Downregulation of Macrophage-Specific Act-1 Intensifies Periodontitis and Alveolar Bone Loss Possibly via TNF/NF-κB Signaling

Abstract

Periodontitis is a chronic inflammatory oral disease that affects almost half of the adult population. NF-κB activator 1 (Act1) is mainly expressed in immune cells, including macrophages, and modulates immune cells' function to regulate inflammation in inflammatory diseases. Macrophages play a vital role in the pathophysiology of periodontitis. However, the effect of macrophage-specific Act1 on periodontitis has not been investigated yet. This study aims to unravel the role of macrophage-specific Act1 on the pathophysiology of periodontitis. The expression of Act1 in healthy and periodontitis periodontal tissue was confirmed by immunohistochemistry. Macrophage-specific Act1 expression downregulated (anti-Act1) mice were developed by inserting anti-Act1 antisense oligonucleotides after the CD68 promoter of C57BL/6 mice. Ligature-induced periodontitis (LIP) was induced in anti-Act1 mice and wildtype mice. Micro-CT, histology, and TRAP staining analyzed the periodontal tissue status, alveolar bone loss, and osteoclast numbers. Immunohistochemistry, RT-qPCR, and ELISA analyzed the inflammatory cells infiltration, expression of inflammatory cytokines, and M1/M2 macrophage polarization. mRNA sequencing of in vitro bacterial lipopolysaccharide (LPS)-treated peritoneal macrophages analyzed the differentially expressed genes in anti-Act1 mice during inflammation. Anti-Act1 mice showed aggravated periodontitis and alveolar bone loss compared to wildtype. Periodontitis-affected periodontal tissue (PAPT) of anti-Act1 mice showed a higher degree of macrophage infiltration, and M1 macrophage polarization compared to wildtype. Levels of pro-inflammatory cytokines (IL-1β, IL-6, and TNFα), and macrophage activity-related factors (CCL2, CCL3, and CCL4) were robustly high in PAPT of anti-Act1 mice compared to wildtype. mRNA sequencing and KEGG analysis showed activated TNF/NF-κB signaling in LPS-treated macrophages from anti-Act1 mice. In vitro studies on LPS-treated peritoneal macrophages from anti-act1 mice showed a higher degree of cell migration and expression of inflammatory cytokines, macrophage activity-related factors, M1 macrophage-related factors, and TNF/NF-κB signaling related P-p65 protein. In conclusion, downregulation of macrophage-specific Act1 aggravated periodontitis, alveolar bone loss, macrophage infiltration, inflammation, and M1 macrophage polarization. Furthermore, LPS-treated macrophages from anti-Act1 mice activated TNF/NF-κB signaling. These results indicate the distinct role of macrophage-specific Act1 on the pathophysiology of periodontitis possibly via TNF/NF-κB signaling.

Keywords: NF-κB activator 1; alveolar bone loss; inflammation; macrophages; periodontitis.

FIGURE 1

Act1 is expressed in healthy and periodontitis gingival tissue. (A) Representative immunohistochemistry images of healthy/periodontitis gingival tissue (human and mice) show Act1 expression. Quantitative analysis of Act1 positive cells in healthy and periodontitis human gingival tissue (B), and mice periodontal tissue (C). Data are presented as mean ± SD (n = 6). WT, wildtype.

FIGURE 2

Periodontitis and alveolar bone loss were aggravated in anti-Act1 mice compared to wildtype. (A) Micro-CT images of the healthy and periodontitis-affected maxilla of wildtype and anti-Act1 mice. Quantitative analysis of CEJ-ABC distance (B), BMD (C) from micro-CT images. (D) H&E stained histological images of PAPT of wild-type and anti-Act1 mice. (E) Quantitative analysis of CEJ-ABC distance from histological images. (F) TRAP stained histological images of PAPT of wild-type and anti-Act1 mice. (G) Quantitative analysis of osteoclasts in PAPT. Data are presented as mean ± SD (n = 6). The significant difference between the groups, ***P < 0.001. CEJ, cement-enamel junction; ABC, alveolar bone crest; Red dot line, CEJ level; Black dot line, ABC level; blue line, CEJ-ABC distance; yellow arrow, osteoclasts; WT, wild-type; and BMD, bone mineral density.

FIGURE 3

Macrophage infiltration in PAPT was enhanced in anti-Act1 mice compared to wildtype. (A) Immunohistochemistry images of PAPT. Quantitative analysis of CD34 expressing MVD (B), CD45 expressing pan leucocytes (C), and F4/80 expressing macrophages (D), n = 6. (E) FACs images of macrophages sorting using FITC-CD11b and PE-F4/80. (F) Quantitative FACs data. Calculated the ratio of CD11b + F4/80+ cells, n = 8. The significant difference between the groups, **P < 0.01, ns, not significant. MVD, Microvessel density; WT, wildtype.

FIGURE 4

(A–F) mRNA level expression of inflammatory cytokines and macrophage migration-related factors in PAPT of wildtype and anti-Act1 mice. (G–I) Protein level expression of inflammatory cytokines in PAPT of wildtype and anti-Act1 mice. Data are presented as mean ± SD (n = 6). The significant difference between the groups, **P < 0.01, ***P < 0.001. WT, wildtype.

FIGURE 5

Lipopolysaccharide -treated peritoneal macrophages from wildtype and anti-Act1 mice showed a differential expression of 2190 mRNAs. (A) Heat map, (B) KEGG pathway analysis, and (C) Volcano map showing differentially expressed mRNAs. Red color intensity indicates upregulation, and blue color intensity indicates the downregulation of mRNA expression.

FIGURE 6

Lipopolysaccharide -treated peritoneal macrophages from anti-Act1 mice differentially upregulate the mRNA expression of TNF-NF-κB signaling pathway-related proteins. (A) Heat map, and (B) bar figures showing differentially upregulated mRNAs. (C–E) Protein level expressions of inflammatory cytokines in LPS-treated peritoneal macrophages from wildtype and anti-Act1 mice (measured by ELISA), n = 6. (F) Representative western bolt images of p65 and P-p65 expression. (G) Quantification of western blots (n = 3). Data are presented as mean ± SD (n = 6). The significant difference between the groups, **P < 0.01, ***P < 0.001. WT, wildtype. Red color intensity indicates upregulation, and blue color intensity indicates the downregulation of gene expression.

FIGURE 7

Lipopolysaccharide -treated peritoneal macrophages from anti-Act1 mice showed higher migration and M1-polarization. (A) Representative images of macrophage migration. (B) Quantitative analysis of macrophage migration, n = 3. (C) CCL2 protein expression (ELISA, n = 6). Heat map (D), and bar figures of differentially expressed macrophage M1-polarization-related genes (E). (F) CD16/32 protein level expression analyzed by ELISA, n = 6. (G) FACs images of mice periodontal macrophages sorting using CD16/32 specific antibody. (H) Quantitative FACs data, n = 6. The significant difference between the groups, **P < 0.01, ***P < 0.001. WT, wildtype. Red color intensity indicates upregulation, and blue color intensity indicates the downregulation of gene expression.

FIGURE 8

Effect of macrophage-specific downregulation of Act1 on the pathophysiology of mice periodontitis.