Human dental follicle stem cell-derived exosomes reduce root resorption by inhibiting periodontal ligament cell pyroptosis

Abstract

Background: To explore the therapeutic effects and mechanisms of the exosomes derived from dental follicle stem cells (DFSC-Exos) in reducing osteoclastogenesis and root resorption (RR) by inhibiting periodontal ligament cell (PDLC) pyroptosis.

Methods: DFSC-Exos, with force stimulation (Force-Exos) or without (Ctrl-Exos), were co-cultured with human PDLCs in vitro and injected into the periodontal ligament (PDL) of rats following the establishment of RR models in vivo. Subsequently, resorption volume, PDLC pyroptotic ratio, and NLRP3-mediated pyroptosis pathway activation were performed to investigate the therapeutic effects of DFSC-Exos on PDLC pyroptosis during RR. Furthermore, the number of M1/M2 macrophages, osteoclast formation, and transwell polarization elucidated the role of Force-Exo treatment in macrophage polarization and osteoclastogenesis by inhibiting pyroptosis. Exosomal miRNA sequencing and bioinformatic analysis were used to identify differentially abundant exosome-derived miRNAs, as well as the dominant biological processes and pathways modulated by miRNA. The administration of miRNA inhibitors further verified the regulation of exosomal miRNA on RR via modulating pyroptosis. Moreover, the potential mechanisms involving candidate miRNAs and relevant pathways were explored.

Results: Exosomes released by force-stimulated DFSCs (Force-Exos) inhibited NOD-like receptor 3 (NLRP3)-mediated PDLC pyroptosis, which impacted M1 macrophage activation and osteoclast formation. Based on exosomal miRNA sequencing, miR-140-3p in Force-Exos were transferred to PDLCs, and the administration of miR-140-3p inhibitors significantly reversed the reduction in PDLC pyroptosis, M1 macrophage polarization, osteoclast number, and resorption volume caused by Force-Exos. More importantly, mechanistic studies demonstrated that miR-140-3p mediated the function of Force-Exos by targeting DNA methyltransferase 1 (DNMT1) to alter the DNA methylation of suppressor of cytokine signaling (SOCS1) and the downstream nuclear factor κB (NF-κB) signaling pathway in PDLCs. Blocking the DNMT1/SOCS1/NFκB axis with DFSC-derived exosomal miR-140-3p downregulated NLRP3-mediated PDLC pyroptosis to impact M1 polarization and osteoclast formation, thereby alleviating RR.

Conclusion: DFSC-Exos downregulated NLRP3-mediated PDLC pyroptosis via miR-140-3p to block DNMT1/SOCS1/NFκB axis, which impacted M1 polarization and osteoclast formation, thereby alleviating RR.

Keywords: Exosome; Macrophage activation; Osteoclastogenesis; Periodontal ligament; Pyroptosis; Root resorption.

Fig. 1

Force-Exos alleviate osteoclast-initiated RR by inhibiting PDLC pyroptosis and M1 polarization. (A) Diagram of the model used for RR and the schedule of exosome administration. Force-Exos and Ctrl-Exos were injected into the PDL of rats induced RR every other day. (B) Micro-CT demonstrates the resorption of the mesial root of the maxillary first molars. After injecting exosomes, the RR volume was significantly decreased and the Force-Exos enhanced the effect. Scale bar: 1 mm. n = 3. (C, D) TRAP staining reveals that the osteoclasts (black arrow, multinucleated TRAP⁺ cells) are predominantly distributed along the mesial compressive side. The number of osteoclasts exhibited a significant difference between Ctrl + PBS group and Force + PBS group. However, the increased osteoclast number was suppressed with the exosome treatment, especially Force-Exos. Scale bar: 50 μm. n = 6. (E) TUNEL staining indicates apoptosis or pyroptosis (green) of PDLCs in the root tip and mesial sides. The dashed line delineates the boundary of the PDL. The count of TUNEL-positive PDLCs was markedly downregulated following the exosomes injection and Force-Exos promoted the effect. Scale bar: 200 μm. n = 5. (F, G) Immunofluorescence staining of TUNEL/NLRP3 (TUNEL-green and NLRP3-red) (F) and TUNEL/caspase-1 (TUNEL-green and Caspase1-red) (G) reveals PDLC pyroptosis. The dashed line delineates the boundary of the PDL. Force-Exos significantly decreased PDLC pyroptosis compared with PBS and Ctrl-Exos. Scale bar: 50 μm. n = 5. (H) Immunohistochemical analysis shows the presence of IL-1β expression on the compression side of the mesial roots. The dashed line delineates the boundary of the PDL. Mechanical force increased the expression of IL-1β, which was substantially attenuated after exosomes were injected and Force-Exos showed a more significant inhibition than Ctrl-Exo. Scale bar: 50 μm. n = 6. (I) Immunohistochemical analysis reveals the expression of IL-18 on the compression side of the mesial roots. The dashed line delineates the boundary of the PDL. Force + Force-Exo group suppressed the expression of IL-18 compared with Force + PBS group and Force + Ctrl-Exo group. Scale bar: 50 μm. n = 6. (J, K) Immunofluorescence staining of CD68/iNOS (CD68-green and iNOS-red) (J) and CD68/CD163 (CD68-green and CD163-red) (K) demonstrates the co-localization of M1 and M2 macrophages. The dashed line delineates the boundary of the PDL. Treatment with exosomes resulted in a reduced number of M1 macrophages and an increased number of M2 macrophages and the effect was promoted by Force-Exos. Scale bar: 50 μm. (L) The quantities of M1 and M2 macrophages were subjected to statistical analysis, as well as the M1/M2 ratio. Force-Exos mitigated the M1/M2 ratio significantly in comparison to other groups. n = 5. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

Force-Exos block NLRP3-mediated PDLC pyroptosis to reduce M1 polarization and osteoclast formation. (A) Scheme of co-culture system. Exosomes were isolated from DFSCs with or without mechanical stimulation and co-cultured with PDLCs. (B) Intracellular localization of exosomes in PDLCs. Exosomes were labeled with PKH26 (red). PDLC cytoskeleton was stained with Phalloidin (green) and nuclei were counterstained with DAPI (blue). Subsequent to co-culturing exosomes with PDLCs, the fluorescence assays indicated the endocytosis of PDLCs on exosomes. Scale bar: 50 μm. (C) Hoechst 33,342 and propidium iodide (PI) fluorescence staining revealed that force upregulated the number of PI-positive cells (pyroptotic cells), which was downregulated by exosomes, especially Force-Exos. Scale bar: 50 μm. n = 5. (D) Flow cytometry showed the number of Annexin V⁺PI⁺ cells (pyroptotic cells) apparently decreased in Force + Force-Exo group, compared with Force + PBS group and Force + Ctrl-Exo group. Scale bar: 50 μm. n = 5. (E) Enzyme-linked immunosorbent assay showed IL-1β levels increased under mechanical stimulation, which was significantly suppressed after the exosome treatment and Force-Exos promoted the effect. n = 4. (F) The Western blot analysis indicated exosomes reduced the protein expression levels of NLRP3, pro-caspase-1, caspase-1, GSDMD, GSDMD-N, pro-IL-1β, IL-1β and IL-18. Force-Exos significantly enhanced the effect. Full‑length blots are presented in Fig. S6: Fig. 2F. (G) Ultrastructural comparison by transmission electron microscopy among Force + PBS and Force + Force-Exo groups. In contrast to the disappeared chromatin and non-viable mitochondria in Force + PBS group (red arrow), cell membrane in Force + Force-Exo group was relatively intact and chromatin condensed (blue arrow). Scale bar: 2 μm. (H) The qRT-PCR demonstrated exosomes significantly decreased the mRNA level of M1 markers including iNOS, TNF-α, IL-1β, whereas the mRNA level of M2 markers including CD163, Arg-1 and IL-4R was increased. Moreover, Force-Exos pronounced the impact, which was consistent with the efficacy of MCC950. n = 5. (I) Flow cytometry showed that the ratio of CD11b⁺ CD86⁺ cells (M1 macrophages) to CD11b⁺ CD206⁺ cells (M2 macrophages) was reduced in the Force + Force-Exo group and Force + MCC950 group, compared with other groups. n = 5. (J) The Western blot analysis revealed that the utilization of Force-Exos markedly diminished the protein expression levels of osteoclast markers (NFAT2, MMP9, CTSK), analogous to the efficacy of MCC950. Full‑length blots are presented in Fig. S6: Fig. 2J. (K) TRAP staining revealed the number of TRAP-positive osteoclasts decreased after treatment with exosomes and MCC950, and Force-Exos promoted the reduction. Scale bar: 200 μm. n = 5. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Force-Exos transfer miR-140-3p to inhibit NLRP3-mediated PDLC pyroptosis. (A) Scheme of exosome extraction and miRNA analysis. (B) Heat map of exosomal miRNA-seq (n = 3). The fluorescence intensity of 25 differently expressed miRNAs (≥ twofold) was represented from high (red) to low (blue). (C) KEGG (Kyoto Encyclopedia of Genes and Genomes) analyzed the 20 most enriched signaling transduction pathways associated with differentially expressed miRNAs, in which NF-κB signaling pathway was observed. (D) Expression of miR-140-3p in DFSCs with or without mechanical stimulation (left), in Ctrl-Exos or Force-Exos derived from DFSC (middle) and in PDLCs induced by PBS/Ctrl-Exos/Force-Exos (right). n = 5. (E) Hoechst 33,342 and PI fluorescence staining showed that the inhibition of miR-140-3p significantly increased PI-positive cells compared with Force + Force-Exo + NC group. Scale bar:50 μm. n = 5. (F) Flow cytometry revealed the PDLC pyroptotic ratio was promoted after miR-140-3p inhibition in Force-Exos, whereas Force-Exos without miR-140-3p inhibitors exhibited an inhibitory effect on PDLC pyroptosis. n = 4. (G) Western blot results indicated that Force-Exos with miR-140-3p inhibition upregulated the protein levels of NF-κB, NLRP3, pro-caspase-1, caspase-1, GSDMD, GSDMD-N, pro-IL-1β, IL-1β and IL-18 in PDLCs, antithetical to the effects of Force-Exos. Full‑length blots are presented in Fig. S6: Fig. 3G. (H) Analysis of cell supernatants for IL-1β levels by ELISA showed IL-1β secretion was upregulated after the inhibition of miR-140-3p in Force-Exos, compared with Force + Force-Exo + NC group. n = 4. (I) The qRT-PCR indicated that Force-Exos with miR-140-3p inhibitors upregulated the mRNA expression of M1 macrophage and downregulated the mRNA expression of M2 macrophage compared with Force-Exos. n = 3. (J) Flow cytometry presented the ratio of M1/M2 macrophages decreased after treatment with Force-Exos, which was upregulated after miR-140-3p inhibition in Force-Exos. (K) Western blot results showed that miR-140-3p inhibitors reversed the inhibitory effect of Force-Exos on the protein level of NFAT2, MMP9 and CTSK (osteoclast markers). Full‑length blots are presented in Fig. S6: Fig. 3K. (L) TRAP staining showed the number of TRAP-positive osteoclasts was significantly increased by Force-Exos with miR-140-3p inhibitors compared with Force + Force-Exo + NC group. Scale bar: 100 μm. n = 5. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

Exosomal miR-140-3p reduces osteoclastogenesis by inhibiting PDLC pyroptosis and reversing M1/M2 ratio. (A) The schedule of administration for miR-140-3p inhibitors and exosomes. (B) Micro-CT demonstrates Force-Exos with miR-140-3p inhibitors significantly increased the RR volume, which was reduced by Force-Exos. Scale bar: 1 mm. n = 4. (C, D) TRAP staining of osteoclasts reveals the. Compared with Force + Force-Exo + NC group, TRAP⁺ osteoclasts were increased by Force-Exos with miR-140-3p inhibitors. Scale bar: 50 μm. n = 6. (E) TUNEL staining indicates apoptosis or pyroptosis (green) of PDLCs in the root tip and mesial sides. The dashed line delineates the boundary of the PDL. The count of TUNEL-positive PDLCs was upregulated following the inhibition of miR-140-3p, while Force-Exos without miR-140-3p inhibitors downregulated apoptosis or pyroptosis of PDLCs. Scale bar: 200 μm. n = 5. (F, G) Immunofluorescence staining for TUNEL/NLRP3 (F) and TUNEL/caspase-1 (G) co-localization shows the PDLC pyroptosis. The dashed line delineates the boundary of the PDL. Force-Exos with miR-140-3p inhibition significantly increased the PDLC pyroptosis compared with Force + Force-Exo + NC group. Scale bar: 50 μm. n = 5. (H, I) The level of IL-1β (H) and IL-18 (I) on the compression side of the mesial roots was increased in Force + Force-Exos + anti-miR-140-3p group, which was decreased in Force + Force-Exo + NC group. Scale bar: 50 μm. n = 6. (J, K) Immunofluorescence staining of CD68/iNOS (CD68-green and iNOS-red) (J) and CD68/CD163 (CD68-green and CD163-red) (K) demonstrates the co-localization of M1 and M2 macrophages. The dashed line delineates the boundary of the PDL. The inhibition of miR-140-3p resulted in more M1 macrophages and fewer M2 macrophages and the effect was reversed by Force-Exos. Scale bar: 50 μm. (L) The quantities of M1 and M2 macrophages were subjected to statistical analysis, as well as the M1/M2 ratio. Force-Exos with miR-140-3p inhibition upregulated the M1/M2 ratio significantly in comparison to Force + Force-Exo + NC group. n = 5. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Exosomal miR-140-3p inhibits PDLC pyroptosis by regulating DNA methylation via DNMT1/SOCS1/NFκB axis. (A) The qRT-PCR showed that mRNA level of SOCS1 in rat PDL was significantly suppressed under mechanical stimulation. n = 6. (B) Western blot results revealed that force downregulated the protein level of SOCS1 and upregulated the expression of NFκB and pNFκB. Moreover, SOCS1 siRNA increased NFκB level, suggesting that SOCS1 was a negative regulator of NF-κB signaling pathway. Full‑length blots are presented in Fig. S7: Fig. 5B. (C) MSP shows the methylation level of SOCS1 promoter in PDLCs. Force upregulated the DNA methylation level of SOCS1. Full-length gels are presented in Fig. S7: Fig. 5C. (D) The qRT-PCR showed that Decitabine (inhibitor of DNA methyltransferase) and DNMT1 siRNA significantly increased mRNA level of SOCS1 in mechanically stimulated PDLCs. n = 6. (E) Western blot analysis indicated that decitabine downregulated DMNT1, NFκB and pNFκB expression, but upregulated the protein level of SOCS1. Full‑length blots are presented in Fig. S7: Fig. 5E. (F) MSP results showed that DNA methylation of SOCS1 was suppressed in Force + Decitabine group compared with Force + PBS group. Full-length gels are presented in Fig. S7: Fig. 5F. (G) Western blot results revealed that si-DNMT1 increased SOCS1 expression but decreased DMNT1, NFκB and pNFκB levels. Full‑length blots are presented in Fig. S7: Fig. 5G. (H) MSP showed that si-DNMT1 administration decreased the DNA methylation level of SOCS1. Full-length gels are presented in Fig. S7: Fig. 5H. (I) Sequence alignment between miR-140-3p and its putative binding sites (in red letters) in the DNMT1. Mutation of the miR-140-3p target sites (in blue letters) was also shown. Luciferase reporter assay for the relative luciferase activities of WT and Mut DNMT1 indicated that luciferase activity was significantly reduced by miR-140-3p overexpression in DNMT1-3’UTR WT group. n = 4. (J) Western blot analysis demonstrated that overexpression of miR-140-3p suppressed both DMNT1, NFκB and pNFκB levels, but upregulated the protein level of SOCS1. Full‑length blots are presented in Fig. S7: Fig. 5J. (K) The qRT-PCR showed decreased mRNA level of DNMT1 and NFκB, as well as increased SOCS1 level with miR-140-3p overexpressed. n = 6. (L) Western blot results showed that exosomes downregulated DMNT1, NFκB and pNFκB levels, and upregulated SOCS1 expression. Force-Exos exhibited a more significant effect than Ctrl-Exos. Full‑length blots are presented in Fig. S7: Fig. 5L. (M) Western blot analysis revealed that the inhibition of miR-140-3p in Force-Exos significantly increased SOCS1 expression but suppressed DMNT1, NFκB and pNFκB levels. Full‑length blots are presented in Fig. S7: Fig. 5M. (N) Schematic illustration of exosomal miR-140-3p playing crucial roles in the prevention and treatment of RR. PDLCs exhibit elevated expression of DNMT1 in response to mechanical stimulation, and increased DNMT1 reduces the protein level of SOCS1 by upregulating DNA methylation to impact transcription. The inhibition of SOCS1 level, as a negative regulator in the NFκB signaling pathway, facilitates the assembly of the NLRP3 inflammasome which promotes the activation of caspase-1. Subsequently, pro-IL-1, pro-IL-18, and GSDMD are cleaved into mature forms by activated caspase-1. The GSDMD-N terminal region facilitates the formation of cell membrane pores, resulting in cell membrane rupture and the release of contents including IL-1β and IL-18, which leads to inflammatory microenvironment that directly stimulates osteoclast formation or indirectly enhances M1 polarization and inhibits M2 polarization to promote osteoclastogenesis. Furthermore, within exosomes from the supernatant of mechanically forced DFSCs (Force-Exo), miRNA-140-3p is significantly overexpressed. When treating force-induced RR with Force-Exo, DFSC-derived exosomal miRNA-140-3p targets DNMT1 to downregulate DNA methylation and upregulate protein expression of SOCS1, which decreases the levels of NFκB and downregulates NLRP3-mediated PDLC pyroptosis to reduce M1 polarization, thereby suppressing osteoclast formation and RR