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, 42 (12), 906-918

Attenuation of Experimental Autoimmune Hepatitis in Mice With Bone Mesenchymal Stem Cell-Derived Exosomes Carrying MicroRNA-223-3p

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Attenuation of Experimental Autoimmune Hepatitis in Mice With Bone Mesenchymal Stem Cell-Derived Exosomes Carrying MicroRNA-223-3p

Feng-Bin Lu et al. Mol Cells.

Abstract

MicroRNA-223-3p (miR-223-3p) is one of the potential microRNAs that have been shown to alleviate inflammatory responses in pre-clinical investigations and is highly encased in exosomes derived from bone mesenchymal stem cells (MSC-exosomes). MSC-exosomes are able to function as carriers to deliver microRNAs into cells. Autoimmune hepatitis is one of the challenging liver diseases with no effective treatment other than steroid hormones. Here, we examined whether MSC-exosomes can transfer miR-223-3p to treat autoimmune hepatitis in an experimental model. We found that MSC-exosomes were successfully incorporated with miR-223-3p and delivered miR-223-3p into macrophages. Moreover, there was no toxic effect of exosomes on the macrophages. Furthermore, treatments of either exosomes or exosomes with miR-223-3p successfully attenuated inflammatory responses in the liver of autoimmune hepatitis and inflammatory cytokine release in both the liver and macrophages. The mechanism may be related to the regulation of miR-223-3p level and STAT3 expression in the liver and macrophages. These results suggest that MSC-exosomes can be used to deliver miR-223-3p for the treatment of autoimmune hepatitis.

Keywords: autoimmune liver disease; exosomes; immunomodulatory; mesenchymal stromal cells.

Conflict of interest statement

Disclosure

The authors have no potential conflicts of interest to disclose.

Figures

Fig. 1
Fig. 1. Characterizations of exosomes derived from bone mesenchymal stem cells
(A) The morphology of MSC-exosomes under an electron microscope and proteins expression in exosomes and MSCs. Arrows indicate exosomes as cup-shaped membrane vesicles of 40 to 100 nm in diameter. Western blot analysis indicates that exosomes are positive for CD9, CD63, CD81, and TSG101, but negative for cytochrome c. (B) The transfected cells under a light microscope and a fluorescent microscope at a magnification of ×200. Scale bars = 100 μm. After transfection, MSCs, which transfected successfully, displayed green fluorescence in fluorescent microscope image. (C) The expression levels of miR-223-3p and miR-223-5p in transfected MSC-exosomes. (D and E) The protein levels of Stat3 and Sema3A, the downstream target of miR-223-3p and miR-223-5p respectively, in transfected MSCs via Western blotting. Group 1, MSCsmiR-223-3p group; Group 2, MSCsmiR-223-3p-C°N group; Group 3, MSCsmiR-223-3p(i) group; Group 4, MSCsmiR-223-3p(i)-C°N group. (F) The effect of lentivirus, miR-223-3p knockin or knockdown on the proliferation viability of MSCs. Group 1, MSCs group; Group 2, MSCsmiR-223-3p-C°N group; Group 3, MSCsmiR-223-3p group; Group 4, MSCsmiR-223-3p(i)-C°N group; Group 5, MSCsmiR-223-3p(i) group. The results showed no difference. (G) The effect of lentivirus itself on the expression of miR-223-3p and miR-223-5p in MSC-exosomes. (H) The effect of MSC-exosomes and transfected MSC-exosomes on the viability of macrophages. The results showed no difference. Data are presented as mean ± SD from three independent experiments. *P < 0.05, #P > 0.05.
Fig. 2
Fig. 2. Liver injury in hepatic S100/CFA-induced AIH mice
The automatic biochemistry analyzer was used to evaluate the serum levels of ALT and AST. (A) The serum levels of ALT and AST in the different groups of mice. Data are presented as mean ± SD from six mice. Group 1, control group; Group 2, model group; Group 3, MSC-exosomes-treated group; Group 4, MSC-exosomesmiR-223-3p-treated group; Group 5, MSC-exosomesmiR-223-3p(i)-treated group; Group 6, drug-treated group (the drug is defined as steroids and azathioprine). (B) The typical liver section stained with H&E at a magnification of ×200. Scale bars = 50 μm. Arrows indicate the infiltration of mononuclear cells in the centrilobular as well as intralobular inflammatory lesions and necrosis. Label a, control group; b, model group; c, MSC-exosomes-treated group; d, MSC-exosomesmiR-223-3p-treated group; e, MSC-exosomesmiR-223-3p(i)-treated group; f, drug-treated group. (C) The histological scoring of each group according to the Ishak grading system. Three histological sections per animal were examined. Group 1, control group; Group 2, model group; Group 3, MSC-exosomes-treated group; Group 4, MSC-exosomesmiR-223-3p-treated group; Group 5, MSC-exosomesmiR-223-3p(i)-treated group; Group 6, drug-treated group. Data are presented as mean ± SD from six mice. *P < 0.05, #P > 0.05.
Fig. 3
Fig. 3. The levels of inflammatory cytokines in mouse serum and liver
ELISA and qRT-PCR were employed to investigate serum and liver levels of inflammatory cytokines. (A-D) The serum levels of IL-1β, IL-6, IL-17, and IL-10, respectively. (E-H) The liver levels of IL-1β, IL-6, IL-17, and IL-10, respectively. For the liver, the data are expressed as fold changes relative to the control group. All histograms are presented as mean ± SD, n = 6. *P < 0.05, #P > 0.05. Group 1, control group; Group 2, model group; Group 3, MSC-exosomes-treated group; Group 4, MSC-exosomesmiR-223-3p-treated group; Group 5, MSC-exosomesmiR-223-3p(i)-treated group; Group 6, drug-treated group (the drug is defined as steroids and azathioprine).
Fig. 4
Fig. 4. Typical flow cytometric plots and different phenotypes of lymphocytes
Flow cytometry was employed to investigate the differentiation of CD4+ lymphocytes. (A and B) The representative flow cytometric plots of CD4+CD25+Foxp3+ Treg and CD4+IL-17+ Th17 cells, respectively, labelled with the corresponding percentage of CD4+ T cells. (C-E) The ratios of Th17/CD4+, ratio of Treg/CD4+ and ratio of Treg/Th17 respectively. Data are presented as mean ± SD, n = 6. *P < 0.05, #P > 0.05. Group 1, control group; Group 2, model group; Group 3, MSC-exosomes-treated group; Group 4, MSC-exosomesmiR-223-3p-treated group; Group 5, MSC-exosomesmiR-223-3p(i)-treated group; Group 6, drug-treated group.
Fig. 5
Fig. 5. Expression of miR-223-3p, p-STAT3, and STAT3 in the livers of different groups of mice
Western blotting and qRT-PCR were used to analyze the expression of miR-223-3p, p-STAT3 and STAT3 in the livers of mice in each group. For the PCR results, the data are expressed as fold changes relative to the control group. (A) The expression of miR-223-3p in the liver. (B) The expression of STAT3 mRNA in the liver. (C) The typical images of Western blots for p-STAT3, STAT3, and GAPDH. (D) The STAT3 levels normalized to GAPDH in the liver. (E) The p-STAT3 levels normalized to GAPDH in the liver. Group 1, control group; Group 2, model group; Group 3, MSC-exosomes-treated group; Group 4, MSC-exosomesmiR-223-3p-treated group; Group 5, MSC-exosomesmiR-223-3p(i)-treated group. Data are presented mean ± SD, n = 6. *P < 0.05, #P > 0.05.
Fig. 6
Fig. 6. Internalization of MSC-exosomes into macrophages and expression of cytokines in macrophages
(A) Confocal laser-scanning microscopy images of macrophages with internalized green dye-labelled MSC-exosomes (×400 magnification). Scale bars = 20 μm. Macrophages and supernatants were collected for cytokine expression analysis via ELISA and qRT-PCR. For the PCR results, the data are expressed as fold changes relative to the control group. (B) The mRNA and protein levels of IL-1β and IL-6. Labels ‘a’ and ‘c’ indicate IL-1β protein in the culture medium and IL-1β mRNA in macrophages, respectively. Labels ‘b’ and ‘d’ indicate IL-6 protein in the culture medium and IL-6 mRNA in macrophages, respectively. Data represent the mean ± SD from six independent experiments. *P < 0.05, #P > 0.05.
Fig. 7
Fig. 7. Regulation of gene expression in macrophages by MSC-exosomes
Western blotting and qRT-PCR were used to analyze the expression of miR-223-3p, p-STAT3 and STAT3 in macrophages in each group. For the PCR results, the data are expressed as fold changes relative to the control group. (A) The expression of miR-223-3p in macrophages in response to different treatments. (B) The STAT3 mRNA levels in macrophages in response to different treatments. (C-E) The protein levels of p-STAT3 and STAT3 in macrophages in response to different treatments. Group 1, control group; Group 2, LPS group; Group 3, LPS+MSC-exosomes group; Group 4, LPS+MSC-exosomesmiR-223-3p group; Group 5, LPS+MSC-exosomesmiR-223-3p(i) group. Data are presented mean ± SD, n = 6. *P < 0.05, #P > 0.05.

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References

    1. Ailawadi S., Wang X., Gu H., Fan G.C. Pathologic function and therapeutic potential of exosomes in cardiovascular disease. Biochim Biophys Acta. 2015;1852:1–11. doi: 10.1016/j.bbadis.2014.10.008. - DOI - PMC - PubMed
    1. Alvarez-Erviti L., Seow Y., Yin H., Betts C., Lakhal S., Wood M.J. Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat Biotechnol. 2011;29:341–345. doi: 10.1038/nbt.1807. - DOI - PubMed
    1. An Haack I., Derkow K., Riehn M., Rentinck M.N., Kuhl A.A., Lehnardt S., Schott E. The role of regulatory CD4 T cells in maintaining tolerance in a mouse model of autoimmune hepatitis. PLoS One. 2015;10:e0143715. doi: 10.1371/journal.pone.0143715. - DOI - PMC - PubMed
    1. Bang C., Batkai S., Dangwal S., Gupta S.K., Foinquinos A., Holzmann A., Just A., Remke J., Zimmer K., Zeug A., et al. Cardiac fibroblast-derived microRNA passenger strand-enriched exosomes mediate cardiomyocyte hypertrophy. J Clin Invest. 2014;124:2136–2146. doi: 10.1172/JCI70577. - DOI - PMC - PubMed
    1. Bettelli E., Carrier Y., Gao W., Korn T., Strom T.B., Oukka M., Weiner H.L., Kuchroo V.K. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature. 2006;441:235–238. doi: 10.1038/nature04753. - DOI - PubMed
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