N6 methyladenosine eraser FTO suppresses Staphylococcus aureus-induced ferroptosis of bone marrow mesenchymal stem cells to ameliorate osteomyelitis through regulating the MDM2/TLR4/SLC7A11 signaling pathway.

Osteomyelitis is a bone destructive inflammatory disease caused by infection. Ferroptosis is closely related to multiple inflammatory diseases, but the role of ferroptosis in Staphylococcus aureus (SA)-induced osteomyelitis remains unknown. In the present study, we found that SA treatment promoted the accumulation of iron, Fe2+ , lipid peroxide, and malondialdehyde, increased TFRC and reduced FTH1 and GPX4 to trigger ferroptosis in rat bone marrow mesenchymal stem cells (BMSCs). Interestingly, increased level of N6 methyl adenosine (m6A) modification along with decreased expression level of m6A eraser FTO were observed in SA-induced BMSCs, while upregulating FTO alleviated SA-triggered ferroptosis and protected cell viability in BMSCs. Mechanistically, MDM2 was identified as a target of FTO-mediated m6A demethylation, and FTO upregulation promoted MDM2 instability to downregulated TLR4 signal and elevate the expression of SLC7A11 and GPX4 in SA-induced BMSCs. Functional recovery experiments verified that overexpressing MDM2 or TLR4 reversed the inhibiting effect of FTO upregulation on ferroptosis in SA-treated BMSCs. Additionally, FTO upregulation restrained ferroptosis and pathological damage to bone tissue in SA-induced osteomyelitis model rats. Altogether, m6A eraser FTO alleviates SA-induced ferroptosis in osteomyelitis models partly through inhibiting the MDM2-TLR4 axis.

DNMT1-mediated DNA methylation in toll-like receptor 4 (TLR4) inactivates NF-κB signal pathway-triggered pyroptotic cell death and cellular inflammation to ameliorate lipopolysaccharides (LPS)-induced osteomyelitis.

Toll-like receptor 4 (TLR4) plays a critical role in various human diseases, and was associated with pyroptotic cell death and inflammatory responses. DNA methylation, which has stable and reversible properties, has been reported to alter the expression of target genes, including TLR4. However, the role of methylated TLR4 in osteomyelitis (OM) and the underlying molecular mechanisms remain unclear. RNA sequencing was used to identify differentially expressed genes and associated signaling pathways. RT-qPCR, Western blot, emzyme-linked immunosorbent assay (ELISA), cell counting kit-8 (CCK-8) and LDH assay kit were used to detect mRNA and protein expression of relevant genes, cell viability and the LDH activity, respectively. TLR4 methylation was detected by methylation-specific PCR (MSP) and verified by Chromatin immunoprecipitation (ChIP). Here, we found that DNA methyltransferase-1 (DNMT1)-mediated TLR4 demethylation significantly suppressed lipopolysaccharides (LPS)-induced pyroptosis and inflammatory response by inhibiting the TLR4/nuclear transcription factor-kappa B (NF-κB) axis. First, we confirmed TLR4 as the study target by mRNA transcriptome sequencing analysis, and TLR4 was observably high-expressed in both OM patients and LPS-treated osteoblastic MC3T3-E1. Then, we found that downregulation of DNMT1 blocked TLR4 promoter methylation modification, resulting in upregulation of TLR4. Simultaneously, functional experiments indicated that suppression of TLR4 or overexpression of DNMT1 promoted cell proliferation and inhibited cell pyroptosis and inflammation in LPS-induced MC3T3-E1, while upregulation of TLR4 restored the effects of DNMT1 silencing on OM progression. In addition, TLR4 elevated phosphorylation of IκB-α and NF-κB p65 in the NF-κB signal pathway, and inhibition of TLR4 or the NF-κB inhibitor PDTC reversed the influence of inhibition of DNMT1. In conclusion, our study demonstrated that DNMT1-mediated TLR4 DNA methylation alleviated LPS-induced OM by inhibiting the NF-κB signaling pathway.