Interleukin-37 ameliorates periodontitis development by inhibiting NLRP3 inflammasome activation and modulating M1/M2 macrophage polarization.

Our study was designed to explore the role of IL-37 in M1/M2 macrophage polarization imbalance in the pathogenesis of periodontitis. Periodontitis is a chronic progressive inflammatory disease featured by gingival inflammation and alveolar bone resorption. Recent research has revealed that regulating macrophage polarization is a viable method to ameliorate periodontal inflammation. IL-37 is an anti-inflammatory cytokine, which has been reported to inhibit innate and adaptive immunity. For in vitro experiment, mouse macrophage RAW264.7 cells were pretreated with 0.1 ng/mL recombinant human IL-37. M1 and M2 polarizations of RAW264.7 cells were induced by 100 ng/mL LPS and 20 ng/mL IL-4, respectively. The expression of M1 (iNOS, TNF-α, and IL-6) and M2 (CD206, Arg1, and IL-10) phenotype markers in RAW264.7 cells was detected by RT-qPCR, western blotting, and immunofluorescence staining. For in vivo experiment, experimental periodontitis mouse models were established by sterile silk ligation (5-0) around the bilateral maxillary second molar of mice for 1 week. H&E staining of the maxillary alveolar bone was used to show the resorption of root cementum and dentin. Alveolar bone loss in mouse models was evaluated through micro-CT analysis. The expression of iNOS and CD206 in gingival tissues was assessed by immunohistochemistry staining. NLRP3 inflammasome activation was confirmed by western blotting. IL-37 pretreatment reduced iNOS, TNF-α, and IL-6 expression in LPS-treated RAW264.7 cells but increased CD206, Arg1, and IL-10 in IL-4-treated RAW264.7 cells. LPS-induced upregulation in NLRP3, GSDMD, cleaved-IL-1β, and cleaved-caspase-1 expression was antagonized by IL-37 treatment. In addition, IL-37 administration ameliorated the resorption of root cementum and dentin in periodontitis mouse models. IL-37 prominently decreased iNOS+ cell population but increased CD206+ cell population in gingival tissues of periodontitis mice. The enhancement in NLRP3, GSDMD, cleaved-IL-1β, and cleaved-caspase-1 expression in the gingival tissues of periodontitis mice was offset by IL-37 administration. IL-37 prevents the progression of periodontitis by suppressing NLRP3 inflammasome activation and mediating M1/M2 macrophage polarization.

Yang L ，Tao W ，Xie C ，Chen Q ，Zhao Y ，Zhang L ，Xiao X ，Wang S ，Zheng X ... -  《-》

A20 inhibits periodontal bone resorption and NLRP3-mediated M1 macrophage polarization.

A20 is involved in inflammation and bone metabolism in periodontitis. Regulation of macrophage polarization may be an effective target for periodontitis treatment, and A20 has a regulatory role in macrophage polarization. This study aimed to explore the effects of A20 on macrophage polarization in periodontitis and the underlying mechanism. Adeno-associated virus (AAV) targeting A20 was exploited to achieve A20 knockdown or overexpression in periodontal tissues of mice with experimental periodontitis. The (AAV-A20-RNAi) +P group showed increased alveolar bone resorption when compared with PBS + P and CON305 + P groups. However, the degree of bone destruction was reduced in the (AAV-A20) +P group relative to PBS + P and CON299 + P groups. A20 knockdown resulted in enhanced inducible nitric oxide synthase (iNOS) expression and decreased CD206 expression in mice periodontal tissues. In addition, higher levels of M1 macrophage polarization markers (iNOS, CD86, TNF-α) and lower CD206 expression were found in THP-1 cells treated with lipopolysaccharide (LPS) from Porphyromonas gingivalis (P. gingivalis) (Pg. LPS) and interferon-γ (IFN-γ) when A20 was silenced. A20 overexpression showed opposite effects on macrophage polarization in vivo and in vitro. Knockdown of A20 was correlated with upregulation of the NLRP3 inflammasome pathway in mice periodontal tissues or THP-1 cells. On the contrary, A20 overexpression inhibited the NLRP3 inflammasome pathway. MCC950 suppressed M1 macrophage polarization aggravated through A20 knockdown in Pg. LPS and IFN-γ stimulated cells. Our data suggested that A20 inhibits periodontal bone resorption and NLRP3-mediated M1 macrophage polarization; A20 is expected to be a novel target for the treatment of periodontitis.

Hou L ，Ye Y ，Gou H ，Tang H ，Zhou Y ，Xu X ，Xu Y ... -  《-》

Leptin Aggravates Periodontitis by Promoting M1 Polarization via NLRP3.

Han Y ，Huang Y ，Gao P ，Yang Q ，Jia L ，Zheng Y ，Li W ... -  《-》

Exploring the effect of Er miao San-containing serum on macrophage polarization through miR-33/NLRP3 pathway.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease. Er miao San (EMS) has been shown to have good anti-inflammatory effects and is widely used in the clinical treatment of RA. However, the exact mechanism is not completely understood. The aim of this study was to explore that EMS-containing serum affects M1/M2 polarization of macrophages and may be mediated through the microRNA (miRNA)-33/NLRP3 pathway, thereby elucidating the molecular mechanism of EMS treatment of RA. We screened for safe concentrations of EMS-containing serum by using CCK-8 measurement. RAW264.7 cells were cultured with lipopolysaccharide (LPS) (100 ng/mL) and interferon-γ (20 ng/mL) for 24 h to induce M1-type macrophages. Adenosine triphosphate (ATP) (5 mM) was added in the last 30 min to activate NLRP3. The content of miR-33 was detected by RT‒qPCR after transfection of the miRNA-33 mimic. The protein expression levels of NLRP3, ASC, caspase-1, Inducible Nitric Oxide Synthase (iNOS) and Arginase-1 (Arg-1) were detected by Western blot. The contents of IL-1β, IL-10, TNF-α, TGF-β and IL-18 in serum and cell supernatant were determined by ELISA. The fluorescence intensity of CD86 and CD206 was detected by immunofluorescence. The results showed that EMS-containing serum promoted the protein expression level of Arg-1 and the secretion levels of TGF-β and IL-10, inhibited the levels of iNOS, IL-1β and TNF-α, and regulated the balance of pro-inflammatory factors and anti-inflammatory factors. RT‒qPCR results showed that EMS-containing serum could reduce the level of miRNA-33. EMS-containing serum could reduce the expression of NLRP3 inflammasome-related proteins and downregulate the expression levels of IL-1β and IL-18. These results suggest that EMS exerts its effect on macrophage polarization through the miRNA-33/NLRP3 pathway. EMS-containing serum inhibits the activation of the NLRP3 inflammasome by downregulating miRNA-33, thus preventing the polarization of M1-type macrophages.

Liu M ，Wang J ，Chen S ，Meng X ，Cheng Z ，Wang J ，Tan Y ，Su W ，Lu Z ，Zhang M ，Jia X ... -  《-》

PGRN is involved in macrophage M2 polarization regulation through TNFR2 in periodontitis.

Progranulin (PGRN), a multifunctional growth factor, plays indispensable roles in the regulation of cancer, inflammation, metabolic diseases, and neurodegenerative diseases. Nevertheless, its immune regulatory role in periodontitis is insufficiently understood. This study attempts to explore the regulatory effects of PGRN on macrophage polarization in periodontitis microenvironment. Immunohistochemical (IHC) and multiplex immunohistochemical (mIHC) stainings were performed to evaluate the expression of macrophage-related markers and PGRN in gingival samples from periodontally healthy subjects and periodontitis subjects. RAW264.7 cells and bone marrow-derived macrophages (BMDMs) were polarized towards M1 or M2 macrophages by the addition of LPS or IL-4, respectively, and were treated with or without PGRN. Real-time fluorescence quantitative PCR (qRT-PCR), immunofluorescence staining (IF), enzyme-linked immunosorbent assay (ELISA), and flow cytometry were used to determine the expressions of M1 and M2 macrophage-related markers. Co-immunoprecipitation was performed to detect the interaction between PGRN and tumor necrosis factor receptor 2 (TNFR2). Neutralizing antibody was used to block TNFR2 to confirm the role of TNFR2 in PGRN-mediated macrophage polarization. The IHC and mIHC staining of human gingival slices showed a significant accumulation of macrophages in the microenvironment of periodontitis, with increased expressions of both M1 and M2 macrophage markers. Meanwhile, PGRN was widely expressed in the gingival tissue of periodontitis and co-expressed mainly with M2 macrophages. In vitro experiments showed that in RAW264.7 cells and BMDMs, M1 markers (CD86, TNF-α, iNOS, and IL-6) substantially decreased and M2 markers (CD206, IL-10, and Arg-1) significantly increased when PGRN was applied to LPS-stimulated macrophages relatively to LPS stimulation alone. Besides, PGRN synergistically promoted IL-4-induced M2 markers expression, such as CD206, IL-10, and Arg1. In addition, the co-immunoprecipitation result showed the direct interaction of PGRN with TNFR2. mIHC staining further revealed the co-localization of PGRN and TNFR2 on M2 macrophages (CD206+). Blocking TNFR2 inhibited the regulation role of PGRN on macrophage M2 polarization. In summary, PGRN promotes macrophage M2 polarization through binding to TNFR2 in both pro- and anti-inflammatory periodontal microenvironments.

Zhang L ，Nie F ，Zhao J ，Li S ，Liu W ，Guo H ，Yang P ... -  《Journal of Translational Medicine》