Exosome-mediated delivery of inflammation-responsive Il-10 mRNA for controlled atherosclerosis treatment

Abstract

Rationale: Tailored inflammation control is badly needed for the treatment of kinds of inflammatory diseases, such as atherosclerosis. IL-10 is a potent anti-inflammatory cytokine, while systemic and repeated delivery could cause detrimental side-effects due to immune repression. In this study, we have developed a nano-system to deliver inflammation-responsive Il-10 mRNA preferentially into macrophages for tailored inflammation control. Methods:Il-10 was engineered to harbor a modified HCV-IRES (hepatitis C virus internal ribosome entry site), in which the two miR-122 recognition sites were replaced by two miR-155 recognition sites. The translational responsiveness of the engineered mRNA to miR-155 was tested by Western blot or ELISA. Moreover, the engineered Il-10 mRNA was passively encapsulated into exosomes by forced expression in donor cells. Therapeutic effects on atherosclerosis and the systemic leaky expression effects in vivo of the functionalized exosomes were analyzed in ApoE^-/- (Apolipoprotein E-deficient) mice. Results: The engineered IRES-Il-10 mRNA could be translationally activated in cells when miR-155 was forced expressed or in M1 polarized macrophages with endogenous miR-155 induced. In addition, the engineered IRES-Il-10 mRNA, when encapsulated into the exosomes, could be efficiently delivered into macrophages and some other cell types in the plaque in ApoE^-/- mice. In the recipient cells of the plaque, the encapsulated Il-10 mRNA was functionally translated into protein, with relatively low leaky in other tissues/organs without obvious inflammation. Consistent with the robust Il-10 induction in the plaque, exosome-based delivery of the engineered Il-10 could alleviate the atherosclerosis in ApoE^-/- mice. Conclusion: Our study established a potent platform for controlled inflammation control via exosome-based systemic and repeated delivery of engineered Il-10 mRNA, which could be a promising strategy for atherosclerosis treatment.

Keywords: Atherosclerosis; exosomes; inflammation-responsive; interleukin-10; internal ribosome entry site.

Figure 1

Engineering of inflammation-responsive Il-10 mRNA. (A) Schematic of miR-155-activated IRES-Il-10 mRNA. Binding to miR-122, HCV-IRES underwent conformational changes that were conducive to translation. The miR-122 recognition region of the HCV-IRES was replaced with a miR-155 recognition sequence (the substituted bases were red) and downstream ligated to the CDS of mouse Il-10 to achieve miR-155-responsive IL-10 translation activation. (B) Schematic of verifying the function of miR-155-activated IRES-Il-10 mRNA at the cellular level by Western blot and qPCR. (C) qPCR analysis of Il-10 mRNA in cells transfected as indicated. GAPDH served as an internal control. (D) Representative Western blot image of IL-10 protein expression in HEK293T cells transfected as indicated. GAPDH served as the loading control. (E) Quantification of Western blot bands by densitometry. Data are presented as mean ± SEM of three independent experiments. *, p < 0.05 by one-way ANOVA. NC, negative control. ND, not determined as Ct value greater than 38.

Figure 2

Preparation and characterization of Exo^IRES-IL-10. (A) Schematic of the exosomes preparation and isolation process. (B) Western blot analysis of the exosomal inclusive markers (TSG101, CD9), exclusive marker (GM130), negative markers to confirm the purity (APOA1) in the isolated exosomes and parental cells. HEK293T cells were treated with PBS or transfected, empty vector, or IRES-Il-10 plasmids. (C) Representative transmission electron microscope (TEM) images of Exo^None, Exo^Empty or Exo^IRES-IL-10. Scale bar = 200 nm. (D) Size distribution of the isolated exosomes as indicated. (E) qPCR analysis of Il-10 mRNA in the isolated exosomes as indicated. GAPDH served as an internal control. (F) qPCR analysis of Il-10 mRNA in Exo^IRES-IL-10 in exosomal RNA degradation assay as indicated. GAPDH served as an internal control. Data are presented as mean ± SEM of three independent experiments. ND, not determined as Ct value greater than 38.

Figure 3

Engineered IRES-Il-10 mRNA in Exosomes is translationally activated by miR-155 in recipient cells. (A) Schematic of exosomes co-cultured with HEK293T cells transfected with miR-155 mimics or NC. (B) qPCR analysis of Il-10 mRNA in HEK293T cells after transfection miR-155 or NC then receiving exosomes as indicated. GAPDH served as an internal control. (C) Western blot analysis of IL-10 protein expression in HEK293T after transfection miR-155 or NC then receiving exosomes as indicated. GAPDH served as the loading control. (D) Quantification of Western blot bands by densitometry. (E) Schematic of exosomes co-cultured with polarized macrophages. (F) qPCR analysis of miR-155 in M0 macrophages and M1 macrophages. (G) qPCR analysis of Il-10 mRNA in polarized macrophages receiving exosomes as indicated. (H) Western blot analysis of IL-10 protein expression in polarized macrophages treated as indicated. GAPDH served as the loading control. (I) Quantification of Western blot bands by densitometry. Data are presented as mean ± SEM of three independent experiments. *, p < 0.05 by student's t test or one-way ANOVA. ns, no significance. ND, not determined as Ct value greater than 38. NC, negative control.

Figure 4

Local inflammation alleviation by Exo^IRES-IL-10 in ApoE^-/- mice. (A) Representative IVIS images of mice injected with PBS, 200 μg DiR labeled exosomes via tail vein. IVIS imaging was performed 4 h after injection. (B) Ex vivo fluorescence imaging analysis of the distribution of the DiR-labeled exosomes in different organs, including the aorta, heart, liver, spleen, lung, and kidney. (C) Representative confocal images of the localization of DiI-labeled exosomes in different organs. Mice were injected with 200 μg DiI-labeled exosomes via tail vein and sacrificed 4 h after injection. Scale bar = 50 µm. (D) Representative confocal images showing the localization of DiI-labeled exosomes in CD68⁺ cells in the atherosclerotic plaques of aortic roots. Scale bar in panorama, 500 µm. Scale bar in magnified image, 100 µm. (E) Schematic of the experimental procedure. ApoE^-/- mice were fed with a high-fat diet for 8 weeks, followed by the injection of 200 µg Exo^IRES-IL-10 or PBS each time, twice a week, for 2 weeks. Then the mice were sacrificed. (F) qPCR analysis of Il-10 mRNA and inflammation cytokine mRNA levels in lesioned aorta. Data are presented as mean ± SD of three independent experiments. *, p < 0.05 by student's t test.

Figure 5

Exo^IRES-IL-10 precisely induce IL-10 in inflamed tissues in ApoE^-/- mice. ELISA measurement of the concentration of IL-10 protein in lesioned aorta (A), liver (B), spleen (C), lung (D), and kidney (E) in Apoe^-/- mice. Data are presented as mean ± SD. *, p < 0.05 by one-way ANOVA. ns, no significance. n = 5 per group.

Figure 6

Therapeutic effects of Exo^IRES-IL-10 on atherosclerotic lesions in ApoE^-/- mice. (A) Schematic of the experimental procedure. ApoE^-/- mice were fed with a high-fat diet for 8 weeks, followed by the injection of 200 µg Exo^IRES-IL-10, 200 µg Exo^IL-10 or PBS each time, twice a week, for 4 weeks. (B) Representative aortic arch view of the atherosclerotic lesions in ApoE^-/- mice treated as indicated. AA, ascending aorta; BA, brachiocephalic artery; LCCA, left common carotid artery; LSA, left subclavian artery; DA, descending aorta. (C) Percentage of the atherosclerotic area in the aortic arch treated as above. (D) Representative images of cross-sectional view of the aortic roots stained with H&E, ORO from ApoE^-/- mice treated as indicated. Scale bars, 500 µm. (E) Statistical data of the Oil-Red-O (ORO) positive plaque area from D. (F) Representative images of ORO staining of the atherogenic lesion areas in mice treated as above. (G) qPCR analysis of inflammatory cytokine mRNA levels in lesioned aorta. Data are presented as mean ± SD. *, p < 0.05 by one-way ANOVA. n = 5 per group.