Intercellular mitochondrial transfer alleviates pyroptosis in dental pulp damage

Abstract

Mitochondrial transfer is emerging as a promising therapeutic strategy for tissue repair, but whether it protects against pulpitis remains unclear. Here, we show that hyperactivated nucleotide-binding domain and leucine-rich repeat protein3 (NLRP3) inflammasomes with pyroptotic cell death was present in pulpitis tissues, especially in the odontoblast layer, and mitochondrial oxidative stress (OS) was involved in driving this NLRP3 inflammasome-induced pathology. Using bone marrow mesenchymal stem cells (BMSCs) as mitochondrial donor cells, we demonstrated that BMSCs could donate their mitochondria to odontoblasts via tunnelling nanotubes (TNTs) and, thus, reduce mitochondrial OS and the consequent NLRP3 inflammasome-induced pyroptosis in odontoblasts. These protective effects of BMSCs were mostly blocked by inhibitors of the mitochondrial function or TNT formation. In terms of the mechanism of action, TNF-α secreted from pyroptotic odontoblasts activates NF-κB signalling in BMSCs via the paracrine pathway, thereby promoting the TNT formation in BMSCs and enhancing mitochondrial transfer efficiency. Inhibitions of NF-κB signalling and TNF-α secretion in BMSCs suppressed their mitochondrial donation capacity and TNT formation. Collectively, these findings demonstrated that TNT-mediated mitochondrial transfer is a potential protective mechanism of BMSCs under stress conditions, suggesting a new therapeutic strategy of mitochondrial transfer for dental pulp repair.

FIGURE 1

Mitochondrial OS mediates NLRP3 inflammasome activation and pyroptosis during pulpitis. (A) Immunofluorescence staining of NLRP3, caspase1 and MitoSOX in the pulp tissue. The nucleus was stained with Hoechst. OD: odontoblast layer. (B,C) Quantitative analysis of NLRP3‐ and MitoSOX‐positive cells in the dental pulp tissue from normal controls (n = 19) and pulpitis patients (n = 53). (D) The relationship between NLRP3‐ and MitoSOX‐positive cells in pulpitis tissue (n = 53). (E) Caspase1 activity in the dental pulp tissue from normal controls (n = 12) and pulpitis patients (n = 12). (F–H) Immunofluorescence staining and quantitative analysis of TMRM and Mitogreen in mDPC6T and LA‐mDPC6T cells (n = 30). (I,J) Immunofluorescence staining and flow cytometry analysis of TMRM and MitoSOX in mDPC6T and LA‐mDPC6T cells (n = 3). (K,L) Immunoblotting analysis of Tom20 and Cyto C protein expression in mDPC6T and LA‐mDPC6T cells (n = 3). (M,N) Immunoblotting analysis of NLRP3, Pink1 and Parkin protein expression in isolated mitochondria from mDPC6T and LA‐mDPC6T cells (n = 3). (O,P) Whole‐cell lysates from mDPC6T and LA‐mDPC6T cells were immunoprecipitated with FUNDC1 and IgG antibodies and then immunoblotted with the indicated antibodies, and the ratio between immunoprecipitated LC3B to FUNDC1 was quantified (n = 4). Data are displayed as the mean ± SD. Statistical significance was determined using Student's t test and one‐way analysis of variance (*p < 0.05; **p < 0.01; ***p < 0.001).

FIGURE 2

Mitochondria from mBMSCs are transferred to mDPC6T cells and the transfer rate is enhanced under stressed conditions. (A) Schematic representation of the coculture experimental design for the detection of mitochondrial transfer. CellTrace CFSE‐labelled mDPC6T cells (mDPC6T‐CFSE) were primed with LPS (1 or 5 μg/mL) for 24 h, followed by incubation with ATP (2 mM) for different times (0, 12 or 24 h). MitoAPC‐labelled mBMSCs (mBMSC‐MitoAPC) were added to mDPC6T cells in the ATP treatment step. The mitochondrial transfer rate was determined by flow cytometry. (B,C) mDPC6T cells were primed with LPS (1 and 5 μg/mL) for 24 h, followed by incubation with both ATP (2 mM) and mBMSCs for 24 h. mDPC6T cells without LPS or ATP treatment were denoted as the control. The mitochondrial transfer rate from mBMSCs to mDPC6T cells was determined by flow cytometry (n = 5). (D,E) mDPC6T cells were primed with LPS (1 μg/mL) for 24 h, followed by incubation with both ATP (2 mM) and mBMSCs for different times (0, 12 or 24 h). The mitochondrial transfer rate from mBMSCs to mDPC6T cells was determined by flow cytometry (n = 5). (F) Immunofluorescence staining of mitochondrial transfer from mBMSC‐MitoRFP to mDPC6T‐CFSE under stressed or normal conditions. The white arrowheads indicate the mitochondria from mBMSCs (red arrowheads) within mDPC6T cells (green arrowheads). Data are displayed as the mean ± SD. Statistical significance was determined using one‐way analysis of variance (**p < 0.01; ***p < 0.001).

FIGURE 3

Mitochondrial transfer of mBMSCs protects mDPC6T cells from mitochondrial dysfunction, NLRP3 inflammasome activation and pyroptosis. (A) Schematic representation of the strategy to capture mDPC6T cells in cocultured cells. mDPC6T‐CFSE cells were subjected to coculture with mBMSCs, followed by cell sorting using FACS. CFSE‐positive cells were collected and then used for further analysis. (B,C) Immunoblotting analysis of Tom20 and Cyto C protein expression in mDPC6T cells and LA‐mDPC6T cells alone or cocultured with (ρ^o)mBMSCs (n = 3). (D–G) Flow cytometry analysis of TMRM and MitoSOX in mDPC6T cells and LA‐mDPC6T cells alone or cocultured with (ρ^o)mBMSCs (n = 3). (H,I) Immunoblotting analysis of NLRP3, caspase1 and IL‐1β protein expression (n = 3), (J) qPCR analysis of IL‐6 and CXCL10 mRNA expression (n = 3) and (K) caspase1 activity in mDPC6T cells and LA‐mDPC6T cells alone or cocultured with (ρ^o)mBMSCs (n = 4). (L) ELISA analysis of IL‐1β levels in the cell culture medium of mDPC6T cells and LA‐mDPC6T cells alone or cocultured with (ρ^o)mBMSCs (n = 3). (M) GSEA results of the NLR pathway in LA‐mDPC6T versus mDPC6T cells and LA‐mDPC6T + mBMSCs versus LA‐mDPC6T cells. (N) Heatmap showing the expression of metabolic genes in mDPC6T cells and LA‐mDPC6T cells alone or cocultured with mBMSCs. (O) GO enrichment analysis of the differentially expressed genes (DEGs) between LA‐mDPC6T cells and LA‐mDPC6T + mBMSCs. Data are displayed as the mean ± SD. Statistical significance was determined using one‐way analysis of variance (*p < 0.05; **p < 0.01; ***p < 0.001).

FIGURE 4

Tunnelling nanotube (TNT) mediates the mitochondrial transfer from mBMSCs to mDPC6T cells and protect mDPC6T cells from injury. Immunofluorescence staining of mitochondrial transfer between (A) mDPC6T cells and mBMSCs, (B) LA‐mDPC6T cells and mBMSCs, and (C) LA‐mDPC6T cells and CB‐mBMSCs. Mitochondria (arrowheads) from mBMSC‐RFP translocate along a TNT toward mDPC6T‐CFSE. (D) Quantitative analysis of the TNT‐forming rate of mBMSCs under the conditions described above (n = 12). (E,F) Immunoblotting analysis of Tom20 and Cyto C protein expression in LA‐mDPC6T cells cocultured with mBMSCs or CB‐mBMSCs (n = 3). (G,H) Flow cytometry analysis of TMRM and MitoSOX in LA‐mDPC6T cells cocultured with mBMSCs or CB‐mBMSCs (n = 3). (I,J) Immunoblotting analysis of NLRP3, caspase1 and IL‐1β protein expression in LA‐mDPC6T cells cocultured with mBMSCs or CB‐mBMSCs (n = 3). Data are displayed as the mean ± SD. Statistical significance was determined using one‐way analysis of variance (*p < 0.05; **p < 0.01; ***p < 0.001).

FIGURE 5

The NF‐κB pathway mediates tunnelling nanotube (TNT) formation and mitochondrial transfer from mBMSCs to mDPC6T cells. (A) RNA sequencing analysis of DEGs in LA‐mDPC6T cells compared to mDPC6T cells and (B) GGI interaction networks of these differentially expressed cytokines and their significantly interacting genes. (C,D) Immunoblotting analysis of NF‐κB pathway‐associated protein expression in mBMSCs, mBMSCs treated with ATP, and mBMSCs cocultured with mDPC6T or LA‐mDPC6T cells (n = 3). Coculture was performed in the transwell system as described in Appendix Figure 4A. (E) Immunofluorescence staining of p65 and TNT in mBMSCs under the indicated transwell conditions and (F,G) quantitative analysis of the p65 nuclear translocation rate and TNT‐forming rate (n = 12). (H,I) Flow cytometry analysis of mitochondrial transfer from mBMSCs to mDPC6T cells under indicated coculture conditions (n = 5). (J,K) Immunoblotting analysis of Tom20 and Cyto C protein expression in LA‐mDPC6T cells alone and LA‐mDPC6T cells cocultured with mBMSCs, ML‐mBMSCs or Bay‐mBMSCs (n = 3). (L,M) Flow cytometry analysis of TMRM and MitoSOX in LA‐mDPC6T cells cocultured with mBMSCs, ML‐mBMSCs or Bay‐mBMSCs (n = 3). (N,O) Immunoblotting analysis of NLRP3, caspase1 and IL‐1β protein expression in LA‐mDPC6T cells alone and LA‐mDPC6T cells cocultured with mBMSCs, ML‐mBMSCs or Bay‐mBMSCs (n = 3). Data are displayed as the mean ± SD. Statistical significance was determined using one‐way analysis of variance (*p < 0.05; **p < 0.01; ***p < 0.001).