collected, resuspended in PBS (pH 7.2) and washed twice for 4 h at 120,000 g and 4 °C. EfSEV pellets were pooled, concentrated in 400 µl of PBS and stored at − 80 °C. Likewise, NC pellets were collected and washed appropriately. The density of EfSEVs was measured from the corresponding ODG layer after appropriate dilution at an optical density of 244 nm [32, 33] with a UV–Visible spectrophotometer (Biomates 3S; Thermo Fisher Scientific) (Additional file 6 : Data set).

Transmission electron microscopy

For transmission electron microscopy (TEM), 10 μl of EfSEVs was added to the a formvar carbon film-coated copper grid for 3 min. Excess liquid was removed with filter paper followed by staining with 3% phosphotungstic acid [34, 35]. An equal volume of NC particles was also stained. The grids were observed in model HT770 transmission electron microscope (Hitachi, Tokyo, Japan).

Nanoparticle tracking analysis

The size of EfSEVs was determined using the Zetasizer Nano-Zs instrument (Malven, Worcestershire, UK). Briefly, 10 μg of EfSEVs and an equal volume of NC pellets were dissolved in an appropriate volume of PBS to make a total volume of 1 ml in each case. The size and distribution of the particles were measured in triplicate [13].

Sodium dodecyl sulfate-polyacryamide gel electrophoresis and liquid chromatography-tandem mass spectrometry

The protein components of the EfSEV and VF fractions were quantified using the BCA kit (Solarbio, Beijing, China). An equivalent of 10 µg of EfSEV and VF fractions, as well as the NC and NCs were separately mixed with lysis buffer (Solarbio, Beijing, China) according to the manufacturer’s instruction, heated in a water bath at 100 °C for 10 min and resolved by 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) [35, 36] at 100 V in a Mini-Cell electrophoresis system (BioRad Laboratories, Hercules, CA, USA). Following electrophoresis, the SDS-PAGE gel was stained with a Coomassie blue stain kit. Subsequently, the EfSEV and VF protein gel lanes were excised and pooled for mass spectrometry (MS).

Liquid chromatography-tandem MS (LC–MS/MS) was performed by the Lu-Ming Biotech Co., Ltd. (Shanghai, China). In brief, excised protein gel lanes were dehydrated and dried in a vacuum. Gel particles were reduced in 10 mM DTT/25 mM NH4HCO3 followed by treatment with alkylation buffer and recovered in 25 mM NH4HCO3. Reduced gel particles were digested in 20 μl of 0.02 μg/μl trypsin in 25 mM NH4HCO3. The supernatant was treated with 50 μl of 5% formic acid/67% acetonitrile followed by elution in 5% formic acid/67% ACN/H2O. After drying using low-pressure centrifugation, the sample was re-dissolved in 40 µl of 0.1% formic acid and centrifuged through a column. The digested peptide elute was adjusted to pH 7 by H3PO4 and the resulting peptides were lyophilized in 150 μl of 100% methanol. MS analyses were performed with a Q-Exactive mass spectrometer (Thermo Fisher Scientific) equipped with Nanospray Flex source (Thermo Fisher Scientific). MS/MS spectra were obtained with a resolution of 17, 500 and an AGC target of 2e5 at a maximum injection time of 50 ms. The Q-E dynamic exclusion was set at 15.0 s in positive mode.

Protein database search and bioinformatics

MaxQuant, a quantitative proteomics software package, was used to search peptide sequences against Eimeria species, Toxoplasma gondii and Plasmodium falciparum proteins in UniProt. Trypsin digestion specificity was used to search databases. For protein quantification, MS1 and MS2 tolerance were set at 10 ppm and 0.02 Da, respectively. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) analyses were also performed.

Western blot

EfSEV and VF protein concentrations were measured using a BCA kit (Solarbio, Beijing, China). An equivalent volume (10 µg) of EfSEV, VF, NC and NCs was separately mixed with 4× Loading Buffer (Solarbio, Beijing, China) and vortexed. The mixture was heated in a water bath at 100 °C for 10 min. The protein components were resolved in a 12% SDS-PAGE gel, and the protein bands were transferred to a polyvinylidene difluoride membrane (Merck-Millipore, Germany). After 1 h of blocking with 0.01% Tween-20 in PBS (PBST) containing 5% skimmed milk, the membrane was incubated with anti-heat shock proteins 70 kDa (Hsp70; Proteintech Group Inc., Rosemont, IL, USA) overnight at 4 °C. Thereafter, the membrane was washed in PBST and incubated with horseradish protein-conjugated antibodies (Proteintech Group) for 1 h at room temperature. The membrane was treated with the WesternBright™ ECL enhanced chemiluminescent substrate kit (Advansta Inc., San Jose, CA, USA) according to the manufacturer’s instruction and visualized using the Luminescent Image Analyzer Amersham Imager 600 series (GE Co., Boston, MA, USA).

Multiplex protein microarray

The MIECs were plated at a density of 1 × 106 in complete medium and incubated overnight at 37 °C and 5% C02. Plated MIECs were subsequently re-cultured in DMEM supplemented with 10% exosome-depleted fetal bovine serum [37] and stimulated with 10, 30, 50 and 100 µg of EfSEVs and PBS (0 µg of EfSEVs) was used as experimental control treatment. Similarly, an equal density of MIECs was treated with 50 µg of EfSEVs for 0, 6, 12 and 18 h. Cell culture supernatants were collected for protein microarrays. IL-1β, IL-6, IL-17, IL-18 and MCP1 proteins were quantified by QAM-INF-1 (Quantibody® Mouse Inflammatory Array 1 Kit; RayBiotech, Peachtree Corners, GA, USA) [38]. Analyses were done in triplicate, and targeted cytokines were tested in quadruplicate. Each cytokine was quantified as mean fluorescence using the InnoScan 300 Microarray Scanner (Innopsys, Carbonne, France) at a wavelength of 532 nm and a resolution of 10 µm. Data were analyzed using QAM-INF-1 analysis software (RayBiotech).

RNA extraction and quantitative PCR

RNA was extracted from EfSEV-stimulated MIECs using TRIzol reagents (Invitrogen, Thermo Fisher Scientific). In parallel, the same density of MIEC was treated with NC particles. A 1-µg aliquot of total RNA was reverse transcribed with the Primescript™ RT Reagent Kit (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. PCR primers for IL-1β, IL-6, IL-17, IL-18, MCP1, caspase 11 and NLRP6 were synthesized by Tsingke Biological Technology (Xi’an, China) (Additional file 5). Real-time quantitative PCR (qPCR) was performed using TB Green pre-mix Ex Taq™ II (Takara Bio Inc.) and the CFX98 real-time PCR detection system (Bio-Rad, Hercules, CA, USA). Each reaction tube contained 1 μl each of forward and reverse primers, 2 μl of cDNA template, 10 μl of TB Green and 6 μl of nuclease-free water. Assays were performed in triplicate, and data were normalized by using β-actin as a reference gene.

Lactate dehydrogenase assay

Approximately 1 × 106 MIECs in DMEM supplemented with 10% exosome-depleted FBS were stimulated with 50 µg of EfSEVs for 0, 6, 12, 18 and 24 h. Likewise, 10, 30, 50, and 100 µg of EfSEVs were used to stimulate MIECs and PBS (0 µg of EfSEVs) as experimental control treatment. Also, an equivalent volume of NC particles was used as a negative control for EfSEV time- and dose-dependent production of lactate dehydrogenase (LDH) by MIECs [39, 40]. The culture plates were incubated at 37 °C and 5% CO2. LDH was measured by Chekine™ LDH assay kit (Abbkine, Wuhan, China) as instructed in the product manual. Absorbance was measured at 450 nm on a SpectraMax M5e Multi-Mode Microplate Reader (Molecular Devices, LLC, San Jose, CA, USA).

Statistical analyses

Data were expressed as the mean with the standard deviation and analyzed by one-way analysis of variance. Turkey’s test was used for multiple comparisons. Linear regression analysis was performed using SPSS software version 21 (SPS IBM Corp., Armonk, NY, USA). Charts were generated in GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). Differences among dose- and time-dependent activities of EfSEVs on MIECs relative to the experimental control were considered to be statistically significant at *P < 0.05, **P < 0.001 and ***P < 0.0001.

Results

E. falciformis sporozoite secretomes during interaction with MIECs

The E. falciformis sporozoite (Fig. 1a) is the motile, invasive stage of the parasite which navigates through the host gut before invading the host cell. The fractions of E. falciformis sporozoite secretome were separated from the culture medium by serial centrifugation and ultracentrifugation steps, and then the scereted EVs were characterized by nanoparticle tracking analysis (NTA) and TEM. Also, EfSEV and VF protein compositions were identified by electrophoresis (Additional file 5) and analyzed by MS (Additional file 6). Our findings revealed that EfSEVs had a buoyant density ranging from 1.12 to 1.179 g/cm3 distributed between the second and the seventh ODG layers. The particles from NC had a buoyant density that ranged between 0.57 and 1.103 g/cm3 (Additional file 5). NTA analysis revealed that EfSEVs were a heterogeneous population of EVs with a diameter of approximately 37.84–1,281 nm (Fig. 1b). TEM observations indicated that EfSEVs were spherical and circular in shape, similar to Toxoplasma gondii and Neospora caninum EVs [35, 41] (Fig. 1c). In addition, TEM revealed that NC particles (Additional file 2) did not contain secreted vesicles, indicating that the observed EVs were formed during E. falciformis sporozoite interaction with MIECs. Additionally, the vesicular property of EfSEVs and depletion of protein in the non-treated control experiment were also confirmed by targeting Hsp70 in EfSEVs, VF, NC, and NCs for western blotting (Fig. 1d).

Fig. 1
figure 1

Eimeria falciformis sporozoite and derived EVs. a E. falciformis sporozoite after excystation from sporocyst. b Bar graph from NTA analysis of 10 µg EfSEVs in 1 ml of PBS. EfSEV mean diameter was 246 ± 2 nm. c TEM image of EfSEV processed through negative staining with 3% phosphotungstic acid. d Western blot analysis of EfSEVs using anti-Hsp70. Abbreviations: EfSEV, E. falciformis sporozoite EVs; EVs, extracellular vesicles; Hsp70, heat shock protein 70; NC, non-treated control particles; NCs, non-treated control supernatant; TEM, transmission microscopy; VF, vesicle-free (see Additional files 1–5)

Full size image

Protein composition of E. falciformis sporozoite EV and VF fractions

Electrophoretic analyses of the EfSEV and VF fractions showed that E. falciformis sporozoites secreted proteins as components of its secretomes (Additional file 6) during interaction with inactivated MIECs. The protein composition of the EfSEV and VF fractions were analyzed by LC–MS/MS. MS/MS spectra were searched in the protein database for avian and bovine Eimeria species, T. gondii and P. falciparum because of the intractable genome of E. falciformis [9]. Forty-two proteins were identified from the E. falciformis sporozoite VF fraction (Additional file 6), including actin (putative), kinases, heat shock proteins (Hsp 70 and Hsp90), vacuolar protein sorting (Vps) 54, elongation factor 1 alpha (EF-1α), early gametocyte enriched phosphoprotein (EGXP), as well as some P. falciparum and T. gondii orthologous proteins (Fig. 2; Additional file 6). GO terms for E. falciformis sporozoite VF protein are shown in Fig. 3a.

Fig. 2
figure 2

EfSEV and VF proteins identified by mass spectrometry. The list and other information on the identified proteins are provided in Additional file 5

Full size image
Fig. 3
figure 3

GO annotation of identified EfSEV and VF proteins. a GO terms for E. falciformis VF proteins, b GO terms for EfSEV proteins. See Additional file 5. Abbreviations: GO, Gene ontology

Full size image

Thirty-seven proteins were identified from EfSEVs along with other unidentified protein peptides (Fig. 2; Additional file 6). Identified EfSEV proteins include refractile body (RB)-associated proteins, Hsp70, EF-1α, cytosol aminopeptidase (putative), aspartyl proteinase (or eimepsin) and proteases. Other proteins from EfSEVs were gliding associated protein (GAP) 45, histones and some orthologous proteins from T. gondii and P. falciparum (Fig. 2; Additional file 6). EfSEV proteins relating to biological processes, cell components and molecular functions are shown in Fig. 3. Also, the protein composition of EfSEV implies the involvement of these proteins in E. falciformis sporozoite binding/fusion to the host cell, endocytosis and metabolic processes (Fig. 3). KEGG analysis revealed that EfSEV proteins were enriched in pathways involving endocytosis, protein processing, RNA transport, spliceosome and ribosome, as found in N. caninum EV proteins [35], as well as the glutathione metabolism pathway, which is associated with gene expression, DNA and protein synthesis, signal transduction, cytokine production and immune responses (Fig. 3; Additional file 5).

EfSEVs induced IL-1β, IL-6, IL-17, IL-18 and MCP1 production in MIECs

Intestinal epithelial cells are known to secret cytokines and chemokines as well as cellular infiltration during inflammatory responses and parasite-host interactions [17, 42]. Also, Eimeria species can induce the secretion of diverse cytokines/chemokines in the host intestinal epithelium, including IL-6, IL-17, IL-18, IL-1β, interferon gamma (INFγ) [17, 44] and MCP1 [43]. However, the reactive antigenicity of Eimeria-derived EVs in host inflammatory responses during infection is not fully understood. Therefore, we examined the secretion of MIEC proinflammatory cytokines after EfSEV stimulation. The results showed that MIECs produced quantifiable IL-1β, IL-18, IL-17 and IL-6 in response to EfSEV dose and time stimulations. While the level of IL-6 significantly increased with increasing time of MIEC stimulation by EfSEVs, the production of IL-17 reduced (Fig. 4). In addition, the production of IL-6 and IL-17 increased at high doses of EfSEVs (Fig. 4). Also, the production of MCP1 significantly increased with increasing EfSEV doses and time of stimulation (Fig. 4). The secretion of IL-18 by EfSEVs-stimulated MIECs was initially low but increased with time and with increasing doses of EfSEVs. Similarly, the production of IL-1β only increased significantly at a high dose of EfSEVs (Fig. 4). Taken together, EfSEV dose enhanced the production of proinflammatory cytokines in MIECs whereas the production of MCP1 was significantly increased with increasing time of stimulation and dosage of EfSEVs (Fig. 4).

Fig. 4
figure 4

Cytokine and chemokine secretions by MIECs. 1 × 106 MIECs were stimulated with 10, 30, 50 and 100 µg of EfSEVs and PBS (0 µg of EfSEVs) for 24 h. Also, 50 µg of EfSEVs were used to stimulate 1 × 106 MIECs for 6, 12, 18 and 0 h as experimental control. IL-6, IL-17, MCP1, IL-1β and IL-18 secretions in cell culture supernatants were measured by protein microarray kit. The signals of the laser scanning map were extracted with GenePix 6.0 microarray analysis software. Quantitative data obtained from the Quantibody-INF-1Q-Analyzer were analyzed using RayBiotech mouse Inflammation Array 1 software. Cytokine concentrations (pg/ml) were determined by mean fluorescence intensities and linear regression standard curves were generated from the manufacturer’s standard. Each spot on the graph represents quantitative mean ± SD values in triplicate experiments. Asterisks indicate statistical significant differences from the control treatments at: *P < 0.05, **P < 0.001, ***P < 0.0001. Abbreviations: IL, Interleukin; MCPI, monocyte chemoattractant protein 1; MIECs, mouse intestinal epithelial cells; PBS, phosphate-buffered saline

Full size image

Gene expression profiles of the selected cytokines and chemokine also indicated steady and significant upregulation of MCP1 and IL-6 mRNA expression with increasing time of stimulation and EfSEV doses, but the increased IL-17 mRNA expression was just significant at high concentration of EfSEVs (Fig. 5). IL-18 mRNA expression was also significantly upregulated with increasing dose of EfSEVs and time of treatment (Fig. 5). However, mRNA expression of INFγ remained unchanged with increasing time and EfSEV doses (data not shown). In essence, EfSEVs are parasite components that modulate proinflammatory cytokines and chemokine genes during Eimeria-host interactions (Fig. 5). The apparent inconsequential change in the expression of the cytokines with NC particles indicated that the observed relative expressions of IL-6, IL-17, IL-18, IL-1β and MCP1 were due to the MIEC responses to EfSEV bioactivities (Fig. 5&6).

Fig. 5
figure 5

Eimeria falciformis sporozoite EVs modified mRNA expressions of IL-6, IL-17, MCP1, IL-1β and IL-18. MIECs (1 × 106) were plated and stimulated with 50 µg of EfSEVs for 6, 12 and 18 h and with 10, 30, 50 and 100 µg of EfSEVs for 24 h. PBS (0 µg of EfSEVs) was used as an experimental control for dose treatments and 0 h was used as control for time-dependent treatments. An equal volume of NC particles was set as negative control. After each time point, RNAs were extracted, and IL-6, IL-17, MCP1, IL-1β and IL-18 expressions were determined by quantitative PCR. Bars represent mean ± standard deviation values in triplicate experiments. Asterisks indicate a statistical significant difference with the experimental control treatment at *P < 0.05, **P < 0.001, ***P < 0.001

Full size image

EfSEVs upregulated MIEC pyroptotic inflammasomes and induced the release of LDH

The eventual outcomes of pathogen-host interactions are sometimes marked by inflammation and host cell death [45]. Given the functional association of IL-18 and IL-1β with inflammatory programmed cell death, we investigated the regulatory activities of caspase 11 and NLRP6 in EfSEV-stimulated MIECs by qPCR. The analysis showed that increasing EfSEV dose and increasing duration of stimulation significantly upregulated the mRNA expression of NLRP6 and caspase 11 in MIECs whereas NC particles showed no distinct effects on inflammasome activation (Fig. 6a, b).

Fig. 6
figure 6

Eimeria falciformis sporozoite EVs upregulate caspase 11 and NLRP6 inflammasomes and release LDH. a, b MIECs (1 × 106) were plated and stimulated with 50 µg of EfSEVs for 6, 12, 18 h and 10, 30, 50 and 100 µg of EfSEVs for 24 h. An equivalent volume of PBS (0 µg of EfSEVs) was used as experimental control for the dose treatment and 0 h was used as control for the time-dependent treatment. An equal volume of NC particles was set as a negative control. RNAs were extracted and analyzed by qPCR for caspase 11 and NLRP6 mRNA expression. Bars represent mean ± standard deviation values in triplicate experiments. The difference in mRNA expression was compared with control treatments and considered to be significant at *P < 0.05, **P < 0.001, ***P < 0.001. c EfSEVs induced secretion of LDH in MIECs. 1 × 106 MIECs were stimulated with increasing doses (10, 20, 30, 50, 100 µg) of EfSEVs. Also, 50 µg of EfSEVs was used to stimulate MIECs at increasing duration (0, 6, 12, 18, 24 h). PBS (0 µg of EfSEVs) was used as experimental control for the dose treatment and 0 h as the control for time-dependent treatment. An equal volume of NC particles was set as a negative control in both cases. MIECs were harvested at the end of the treatment and assayed for LDH production using the Chekine™ LDH assay kit in triplicate. Differences in the time- and dose-dependent release of LDH were compared with the control treatments and considered to be significant at *P < 0.05, **P < 0.001, ***P < 0.001. Abbreviations: LDH, Lactate dehydrogenase; NLRP6, nucleotide-oligomerization domain (NOD)-like receptor pyrin 6

Full size image

Cytosolic protein ligands, such as pathogen-associated molecular patterns (PAMPs), have been shown to activate pyroptosis-dependent IEC death. Specifically, pyroptosis of IECs can occur via the activation of caspase 11 and NLRP6 with a concomitant secretion of IL-18 and IL-1β [46] with the release of cytosolic LDH [39]. Therefore, LDH production by EfSEV-stimulated MIECs was investigated. The increased production of LDH by MIECs was significant from 18 h of EfSEV treatment and with increasing doses of EfSEVs. The result indicated that EfSEVs induced significant production of LDH as a marker for proinflammatory MIEC death. This finding was supported by the observation that NC particles showed no significant involvement in the production of LDH by MIECs (Fig. 6c).

Discussion

Previous studies have demonstrated that protozoan parasite secretomes include vesicle-bound and VF molecules such as nucleic acids, carbohydrates or glycocnjugates, lipids and proteins [6, 7, 47]. In this study, E. falciformis sporozoite EV and VF proteins were resolved from the total secretome formed while the parasite interacted with inactivated MIECs, and the protein components were analyzed by gel electrophoresis and LC–MS/MS. The electrophoretic profiles revealed protein bands in EfSEV and VF gel lanes, but no specific band was visible from NC and NCs, indicating that EfSEVs and VF preparations were free from MIEC secretions (Fig. 1d) (Additional file 5). This observation is in conformity with T. gondii tachyzoites which were unable to secret vesicles after being treated with aldehyde [48]. Notwithstanding, EfSEVs are subpopulations of self-assembled spherical and circular-shaped EVs with an expression of Hsp70 as membrane marker (Fig. 1c, d).

The proteomic dataset showed that EfSEVs and the VF fraction comprised membrane-associated and bioactive proteins (Additional file 6). Functionally, the gliding motility of apicomplexan parasites requires actin polymerization [49], and E. falciformis sporozoite VF Hsp90 might be involved in host cell invasion, host-parasite interaction, signal transduction and parasite development similarly as found in Eimeria tenella, T. gondii, and P. falciparum Hsp90 [50]. Also, the Vps51-54 complex has been suggested to mediate host endocytosis of T. gondii and P. falciparum [51, 52]. Vps54 would possibly play a similar role during E. falciformis sporozoite invasion (Fig. 2). Additionally, Cryptosporidium parvum EF-1α has been associated with host cell invasion [53], and E. tenella protein kinases are immunogens [54] known to inhibit apoptosis and play major roles during stress [55, 56]. Congruently, E. falciformis VF proteins are likely to be important for sporozoite motility, survival, invasion and pathogenesis.

Moreover, EF-1α and Hsp70 have been identified as EV markers of Leishmania major and other parasitic protozoa [57]. The identification of Hsp70 and EF-1α in EfSEV and VF proteins (Fig. 2) suggests that specific protein could be secreted via two or more secretory pathways during host-parasite interactions. The secretion of Hsp70 by Eimeria species is associated with sporozoite formation, pathogenicity, protective immunity and response to stress [5, 58, 59]. Hsp70 has also been reported among EV proteins from C. parvum, N. caninum, T. gondii, Leishmania and Trypanosoma species [7]. EfSEV proteases are likely to play important roles in E. falciformis sporozoite invasion, evasion of immune cells [60], developmental regulation and virulence, as revealed in other Apicomplexa [61], as well as degrading the cell matrix of host tissue [62]. Also, proteases are transcriptional regulators of E. tenella, Plasmodium and T. gondii life-cycles [5, 63] and, more importantly, the invasion of Eimeria sporozoites is accompanied by proteolytic shedding of surface adhesins mediated by protease [64] (Fig. 2).

Refractile body (RB)-derived functional molecules include a number of proteins/enzymes, such as aspartyl proteinase (eimepsin), RB antigen (SO7), and several haloacid dehalogenases such as hydrolase, subtilase, transhydrogenase and ubiquitin, as well as some carbohydrates [65, 66]. Eimepsin is an Eimeria-specific antigenic protein [67, 68] that is abundantly secreted in the sporozoite stage [5]. SO7 is also highly immunogenic with conserved antigenic epitopes among Eimeria species infecting chicken [69]. SO7 has been reported to effectively stimulate the immune response compared with single fragment vaccine [70], and it also plays important roles in host cell invasion, secretion of microneme proteins and Eimeria parasite intracellular survival [69]. In addition, the E. falciformis and E. tenella leucine aminopeptidase is an enzyme which preferentially catalyzes the hydrolysis of leucine residues at the N-termnus of peptides and proteins [71], which could be crucial for invasion, immune response, parasite excystment [72] and functions similar to the EV-bound aminopeptidase and serine protease of Acanthoamoeba castellanii [6, 73]. Identification of EfSEV GAP45 suggests that the E. falciformis sporozoite has a trail of EVs during gliding motility [74, 75] as reported in C. parvum infection [76]. Also, histones enclosed in EVs secreted by Leishmania donovani, P. falciparum, Trypanosoma brucei and T. cruzi could have functions in parasite viability and virulence [77,78,79], and EfSEV histones could have similar roles. Although EfSEV RNA content was low (data not shown), attending EfSEV histones suggest that E. falciformis EV cargo contained nucleic acids. Also, inactivation might have resulted in the loss of MIEC receptors [10] for E. falciformis sporozoite attachment, which in turn could have affected the number and composition of proteins in E. falciformis sporozoite secretomes.

IL-1β, IL-6, IL-17, IL-18 and MCP1 secretions are known to play crucial roles in E. tenella, C. parvum and T. gondii infections [80, 81]. IEC IL-18 is constitutive and links innate and adaptive immunity during infection [82]. Similarly, MCP1 recruits monocytes and macrophages and co-regulates IL-1β in inflamed IECs [83]. Therefore, the expression of MCP1 (Figs. 4, 5) indicated that EfSEVs could play critical roles in the transcytosis of monocytes to the site of E. falciformis infection [21]. Also, the production of IL-1β could accelerate IL-17 response to PAMPs and stimulate chemokines recruiting leukocytes, T lymphocytes and neutrophils to the intestinal epithelium [84]. Then, E. falciformis sporozoites possibly secrete EVs to prevent early infiltration of cytotoxic CD4 + T cells to the site of infection [84] as MIEC IL-1β and IL-17 expressions were low in time-dependent responses to EfSEVs (Figs. 4, 5) and it is comparable  to the expressions of the IL-17 subset and neutrophils in T. cruzi-infected mice [85]. However, at a later time and high concentrations, EfSEVs induced significant expression of IL-17, as reported in mice infected with Leishmania major [86], suggesting that EfSEVs are potent, dose-dependent activators of inflammation and that the observed latent inflammatory effect of EfSEVs on MIEC might be associated with E. falciformis sporozoite behavior for intracellular survival.

It has been previously reported that Trichomonas vaginalis, Leishmania infantum, and P. falciparum EVs induced secretion of IL-6 from immune cells [7]. However, the secretion of IL-6 could be related to anti-inflammatory and anti-apoptotic responses in IECs [87, 88]. It is possible that E. falciformis sporozoites secrete EVs to suppress MIEC apoptotic activities because it has been suggested that T. vaginalis exosomes upregulate IL-6 to suppress other proinflammatory cytokines [89]. This is in contrast to extracellular acidosis that has been reported to transiently modulate IL-6 expression in rat epithelial cell [90]. Nevertheless, EfSEVs enclosed potential danger/damage/stress-associated ligands that activated the release of endogenous IL-18 and IL-1β as proinflammatory cytokines associated with inflammasome complexes [91] (Figs. 5, 6). Also, EfSEVs [35, 41] were able to bind green fluorescent dye and aggregate around the MIEC membrane with some degree of internalization at the later experimental time, as well as involvemnet  in MIEC membrane disruption or sphericalization (Additional file 5). The cytoplasmic incorporation of EfSEVs by MIECs might render them as cytosolic ligands that subsequently activate inflammasome complexes [92].

Inflammasomes are vital innate immune complexes in protozoa-host interactions [93] and are activated by intracellular pattern-recognition receptors (or sensors) that recognize pathogen- danger- [94] and stress-associated ligands [95]. Inflammasomes include NLRPs, AIM2 and caspases [93, 96]. NLRP inflammasome families contribute to intestinal disease pathogenesis while caspases participate in proinflammatory processes and pathophysiology of enteric pathogens [20, 97, 98]. In the present study, upregulation of MIEC caspase 11 and NLRP6, as well as dose-dependent upregulation of IL-18 and IL-1β (Figs. 5, 6) are comparable with inflammatory cascades and NLRP6 activation during mice colonic inflammation and tumorigenesis [46, 96]. Likewise, the activation of NLRP6 during Cryptosporidium tyzzeri infection in mice caused a luminal release of IL-18 [99, 100]. It is therefore not unlikely that C. tyzzeri-derived EVs, though not characterized, might have played pivotal role in the activation of NLRP6 in C. tyzzeri-infected mice, as observed in this study (Fig. 6a, b). Mechanistically, non-canonical caspase 11 is activated when toxins (e.g. bacterial lipopolysaccharide [LPS]) find their way into the host cell cytoplasm [97, 101]. In the absence of the bacteria, Brucella melitensis LPS induced caspase 11 in murine enterocytes [102] like EfSEVs in this study. Moreover, the production of IL-1β, IL-18 and caspase 11 have been posited as markers for intestinal inflammation in response to pathogens [92, 103].

Additionally, caspase 11-dependent pyroptosis, in association with IL-18, is a particularly important host immune mechanism against intracellular pathogens [92, 102]. Also, the production of cytosolic LDH has been defined as a marker for inflammasome-dependent pyroptosis [98, 104, 105]. Upregulation of caspase 11 and NLRP6 with a significant release of LDH as well as dose- and time-dependent secretion of IL-1β and IL-18 [106] signified that EfSEVs are potential activators of pyroptosis in MIECs (Figs. 5, 6). Comparatively, the MIEC inflammatory response and the release of the cell death signal induced by EfSEVs were synchronous with the time of E. falciformis sporozoite intracellular egress [30]. However, the mechanism by which intracellular parasites secrete a regulated amount of EVs to temporarily delay host responses until the completion of intracellular development requires further investigation. It is therefore possible that inflammatory responses and host cell death during Eimeria infection are not entirely passive due to intraepithelial distress, but are prefigured by Eimeria parasite behavior for survival and successful completion of the life-cycle.

Conclusion

This study emphasized host-parasite interaction as an additional mechanism by which the protozoan parasite E. falciformis secretes EVs and VF molecules. The study unveils the protein compositions of E. falciformis sporozoite secretome as well as the MIEC inflammatory response to E. falciformis sporozoite EVs. Specifically, E. falciformis sporozoite EVs contained important antigenic and immunogenic proteins as well as other apicomplexan proteins that are implicated in motility, invasion, pathogenesis, survival and host cell degradation. Also, E. falciformis VF proteins encompassed proteins involved in pathogenesis, motility and vesicle formation. Furthermore, E. falciformis sporozoite EVs induced the release of proinflammatory molecules such as IL-6, IL-17, IL-1β and IL-18 as well as the upregulation of pyroptosis-dependent caspase 11 and NLRP6, at the least, in a dose-dependent manner. Essentially, E. falciformis EVs are components of danger-associated molecular patterns (DAMPs) with protein- and gene-regulatory activities that support pyroptosis-dependent response. Unlike parasite protein characterization through extraneous stimuli, the model of the study could be adapted to ascertain secretory motifs and constitutive antigens/immunogens in Eimeria species for the development of vaccines against poultry coccidiosis. Also, it will benefit our understanding to further describe the classical components of NLRP6- and caspase 11-mediated pyroptosis during E. falciformis infection.

Abbreviations

AIM2:

Absent in melanoma 2

ECL:

Enhanced chemiluminescence

EfSEVs:

E. falciformis sporozoite EVs

EVs:

Extracellular vesicles

IL:

Interleukin

LC–MS/MS:

Liquid chromatography–tandem mass spectrometry

LPS:

Lipopolysaccharide

MCP1:

C–C motif chemokine 2/monocyte chemoattractant protein 1

MIECs:

Mouse intestinal epithelial cells

NLRP6:

Nucleotide-oligomerization domain (NOD)-like receptor pyrin 6

PAMP:

Pathogen-associated molecular pattern

PBS:

Phosphate-buffered saline

ODG:

OptiPrepâ„¢ density gradient

SDS-PAGE:

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis

VF:

Vesicle-free

References

  1. Montaner S, Galiano A, Trelis M, Martin-jaular L, Portillo HA, Bernal D, et al. The role of extracellular vesicles in modulating the host immune response. Front Immunol. 2014;5:1–9.

    Article  CAS  Google Scholar 

  2. Marcilla A, Trelis M, Cortés A, Sotillo J, Cantalapiedra F, Minguez MT, et al. Extracellular vesicles from parasitic helminths contain specific excretory/secretory proteins and are internalized in intestinal host cells. PLoS ONE. 2012;7:e45974.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Pope SM, Lässer C, Lasser C. Toxoplasma gondii infection of fibroblasts causes the production of exosome-like vesicles containing a unique array of mRNA and miRNA transcripts compared to serum starvation. J Extracell Vesicles. 2013;1:1–8.

    Google Scholar 

  4. Graewe S, Rankin KE, Lehmann C, Deschermeier C, Hecht L, Froehlke U, et al. Hostile takeover by Plasmodium: reorganization of parasite and host cell membranes during liver stage egress. PLoS Pathog. 2011;7:e1002224.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Olajide JS, Qu Z, Yang S, Oyelade OJ, Cai J. Eimeria proteins: order amidst disorder. Parasit Vectors. 2022;15:1–16.

    Article  Google Scholar 

  6. Gonçalves DDS, Ferreira S, Liedke SC, Gomes KX, De OGA, Lopes PE, et al. Extracellular vesicles and vesicle-free secretome of the protozoa Acanthamoeba castellanii under homeostasis and nutritional stress and their damaging potential to host cells. Virulence. 2018;5594:1–83.

    Google Scholar 

  7. Olajide JS, Cai J. Perils and promises of pathogenic protozoan extracellular vesicles. Front Cell Infect Microbiol. 2020;10:1–17.

    Article  CAS  Google Scholar 

  8. Castelli G, Bruno F, Saieva L, Alessandro R, Galluzzi L, Diotallevi A, et al. Exosome secretion by Leishmania infantum modulate the chemotactic behavior and cytokinic expression creating an environment permissive for early infection. Exp Parasitol. 2019;198:39–45.

    Article  CAS  PubMed  Google Scholar 

  9. Heitlinger E, Spork S, Lucius R, Dieterich C. The genome of Eimeria falciformis—reduction and specialization in a single host apicomplexan parasite. BMC Genomics. 2014;15:1–17.

    Article  CAS  Google Scholar 

  10. Li W, Wang M, Chen Y, Chen C, Liu X, Sun X, et al. EtMIC3 and its receptors BAG1 and ENDOUL are essential for site-specific invasion of Eimeria tenella in chickens. Vet Res. 2020;51:1–15.

    Article  CAS  Google Scholar 

  11. Deolindo P, Evans-Osses I, Ramirez MI. Microvesicles and exosomes as vehicles between protozoan and host cell communication. Biochem Soc Trans. 2013;41:252–7.

    Article  CAS  PubMed  Google Scholar 

  12. Szempruch AJ, Dennison L, Kieft R, Harrington JM, Hajduk SL. Sending a message: extracellular vesicles of pathogenic protozoan parasites. Nat Rev Microbiol. 2016;14:669–75.

    Article  CAS  PubMed  Google Scholar 

  13. Silva VO, Maia MM, Torrecilhas AC, Taniwaki NN, Namiyama GM, Oliveira KC, et al. Extracellular vesicles isolated from Toxoplasma gondii induce host immune response. Parasite Immunol. 2018;40:1–11.

    Article  CAS  Google Scholar 

  14. Wu Z, Wang L, Li J, Wang L, Wu Z, Sun X. Extracellular vesicle-mediated communication within host-parasite interactions. Front Immunol. 2018;9:3066.

    Article  CAS  PubMed  Google Scholar 

  15. Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007;9:654–9.

    Article  CAS  PubMed  Google Scholar 

  16. Huang G, Zhang S, Zhou C, Tang X, Li C, Wang C, et al. Influence of Eimeria falciformis infection on gut microbiota and metabolic pathways in mice. Infect Immun. 2018;86:1–11.

    Article  Google Scholar 

  17. Schmid M, Heitlinger E, Spork S, Mollenkopf HJ, Lucius R, Gupta N. Eimeria falciformis infection of the mouse caecum identifies opposing roles of IFNγ-regulated host pathways for the parasite development. Mucosal Immunol. 2014;7:969–82.

    Article  CAS  PubMed  Google Scholar 

  18. Ehret T, Spork S, Dieterich C, Lucius R, Heitlinger E. Dual RNA-seq reveals no plastic transcriptional response of the coccidian parasite Eimeria falciformis to host immune defenses. BMC Genomics. 2017;18:1–17.

    Article  CAS  Google Scholar 

  19. Straub KW, Cheng SJ, Sohn CS, Bradley PJ. Novel components of the Apicomplexan moving junction reveal conserved and coccidia-restricted elements. Cell Microbiol. 2009;11:590–603.

    Article  CAS  PubMed  Google Scholar 

  20. Fink SL, Cookson BT. Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells. Infect Immun. 2005;73:1907–16.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Al Klifeh E, Balard A, Jarquín-Diaz VH, Weyrich A, Wibbelt G, Heitlinger E. Eimeria falciformis BayerHaberkorn1970 and novel wild derived isolates from house mice : differences in parasite lifecycle, pathogenicity and host immune reactions. bioRxiv. 2019;8:1–33.

    Google Scholar 

  22. Pogonka T, Schelzke K, Stange J, Papadakis K, Steinfelder S, Liesenfeld O, et al. CD8+ cells protect mice against reinfection with the intestinal parasite Eimeria falciformis. Microbes Infect. 2010;12:218–26.

    Article  CAS  PubMed  Google Scholar 

  23. Suresh P, Rehg JE. Comparative evaluation of several techniques for purification of Cryptosporidium parvum oocysts from rat feces. J Clin Microbiol. 1996;34:38–40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Cai X, Lorraine Fuller A, McDougald LR, Tan X, Cai J, Wang F, et al. Biochemical characterization of enoyl reductase involved in Type II fatty acid synthesis in the intestinal coccidium Eimeria tenella (Phylum Apicomplexa). FEMS Microbiol Lett. 2007;272:238–44.

    Article  CAS  PubMed  Google Scholar 

  25. El-ashram S, Suo X. Electrical cream separator coupled with vacuum filtration for the purification of eimerian oocysts and trichostrongylid eggs. Sci Rep. 2017;7:1–14.

    CAS  Google Scholar 

  26. Iwamoto T, Yamada K, Shimizu M, Totsuka M. Establishment of intestinal epithelial cell lines from adult mouse small and large intestinal crypts. Biosci Biotechnol Biochem. 2011;75:925–9.

    Article  CAS  PubMed  Google Scholar 

  27. Gvan N, Raposo A, Candalh C, Boussac M, Hershberg R, Cerf-Bensussan N, et al. Intestinal epithelial cells secrete exosome-like vesicles. Gastroenterology. 2001;121:337–49.

    Article  Google Scholar 

  28. Pérez-Cabezas B, Santarém N, Cecílio P, Silva C, Silvestre R, Catita J, et al. More than just exosomes: distinct Leishmania infantum extracellular products potentiate the establishment of infection. J Extracell Vesicles. 2018. https://doi.org/10.1080/20013078.2018.1541708.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Lutz K, Taubert A, Zahner H, Menge C, Hermosilla C, Stamm I. Fluorescent Eimeria bovis sporozoites and meront stages in vitro: a helpful tool to study parasite–host cell interactions. Parasitol Res. 2008;102:777–86.

    Article  PubMed  Google Scholar 

  30. Mesfin GM, Bellamy JEC. The life cycle of Eimeria falciformis var. Pragensis (Sporozoa: Coccidia) in the mouse, Mus musculus. J Parasitol. 1978;64:696.

    Article  CAS  PubMed  Google Scholar 

  31. Ribeiro KS, Vasconcellos CI, Soares RP, Mendes MT, Ellis CC, Aguilera-Flores M, et al. Proteomic analysis reveals different composition of extracellular vesicles released by two Trypanosoma cruzi strains associated with their distinct interaction with host cells. J Extracell Vesicles. 2018;7:1463779.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Tauro BJ, Mathias RA, Greening DW, Gopal SK, Ji H, Kapp EA, et al. Oncogenic H-Ras reprograms madin-darby canine kidney (MDCK) cell-derived exosomal proteins following epithelial-mesenchymal transition. Mol Cell Proteomics. 2013;12:2148–59.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Caeiro LD, Alba-Soto CD, Rizzi M, Solana ME, Rodriguez G, Chidichimo AM, et al. The protein family TcTASV-C is a novel Trypanosoma cruzi virulence factor secreted in extracellular vesicles by trypomastigotes and highly expressed in bloodstream forms. PLoS Negl Trop Dis. 2018;12:1–26.

    Article  CAS  Google Scholar 

  34. Li Y, Xiu F, Mou Z, Xue Z, Du H, Zhou C. Exosomes derived from Toxoplasma gondii stimulate an inflammatory response through JNK signaling pathway. Nanomedicine. 2018;13:1157–68.

    Article  CAS  PubMed  Google Scholar 

  35. Li S, Gong P, Tai L, Li X, Wang X, Zhao C, et al. Extracellular vesicles secreted by Neospora caninum are recognized by toll-like receptor 2 and modulate host cell innate immunity through the MAPK signaling pathway. Front Immunol. 2018;9:1–14.

    CAS  Google Scholar 

  36. Wowk PF, Zardo ML, Miot HT, Goldenberg S, Carvalho PC, Mörking PA. Proteomic profiling of extracellular vesicles secreted from Toxoplasma gondii. Proteomics. 2017;17:15–6.

    Article  CAS  Google Scholar 

  37. Huang T, He J. Characterization of extracellular vesicles by size-exclusion high-performance liquid chromatography (HPLC). Methods Mol Biol. 2017;1660:191–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Wolff BS, Alshawi SA, Feng LR, Juneau PL, Saligan LN. Inflammation plays a causal role in fatigue-like behavior induced by pelvic irradiation in mice. Brain Behav Immun Health. 2021;15:1–9.

    Google Scholar 

  39. den Hartigh AB, Fink SL. Pyroptosis Induction and detection. Curr Protoc Immunol. 2018;122:1–22.

    Google Scholar 

  40. Rayamajhi M, Zhang Y, Miao E. Detection of pyroptosis by measuring released lactate dehydrogenase activity. Methods Mol Biol. 2013;1040:85–90.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Li Y, Liu Y, Fangming X, Jianing W, Cong H, He S, et al. Characterization of exosomes derived from Toxoplasma gondii and their functions in modulating immune responses. Int J Nanomed. 2018;13:467–77.

    Article  CAS  Google Scholar 

  42. Stadnyk AW. Intestinal epithelial cells as a source of inflammatory cytokines and chemokines. Can J Gastroenterol. 2002;16:241–6.

    Article  PubMed  Google Scholar 

  43. Hong YH, Lillehoj HS, Lee SH, Dalloul RA, Lillehoj EP. Analysis of chicken cytokine and chemokine gene expression following Eimeria acervulina and Eimeria tenella infections. Vet Immunol Immunopathol. 2006;114:209–23.

    Article  CAS  PubMed  Google Scholar 

  44. Hong YH, Lillehoj HS, Lillehoj EP, Lee SH. Changes in immune-related gene expression and intestinal lymphocyte subpopulations following Eimeria maxima infection of chickens. Vet Immunol Immunopathol. 2006;114:259–72.

    Article  CAS  PubMed  Google Scholar 

  45. Malireddi RKS, Kanneganti TD. Role of type I interferons in inflammasome activation, cell death, and disease during microbial infection. Front Cell Infect Microbiol. 2013;3:77.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Ghimire L, Paudel S, Jin L, Jeyaseelan S. The NLRP6 inflammasome in health and disease. Mucosal Immunol. 2020;13:388–98.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Nievas YR, Coceres VM, Midlej V, de Souza W, Benchimol M, Pereira-Neves A, et al. Membrane-shed vesicles from the parasite Trichomonas vaginalis: characterization and their association with cell interaction. Cell Mol Life Sci. 2018;75:2211–26.

    Article  CAS  PubMed  Google Scholar 

  48. Ramírez-Flores CJ, Cruz-Mirón R, Mondragón-Castelán ME, González-Pozos S, Ríos-Castro E, Mondragón-Flores R. Proteomic and structural characterization of self-assembled vesicles from excretion/secretion products of Toxoplasma gondii. J Proteomics. 2019;208:103490.

    Article  PubMed  CAS  Google Scholar 

  49. Marq J, Stratmann R, Limenitakis J. Article functional dissection of the apicomplexan glideosome molecular architecture. Cell Host Microbe. 2010;8:343–57.

    Article  PubMed  CAS  Google Scholar 

  50. Péroval M, Péry P, Labbé M. The heat shock protein 90 of Eimeria tenella is essential for invasion of host cell and schizont growth. Int J Parasitol. 2006;36:1205–15.

    Article  PubMed  CAS  Google Scholar 

  51. Jimenez-ruiz E, Morlon-guyot J, Daher W, Meissner M. Vacuolar protein sorting mechanisms in apicomplexan parasites. Mol Biochem Parasitol. 2016;01:1–8.

    Google Scholar 

  52. Spielmann T, Gras S, Sabitzki R, Meissner M. Endocytosis in Plasmodium and Toxoplasma parasites. Trends Parasitol. 2020;1988:1–13.

    Google Scholar 

  53. Matsubayashi M, Teramoto-kimata I, Uni S, Lillehoj HS, Matsuda H. Elongation Factor-1 is a novel protein associated with host cell invasion and a potential protective antigen of Cryptosporidium parvum. J Biol Chem. 2013;288:34111–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Zhang Z, Wang S, Li C, Liu L. Immunoproteomic analysis of the protein repertoire of unsporulated Eimeria tenella oocysts. Parasite. 2017;24:1–10.

    Article  PubMed  PubMed Central  Google Scholar 

  55. Diallo MA, Sausset A, Gnahoui-David A, Silva ARE, Brionne A, Le Vern Y, et al. Eimeria tenella rop kinase etrop1 induces g0/g1 cell cycle arrest and inhibits host cell apoptosis. Cell Microbiol. 2019;21:1–14.

    Article  CAS  Google Scholar 

  56. Sullivan WJ, Narasimhan J, Bhatti MM, Wek RC. Parasite-specific eIF2 ( eukaryotic initiation factor-2) kinase required for stress-induced translation control. Biochem J. 2004;380:523–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. de Souza W, Barrias ES. Membrane-bound extracellular vesicles secreted by parasitic protozoa: cellular structures involved in the communication between cells. Parasitol Res Parasitol Res. 2020;119:2005–23.

    Article  PubMed  Google Scholar 

  58. del Cacho E, Gallego M, López-Bernad F, Quílez J, Sánchez-Acedo C. Differences in Hsp70 expression in the sporozoites of the original strain and precocious lines of Eimeria tenella. J Parasitol. 2005;91:1127–31.

    Article  PubMed  Google Scholar 

  59. Bogado ALG, Martins GF, Sasse JP, Guimarães JDS, Garcia JL. Molecular cloning, sequencing, and expression of Eimeria tenella HSP70 partial gene. Genet Mol Res. 2017;16:1–9.

    Article  CAS  Google Scholar 

  60. Benns HJ, Tate EW, Child MA. Activity-based protein profiling for the study of parasite biology. In: Tate HJ, editor. Curr Top Microbiol Immunol. Cham: Springer; 2018.

    Google Scholar 

  61. Liu R, Ma X, Liu A, Zhang L, Cai J, Wang M. Identification and characterization of a cathepsin-L-like peptidase in Eimeria tenella. Parasitol Res. 2014;113:4335–48.

    Article  PubMed  Google Scholar 

  62. Pi˜na-Vazquez C, Reyes-Lopez M, Ortız-Estrada G, de la Garza M, Serrano-Luna J. Host-parasite interaction : parasite-derived and -induced proteases that degrade human extracellular matrix. J Parasitol Res. 2012;748:1–25.

    Article  CAS  Google Scholar 

  63. Matsubayashi M, Kawahara F, Hatta T, Yamagishi J, Miyoshi T, Anisuzzaman, et al. Transcriptional profiles of virulent and precocious strains of Eimeria tenella at sporozoite stage, novel biological insight into attenuated asexual development. Infect Genet Evol. 2016;40:54–62.

    Article  PubMed  Google Scholar 

  64. Zheng J, Gong P, Jia H, Li M, Zhang G, Zhang X, et al. Eimeria tenella rhomboid 3 has a potential role in microneme protein cleavage. Vet Parasitol. 2014;201:146–9.

    Article  CAS  PubMed  Google Scholar 

  65. de Venevelles P, François Chich J, Faigle W, Lombard B, Loew D, Péry P, et al. Study of proteins associated with the Eimeria tenella refractile body by a proteomic approach. Int J Parasitol. 2006;36:1399–407.

    Article  PubMed  CAS  Google Scholar 

  66. Vermeulen AN, Kok JJ, Van den Boogaart P, Dijkema R, Claessens JA. Eimeria refractile body proteins contain two potentially functional characteristics : transhydrogenase and carbohydrate transport. FEMS Microbiol Lett. 1993;110:223–9.

    Article  CAS  PubMed  Google Scholar 

  67. Jean L, Grosclaude J, Labbé M, Tomley F, Péry P. Differential localisation of an Eimeria tenella aspartyl proteinase during the infection process. Int J Parasitol. 2000;30:1099–107.

    Article  CAS  PubMed  Google Scholar 

  68. De Venevelles P, Chich JF, Faigle W, Loew D, Labbé M, Girard-Misguich F, et al. Towards a reference map of Eimeria tenella sporozoite proteins by two-dimensional electrophoresis and mass spectrometry. Int J Parasitol. 2004;34:1321–31.

    Article  PubMed  CAS  Google Scholar 

  69. Rafiqi SI, Garg R, Reena KK, Ram H, Singh M, Banerjee PS. Immune response and protective efficacy of Eimeria tenella recombinant refractile body protein, EtSO7, in chickens. Vet Parasitol. 2018;258:108–13.

    Article  CAS  PubMed  Google Scholar 

  70. Song X, Xu L, Yan R, Huang X, Li X. Construction of Eimeria tenella multi-epitope DNA vaccines and their protective efficacies against experimental infection. Vet Immunol Immunopathol. 2015;166:79–87.

    Article  CAS  PubMed  Google Scholar 

  71. Li JG, Gu WY, Tao JP, Liu ZP. The effects of S-nitroso-glutathione on the activities of some isoenzymes in Eimeria tenella oocysts. Vet Parasitol. 2009;162:236–40.

    Article  CAS  PubMed  Google Scholar 

  72. Fetterer RH, Miska KB, Barfield RC. Partial purification and characterization of an aminopeptidase from Eimeria tenella. J Parasitol. 2006;91:1280–6.

    Article  Google Scholar 

  73. Lin WC, Tsai CY, Huang JM, Wu SR, Chu LJ. Quantitative proteomic analysis and functional characterization of Acanthamoeba castellanii exosome—like vesicles. Parasit Vectors. 2019;12:1–12.

    Article  Google Scholar 

  74. Roditi I. The languages of parasite communication. Mol Biochem Parasitol. 2016;208:16–22.

    Article  CAS  PubMed  Google Scholar 

  75. Sloves P, Morelle W, Alayi TD, Slomianny C, Werkmeister E, Schaeffer C, et al. Unusual N -glycan structures required for trafficking Toxoplasma gondii GAP50 to the inner membrane complex regulate host cell entry through parasite motility. Mol Cell Proteomics. 2011;10:1–17.

    Google Scholar 

  76. Gong AY, Hu G, Zhou R, Liu J, Feng Y, Soukup GA, et al. MicroRNA-221 controls expression of intercellular adhesion molecule-1 in epithelial cells in response to Cryptosporidium parvum infection. Int J Parasitol. 2011;41:1.

    Article  CAS  Google Scholar 

  77. Silverman JM, Clos J, Horakova E, Wang AY, Wiesgigl M, Kelly I, et al. Leishmania exosomes modulate innate and adaptive immune responses through effects on monocytes and dendritic cells. J Immunol. 2010;185:5011–22.

    Article  CAS  PubMed  Google Scholar 

  78. Sisquella X, Regev-Rudzki N, Gerlic M, Schofield L, Sampaio NG, Hansen DS, et al. Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors. Nat Commun. 2017;8:1–16.

    Article  CAS  Google Scholar 

  79. Sullivan WJ Jr, Naguleswaran A, Angel SO. Histones and histone modifications in protozoan parasites. Cell Microbiol. 2006;8:1850–61.

    Article  CAS  PubMed  Google Scholar 

  80. Buzoni-Gatel D, Schulthess J, Menard LC, Kasper LH. Mucosal defences against orally acquired protozoan parasites, emphasis on Toxoplasma gondii infections. Cell Microbiol. 2006;8:535–44.

    Article  CAS  PubMed  Google Scholar 

  81. Zhang L, Liu R, Song M, Hu Y, Pan B, Cai J, et al. Eimeria tenella: Interleukin 17 contributes to host immunopathology in the gut during experimental infection. Exp Parasitol. 2013;133:121–30.

    Article  CAS  PubMed  Google Scholar 

  82. van de Veerdonk FL, Netea MG, Dinarello CA, Joosten LAB. Inflammasome activation and IL-1β and IL-18 processing during infection. Trends Immunol. 2011;32:110–6.

    Article  PubMed  CAS  Google Scholar 

  83. Reinecker HC, Loh EY, Ringler DJ, Mehta A, Rombeau JL, MacDermott RP. Monocyte-chemoattractant protein 1 gene expression in intestinal epithelial cells and inflammatory bowel disease mucosa. Gastroenterology. 1995;108:40–50.

    Article  CAS  PubMed  Google Scholar 

  84. Ge Y, Huang M, Yao YM. Biology of Interleukin-17 and its pathophysiological significance in sepsis. Front Immunol. 2020;11:1–13.

    Article  CAS  Google Scholar 

  85. Erdmann H, Roßnagel C, Böhme J, Iwakura Y, Jacobs T, Schaible UE, et al. IL-17A promotes macrophage effector mechanisms against Trypanosoma cruzi by trapping parasites in the endolysosomal compartment. Immunobiology. 2013;218:910–23.

    Article  CAS  PubMed  Google Scholar 

  86. Gonzalez-Lombana C, Gimblet C, Bacellar O, Oliveira WW, Passos S, Carvalho LP, et al. IL-17 mediates immunopathology in the absence of il-10 following Leishmania major infection. PLoS Pathog. 2013;9:e1003243.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Chen L, Deng H, Cui H, Fang J, Zuo Z, Deng J, et al. Inflammatory responses and inflammation-associated diseases in organs. Oncotarget. 2018;9:7204–18.

    Article  PubMed  Google Scholar 

  88. Hu B, Elinav E, Huber S, Strowig T, Hao L, Hafemann A, et al. Microbiota-induced activation of epithelial IL-6 signaling links inflammasome-driven inflammation. Proc Natl Acad Sci USA. 2013;110:9862–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Twu O, De MN, Lustig G, Stevens GC, Vashisht AA, Wohlschlegel JA, et al. Trichomonas vaginalis exosomes deliver cargo to host cells and mediate host : parasite interactions. PLoS Pathog. 2013;9:22–4.

    Article  CAS  Google Scholar 

  90. Riemann A, Reime S, Gießelmann M, Thews O. Extracellular acidosis regulates the expression of inflammatory mediators in rat epithelial cells. Adv Exp Med Biol. 2020;1232:277–82.

    Article  CAS  PubMed  Google Scholar 

  91. Redpath SA, Fonseca NM, Perona-Wright G. Protection and pathology during parasite infection: IL-10 strikes the balance. Parasite Immunol. 2014;36:233–52.

    Article  CAS  PubMed  Google Scholar 

  92. Lei-Leston AC, Murphy AG, Maloy KJ. Epithelial cell inflammasomes in intestinal immunity and inflammation. Front Immunol. 2017;8:1–17.

    Article  CAS  Google Scholar 

  93. de Carvalho RVH, Zamboni DS. Inflammasome activation in response to intracellular protozoan parasites. Trends Parasitol. 2020;36:459–72.

    Article  PubMed  CAS  Google Scholar 

  94. Man SM. Inflammasomes in the gastrointestinal tract: infection, cancer and gut microbiota homeostasis. Nat Rev Gastroenterol Hepatol. 2018;15:721–37.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Gagliani N, Palm NW, de Zoete MR, Flavell RA. Inflammasomes and intestinal homeostasis: regulating and connecting infection, inflammation and the microbiota. Int Immunol. 2014;26:495–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Chen GY, Liu M, Wang F, Bertin J, Núñez G. A Functional Role for Nlrp6 in intestinal inflammation and tumorigenesis. J Immunol. 2011;186:7187–94.

    Article  CAS  PubMed  Google Scholar 

  97. de Gassart A, Martinon F. Pyroptosis: caspase-11 unlocks the gates of death. Immunity. 2015;43:835–7.

    Article  PubMed  CAS  Google Scholar 

  98. Yin J, Sheng B, Yang K, Sun L, Xiao W, Yang H. The protective roles of NLRP6 in intestinal epithelial cells. Cell Prolif. 2019;52:1–6.

    Article  CAS  Google Scholar 

  99. Placea DE, Kanneganti T-D. Recent advances in inflammasome biology. Physiol Behav. 2017;176:139–48.

    CAS  Google Scholar 

  100. Sateriale A, Gullicksrud JA, Engiles JB, McLeod BI, Kugler EM, Henao-Mejia J, et al. The intestinal parasite Cryptosporidium is controlled by an enterocyte intrinsic inflammasome that depends on NLRP6. Proc Natl Acad Sci USA. 2021. https://doi.org/10.1073/pnas.2007807118.

    Article  PubMed  Google Scholar 

  101. Zamboni DS, Sacks DL. Inflammasomes and Leishmania: in good times or bad, in sickness or in health. Curr Opin Microbiol. 2019;52:70–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Lacey CA, Mitchell WJ, Dadelahi AS. caspase-1 and caspase-11 mediate pyroptosis, inflammation, and control of Brucella joint infection. Infect Immun. 2018;86:1–15.

    Article  CAS  Google Scholar 

  103. Lopez-Castejon G, Brough D. Understanding the mechanism of IL-1β secretion. Cytokine Growth Factor Rev. 2011;22:189–95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. Winsor N, Krustev C, Bruce J, Philpott DJ, Girardin SE. Canonical and noncanonical inflammasomes in intestinal epithelial cells. Cell Microbiol. 2019;21:1–13.

    Article  CAS  Google Scholar 

  105. Huang X, Feng Y, Xiong G, Whyte S, Duan J, Yang Y, et al. Caspase-11, a specific sensor for intracellular lipopolysaccharide recognition, mediates the non-canonical inflammatory pathway of pyroptosis. Cell Biosci. 2019;9:1–11.

    Article  PubMed  PubMed Central  Google Scholar 

  106. Frank D, Vince JE. Pyroptosis versus necroptosis: similarities, differences, and crosstalk. Cell Death Differ. 2019;26:99–114.

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

Our sincere gratitude is given to Prof. Xian-Ming Chen, Rush University, Chicago and Dr. Goku Hu, Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE for their assessment of TEM images and useful suggestions on experimental procedures. Special thanks also go to Li Yong, Li Miaomiao, Qi Shuyun and Gong Zhenli for their assistance on ultracentrifugation and microscopy, and to Xinming Zhang, Wang Heng and Zhenxing Gong for their assistance with cell culture and oocyst sporulation.

Funding

This work was supported by the Key Technologies Research and Development Program (Key Technologies R&D Program) 2017YFD050040320 and the Innovative Special Project of Agricultural Science and Technology (Grant No. CAAS-ASTIP2014-LVRI-09).

Author information

Authors and Affiliations

  1. State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, 730046, China

    Joshua Seun Olajide, Ling Xiong, Shunli Yang, Zigang Qu, Xiao Xu, Bin Yang, Jing Wang, Baohong Liu, Xueting Ma & Jianping Cai

  2. Centre for Distance Learning, Obafemi Awolowo University, Ile-Ife, Nigeria

    Joshua Seun Olajide

Authors
  1. Joshua Seun Olajide
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ling Xiong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shunli Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zigang Qu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiao Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bin Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jing Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Baohong Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Xueting Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jianping Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

JC designed the study. QZ, SY and JO conceptualized the study. SY, QZ, XX, XL, JW, BL and MX, provided briefings on protocols. YB processed the proteomic data. JO carried out the experiments. JC and SY appraised the experimental results. JO drafted the manuscript and JC revised the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Jianping Cai.

Ethics declarations

Ethics approval and consent to particpate

Balb/c mice used in this work were handled in strict compliance with Animal Ethics Procedures and Guidelines of the People’s Republic of China as approved by the Animal Administration and Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (LVRIAEC-2020-035).

Consent for publication

The authors agree to the publication of the study.

Competing interests

All authors declare that there is no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: Non-treated control before incubation.

Additional file 2: Parasite and inactivated MIECs before incubation.

Additional file 3: Non-treated control after incubation.

Additional file 4: Parasite and inactivated MIECs after incubation.

Additional file 5:

Electrophoretic gel, bioinformatic result, and result from non-treated control.

Additional file 6:

Mass spectrometry data set for identified proteins.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Olajide, J.S., Xiong, L., Yang, S. et al. Eimeria falciformis secretes extracellular vesicles to modulate proinflammatory response during interaction with mouse intestinal epithelial cells. Parasites Vectors 15, 245 (2022). https://doi.org/10.1186/s13071-022-05364-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13071-022-05364-x

Keywords

Parasites & Vectors

ISSN: 1756-3305

Contact us