The miR-21-5p enriched in the apoptotic bodies of M2 macrophage-derived extracellular vesicles alleviates osteoarthritis by changing macrophage phenotype

Abstract

Macrophages (Mφs) play a crucial role in the pathological progression of osteoarthritis (OA) by regulating inflammation and tissue repair. Decreasing pro-inflammatory M1-Mφs and increasing anti-inflammatory M2-Mφs can alleviate OA-related inflammation and promote cartilage repair. Apoptosis is a natural process associated with tissue repair. A large number of apoptotic bodies (ABs), a type of extracellular vesicle, are produced during apoptosis, and this is associated with a reduction in inflammation. However, the functions of apoptotic bodies remain largely unknown. In this study, we investigated the role of M2-Mφs-derived apoptotic bodies (M2-ABs) in regulating the M1/M2 balance of macrophages in a mouse model of OA. Our data show that M2-ABs can be targeted for uptake by M1-Mφs, and this reprograms M1-to-M2 phenotypes within 24 h. The M2-ABs significantly ameliorated the severity of OA, alleviated the M1-mediated pro-inflammatory environment, and inhibited chondrocyte apoptosis in mice. RNA-seq revealed that M2-ABs were enriched with miR-21-5p, a microRNA that is negatively correlated with articular cartilage degeneration. Inhibiting the function of miR-21-5p in M1-Mφs significantly reduced M2-ABs-guided M1-to-M2 reprogramming following in vitro cell transfection. Together, these results suggest that M2-derived apoptotic bodies can prevent articular cartilage damage and improve gait abnormalities in OA mice by reversing the inflammatory response caused by M1 macrophages. The mechanism underlying these findings may be related to miR-21-5p-regulated inhibition of inflammatory factors. The application of M2-ABs may represent a novel cell therapy, and could provide a valuable strategy for the treatment of OA and/or chronic inflammation.

Keywords: Apoptotic body; Extracellular vesicles; Macrophage phenotype switch; MicroRNA-21; Osteoarthritis.

Figure 1

Establishment of M1 and M2 Mφs and characterization of Mφ-ABs. (A) Schematic illustration of apoptotic bodies-guided macrophage reprogramming. M2 Mφ-derived apoptotic bodies (M2-ABs) can trigger the switch of M1 to M2 Mφs transformation and alleviate the progression of osteoarthritis. (B) Schematic diagram of obtaining M1 and M2 Mφs. (C) Western blot analysis showed the expression of Mφ markers 24 h after polarization. (D) Representative SEM image of ABs. Scale bar, 500 nm. (E) Size distribution of ABs measured by DLS. (F) Western blot analysis specific protein markers of Mφs and Mφs-ABs. (G) Confocal images of M1 Mφs incubation with 10, 25, 50, and 100 μg/mL of DiD-labeled M2-ABs in 4 or 6 h, respectively (Red: DiD-labeled M2-ABs; Blue: cell nuclear). Scale bars, 50 μm. (H) Relative fluorescence intensity of DiD-labeled M2-ABs internalized in M1 Mφs.

Figure 2

M2-ABs guided reprogramming of M1 Mφs to M2 Mφs. (A) Immunostaining of iNOS and Arginase in M1 Mφs after 24 h, 48 h and 72 h incubation with 50 μg/mL of M2-ABs, respectively. (B) Western blot analysis of M1 Mφs treated with 50 μg/mL of M2-ABs over time. (C) Relative gray value of M1 and M2 Western blot markers in Mφs. (D) Heat map for expression analysis (using RT-qPCR) of M1 and M2 macrophages marker genes (n = 3). (E) FACS histogram showed reprogramming efficiency of M1 Mφs treated with 50 μg/mL of M2-ABs over time.

Figure 3

In vitro anti-inflammatory and chondrocyte protective effects of RM2 Mφs. (A) The concentrations of macrophage serum-free medium cytokines were determined using the Bio-Plex mouse cytokine 23-Plex panel. (B)a. Schematic diagram of RM2 co-culture with chondrocytes; b. EdU proliferation assays of chondrocytes exposed to classically activated M1-Mφs, alternatively activated M2-Mφs and RM2 for 24 h were performed; c. EdU positive fluorescence intensity analysis. ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05.

Figure 4

Micro-RNA sequencing (miRNA-seq) analysis and molecular mechanism of M2-ABs. (A) Top 50 known miRNAs that were detected in M2-ABs. (B) Co-localization of macrophages and miR-21a-5p. (C) Western blot analysis of the effect of miR-21a-5p inhibitors on M2-ABs guided M1-Mφs reprogramming to M2-Mφs. (D) Flow cytometry (FACS) histogram shows the effect of miR-21a-5p inhibitors on M2-ABs guided M1-Mφs reprogramming to M2-Mφs.

Figure 5

In vivo biodistribution of M2-ABs. (A) Real-time in vivo imaging of Cy7 NHS labeled M2-ABs. The mice were analyzed at the indicated times after knee injection of phosphate-buffered saline (PBS) and 50 μg/μl of M2-ABs. (B)Ex vivo imaging of major organs at day 3 after mice had been treated with M2-ABs. (C) Viscera distribution of fluorescent at day 3 after knee injection of Cy7-NHS labeled M2-ABs.

Figure 6

μCT evaluations of M2-ABs treated OA induced by ACLT. (A) Three-dimensional μCT images of frontal views of the knee joints at 5 weeks after sham operation or ACLT operation. (B) Sagittal views of medial compartment subchondral bone. (C) Quantitative analysis of BV, BV/TV, Tb.Sp, Tb.n and Tb.Th. ∗P < 0.05, ∗∗P < 0.01. Scale = 1 mm.

Figure 7

M2-ABs alleviated the disease progression of OA in vivo (n = 5 for each group). (A) Representative images of H&E staining and (B) Safranin O-fast green staining from each group. (C) Representative immunostaining images of M2 (Arginase, red) and (D) M1 (iNOS, green) Mφ in synovial tissues. (E) Depth of cartilage. (F) Total Mankin scores of articular cartilage. (G) Fluorescence intensity analysis of CD206 and (H) CD86 in synovial tissue. ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05.