Exosomes derived from microRNA-540-3p overexpressing mesenchymal stem cells promote immune tolerance via the CD74/nuclear factor-kappaB pathway in cardiac allograft

Figure 1 Overexpression of microRNA-540-3p in dendritic cells modulates surface markers and cytokine production. A: Ten immune-related microRNAs (miRNAs) in indoleamine 2,3-dioxygenase-overexpressing bone marrow mesenchymal stem cell exosomes compared to those in bone marrow mesenchymal stem cell exosomes; B: Quantification of immune-related miRNA expression by quantitative real-time polymerase chain reaction; C: Expression of miR-540-3p in dendritic cells (DCs) at 48, 72, and 96 hours after transfection; D-G: Flow cytometry analysis of the positive rate of cell surface markers in DCs (major histocompatibility complex II, CD80, CD86, CD274) and DCs after co-culture with T cells for 72 hours; H-K: Enzyme-linked immunosorbent assay-based quantitative analysis of cytokine production (interleukin-1β, interferon-γ, interleukin-10, transforming growth factor β1) in DCs and DCs after co-culture with T cells for 72 hours. ^aP < 0.05, ^dP < 0.0001. BMSC: Bone marrow mesenchymal stem cell; NC: Negative control; IDO: Indoleamine 2,3-dioxygenase; DC: Dendritic cell; LPS: Lipopolysaccharide; IL: Interleukin; TGF: Transforming growth factor; IFN: Interferon.

Figure 2 Overexpression of microRNA-540-3p in T cells modulates cell subtypes and cytokine production. A: Expression of microRNA-540-3p in T cells at 48, 72, and 96 hours post-plasmid transfection; B-D: Flow cytometry analysis of the positive rate of T cells (CD4⁺ T, CD8⁺ T, and T regulatory cell) and T cells after co-culture with dendritic cells for 72 hours; E-H: Enzyme-linked immunosorbent assay-based quantitative analysis of cytokine production (interleukin-1β, interferon-γ, interleukin-10, and transforming growth factor β1) in T cells and T cells after co-culture with dendritic cells for 72 hours. ^aP < 0.05, ^dP < 0.0001. NC: Negative control; LPS: Lipopolysaccharide; IL: Interleukin; TGF: Transforming growth factor; IFN: Interferon.

Figure 3 Production and characterization of bone marrow mesenchymal stem cell^{miR-540-3p-mimic} exosomes. A: Transmission electron microscopy image of isolated exosomes; B: Nanosight analysis for exosome size and concentration; C: Expression of microRNA-540-3p (miR-540-3p) in bone marrow mesenchymal stem cell-derived exosomes at 48, 72, and 96 hours after plasmid transfection; D: Expression of miR-540-3p in dendritic cells; E: Expression of miR-540-3p in T cells. ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. BMSC: Bone marrow mesenchymal stem cell; IDO: Indoleamine 2,3-dioxygenase; DC: Dendritic cell; LPS: Lipopolysaccharide; exo: Exosomes.

Figure 4 Bone marrow mesenchymal stem cell^{miR-540-3p-mimic} exosomes reduces immune resistance in dendritic cells. A-D: Flow cytometry analysis of the positive rate of cell surface markers in dendritic cells (major histocompatibility complex II, CD80, CD86, and CD274) for 72 hours; E-H: Enzyme-linked immunosorbent assay-based quantitative analysis of cytokine production (interleukin-1β, interferon-γ, interleukin-10, transforming growth factor β1) in dendritic cells. ^aP < 0.05, ^dP < 0.0001. MHC: Major histocompatibility complex; BMSC: Bone marrow mesenchymal stem cell; IDO: Indoleamine 2,3-dioxygenase; DC: Dendritic cell; LPS: Lipopolysaccharide; exo: Exosomes; IL: Interleukin; TGF: Transforming growth factor; IFN: Interferon.

Figure 5 Bone marrow mesenchymal stem cell^{miR-540-3p-mimic} exosomes reduces immune resistance in T cells. A-C: Flow cytometry analysis of the positive rate of CD4⁺ T cells, CD8⁺ T cells, and regulatory T cells for 72 hours; D-G: Enzyme-linked immunosorbent assay-based quantitative analysis of cytokine production (interleukin-1β, interferon-γ, interleukin-10, and transforming growth factor β1) in T cells. ^dP < 0.0001. BMSC: Bone marrow mesenchymal stem cell; LPS: Lipopolysaccharide; exo: Exosomes; IL: Interleukin; TGF: Transforming growth factor; IFN: Interferon.

Figure 6 Effect of bone marrow mesenchymal stem cell^{miR-540-3p-mimic} exosomes on dendritic cells after co-culturing with T cells. A-D: Flow cytometry analysis of the positive rate of cell surface markers (major histocompatibility complex II, CD80, CD86, and CD274) in dendritic cells cultured with T cells for 72 hours; E-G: Flow cytometry analysis of the positive rate of T cells (CD4⁺ T, CD8⁺ T, and T regulatory cells) and T cells after co-culture with dendritic cells; H-K: Enzyme-linked immunosorbent assay-based quantitative analysis of cytokine production (interleukin-1β, interferon-γ, interleukin-10, and transforming growth factor β1) in dendritic cell-T cells co-cultures. ^aP < 0.05, ^dP < 0.0001. MHC: Major histocompatibility complex; BMSC: Bone marrow mesenchymal stem cell; IDO: Indoleamine 2,3-dioxygenase; DC: Dendritic cell; LPS: Lipopolysaccharide; exo: Exosomes; IL: Interleukin; TGF: Transforming growth factor; IFN: Interferon; Treg: T regulatory cell.

Figure 7 CD74 is a target of microRNA-540-3p and regulates P65 expression. A: Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses of microRNA-540-3p (miR-540-3p) targets; B: A dual-luciferase assay was used to detect the binding of miR-540-3p to CD74; C: Co-IP was used to detect the binding of CD74 and nuclear factor-kappaB (NF-κB) in dendritic cells (DCs); D: Co-IP was used to detect the binding of CD74 and NF-κB in T cells; E-G: Western blot analysis of the expression of CD74 and NF-κB in DCs; H-J: Western blot analysis of the expression of CD74 and NF-κB in T cells; K-M: Western blot analysis of CD74 and NF-κB expression in DCs after co-culture with T cells; N-P: Western blot analysis of the expression of CD74 and NF-κB in T cells after co-culture with DCs; Q-S: Western blot analysis of the expression of CD74 and NF-κB in DCs after plasmid transfection; T-V: Western blot analysis of CD74 and NF-κB expression in T cells after plasmid transfection. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. Mut: Mutant; BMSC: Bone marrow mesenchymal stem cell; IDO: Indoleamine 2,3-dioxygenase; DC: Dendritic cell; LPS: Lipopolysaccharide; exo: Exosomes; NF-κB: Nuclear factor-kappaB.

Figure 8 Exosome-derived miR-540-3p alleviates immune resistance after heterotopic heart transplantation. A: Histopathological observation of the heart after heterotopic heart transplantation using hematoxylin and eosin staining; B: Detection and calculation of the difference in ejection fraction before and after heterotopic heart transplantation; C: Detection and calculation of differences in the percentage of fractional shortening before and after heterotopic heart transplantation; D-G: Enzyme-linked immunosorbent assay-based quantitative analysis of cytokine production (interleukin-1β, interferon-γ, interleukin-10, transforming growth factor β1) in serum; H: Expression of microRNA-540-3p in extracted dendritic cells (DCs); I: Expression of microRNA-540-3p in extracted T cells; J-M: Flow cytometric analysis of the positive rate of cell surface markers in DCs (major histocompatibility complex II, CD80, CD86, and CD274); N-P: Flow cytometric analysis of the positive rate of T cells (CD4⁺ T, CD8⁺ T, and T regulatory cells); Q-S: Western blot analysis of the expression of CD74 and nuclear factor-kappaB (NF-κB) in extracted DCs; T-V: Western blot analysis of CD74 and NF-κB expression in the extracted T cells; W: Co-IP was used to detect the binding of CD74 and NF-κB in extracted DCs; X: Co-IP was used to detect the binding of CD74 and NF-κB in extracted T cells. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. BMSC: Bone marrow mesenchymal stem cell; IDO: Indoleamine 2,3-dioxygenase; DC: Dendritic cell; LPS: Lipopolysaccharide; NF-κB: Nuclear factor-kappaB; MHC: Major histocompatibility complex; IL: Interleukin; TGF: Transforming growth factor; IFN: Interferon.

Figure 9 Exosomes derived from microRNA-540-3p overexpressing bone marrow mesenchymal stem cells promote immune tolerance to cardiac allograft via the CD74/nuclear factor-kappaB pathway. BMSC: Bone marrow mesenchymal stem cell; IDO: Indoleamine 2,3-dioxygenase; IL: Interleukin; TGF: Transforming growth factor; IFN: Interferon; NF-κB: Nuclear factor-kappaB.