Dec2 inhibits macrophage pyroptosis to promote periodontal homeostasis

Abstract

Purpose: Macrophages play crucial roles as early responders to bacterial pathogens and promote/ or impede chronic inflammation in various tissues. Periodontal macrophage-induced pyroptosis results in physiological and pathological inflammatory responses. The transcription factor Dec2 is involved in regulating immune function and inflammatory processes. To characterize the potential unknown role of Dec2 in the innate immune system, we sought to elucidate the mechanism that may alleviate macrophage pyroptosis in periodontal inflammation.

Methods: Porphyromonas gingivalis lipopolysaccharide (LPS) was used to induce pyroptosis in RAW 264.7 macrophages. Subsequently, we established an LPS-stimulated Dec2 overexpression cellular model in macrophages. Human chronic periodontitis tissues were employed to evaluate potential changes in inflammatory marker expression and pyroptosis. Finally, the effects of Dec2 deficiency on inflammation and pyroptosis were characterized in a P. gingivalis-treated experimental periodontitis Dec2-knockout mouse model.

Results: Macrophages treated with LPS revealed significantly increased messenger RNA expression levels of Dec2 and interleukin (IL)-1β. Dec2 overexpression reduced IL-1β expression in macrophages treated with LPS. Overexpression of Dec2 also repressed the cleavage of gasdermin D (GSDMD), and the expression of caspase-11 was concurrently reduced in macrophages treated with LPS. Human chronic periodontitis tissues showed significantly higher gingival inflammation and pyroptosis-related protein expression than non-periodontitis tissues. In vivo, P. gingivalis-challenged mice exhibited a significant augmentation of F4/80, tumor necrosis factor-α, and IL-1β. Dec2 deficiency markedly induced GSDMD expression in the periodontal ligament of P. gingivalis-challenged mice.

Conclusions: Our findings indicate that Dec2 deficiency exacerbated P. gingivalis LPS-induced periodontal inflammation and GSDMD-mediated pyroptosis. Collectively, our results present novel insights into the molecular functions of macrophage pyroptosis and document an unforeseen role of Dec2 in pyroptosis.

Keywords: Dec2; Macrophage; Periodontal inflammation; Porphyromonas gingivalis; Pyroptosis.

Figure 1. Reverse transcription-polymerase chain reaction analysis showing that the transcription factor Dec2 is elevated following treatment with P. gingivalis LPS for 24 hours. (A) LPS treatment significantly induced mRNA expression of IL-1β in RAW 264.7 macrophages. (B) The mRNA expression level of Dec2 was also significantly elevated. The data shown represent means±standard deviation. All results are representative of at least 3 independent experiments.

IL: interleukin, LPS: lipopolysaccharide, mRNA: messenger RNA. ^a)P<0.05.

Figure 2. Reverse transcription-polymerase chain reaction analysis showing that Dec2 controls P. gingivalis LPS-induced pyroptosis. (A) The messenger RNA level of Dec2 was highly upregulated after transfection with the Dec2 expression vector. (B) IL-1β expression was suppressed by the overexpression of Dec2 after treatment with 10 μg/mL P. gingivalis LPS. FLAG denotes an empty vector. The data shown represent means±standard deviation. All results are representative of at least 3 independent experiments.

IL: interleukin, LPS: lipopolysaccharide, NS: non-significant. ^a)P<0.05, ^b)P<0.01.

Figure 3. Western blot analysis showing that Dec2 reduces the P. gingivalis LPS-induced pyroptosis. (A, B) Treatment with 10 μg/mL P. gingivalis LPS led to significant increases of GSDMD and pro- and cleaved caspase-11. The overexpression of Dec2 alleviated the expression of cell pyroptosis markers and controlled cell pyroptosis under inflammatory conditions. FLAG denotes an empty vector. The data shown represent means±standard deviation. All results are representative of at least 3 independent experiments.

GSDMD: gasdermin D, LPS: lipopolysaccharide, NS: non-significant. ^a)P<0.05.

Figure 4. Immunohistochemistry showing that Dec2 is upregulated in chronic periodontitis tissues. The expression of Dec2 was higher in periodontitis tissue than in non-periodontitis tissue. The expression of Dec2, GSDMD, and CD68 was also consistently induced in periodontitis tissues. The data shown represent means±standard deviation. All results are representative of at least 3 independent experiments.

H&E: hematoxylin and eosin, GSDMD: gasdermin D. ^a)P<0.001.

Figure 5. Immunohistochemistry showing that inflammation is induced in chronic periodontitis tissue. The expression levels of PMNs, TNF-α, and IL-1β were significantly elevated in chronic periodontitis tissue. The data shown represent means±standard deviation. All results are representative of at least 3 independent experiments.

PMN: polymorphonuclear neutrophil, TNF-α: tumor necrosis factor-α, IL-1β: interleukin-1β. ^a)P<0.001.

Figure 6. Immunohistochemistry showing that a deficiency of Dec2 facilitates the infiltration of inflammatory cells and osteoclast differentiation in periodontitis models. The expression of GSDMD was upregulated in Dec2KO mice compared with WT mice, and consistently, the infiltration of inflammatory cells was increased more in Dec2KO mice compared with WT mice, as well as the inflammatory markers, which included IL-1β and TNF-α. Notably, a higher number of TRAP-positive osteoclasts was detected in Dec2KO mice than in WT mice; black arrows indicate TRAP-positive cells. The data shown represent means±standard deviation. All results are representative of at least 3 independent experiments.

H&E: hematoxylin and eosin, GSDMD: gasdermin D, PMN: polymorphonuclear neutrophil, Dec2KO: Dec2-knockout, WT: wild-type, TNF-α: tumor necrosis factor-α, IL-1β: interleukin-1β, TRAP: tartrate-resistant acid phosphatase, NS: not significant. ^a)P<0.05, ^b)P<0.01, ^c)P<0.001.