Exosomes derived from 3D-cultured MSCs improve therapeutic effects in periodontitis and experimental colitis and restore the Th17 cell/Treg balance in inflamed periodontium

Abstract

Although mesenchymal stem cell-derived exosomes (MSC-exos) have been shown to have therapeutic effects in experimental periodontitis, their drawbacks, such as low yield and limited efficacy, have hampered their clinical application. These drawbacks can be largely reduced by replacing the traditional 2D culture system with a 3D system. However, the potential function of MSC-exos produced by 3D culture (3D-exos) in periodontitis remains elusive. This study showed that compared with MSC-exos generated via 2D culture (2D-exos), 3D-exos showed enhanced anti-inflammatory effects in a ligature-induced model of periodontitis by restoring the reactive T helper 17 (Th17) cell/Treg balance in inflamed periodontal tissues. Mechanistically, 3D-exos exhibited greater enrichment of miR-1246, which can suppress the expression of Nfat5, a key factor that mediates Th17 cell polarization in a sequence-dependent manner. Furthermore, we found that recovery of the Th17 cell/Treg balance in the inflamed periodontium by the local injection of 3D-exos attenuated experimental colitis. Our study not only showed that by restoring the Th17 cell/Treg balance through the miR-1246/Nfat5 axis, the 3D culture system improved the function of MSC-exos in the treatment of periodontitis, but also it provided a basis for treating inflammatory bowel disease (IBD) by restoring immune responses in the inflamed periodontium.

Conflict of interest statement

The authors declare no competing interests.

© 2021. The Author(s).

Figures

Fig. 1

Characterization of 2D-exos and 3D-exos. a Brightfield images of 3D-cultured or 2D-cultured DPSCs. Scale bar = 100 μm. b TEM images showing the morphology of 2D-exos and 3D-exos. Scale bar = 100 nm. c Flow cytometric analysis showing the expression of CD63 and CD9 in 2D-exos and 3D-exos. d Representative western blot image showing the expression of exosome markers (CD9, CD63, and TSG101) and a cytosolic marker (GM130) in 2D-exos and 3D-exos. e Particle sizes and numbers of 2D-exos and 3D-exos were analyzed by using NTA. f BCA assays determined the yields of 2D-exos and 3D-exos

Fig. 2

3D-exos exerted enhanced effects in ameliorating periodontitis and commensal pathobiont-driven colitis. a A flow diagram showing the time of ligature-induced periodontitis and DSS-induced colitis induction, periodontal injection and specimen collection. b 3D reconstructions of maxillae of the PBS-, 2D-exo- and 3D-exo-treated groups (n = 6 per group) were generated by micro-CT. The vertical line extends from the CEJ to the ABC. The CEJ-ABC distance was measured at six predetermined sites on both the buccal and palatal sides. Scale bar = 500 μm. c Statistical analysis of the CEJ-ABC distance in each group (n = 6) as determined by micro-CT. The error bars represent the SEM. *P < 0.05. d, e Analysis of body weights and disease activity index (DAI) in each group (n = 6). f Representative colon pictures of the mice in each group. g Statistical analysis of the colon length in each group (n = 6 per group). The error bars represent the SEM. *P < 0.05. h Histopathological changes in the colon were analyzed by haematoxylin and eosin (HE) staining. Scale bar = 200 μm. i Statistical analysis of histological scores in each group (n = 6). j RT-qPCR analysis of the expression levels of the TNF-α, IL-1β and IL-6 genes in the colon in each group (n = 6). The error bars represent the SEM. *P < 0.05

Fig. 3

3D-exos exerted an enhanced anti-inflammatory effect in DSS-P mice. a Heatmap of DEGs in the gingiva of the 2D-exo- and 3D-exo-treated groups vs. the gingiva of the PBS-treated group (n = 3 per group). b Volcano plots show DEGs between the PBS- and 3D-exo-treated groups. c Gene Ontology (GO) functional analysis of the top 200 downregulated genes in the gingiva of the 3D-exo-treated group compared with the PBS-treated group. d Volcano plots show DEGs between the 2D-exo- and 3D-exo-treated groups. e GO functional analysis of the top 200 downregulated genes in the gingiva of the 3D-exo-treated group compared with the 2D-exo-treated group

Fig. 4

3D-exos showed an enhanced ability to reduce the ratio of Th17 cells to Treg cells in both the inflamed gingiva and the colon. a A flow diagram showing the time of ligature-induced periodontitis and DSS-induced colitis induction, periodontal injection, and specimen collection. b–e Flow cytometric profiles of Foxp3 and IL-17 expression in CD4+ cells in the inflamed gingiva of the PBS-, 2D-exo- and 3D-exo-treated groups. Numerical values denote the mean percentage of CD4+ cells expressing Foxp3 and IL-17 in each group (n = 6). The error bars represent the SEM. *P < 0.05. f–i Flow cytometric profiles of Foxp3 and IL-17 expression in CD4+ cells in the inflamed colon of the PBS-, 2D-exo- and 3D-exo-treated groups. Numerical values denote the mean percentage of CD4+ cells expressing Foxp3 and IL-17 in each group (n = 6). The error bars represent the SEM. *P < 0.05

Fig. 5

3D-exos showed an enhanced ability to inhibit Nfat5 in T cells by miR-1246. a Heatmap of the relative proportion of miRNAs in the total miRNA reads of the 3D-exo group vs. the 2D-exo group (n = 3 per group). b–d CD4+ naive T cells were stimulated with the indicated cytokines to induce Treg cells and Th17 cells and then treated with PBS, NCI-3D-exos or miR1246I-3D-exos in vitro. Flow cytometric profiles of Foxp3 and IL-17 expression in the above CD4+ T cells. Numerical values denote the mean percentage of CD4+ cells expressing Foxp3 and IL-17 in each group (n = 6). The error bars represent the SEM. *P < 0.05. e RT-qPCR analysis of the expression of the Treg-associated gene Foxp3 and the Th17 cell-associated gene ROR-γt in CD4+ T cells of the PBS-, NCI-3D-exo- and miR-1246I-exo-treated groups. f Binding sites in the Nfat5 3′-UTR targeted by miR-1246. g The wild-type binding site sequence of Nfat5 was cotransfected with miR-1246, which led to decreased luciferase activity, verifying their targeted relationship. h RT-qPCR analysis of the mRNA expression of Nfat5 in CD4+ T cells after transfection of the miR-1246 mimic and miR-1246 inhibitor. i Western blot analysis of the expression of Nfat5 in CD4+ T cells after transfection with the miR-1246 mimic and miR-1246 inhibitor

Fig. 6

mir-1246 antagomir reversed the effects of 3D-exos in ameliorating periodontitis and commensal pathobiont-driven colitis. a A flow diagram showing the time of animal model induction, periodontal injection, and specimen collection. b 3D reconstructions of maxillae from the PBS-, NCI-3D-exo-, and miR1246I-3D-exo-treated groups (n = 6 per group) were generated by micro-CT. The CEJ-ABC distance was measured. Scale bar = 500 μm. c Statistical analysis of the CEJ-ABC distance in each group (n = 6 per group) as determined by micro-CT. The error bars represent the SEM. *P < 0.05. d, e Statistical analysis of body weights and DAI scores of the mice in each group (n = 6). f Representative images of colons from experimental mice in each group. g Statistical analysis of the colon length in each group (n = 6). The error bars represent the SEM. *P < 0.05. h Histopathological changes in colon tissues were analyzed by haematoxylin and eosin (HE) staining. Scale bar = 200 μm. i Statistical analysis of the histological score in each group (n = 6). j RT-qPCR was used to analyze expression levels of the TNF-α, IL-1β, and IL-6 genes in each group (n = 6). The error bars represent the SEM. *P < 0.05

Fig. 7

mir-1246 antagomir reversed the effects of 3D-exos, reducing the ratio of Th17 cells to Treg cells in both the inflamed gingiva and colon. a–d Flow cytometric profiles of Foxp3 and IL-17 expression in CD4+ cells in the inflamed gingiva of PBS-, 3D-exo– and 3D-exo+ antagomir 1246-treated groups. Numerical values denote the mean percentage of CD4+ cells expressing Foxp3 and IL-17 in each group (n = 6). The error bars represent the SEM. *P < 0.05. e–h Flow cytometric profiles of Foxp3 and IL-17 expression in CD4+ cells in the inflamed colon of the PBS-, NCI-3D-exo-, and miR1246I-3D-exo-treated groups. Numerical values denote the mean percentage of CD4+ cells expressing Foxp3 and IL-17 in each group (n = 6). The error bars represent the SEM. *P < 0.05