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Exosomal miR-223 Contributes to Mesenchymal Stem Cell-Elicited Cardioprotection in Polymicrobial Sepsis

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Exosomal miR-223 Contributes to Mesenchymal Stem Cell-Elicited Cardioprotection in Polymicrobial Sepsis

Xiaohong Wang et al. Sci Rep.

Abstract

Mesenchymal stem cells (MSCs) have been shown to elicit cardio-protective effects in sepsis. However, the underlying mechanism remains obscure. While recent studies have indicated that miR-223 is highly enriched in MSC-derived exosomes, whether exosomal miR-223 contributes to MSC-mediated cardio-protection in sepsis is unknown. In this study, loss-of-function approach was utilized, and sepsis was induced by cecal ligation and puncture (CLP). We observed that injection of miR-223-KO MSCs at 1 h post-CLP did not confer protection against CLP-triggered cardiac dysfunction, apoptosis and inflammatory response. However, WT-MSCs were able to provide protection which was associated with exosome release. Next, treatment of CLP mice with exosomes released from miR-223-KO MSCs significantly exaggerated sepsis-induced injury. Conversely, WT-MSC-derived-exosomes displayed protective effects. Mechanistically, we identified that miR-223-KO exosomes contained higher levels of Sema3A and Stat3, two known targets of miR-223 (5p &3p), than WT-exosomes. Accordingly, these exosomal proteins were transferred to cardiomyocytes, leading to increased inflammation and cell death. By contrast, WT-exosomes encased higher levels of miR-223, which could be delivered to cardiomyocytes, resulting in down-regulation of Sema3A and Stat3. These data for the first time indicate that exosomal miR-223 plays an essential role for MSC-induced cardio-protection in sepsis.

Figures

Figure 1
Figure 1. Absent of miR-223 impairs MSC-induced protection against sepsis-triggered cardiac injury.
(AH) Characterization of MSCs derived from bone marrow of WT mice (AD) and miR-223 KO mice (EH). (I) Total RNA was isolated from MSCs and performed RT-PCR analysis, and showed that miR-223-5p and -3p both are absent in KO-MSCs. U6 snRNA was used as internal control. (J) Injection of miR-223-KO MSCs (n = 14) at 1 h post-CLP did not improve animal survival, whereas the survival rate was significantly improved in WT-MSC-treated mice (n = 12), compared with PBS controls (n = 10, *p < 0.05). (KM) CLP-induced cardiac depression, measured by echocardiography (K), was significantly attenuated in WT-MSC-treated mice, but not in KO-MSC-treated mice. *p < 0.05, vs. sham group; #p < 0.05, vs. CLP+PBS control group; n = 6 for sham group, n = 10 for CLP + PBS, n = 12 for WT- and KO-MSC-treated groups. (N) Representative TUNEL staining for detection of apoptotic cardiomyocytes (green dots indicated by arrows, upper row). Cardiomyocytes were stained with α-actin (Red) and nuclei were stained with DAPI (blue), which were merged together, indicated in the lower row. (O) Quantitative results of TUNEL staining, which was further confirmed by ELISA measurement of histone-associated DNA fragmentation (P). (n = 5 hearts, *p < 0.05, vs. shams; #p < 0.05 vs. CLP+PBS control group).
Figure 2
Figure 2. Loss of miR-223 in MSCs negates MSC-induced inhibitory effects on CLP-triggered systemic inflammatory response.
Injection of WT-MSCs into mice at 1h post-CLP significantly reduced the circulating levels of inflammatory cytokines: (A) TNF-α, (B) IL-1β, and (C) IL-6, whereas they were not reduced in KO-MSC-treated mice, compared with PBS-treated samples. *p < 0.05, n = 4 for shams, n = 6–8 for CLP mice.
Figure 3
Figure 3. MiR-223 is critical for MSC-mediated inhibitory effects on the cytokine production in macrophages upon LPS challenge.
(A) A diagram of cell co-culture system in which macrophages (RAW264.7 cells) were cultured in the lower chamber and MSCs were cultured in the upper chamber of a 12-well insert. (BD) Inhibition of LPS-triggered TNF-α (B), IL-1β (C), and IL-6 (D) production was more remarkable in macrophages co-cultured with WT-MSCs than those co-cultured with KO-MSCs. (n = 3 wells, *p < 0.05, vs. Medium controls and KO-MSC samples). Similar results were observed in two additional, independent experiments.
Figure 4
Figure 4. Blockade of exosome release negates the inhibitory effects of WT-MSCs on LPS-triggered inflammatory cytokine production in macrophages and LPS-induced cardiomyocyte death.
WT-MSCs significantly suppressed the production of TNF-α (A), IL-1β (B), and IL-6 (C) in co-cultured RAW264.7 cells upon LPS challenge. Such inhibitory effects were offset by addition of GW4869 (20 μM). n = 3 wells, *p < 0.05, vs. RAW264.7 cultured alone. Similar results were observed in three additional, independent experiments. (D) A diagram of cell co-culture system in which cardiomyocytes were cultured in the lower chamber of a 12-well plate pre-coated with laminin (10 μg/ml) and MSCs were cultured in the upper chamber of a 12-well insert. (E) LPS exposure significantly decreased cardiomyocyte survival, whereas it is greatly improved by co-culturing with WT-MSCs. n = 3 wells, *p < 0.05, vs. cardiomyocytes cultured alone. Addition of GW4869 (20 μM) offset WT-MSC-elicited protective effects on LPS-triggered myocyte death. Similar results were observed in two additional, independent experiments.
Figure 5
Figure 5. Characterizations of exosomes derived from MSCs and their functional roles in macrophages and cardiomyocytes upon LPS challenge.
(A,B) The size of exosomes derived from (A) WT-MSCs and (B) miR-223 KO-MSCs, measured using a Zetasizer Nano ZS instrument. (C) Protein levels of CD63 and CD81 were similarly encased in WT-exosomes and KO-exosomes. Figure represents truncated western blot images for simplicity. Whole membrane images are shown in Supplementary Figure S2. (D) Both strands of miR-223 were included in WT-exosomes and null in KO-exosomes, 100 bp-DNA lander was used as a gel loading marker. MiR-320 was used as an internal control for RT-PCR. n = 3 independent experiments for A–D. (EG) Addition of WT-exosomes (20 μg/ml) to cultured RAW264.7 cells significantly inhibited LPS-triggered secretion of TNF-α (E), IL-1β (F), and IL-6 (G). Remarkably, KO-exosomes (20 μg/ml) promoted RAW264.7 cell secretion of TNF-α (E), IL-1β (F), and IL-6 (G) upon LPS challenge (100 ng/ml). n = 3 wells for each group; *p < 0.05, vs. medium controls; #p < 0.05, vs. medium controls. Similar results were observed in other two additional, independent experiments. (HJ) LPS-induced cardiomyocyte death/apoptosis was significantly mitigated by treatment with WT-exosomes (20 μg/ml) and remarkably promoted by addition of KO-exosomes (20 μg/ml). Representative images of cardiomyocytes in the absent and present of LPS plus WT-exosomes or KO-exosomes were shown in (H). Survival rate was determined by MTS incorporation (I), and cardiomyocyte apoptosis (DNA fragmentation) was determined using an ELISA kit (J). n = 3 wells for each group; *p < 0.05, vs. medium controls; #p < 0.05, vs. medium controls. Similar results were observed in other three additional, independent experiments.
Figure 6
Figure 6. The effects of WT-exosomes and miR-223-KO exosomes on CLP-induced inflammatory response, cardiac dysfunction and animal mortality.
(AC) CLP-mice treated with WT-exosomes (n = 11) showed lower levels of serum TNF-α (A), IL-1β (B), and IL-6 (C), whereas CLP-mice injected with KO-exosomes (n = 11) exhibited higher levels of circulating TNF-α (A), IL-1β (B), and IL-6 (C), compared with those treated with incomplete DMEM medium (n = 10) (^p < 0.05 vs. shams; *p < 0.05 vs. CLP + medium; #p < 0.05 vs. CLP + medium). (D) Results of echocardiography measurement showed that values of the left ventricular ejection fraction (EF%, E) and the fractional shortening (FS%, F) were significantly decreased in CLP mice injected with incomplete DMEM medium (n = 10), compared with shams (n = 8). Remarkably, the reduction of EF% and FS% was attenuated in WT-exosome-treated CLP mice (n = 11); whereas it was aggravated in CLP mice administrated with miR-223-KO exosomes (n = 11) (^p < 0.05 vs. shams; *p < 0.05 vs. CLP + medium; #p < 0.05 vs. CLP + medium). (G) The survival of CLP-mice was significantly improved by WT-exosome treatment, whereas it was worse by miR-223-KO exosome injection (n = 8, *p < 0.05 vs. CLP + medium).
Figure 7
Figure 7. miR-223-KO exosomes can deliver Sema3A and Stat3, whereas WT-exosomes can deliver miR-223 to the myocardium in vivo.
(A) A diagram shows that both strands of pre-miR-223 can be processed to be mature miRNA and target Sema3A and Stat3, respectively. (B,C) Sema3A and Stat3 both were up-regulated in miR-223-KO MSCs (n = 4, *p < 0.05 vs. WTs). (D,E) Sema3A and Stat3 both proteins were highly enriched in exosomes released from miR-223-KO MSCs (n = 4, *p < 0.05 vs. WTs). (FH) Red dye PKH26-labeled WT- and KO-exosomes both were detected in the myocardium after i.v. injection. Cardiomyocytes were stained with Alexa-Fluor 488 labeled α–actinin antibody. (I,J) Higher levels of Sema3A and Stat3 encased in miR-223-KO exosomes were effectively transported to the heart, whereas WT-exosome-treated hearts displayed lower levels of Sema3A and Stat3, compared to PBS-injected control hearts, respectively (n = 4, *p < 0.05 vs. PBS-treated samples). α-actin was used as a loading control. AU: arbitrary unit. The gel had been run under the same experimental conditions. The full-length blots are shown in Supplementary Figure S3. (K) The levels of miR-223-5p and -3p were significantly increased in WT-exosome-treated myocardium, whereas they were not altered in KO-exosome-treated hearts. U6 snRNA was used as an internal control for qRT-PCR analysis (n = 4, *p < 0.05 vs. PBS controls).
Figure 8
Figure 8. A work model elucidating that miR-223 contributes to MSC-elicited protective effects against sepsis through the exosome-mediated transfer of miR-223 to other types of cells (i.e. macrophages and cardiomyocytes), leading to attenuation of inflammatory response and inhibition of cell death in recipient cells.

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