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.
Highlights
SLIT2 protein, a member of neuronal guidance cues, is expressed in extraneuronal tissues, including kidney, lung, heart, and immune cells (Yuan et al, 1999; Wu et al, 2001; Wong et al, 2002)
A 3.9-fold higher expression of SLIT2 protein was observed in Slit2-Tg mice Periodontitis-affected periodontal tissue (PAPT) compared to healthy periodontal tissue (Figure 1B)
The serum of wild-type periodontitis mice showed 5.0-fold higher expression of SLIT2 protein compared to wild-type healthy mice (Figure 1C)
Summary
SLIT2 protein, a member of neuronal guidance cues, is expressed in extraneuronal tissues, including kidney, lung, heart, and immune cells (Yuan et al, 1999; Wu et al, 2001; Wong et al, 2002). SLIT2 is aberrantly expressed in various cancers and plays a role in the regulation of cancer cell apoptosis, cancer metastasis, tumor-associated inflammation, and tumor progression (Wang et al, 2003, 2008; Schmid et al, 2007; Avci et al, 2008; Yang et al, 2010; Zhou et al, 2011; Chang et al, 2015; Liu et al, 2018). The SLIT2 has been reported to aberrantly regulate the inflammation in different inflammatory diseases and cell types (Zhao et al, 2014; Zhou et al, 2017; Fernando et al, 2018). Binding of SLIT2 with specific ROBO receptor regulates particular cell functions. The expression pattern of SLIT2 in periodontal tissue during periodontitis and its’ role in the pathophysiology of periodontitis is still unknown
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