Int J Stem Cells.  2021 Aug;14(3):331-340. 10.15283/ijsc20156.

Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells Regulate Macrophage Polarization to Attenuate Systemic Lupus Erythematosus-Associated Diffuse Alveolar Hemorrhage in Mice

• Chen X
• Wei Q
• Sun H
• Zhang X
• Yang C
• Tao Y
• Nong G

• KMID: 2519375
• DOI: http://doi.org/10.15283/ijsc20156

Abstract

Background and Objectives
To investigate the effect and the underlying mechanism of exosomes secreted by human umbilical cord mesenchymal stem cells (hUCMSCs) on diffuse alveolar hemorrhage (DAH) in murine lupus.
Methods and Results
Exosomes were extracted from cultured hUCMSCs by ultracentrifugation. The expressions of exosome markers (Alix, CD63 and TSG101) were measured for identification of hUCMSC-derived exosomes (hUCMSC-exosomes). The alveolar hemorrhage of DAH mice was revealed by H&E staining. The primary alveolar macrophages were isolated from bronchoalveolar lavage fluid (BALF) of DAH mice. The expressions of M1 macrophage markers (iNOS, IL-6, TNF-α and IL-1β ) and M2 macrophage markers (Arg1, IL-10, TGF-β and chi3l3) were detected. Flow cytometry measured the ratio of M1/M2 macrophages. ELISA measured the secretion of pro-inflammatory cytokines (IL-6 and TNF-α) and anti-inflammatory cytokines (IL-10 and TGF-β ). DAH mice had hemorrhage and small-vessel vasculitis in the lung, with neutrophil and monocyte infiltration observed around the capillary and small artery. Furthermore, increases of IL-6 and TNF-α, and decreases of IL-10 and TGF-β were detected in the BALF of DAH mice. M1 makers were overexpressed in alveolar macrophages of DAH mice while M2 makers were lowly expressed. DAH mice had a higher proportion of M1 macrophages than M2 macrophages. After hUCMSC-exosome or methylprednisolone treatment in DAH mice, the alveolar injuries and inflammatory responses were attenuated, and the proportion of M2 macrophages was increased.
Conclusions
hUCMSC-exosomes attenuate DAH-induced inflammatory responses and alveolar hemorrhage by regulating macrophage polarization.

Keyword

Human umbilical cord mesenchymal stem cells; Exosome; Systemic lupus erythematosus; Diffuse alveolar hemorrhage; M1 macrophage; M2 macrophage

Figure

• 

  Fig. 1 Identification of hUCMSC-exosomes. (A) The ultrastructure of exosomes was observed under a transmission electron microscope; (B) expressions of exosome markers were tested by Western blotting; (C) exosome size was measured by the nanoparticle tracking analyzer Zetaview. hUCMSC, human umbilical cord mesenchymal stem cell.

• 

  Fig. 2 Establishment of DAH models on mice. (A) H&E staining detected the pathological conditions of mouse lung; (B∼E) ELISA measured the expressions of IL-6, TNF-α, IL-10 and TGF-β in mouse BALF. Each group had ten mice. **p< 0.01 compared to the control group; DAH, diffuse alveolar hemorrhage; BALF, bronchoalveolar lavage fluid; ELISA, enzyme-linked immunosorbent assay.

• 

  Fig. 3 hUCMSC-exosomes alleviate pathological symptoms of DAH mice. After hUCMSC-exosome or methylprednisolone treatment, (A) H&E staining assessed the morphology of mouse lung; (B∼E) ELISA measured the expressions of IL-6, TNF-α, IL-10 and TGF-β in mouse BALF. Each group had ten mice. **p<0.01 compared to the control group; hUCMSC, human umbilical cord mesenchymal stem cell; DAH, diffuse alveolar hemorrhage; BALF, bronchoalveolar lavage fluid; ELISA, enzyme-linked immunosorbent assay.

• 

  Fig. 4 hUCMSC-exosomes facilitate the transformation of macrophages from M1 into M2 phenotype. (A∼H) The expressions of iNOS, IL-6, TNF-α, IL-1β, Arg1, IL-10, TGF-β and chi3l3 in mouse macrophages were tested by qRT-PCR; (I∼K) the proportions of M1 and M2 macrophages were measured by flow cytometry; (L) phagocytosis was assessed by flow cytometry. n=10; *p<0.05, **p<0.01 compared to the control group; #p<0.05, ##p<0.01 compared to the DAH group; hUCMSC, human umbilical cord mesenchymal stem cell; DAH, diffuse alveolar hemorrhage.

Reference

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