microRNA-27b shuttled by mesenchymal stem cell-derived exosomes prevents sepsis by targeting JMJD3 and downregulating NF-κB signaling pathway

Abstract

Background: Exosomal microRNAs (miRs) derived from mesenchymal stem cells (MSCs) have been shown to play roles in the pathophysiological processes of sepsis. Moreover, miR-27b is highly enriched in MSC-derived exosomes. Herein, we aimed to investigate the potential role and downstream molecular mechanism of exosomal miR-27b in sepsis.

Methods: Inflammation was induced in bone marrow-derived macrophages (BMDMs) by lipopolysaccharide (LPS), and mice were made septic by cecal ligation and puncture (CLP). The expression pattern of miR-27b in MSC-derived exosomes was characterized using RT-qPCR, and its downstream gene was predicted by in silico analysis. The binding affinity between miR-27b, Jumonji D3 (JMJD3), or nuclear factor κB (NF-κB) was characterized to identify the underlying mechanism. We induced miR-27b overexpression or downregulation, along with silencing of JMJD3 or NF-κB to examine their effects on sepsis. The production of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 was detected by ELISA.

Results: miR-27b was highly expressed in MSC-derived exosomes. Mechanistic investigations showed that miR-27b targeted JMJD3. miR-27b decreased expression of pro-inflammatory genes by inhibiting the recruitment of JMJD3 and NF-κB at gene promoter region. Through this, MSC-derived exosomal miR-27b diminished production of pro-inflammatory cytokines in LPS-treated BMDMs and septic mice, which could be rescued by upregulation of JMJD3 and NF-κB. Besides, in vitro findings were reproduced by in vivo findings.

Conclusion: These data demonstrated that exosomal miR-27b derived from MSCs inhibited the development of sepsis by downregulating JMJD3 and inactivating the NF-κB signaling pathway.

Keywords: Exosome; Jumonji D3; Mesenchymal stem cells; MicroRNA-27b; Nuclear factor κB/p65; Sepsis.

Fig. 1

Exosomal miR-27b in mouse BMMSCs inhibits CLP-induced sepsis. a Isolation and identification of mouse MSCs, bright field (BF), CD29, Sca-1 and CD34 (green), DAPI (blue). b TEM micrographs of mouse MSC-derived exosomes. c Size distribution and concentration of particles as quantified by NTA. d The expression of miR-27b in mouse MSC-derived exosomes analyzed by RT-qPCR. *p < 0.05 vs. exosomes derived from mouse fibroblasts L929. e Measurements of a 7-day survival of mice by Kaplan-Meier method. n = 10 for mice following each treatment. f The production of TNF-α, IL-1β, IL-10, and IL-6 in serum of CLP-induced septic mice injected with MSC-EXO, MSC-miR-27b-inhibitor-EXO, and MSC-NC-inhibitor-EXO examined by ELISA. g Detection of ALT, AST, and SCr levels in the serum of mice. h Bacterial colonies in peritoneal fluid and serum of mice. *p < 0.01 vs. CLP-induced mice; #p < 0.001 vs. mice with sham operation; &p < 0.01 vs. CLP-induced septic mice treated with MSC-miR-27b-inhibitor-EXO. n = 10 for mice following each treatment. Quantitative data were presented as mean ± standard deviation. Unpaired data in compliance with normal distribution and equal variance between two groups were compared using unpaired t test. Comparisons among multiple groups were analyzed by one-way ANOVA with Tukey’s post hoc test. p < 0.05 indicated significant difference

Fig. 2

miR-27b prevents LPS-mediated BMDM inflammation through suppression of JMJD3. a Target genes of miR-27b predicted by the Starbase website. b, c Binding sites (b) of miR-27b to JMJD3 and verification (c) by dual-luciferase reporter gene assay. *p < 0.01 vs. BMDMs transfected with NC-mimic. d Images of BMDMs uptaking exosomes visualized under a LSCM. e The expression of miR-27b in BMDMs treated with LPS or MSC-NC-mimic-EXO and MSC-miR-27b-mimic-EXO analyzed by RT-qPCR. f The expression of JMJD3 and H3K27me3 in BMDMs treated with LPS or MSC-NC-mimic-EXO and MSC-miR-27b-mimic-EXO analyzed by western blot. In panel 2 e, f, *p < 0.05 vs. PBS-treated BMDMs; #p < 0.05 vs. LPS-treated BMDMs; &p < 0.05 vs. BMDMs treated with MSC-NC-mimic-EXO. The experiment was repeated 3 times independently. Quantitative data were presented as mean ± standard deviation. Unpaired data in compliance with normal distribution and equal variance between two groups were compared using unpaired t test. Comparisons among multiple groups were analyzed by one-way ANOVA with Tukey’s post hoc test. p < 0.05 indicated significant difference

Fig. 3

JMJD3 interacting with NF-κB/p65 elevates expression of the pro-inflammatory cytokines in LPS-induced BMDMs. a–c The recruitment of JMJD3, p65, H3K27me3, and H3K27me1 in the promoter region of pro-inflammatory cytokines TNF-α (a), IL-1β (b), and IL-6 (c) in BMDMs analyzed by CHIP-qPCR. #p < 0.05 vs. PBS-treated BMDMs. d–f The expression of LPS-induced pro-inflammatory factors TNF-α (d), IL-1β (e), and IL-6 (f) after knockdown of JMJD3 and p65. *p < 0.05 vs. PBS-treated BMDMs in response to si-NC; #p < 0.05 vs. BMDMs in response to si-NC. g–i The expression of pro-inflammatory factors TNF-α (g), IL-1β (h), and IL-6 (i) in BMDMs after 1-h treatment with GSK-J4 (4 umol/L, pharmacological inhibitor of JMJD3). *p < 0.05 vs. LPS-treated BMDMs in response to GSK-J4; #p < 0.05 vs. BMDMs in response to GSK-J4. The experiment was repeated 3 times independently. Quantitative data were presented as mean ± standard deviation. Unpaired data in compliance with normal distribution and equal variance between two groups were compared using unpaired t test. Comparisons among multiple groups were analyzed by one-way ANOVA with Tukey’s post hoc test. p < 0.05 indicated significant difference

Fig. 4

MSC-derived exosomal miR-27b inhibits JMJD3 and NF-κB/p65 axis to restrain LPS-induced pro-inflammatory response. a–c The recruitment of JMJD3, p65, H3K27me3, and H3K27me1 in the promoter region of pro-inflammatory cytokines TNF-α (a), IL-1β (b), and IL-6 (c) in LPS-treated BMDMs transfected with MSC-miR-27b-inhibitor-EXO analyzed by CHIP-qPCR. *p < 0.05 vs. LPS-treated BMDMs transfected with MSC-NC-inhibitor-EXO. d–f The expression of pro-inflammatory cytokines TNF-α (d), IL-1β (e), and IL-6 (f) in BMDMs after overexpression of JMJD3 or p65. *p < 0.05 vs. PBS-treated BMDMs. The experiment was repeated 3 times independently. Quantitative data were presented as mean ± standard deviation. Unpaired data in compliance with normal distribution and equal variance between two groups were compared using unpaired t test. Comparisons among multiple groups were analyzed by one-way ANOVA with Tukey’s post hoc test. p < 0.05 indicated significant difference

Fig. 5

Exosomal miR-27b from MSCs suppresses the JMJD3/NF-κB/p65 axis to inhibit the development of CLP-induced sepsis. a The expression of JMJD3 and H3K27me3 in mice subjected to sham operation or CLP in response to MSC-miR-27b-mimic-EXO analyzed by Western blot. *p < 0.05 vs. mice subjected to CLP; #p < 0.05 vs. mice subjected to sham operation. b Results of a 7-day survival in mice. n = 10 for mice following each treatment. c The production level of TNF-α, IL-1β, IL-10, and IL-6 in mouse serum examined by ELISA. d Bacterial colonies in peritoneal fluid and serum of mice. e Detection of the liver and kidney function in mice. *p < 0.05 vs. mice subjected to CLP. n = 10 for mice following each treatment. Cellular experiments were repeated 3 times independently. Quantitative data were presented as mean ± standard deviation. Comparisons among multiple groups were analyzed by one-way ANOVA with Tukey’s post hoc test. p < 0.05 indicated significant difference

Fig. 6

Schematic diagram of the function and mechanism of exosomal miR-27b in sepsis. JMJD3 can be transferred into nucleus through cytoplasm and nucleus and promote the demethylation of H3K27me3 by recruiting NF-κB, thus activating the transcription of TNF-α/IL-β/IL-6 to increase the expression of these target proteins. However, miR-27b loaded in the exosomes secreted by MSCs binds to the mRNA of JMJD3 in the target cells after membrane fusion and inhibits the expression of JMJD3 protein, thus suppressing the expression of inflammatory factors. GSK-J4, a pharmacological inhibitor of JMJD3, has similar effects