Background: The inflammation and regeneration process may be accompanied by the shift in the M1/M2 polarization of macrophages to adapt to extracellular signals. How the macrophages responded to the altered immunological environment in the periodontal niche after stem cell transplantation has never been explored. The purpose of present study is to investigate whether M1/M2 polarization of macrophages participated in the tissue homeostasis and wound healing during periodontal ligament stem cell (PDLSC)-based periodontal regeneration.Methods: A rat periodontal defect model was utilized to observe the regeneration process in the PDLSC transplantation-enhanced periodontal repair. Dynamic changes in the markers of M1/M2 macrophages were observed on days 3, 7, and 21 post surgery. In addition, the outcome of regeneration was analyzed on day 21 after surgery. To further investigate the effect of PDLSCs on macrophage polarization, the conditioned medium of PDLSCs was utilized to treat M0, M1, and M2 macrophages for 24 h; markers of M1/M2 polarization were evaluated in macrophages.Results: Elevated bone volume and average thickness of bone trabecular was observed in the PDLSC-treated group by micro-computed tomography on day 21. In addition, enhanced periodontal regeneration was observed in the PDLSC-treated group with cementum-like structure regeneration and collagen fiber formation, which inserted into the newly formed cementum. On day 3, PDLSC transplantation increased IL-10 level in the periodontal tissue, while decreased TNF-α in the early stage of periodontal regeneration. On day 7, enhanced CD163+ cell infiltration and heightened expression of markers of M2 macrophages were observed. Furthermore, conditioned medium from PDLSC culture induced macrophage polarization towards the anti-inflammatory phenotype by downregulating TNF-α and upregulating IL-10, Arg-1, and CD163 in vitro.Conclusions: PDLSCs could induce macrophage polarization towards the M2 phenotype, and the shift in the polarization towards M2 macrophages in the early stage of tissue repair contributed to the enhanced periodontal regeneration after stem cell transplantation. Therefore, signals from the transplanted PDLSCs might alter the immune microenvironment to enhance periodontal regeneration.

Cell survival or death is one critical issue in inflammatory responses. Ferroptosis, which is characterized by iron-dependent lethal lipid peroxidation, has been found to participate in the development of cancers, degenerative brain diseases and ischemia-reperfusion injuries. Incorporation of polyunsaturated fatty acids (PUFAs) into cellular membranes represents a vulnerability to invasion of microbials and sterile stimuli. In addition, the competition for iron in the battle between microbials and host cells underlies infection development. Although host cells have been equipped with complex antioxidant systems to combat lethal accumulation of lipid peroxidation, emerging evidence suggests several pathogens may target PUFAs in the cell membrane, and manipulate ferroptosis as a way for pathogen propagation. Moreover, ferroptosis takes part in the progression of sterile inflammations, such as cigarette smoke-induced chronic obstructive pulmonary disease, stroke and ischemia-reperfusion injuries. As iron-dependent oxidative stress and lipid peroxidation are common features for ferroptosis and inflammatory diseases, underlying mechanisms linking such pathological conditions will be discussed in this review. Progress in the research of ferroptosis may shed more light on the etiology and treatment of inflammatory diseases.

Loss of periodontal ligament fibroblasts (PDLFs) is one critical issue for regenerating lost periodontal tissues. A wide variety of regulated cell death pathways, such as apoptosis, pyroptosis, and necroptosis have been proposed in the periodontitis development. The aim of the present study was to explore whether long-term periodontitis-level butyrate may trigger ferroptosis, a newly characterized iron-dependent regulated cell death in PDLFs. Here, we showed that long-term treatment of butyrate, an important short-chain fatty acid in the periodontal pocket, induces the cargo receptor nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy and ferroptosis in PDLFs. Butyrate-induced iron accumulation, reactive oxygen species (ROS) generation, glutathione depletion and lipid peroxidation in PDLFs, and the butyrate-induced ferroptosis can be blocked by the lipid peroxide scavenger ferrostatin-1. The NCOA4-mediated ferritinophagy is dependent on p38/hypoxia inducible factor-1α (HIF-1α) pathway activation as well as Bromodomain-containing protein (BRD) 4 and cyclin-dependent kinase 9 (CDK9) coordination. These lines of evidence provide a new mechanistic insight into the mechanism of loss of PDLFs during periodontitis development, showing that periodontitis-level butyrate disrupted iron homeostasis by activation of NCOA4-mediated ferritinophagy, leading to ferroptosis in PDLFs.

Periodontal homeostasis is maintained by the dynamic equilibrium between cell death, differentiation and proliferation of resident cells in the periodontal microenvironment. Loss of resident periodontal ligament fibroblasts (PDLFs) has been a major challenge in the periodontal treatment. This study aimed to investigate the exact role of necroptotic cell death in periodontal diseases. Elevated levels of receptor-interacting protein serine-threonine kinases -1 (RIPK1), phosphorylated RIPK3, mixed lineage kinase domain-like protein (MLKL), phosphorylated MLKL and FLIP_L were observed in gingival tissues collected from patients with untreated chronic periodontitis; whereas no difference in caspase 8 was observed between the periodontitis and healthy control group. In contrast to the high incidence of necroptotic cell death in monocytes during live P. gingivalis infection with a low multiplicity of infection (MOI), necroptosis was only observed in PDLFs with a high MOI. Priming PDLFs with frozen thawed monocytes enhanced proinflammatory responses to P. gingivalis infection; moreover, frozen thawed monocytes stimulation triggered RIPK1, RIPK3 and MLKL-mediated-necroptotic cell death in PDLFs. These results indicated that RIPK3 and MLKL-mediated-necroptotic cell death participated in the pathogenesis of periodontitis, and DAMPs released from monocytes after P. gingivalis stimulation by necroptosis triggered not only inflammatory responses, but also necroptosis of PDLFs.

Metabolic reprogramming from oxidative phosphorylation to glycolysis have been implicated in the pathogenesis of inflammatory diseases, such as pulmonary hypertension, rheumatoid arthritis and sepsis. Whether metabolic reprogramming participates in the progression of bacteriogenic periodontitis has never been reported. In the present study, we explored metabolic changes in periodontal ligament cells (PDLSCs) in response to Porphyromonas gingivalis. (P. gingivalis)-infected PDLSCs showed distinct metabolomics with metabolic reprogramming from oxidative phosphorylation to glycolysis. In addition, bacteria invasion triggered fundamental changes in glycolysis and tricarboxylate acid (TCA) cycle-related genes, such as the hexokinase (HK), isocitrate dehydrogenase (IDH) and succinate dehydrogenase (SDH). Moreover, P. gingivalis-infected PDLSCs showed accumulation of succinate, elevation in succinate dehydrogenase activity, pileup of reactive oxygen species and activation of hypoxia inducible factor-1α (HIF-1α) pathway. HIF-1α and succinate inhibitors, as well as SDH knockdown alleviated proinflammatory cytokine expression in P. gingivalis-infected PDLSCs. Therefore, targeting metabolic reprogramming by regulating the succinate-SDH-HIF-1α axis may facilitate host modulation therapy of chronic periodontitis.