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. 2022 Jul 8;15(1):245.
doi: 10.1186/s13071-022-05364-x.

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

Joshua Seun Olajide  1   2 , Ling Xiong  1 , Shunli Yang  1 , Zigang Qu  1 , Xiao Xu  1 , Bin Yang  1 , Jing Wang  1 , Baohong Liu  1 , Xueting Ma  1 , Jianping Cai  3
Affiliations

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

Joshua Seun Olajide et al. Parasit Vectors. .

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.

Keywords: Eimeria falciformis; Extracellular vesicles; Host-parasite interactions; Inflammasomes; Pyroptosis; Secretome.

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Conflict of interest statement

All authors declare that there is no conflict of interest.

Figures

Fig. 1
Fig. 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)
Fig. 2
Fig. 2
EfSEV and VF proteins identified by mass spectrometry. The list and other information on the identified proteins are provided in Additional file 5
Fig. 3
Fig. 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
Fig. 4
Fig. 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
Fig. 5
Fig. 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
Fig. 6
Fig. 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
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