[M2 macrophage-derived exosomal lncRNA NR_028113.1 promotes macrophage polarization possibly by activating the JAK2/STAT3 signaling pathway]

Abstract

Objective: To explore the effect of M2 macrophage-derived exosomal lncRNA NR_028113.1 on macrophage polarization and its possible mechanism.

Methods: Bone marrow-derived macrophages (BMDMs) from BALB/c mice were isolated and cultured in vitro. After IL-4 treatment to induce M2 macrophage polarization, exosomes (M2-exo) were extracted from the supernatant of M2 macrophages and identified. The expression of lncRNA in M2-exo was detected by qRT-PCR. BMDMs were co-cultured with M2-exo (100 μg/mL) or PBS for 48 h, and the changes in cellular expression levels of Arg1, YM-1, FIZZ1, iNOS and TNF-α were detected using qRT-PCR and Western blotting. The percentage of CD206⁺ cells was analyzed using flow cytometry, and the phosphorylation levels of JAK2/STAT3 proteins were detected using Western blotting. A lncRNA smart silencer was designed to specifically inhibit the expression of lncRNA NR_028113.1 in the M2 macrophages, from which exosomes were extracted and co-cultured with BMDMs for 48 h. The mRNA expression levels of Arg1, YM-1, FIZZ1, iNOS and TNF-α, CD206⁺ cell percentage and the phosphorylation levels of JAK2/STAT3 proteins were detected using qRT-PCR, flow cytometry and Western blotting.

Results: LncRNA NR_028113.1 was highly expressed in the exosomes of M2 macrophages (P < 0.05). Co-culture with M2-exo significantly increased mRNA expressions of M2 macrophage marker genes Arg1, YM-1 and FIZZ1 (P < 0.05), lowered the expressions of iNOS and TNF-α (P < 0.05), and increased CD206⁺ cell percentage and JAK2/STAT3 protein phosphorylation level in BMDMs (P < 0.05). After inhibiting the expression of lncRNA NR_028113.1 in M2 macrophages, the extracted M2-exo caused significant down-regulation of the mRNA expressions of Arg1, YM-1 and FIZZ1 and up-regulation of iNOS and TNF-α mRNA (P < 0.05), resulting also in signi-ficantly reduced CD206⁺ cell percentage and lowered phosphorylation levels of JAK2/STAT3 proteins in co-cultured BMDM (P < 0.05).

Conclusions: M2 macrophage-derived exosomal lncRNA NR_028113.1 can significantly promote M2 polarization of macrophages possibly by activating the JAK2/STAT3 signaling pathway.

Keywords: JAK2; STAT3; exosomes; long non-coding RNA; macrophage polarization.

图 1

体外极化M2巨噬细胞的鉴定 Identification of polarized M2 macrophages in vitro. A: Surface markers of M2-type macrophages observed by immunofluorescence microscope (Original magnification: ×200). B: Detection of Arg1, YM-1 and FIZZ1 mRNA expressions on M2 macrophages by RT-qPCR. ^*P < 0.05 vs control group.

图 2

M2-Exo外泌体的鉴定 Identification of M2 exosomes. A: Morphology of exosomes observed by transmission electron microscope (×30 000). B: Particle size distribution of M2 exosomes measured by nanoparticle tracking analysis. C: Western blotting of expressions of exosome marker proteins.

图 3

激光共聚焦显微镜下观察外泌体的摄取 Uptake of exosomes observed under laser confocal microscope (×400).

图 4

M2外泌体对巨噬细胞极化的影响 Effect of M2 exosomes on polarization of macrophages. A: Expression of lncRNA in M2 exosomes detected by RTqPCR. B: Detection of Arg1, YM-1, FIZZ1, iNOS, and TNF-α mRNA expressions in M2 macrophages by RT-qPCR. C: Detection of Arg1, YM-1, and FIZZ1 protein expression in M2 macrophages by Western blotting. D: Expression levels of CD206 detected by flow cytometry. ^*P < 0.05, ^**P < 0.01 vs control group.

图 5

M2外泌体lncRNANR_028113.1对巨噬细胞极化的影响 Effect of M2 exosome-derived lncRNA NR_028113.1 on polarization of macrophages. A: Detection of M2 macrophage Arg1, YM-1, FIZZ1, iNOS, and TNF-α mRNA expressions by RT-qPCR. B: Detection of M2 macrophage Arg1, YM-1, and FIZZ1 protein expressions by Western blotting. C: Expression levels of CD206 detected by flow cytometry. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs exo+NC group.

图 6

M2巨噬细胞外泌体lncRNANR_028113.1激活JAK2/STAT3信号通路 M2 macrophage exosomal lncRNA NR_0281131.1 activates JAK2/STAT3 signaling pathway. A: Effect of macrophage exosomes on JAK2 and STAT3 phosphorylation detected by Western blotting (^*P < 0.05 vs control group). B: Effect of inhibition of exosomal lncRNA NR_0281131.1 on phosphorylation of JAK2 and STAT3 detected by Western blotting (^*P < 0.05 vs exo+ NC group).