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. 2021 Aug 11;12:4851. doi: 10.1038/s41467-021-24997-7

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© The Author(s) 2021

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — A RT-PCR analysis of CXCL10, CCL5, IFNA, and IFNB normalized to HPRT1 of THP-1 cells treated with, trophozoite(TR)-derived EVs, ring-derived EVs or not treated (NT) for 6, 12, or 24 h. n = 3 biologically independent experiments, SEM, one-way ANOVA followed by Dunnett’s test, (1 h- CCL5; Ring EVs – NT P < 0.001 *** TR EVs – NT **P = 0.00783. CXCL10; Ring EVs – NT ***P < 0.001. IFNA and IFNB ns not significant. 6 h- CCL5; Ring EVs - ***P < 0.001, TR EVs - NT **P = 0.00514. CXCL10; Ring EVs - NT ***P < 0.001, TR EVs - NT *P = 0.0338. IFNB; Ring EVs - NT ***P < 0.001, TR EVs - NT **P = 0.00597, uRBC EVs - NT **P = 0.00907. IFNA; Ring EVs - NT *P = 0.0229. 24 h- CCL5; Ring EVs - NT ***P < 0.001. CXCL10; Ring EVs - NT ***P < 0.001, TR EVs - NT *P = 0.0417. IFNB; Ring EVs - NT ***P < 0.001. IFNA; Ring EVs - NT ***P < 0.001). B THP-1 cells were incubated with Pf-ring-stage-, Pf-trophozoite-stage- or uRBC-derived vesicles for 1, 6, and 24 h. An ELISA assay was performed on the cells’ media to detect secreted CCL5 and CXCL10. HEK blue IFNα/β assay was performed. n = 3 biologically independent experiments, SEM, one-way ANOVA followed by Dunnett’s test, CXCL10; ns. CCL5; 1-h-ns, 6 h- Ring EVs - uRBC EVs ***P < 1e-10, 24 h- Ring EVs - uRBC EVs ***P = 1.03e-10. IFNα/β- 1 h- Ring EVs - uRBC ***P = 4.17e-06, TR EVs - uRBC EVs *P = 0.0138. 6 h- Ring EVs - uRBC EVs ***P = 1.24e-08, TR EVs - uRBC EVs ***P = 0.000847, 24 h- Ring EVs - uRBC EVs ***P = 9.66e-08, TR EVs - uRBC EVs ***P = 0.000459). C Western Blot assay of CXCL10 and HSP90 (loading control) was performed on THP-1 cell lysate. THP-1 cells were incubated with Pf-ring-stage-derived EVs and then transfected with poly(dA:dT) or not treated (NT) for 16 h before harvesting. Results are representative of at least three independent biological replicates. D THP-1 cells were incubated with Pf-ring-stage-derived EVs and then transfected with poly(dA:dT) or NT for 16, 20 or 24 h. An ELISA assay was performed on the cell lysate for CXCL10. n = 3 biologically independent experiments, SEM, one-way ANOVA followed by Dunnett’s test, (16 h dAdT - NT ***P = 1.68e-05. 20 hours dAdT - NT ***P = 6.28e-07. 24 h dAdT - NT ***P = 2.44e-06). E Western Blot analysis of THP-1 cell lysate. THP-1 cells were incubated with Pf-ring-stage-derived EVs, transfected with poly(dA:dT) and treated with Pf-ring-stage-derived EVs for 1 h before being transfected with poly(dA:dT). The cells were harvested 16, 20, and 24 h post treatment. Antibodies were used against CXCL10 and HSP90 (loading control). NT not treated. Results are representative of at least three independent biological replicates. F ELISA assay for CXCL10 was performed on cell lysate. THP-1 cells were incubated with Pf-ring-stage-derived EVs, transfected with poly(dA:dT), and then treated with Pf-ring-stage-derived EVs for 1 h before being transfected with poly(dA:dT). Cells were harvested 16, 20, and 24 h post treatment NT-not treated. n = 3 biologically independent experiments, SEM. One-way ANOVA followed by Dunnett’s test, (16 h- dAdT - NT ***P < 1e-04, Pf EVs+dAdT - NT ***P = 0.000245. 20 h- dAdT - NT ***P < 0.001, Pf EVs+dAdT - NT **P = 0.00475. 24 h- dAdT - NT ***P < 0.001, Pf EVs+dAdT - NT P < 0.001 ***). G ELISA assay for detection of CXCL10 in PBMC-derived monocytes. CD14+ primary cells (monocytes) isolated from three naive healthy donors. The cells were incubated with Pf-ring-stage-derived EVs or transfected with poly(dA:dT) and then analyzed by ELISA. n = 3 biologically independent experiments, SEM, one-way ANOVA followed by Dunnett’s test, (dAdT - NT ***P = 1.29e-07) Source data are provided as a Source Data file. .