Effective delivery of miR-511-3p with mannose-decorated exosomes with RNA nanoparticles confers protection against asthma

Abstract

Our previous studies have shown that miR-511-3p treatment has a beneficial effect in alleviating allergic airway inflammation. Here, we sought to explore its therapeutic potential in animal models and gain a deeper understanding of its therapeutic value for asthma. miR-511-3p knockout mice (miR-511-3p^-/-) were generated by CRISPR/Cas and showed exacerbated airway hyper-responsiveness and Th2-associated allergic airway inflammation compared with wild-type (WT) mice after exposed to cockroach allergen. RNA nanoparticles with mannose decorated EV-miR-511-3p were also created by loading miR-511-3p mimics into the mannose decorated EVs with engineered RNA nanoparticle PRNA-3WJ (Man-EV-miR-511-3p). Intra-tracheal inhalation of Man-EV-miR-511-3p, which could effectively penetrate the airway mucus barrier and deliver functional miR-511-3p to lung macrophages, successfully reversed the increased airway inflammation observed in miR-511-3p^-/- mice. Through microarray analysis, complement C3 (C3) was identified as one of the major targets of miR-511-3p. C3 was increased in LPS-treated macrophages but decreased after miR-511-3p treatment. Consistent with these findings, C3 expression was elevated in the lung macrophages of an asthma mouse model but decreased in mice treated with miR-511-3p. Further experiments, including miRNA-mRNA pulldown and luciferase reporter assays, confirmed that miR-511-3p directly binds to C3 and activates the C3 gene. Thus, miR-511-3p represents a promising therapeutic target for asthma, and RNA nanotechnology reprogrammed EVs are efficient carriers for miRNA delivery for disease treatment.

Keywords: Asthma; Complement C3; Inflammation; Macrophage; miRNA.

Fig. 1.. Generation of miR-511-3p knockout mice using the CRISPR-Cas9 system.

(A) Schematic illustration of the process for the generation of miR-511-3p^−/− mice by using CRISPR/Cas9 system with guide RNA (gRNA) sequences flanking the murine miR-511-3p. (B-C) Confirmation of the newly generated miR-511-3p^−/− mice by Sanger Sequencing (B) and genotyping (C). (D) RT-PCR confirmation of miR-511-3p deletion in the lung tissues of miR-511-3p^−/− mice (n=10/group). (E-F) Expression of Mrc1 in lung tissue macrophages of wild-type (WT) and miR-511-3p^−/− mice analyzed by flow cytometry analysis (E) and co-immunostaining with Mrc1 and F4/80 (F). (G) Representative of FITC-CRE (green) in macrophages (red) of lung tissues from WT and miR-511-3p^−/− mice (n = 3 per group). All data were expressed as the means ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey’s post-hoc test. ***P < 0.001.

Fig. 2.. miR-511-3p^−/− mice show exacerbation of cockroach allergen-induced airway hyper-responsiveness and lung inflammation.

(A) Experimental protocol for the generation of cockroach allergen-induced mouse model of asthma with WT and miR-511-3p^−/− mice. (B) Lung resistance in response to increasing concentrations of methacholine using the forced oscillation technique (FlexiVent, SCIREQ) (n=5/group). (C) Histological examination of mouse paraffin lung sections stained with hematoxylin and eosin (H&E, upper panel) and Periodic acid–Schiff (PAS, lower panel) staining. (D) Bronchoalveolar lavage (BAL) fluid total and eosinophil cell counts as assessed by flow cytometry. (E) Serum levels of cockroach allergen-specific IgE and IgG1. (F) Levels of cytokines in BAL fluids. D-F, n=6–16/group. All data were expressed as the means ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey’s post-hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. CRE: cockroach extract. WT: wild-type.

Fig. 3.. Lack of miR-511-3p leads macrophage polarization to M1 phenotypes.

(A) Schematic of preparation of single cells from lung tissues for flow cytometry analysis. (B) Gating strategies for the flow cytometry analysis of M1/M2 macrophages in the lung tissues of wild-type (WT) and miR-511-3p^−/− mice. (C-D) Quantitation of lung tissue M1 and M2 macrophages as indicated by the percentage of F4/80⁺CD11c⁺iNOS⁺ cells (%M1, C) and F4/80⁺CD11c⁺Arg-1⁺ cells (%M2, D) among total lung single cells. n=4/group. (E) Representative image of immune fluorescence staining with the primary antibodies against iNOS. Nuclei were counterstained with 4’-6-diamidino-2-phenylindole dihydrochloride (DAPI). Right panel: quantification of immunostainings for F4/80⁺iNOS⁺ in total F4/80⁺ cells. n=5/group. (F) Representative image of immune fluorescence staining with the primary antibodies against Arg-1. Right panel: quantification of immunostainings for F4/80⁺Arg-1⁺ in total F4/80⁺ cells. n=5. (G-H) Quantitative RT-PCR analyses for M1 (G) and M2 (H)-associated genes in bone marrow-derived macrophages (BMDMs) from WT and miR-511-3p^−/− mice cultured under M1 or M2 condition. n=4. Data represent means ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey’s post-hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 4.. Generation of RNA nanoparticles with mannose decorated extracellular vesicle-miR-511-3p for targeted delivery.

(A) Schematic of generation of RNA nanoparticles with mannose decorated extracellular vesicle-miR-511-3p for specific delivery of miR-511-3p. (B) Schematic of the mannose decorated EVs with miR-511-3p targeting macrophages through mannose receptor Mrc1. (C) 3D structure of Man-EV-miR-511-3p with EVs (blue) and RNA nanoparticles harboring mannose (yellow). (D) Size and concentration of Man-EV-miR-511-3p detected by Nanoparticle tracking analysis (NTA). (E) Actual morphology was observed with transmission electron microscope. (F)Representative confocal images show miR-511-3p uptake by RAW 264.7 cells. Nucleus (blue), cytoplasm (green), and miR-511-3p (red). (G) Schematic of a luciferase-based miR-511-3p reporter system in which a binding sequence of miR-511-3p was cloned into the 3’-UTR of Renila luciferase gene (psi-Check2-anti-miR-511-3p). (H) Dual luciferase assay to test the delivery of miR-511-3p into RAW 264.7 cells and shows a better inhibition as assessed by renilla/firefly luciferase ratio.

Fig. 5.. Man-EV-miR-511-3p recovers the increased airway inflammation caused by miR-511-3p deficiency.

(A) Experimental protocol for the use of Man-EV-miR-511-3p in asthma mouse model. (B) (C) Histological examination of mouse paraffin lung sections stained with hematoxylin and eosin (H&E, upper panel) and Periodic acid-Schiff (PAS, lower panel) staining. (C) Flow cytometry analyses of inflammatory cells in bronchoalveolar lavage (BAL) fluids. (D) ELISA analyses of cockroach allergen-specific IgE and IgG1 levels in serum. (E) ELISA analyses of cytokine levels in BAL fluids. C-E, n=6–11/group. All data were expressed as the means ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey’s post-hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. CRE: cockroach extract. WT: wild-type. ManEV: mannose decorated extracellular vesicle.

Fig. 6.. Man-EV-miR-511-3p polarizes macrophages into M2 phenotypes.

(A) Schematic of delivered Man-EV-miR-511-3p targeting lung macrophages via Mrc1 to modulating macrophage polarization. (B) Gating strategies for the flow cytometry analysis of M1/M2 macrophages in the lung tissues. (C-D) Quantitation of lung tissue M1 and M2 macrophages as indicated by the percentage of F4/80⁺CD11c⁺iNOS⁺ cells (%M1, C) and F4/80⁺CD11c⁺Arg-1⁺ cells (%M2, D) among total lung single cells. n=5/group. (E) Representative image of immune fluorescence staining with the primary antibodies against iNOS. Nuclei were counterstained with 4’-6-diamidino-2-phenylindole dihydrochloride (DAPI). Right panel: uantification of immunostainings for F4/80⁺iNOS⁺ in total F4/80⁺ cells. n=3–4/group (F) Representative image of immune fluorescence staining with the primary antibodies against Arg-1. Right panel: uantification of immunostainings for F4/80⁺Arg-1⁺ in total F4/80⁺ cells. n=3–4/group (G-H) Quantitative RT-PCR analyses for M1 (G) and M2 (H)-associated genes in Man-EV-miR-511-3p pre-treated bone marrow-derived macrophages (BMDMs) cultured under M1 or M2 condition. n=3/group. Data represent means ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey’s post-hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. ManEV: mannose decorated extracellular vesicle.

Fig. 7.. Microarray analysis identifies C3 as one of the major miR-511-3p target genes.

(A) Schematic of microarray analysis for miR-511-3p pre-treated macrophages under M1 culture condition using the mouseRef-8 v2.0 BeadChip. (B-C) Volcano plot of differentially expressed genes in bone marrow-derived macrophages (BMDMs) cultured with (M1) or without (M0) LPS (B) or LPS-treated BMDMs with or without miR-511-3p pretreatment (C). The logarithms of the fold changes of individual genes (x axis) are plotted against the negative logarithm of false discover rate (FDR) to base 10 (y axis). (D-E) Heatmap (D) and gene expression (E) of complement C3 gene in undifferentiated BMDMs and M1 macrophages with or without miR-511-3p pretreatment. n=3/group. (F-G) qPCR analysis of C3 expression in BMDMs (F) and lung tissues of asthma mouse model (G) with or without AAV-miR-511-3p treatment. n=6/group. (H) Expression of C3 in macrophages defined by the representative image of C3 (red) co-immune fluorescence staining with F4/80 (green). Nuclei were counterstained with 4’-6-diamidino-2-phenylindole dihydrochloride (DAPI). Right panel: quantification of double immunostainings for C3 and F4/80. n=9–12/group. (I) Expression of C3 in macrophages defined by the representative image of C3 (red) co-immune fluorescence staining with F4/80 (green) in miR-511-3p^−/− mice treated with/without ManEV-miR-511-3p. Nuclei were counterstained with 4’-6-diamidino-2-phenylindole dihydrochloride (DAPI). Right panel: quantification of double immunostainings for C3 and F4/80. n=4–6/group. All data were expressed as the means ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey’s post-hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. CRE: cockroach extract. ManEV: mannose decorated extracellular vesicle.

Fig. 8.. miR-511-3p illustrates a direct binding to C3 and regulates C3 activity.

(A) Schematic representation of miR-511-3p-C3 mRNA biotin pulldown assay. (B) qPCR analysis of C3 expression in miR-511-3p-C3 mRNA complexes. n=4. (C) In silico prediction of miR-511-3p binding site in C3 mRNA by BiBiServ RNAhybrid. (D-F) Dual-luciferase assay in HEK293T cells transfected with C3-psicheck2 plasmid containing the binding site (D), mutant binding site (E), and control (F) and then treated with either NC-mimics or miR-511-3p mimics. n=3. All data were expressed as the means ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey’s post-hoc test. *P < 0.05, ***P < 0.001.