SLIT2 Overexpression in Periodontitis Intensifies Inflammation and Alveolar Bone Loss, Possibly via the Activation of MAPK Pathway

Abstract

SLIT2, a member of neuronal guidance cues, has been reported to regulate inflammation and cancer progression. Periodontitis is an oral inflammatory disease that degenerates periodontal tissue, alveolar bone and tooth. This study aims to explore the expression pattern of SLIT2 in periodontitis and its role in disease progression and bone loss. Gingival tissue of 20 periodontitis patients and 20 healthy-controls was obtained. Ligature-induced periodontitis (LIP) mice-model was developed in Slit2-Tg and wild-type mice. The effect of SLIT2 on inflammation, immune cell infiltration, M1 macrophage polarization, and alveolar bone loss in periodontitis was analyzed extensively. In periodontitis-affected gingival-tissue, SLIT2 expression was 4.4-fold higher compared to healthy-volunteers. LIP enhanced SLIT2 expression in mice periodontitis-affected periodontal tissue (PAPT) and blood circulation of wild-type mice by 4. 6-, and 5.0-fold, respectively. In Slit2-Tg-mice PAPT, SLIT2 expression was 1.8-fold higher compared to wild-type mice. Micro-CT and histomorphometric analysis revealed a 1.3-fold higher cement-enamel-junction to the alveolar-bone-crest (CEJ-ABC) distance and alveolar bone loss in LIP Slit2-Tg-mice compare to LIP wild-type mice. Results from RNA-sequencing, RT-qPCR, and ELISA showed a higher expression of Cxcr2, Il-18, TNFα, IL-6, and IL-1β in Slit2-Tg-mice PAPT compared to wild-type-mice. Slit2-Tg-mice PAPT showed a higher number of osteoclasts, M1 macrophages, and the upregulation of Robo1 expression. Slit2-Tg-mice PAPT showed upregulation of M1 macrophage marker CD16/32 and osteoclastogenic markers Acp5 , Ctsk , and Nfatc1 , but osteogenic markers ( Alp, Bglap ) remained unchanged. Immunohistochemistry unveiled the higher vasculature and infiltration of leucocytes and macrophages in Slit2-Tg-mice PAPT. RNA-sequencing, GO-pathway enrichment analysis, and western blot analysis revealed the activation of the MAPK signaling pathway in Slit2 -Tg mice PAPT. In conclusion, SLIT2 overexpression in periodontitis intensifies inflammation, immune cells infiltration, M1 macrophage polarization, osteoclastogenesis, and alveolar bone loss, possibly via activation of MAPK signaling, suggesting the role of SLIT2 on exacerbation of periodontitis and alveolar bone loss.

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

“…Alveolar bone resorption, periodontal tissue degeneration, higher CEJ-ABC, severe alveolar bone loss, and an increased number of osteoclasts were observed in anti-Act1 periodontitis mice compare to wildtype periodontitis mice. A higher degree of angiogenesis and infiltration of leucocyte/macrophages in PAPT have been reported in severe cases of periodontitis ( Shi et al, 2008 ; Parisi et al, 2018 ; Wang L. et al, 2020 ; Zhang Z. et al, 2020 ). In this study, a similar extent of neo-vasculogenesis and leucocyte infiltration was observed in anti-Act1 and wildtype LIP mice.…”

“…However, macrophage infiltration was highly enhanced in PAPT of anti-Act1 mice compared to wildtype. M1 macrophage polarization is associated with the severity of periodontitis ( He et al, 2020 ; Wang L. et al, 2020 ). In this study, M1 macrophage polarization was enhanced in PAPT of anti-Act1 mice compared to wildtype.…”

“…In this study, M1 macrophage polarization was enhanced in PAPT of anti-Act1 mice compared to wildtype. M1 macrophages release the pro-inflammatory cytokines amplifying inflammation in the PAPT milieu ( He et al, 2020 ; Wang L. et al, 2020 ). The higher levels of pro-inflammatory cytokines in PAPT aggravate periodontitis and alveolar bone loss ( Fredriksson et al, 2002 ; Azuma et al, 2014 ; Sakalauskiene et al, 2016 ; Wang D. et al, 2020 ).…”

“…Enzyme-linked immunosorbent assay (ELISA) was performed to measure the protein expression in periodontal tissue protein extract, conditioned medium of mice macrophages culture, and mice macrophages cell lysates. The macrophages were treated with 100 ng/ml LPS for 24 h. The periodontal tissue cell lysate was prepared as described previously ( Wang L. et al, 2020 ). Mice peritoneal macrophages were isolated as described previously ( Kheirandish et al, 2018 ).…”

“…The cell suspension was prepared from mice periodontal tissue, including gingiva, periodontal ligament, and a part of alveolar bone as described previously (Wang L. et al, 2020). The cell suspension was used for flow cytometry analysis (Mizraji et al, 2013 cells for 30 min on ice in the dark.…”