Human umbilical cord mesenchymal stem cell-derived exosomal miR-146a-5p reduces microglial-mediated neuroinflammation via suppression of the IRAK1/TRAF6 signaling pathway after ischemic stroke - PubMed (original) (raw)

. 2021 Jan 21;13(2):3060-3079.

doi: 10.18632/aging.202466. Epub 2021 Jan 21.

Xiaoxiong Zou 1, Run Zhang 1, Yu Xie 1, Zhiming Feng 1, Feng Li 1, Jianbang Han 1, Haitao Sun 1, Qian Ouyang 1, Shiting Hua 1, Bingke Lv 1, Tian Hua 1, Zhizheng Liu 1, Yingqian Cai 1, Yuxi Zou 1, Yanping Tang 1, Xiaodan Jiang 1 2

Affiliations

Human umbilical cord mesenchymal stem cell-derived exosomal miR-146a-5p reduces microglial-mediated neuroinflammation via suppression of the IRAK1/TRAF6 signaling pathway after ischemic stroke

Zhongfei Zhang et al. Aging (Albany NY). 2021.

Abstract

To investigate the therapeutic mechanism of action of transplanted stem cells and develop exosome-based nanotherapeutics for ischemic stroke, we assessed the effect of exosomes (Exos) produced by human umbilical cord mesenchymal stem cells (hUMSCs) on microglia-mediated neuroinflammation after ischemic stroke. Our results found that injected hUMSC-Exos were able to access the site of ischemic damage and could be internalized by cells both in vivo and in vitro. In vitro, treatment with hUMSC-Exos attenuated microglia-mediated inflammation after oxygen-glucose deprivation (OGD). In vivo results demonstrated that treatment with hUMSC-Exos significantly reduced infarct volume, attenuated behavioral deficits, and ameliorated microglia activation, as measured three days post-transient brain ischemia. Furthermore, miR-146a-5p knockdown (miR-146a-5p k/d Exos) partially reversed the neuroprotective effect of hUMSC-Exos. Our mechanistic study demonstrated that miR-146a-5p in hUMSC-Exos reduces microglial-mediated neuroinflammatory response through IRAK1/TRAF6 pathway. We conclude that miR-146a-5p derived from hUMSC-Exos can attenuate microglia-mediated neuroinflammation and consequent neural deficits following ischemic stroke. These results elucidate a potential therapeutic mechanism of action of mesenchymal stem cells and provide evidence that hUMSC-Exos represent a potential cell-free therapeutic option for ischemic stroke.

Keywords: exosomes; ischemic stroke; mesenchymal stem/stromal cell; microRNA; neuroinflammation.

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Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare that they have no conflicts of interest.

Figures

Figure 1

Figure 1

Analysis of human umbilical mesenchymal stem cells (hUMSCs) and hUMSC-derived exosomes (hUMSC-Exos). (A) Representative micrographs of cultured hUMSCs at passage 3 (P3). Scale bar: 200 μm. (B) Flow cytometry analysis of hUMSC CD73, CD105, CD90, CD11b, CD19, CD34, CD45, and HLA-DR expression. (C) Representative electron micrographs of hUMSC-Exos. Scale bar: 200 nm. (D) Exosome particle size and concentration. (E) Western blot analysis of Exos-specific markers CD9, ALIX, and TSG101. Each blot represents three independent experiments of two samples each.

Figure 2

Figure 2

Treatment with hUMSC-Exos attenuates microglia-mediated inflammation and neurological deficits after ischemic stroke. (A) Schematic of the protocol. (B) Representative photomicrographs of TTC-stained tissue from the control, vehicle-only, and experimental groups, with associated infarct size as calculated using ImageJ software. Data are expressed as mean ± SEM (n = 12 per group). Significant differences are indicated (_*p < 0.05). (C) Neurological deficit scores in the vehicle-only and experimental groups 72 hours post-reperfusion. Data are expressed as mean ± SEM (n = 12 per group). Significant differences are indicated (*_p < 0.05, _**p < 0.01). (D) The red box indicates the cerebral ischemic penumbra. (E) H&E staining. Scale bar: 50 μm. (F) Representative photomicrographs of IL-6 and NFκB in the ischemic penumbra 72 hours post-reperfusion, with associated relative intensities as calculated using ImageJ software. Scale bar: 50 μm. Data are expressed as mean ± SEM (n = 6 per group). Significant differences are indicated (*_p < 0.05).

Figure 2

Figure 2

Treatment with hUMSC-Exos attenuates microglia-mediated inflammation and neurological deficits after ischemic stroke. (G) Red fluorescence indicates PKH26-labeled exosomes which have accessed the site of cerebral damage. Scale bar: 50 μm. (H) Microglial M1 markers IBA-1 and CD16 in the ischemic penumbra 3 days following ischemic stroke, in the control, vehicle-only, and experimental groups. Scale bar: 50 μm. Associated M1 counts are shown (A, B). (I) Microglial M2 markers IBA-1 and CD206 in the ischemic penumbra 3 days following ischemic stroke, in the control, vehicle-only, and experimental groups. Scale bar: 50 μm. Associated M2 counts - from the same animals in which M1 counts were determined - are shown (C, D). Significant differences are indicated (_*_p < 0.05). (J) Lower protein levels of pro-inflammatory cytokines IL-6, TNF-α, and IL-1β in the experimental group. Data are expressed as mean ± SEM (experiments were performed in triplicate). Significant differences are indicated (*p < 0.05, **p < 0.01).

Figure 3

Figure 3

Treatment with hUMSC-Exos reduces microglial pro-inflammatory activity in vitro. (A) Confocal imaging demonstrating uptake of PKH-26-labeled exosomes (red) by BV2 microglia. Scale bar: 50 μm. (B) Lower protein levels of pro-inflammatory cytokines IL-6, TNF-α, and IL-1β in the hUMSC-Exos treatment group. (C) Levels of IL-6, TNF-α, and IL-1β mRNA as detected using qRT-PCR. Data are expressed as mean ± SEM (experiments were performed at least in triplicate). Significant differences are indicated (*p < 0.05, **p < 0.01).

Figure 4

Figure 4

Exosomal miRNAs are implicated in hUMSC-Exos-mediated attenuation of microglial pro-inflammatory activity. (A, B) After 24 hours’ siRNA-Drosha transfection, hUMSC Drosha knockdown efficiency was evaluated by qPCR quantitation of Drosha mRNA and western blot-based quantitation of Drosha protein. Western blots are representative of three independent experimental replicates. (C, D) Exosomal miR-146a-5p and miR-21-5p content was significantly decreased after Drosha knockdown. (E) Protein levels of the pro-inflammatory cytokines IL-6, TNF-α, and IL-1β in hUMSC-Exos were decreased after Drosha knockdown. (F) Detection of IL-6, TNF-α, and IL-1β mRNA levels via qRT-PCR. Data are expressed as mean ± SEM. (A-F) Each experiment is representative of n = 3 per group. Significant differences are indicated (*p < 0.05, **p < 0.01).

Figure 5

Figure 5

Exosomal miR-146a-5p decreases microglial pro-inflammatory activity by suppressing the IRAK1/TRAF6 signaling pathway in vitro. (A) Expression levels of the top ten hUMSC-Exosomal miRNAs, including MiR-146a-5p. (B) After post-OGD exposure to hUMSC-Exos, BV2 microglia exhibited significantly increased miR-146a-5p content. Data were normalized to levels of U6. (C) In vitro experimental scheme. (D) Expression of pro-inflammatory cytokines IL-6, TNF-α, and IL-1β, as well as signaling pathway IRAK1, TRAF6, and NFκB (p65) in microglia treated with wild-type versus miR-146a-5p knockdown hUMSC-Exos. (E) Determination of IL-6, TNF-α, and IL-1β mRNA levels via qRT-PCR. (F) Determination of supernatant IL-6, TNF-α, and IL-1β protein levels via ELISA. Data are expressed as mean ± SEM. (A–F) Each experiment is representative of n = 3 per group. Significant differences are indicated (*p < 0.05, **p < 0.01, ***p < 0,001).

Figure 6

Figure 6

Treatment with hUMSC-Exos decreases neuroinflammation and is neuroprotective by down-regulating IRAK1/TRAF6 signaling pathway activity in vivo. (A) Representative photomicrographs of TTC-stained tissue from wild-type versus miR-146a-5p knockdown hUMSC-Exos groups, with infarct size as calculated using ImageJ software. Data are expressed as mean ± SEM (n = 6 per group). Significant differences are indicated (*p < 0.05). (B, C) Neurological deficit scores in vehicle-only versus experimental groups at 72 hours post-reperfusion. Data are expressed as mean ± SEM (n = 12 per group). Significant differences are indicated (_*_p < 0.05, _**p < 0.01). (D, E) Representative photomicrographs of IL-6 and NFκB in the ischemic penumbra 72 hours post-reperfusion, with associated relative intensities as calculated using ImageJ software. Scale bar: 50 μm. Data are expressed as mean ± SEM (n = 6 per group). Significant differences are indicated (*_p < 0.05). (F) Microglial M1 markers IBA-1 and CD16 in the ischemic penumbra 3 days following ischemic stroke. (G) Expression of signaling pathway IRAK1, TRAF6, and NFκB (p65) in the wild-type versus miR-146a-5p knockdown groups. (H) Determination of IL-6, TNF- α, and IL-1β protein levels via ELISA. Data are expressed as mean ± SEM (experiments were performed in triplicate). Significant differences are indicated (*p < 0.05, **p < 0.01, ***p < 0,001).

Figure 7

Figure 7

A potential mechanism contributing to the hUMSC-Exos-induced decrease in microglia-mediated neuroinflammation after ischemic stroke. After injection of hUMSC-Exos into the tail vein of the murine ischemic stroke model, they traversed the blood-brain barrier and were internalized by microglia at the site of cerebral injury. Exosomal miR-146a-5p may decrease microglia-mediated neuroinflammation by suppressing the IRAK1/TRAF6 signaling pathway.

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References

    1. Feigin VL, Nguyen G, Cercy K, Johnson CO, Alam T, Parmar PG, Abajobir AA, Abate KH, Abd-Allah F, Abejie AN, Abyu GY, Ademi Z, Agarwal G, et al., and GBD 2016 Lifetime Risk of Stroke Collaborators. Global, regional, and country-specific lifetime risks of stroke, 1990 and 2016. N Engl J Med. 2018; 379:2429–37. 10.1056/NEJMoa1804492 - DOI - PMC - PubMed
    1. Yang JL, Yang YR, Chen SD. The potential of drug repurposing combined with reperfusion therapy in cerebral ischemic stroke: a supplementary strategy to endovascular thrombectomy. Life Sci. 2019; 236:116889. 10.1016/j.lfs.2019.116889 - DOI - PubMed
    1. Wang J, Zhang W, Lv C, Wang Y, Ma B, Zhang H, Fan Z, Li M, Li X. A novel biscoumarin compound ameliorates cerebral ischemia reperfusion-induced mitochondrial oxidative injury via Nrf2/Keap1/ARE signaling. Neuropharmacology. 2020; 167:107918. 10.1016/j.neuropharm.2019.107918 - DOI - PubMed
    1. Chen H, Guan B, Wang B, Pu H, Bai X, Chen X, Liu J, Li C, Qiu J, Yang D, Liu K, Wang Q, Qi S, Shen J. Glycyrrhizin prevents hemorrhagic transformation and improves neurological outcome in ischemic stroke with delayed thrombolysis through targeting peroxynitrite-mediated HMGB1 signaling. Transl Stroke Res. 2020; 11:967–82. 10.1007/s12975-019-00772-1 - DOI - PubMed
    1. Xue J, Yu Y, Zhang X, Zhang C, Zhao Y, Liu B, Zhang L, Wang L, Chen R, Gao X, Jiao P, Song G, Jiang XC, Qin S. Sphingomyelin synthase 2 inhibition ameliorates cerebral ischemic reperfusion injury through reducing the recruitment of toll-like receptor 4 to lipid rafts. J Am Heart Assoc. 2019; 8:e012885. 10.1161/JAHA.119.012885 - DOI - PMC - PubMed

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