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.

Keywords: MAPK signaling; SLIT2; alveolar bone loss; osteoclasts; periodontitis.

FIGURE 1

Periodontitis-induced SLIT2 aggravates periodontitis and alveolar bone loss. Expression pattern of SLIT2 protein in, (A) Gingival tissue of periodontitis patients and healthy individuals (n = 20), and (B) periodontal tissue of healthy and periodontitis wild-type or Slit2-Tg mice. (C) Serum level of SLIT2 protein in wild-type mice (n = 5). (D) Micro-CT images of healthy and periodontitis-affected alveolar bone with the intact tooth of wild-type and Slit2-Tg mice. (E) Quantitative data of CEJ-ABC distance analyzed from micro-CT images. (F) H&E stained histological images of PAPT of wild-type and Slit2-Tg mice. (G) Quantitative data of alveolar bone loss around the periodontitis-affected tooth analyzed from histological images. Data are presented as mean ± SD (n = 6). The significant difference among the groups, *P < 0.05, **P < 0.01, ***P < 0.001. CEJ: cement-enamel junction, ABC: alveolar bone crest, Red dot line: CEJ level, Black dot line: ABC level, al: alveolar bone, de: dentine, blue arrow: inflammatory infiltration of the gingival epithelium, black arrow: alveolar bone resorption, double arrowhead (blue): CEJ-ABC distance, and WT: wild-type.

FIGURE 2

SLIT2 overexpression augments pathological changes in both periodontal milieu soft and bone tissues during periodontitis. (A) Representative histological images of PAPT sections showing TRAP+ osteoclasts. (B) Representative CD34 and CD45 immunohistochemistry images of PAPT sections. (C) Quantitative analysis of osteoclasts from TRAP-stained histological tissue sections (n = 6). (D) Quantification of micro-vessel density from CD34 immunohistochemistry images (n = 5). (E) Quantification of CD45 expressing cells from CD45 immunohistochemistry images (n = 6). The significant difference among the groups, *P < 0.05, **P < 0.01. Black arrow: TRAP+ multinucleated osteoclasts. Black triangle arrowhead: microvessel. WT: wild-type.

FIGURE 3

SLIT2 overexpression triggers up-regulation of periodontal inflammatory factors. (A (i)) Relative mRNA expression, and (A (ii)) Heat map showing the differential expression pattern of inflammatory markers in PAPT analyzed by RNA sequencing (n = 3). Relative mRNA expression of (B) Il6, (C) Il1β, and (D) Tnfα in periodontitis-affected tissue analyzed by RT-qPCR. Protein expression of, (E) IL-6, (F) IL-1β, and (G) TNF-α in periodontitis-affected tissue analyzed by ELISA. Data are presented as mean ± SD (n = 6). Data are presented as mean ± SD. The significant difference among the groups, *P < 0.05, **P < 0.01, ***P < 0.001. Red color intensity indicates upregulation, and blue color intensity indicates the downregulation of gene expression.

FIGURE 4

SLIT2 overexpression activates osteoclastogenesis-related signaling pathways in periodontitis. (A (i)) Relative mRNA expression, and (A (ii)) Heat map showing the differential expression pattern of osteoclastogenesis related genes analyzed by RNA-seq (n = 3). Relative mRNA expression of (B) Acp5, (C) Ctsk, and (D) Nfatc1 in periodontal tissue analyzed by RT-qPCR (n = 6). (E) Relative mRNA expression of osteoblastogenesis related genes analyzed by RNA sequencing (n = 3). Data are presented as mean ± SD. The significant difference among the groups, *P < 0.05, **P < 0.01, ***P < 0.001. WT: wild-type.

FIGURE 5

SLIT2 overexpression induces M1 macrophage polarization in PAPT. (A (i)) Representative F4/80 immunohistochemistry images of the PAPT section. (A (ii)) Quantification of F4/80 expressing cells in a PAPT section from immunohistochemistry images (n = 6). (B (i)) Flow cytometry analysis of F4/80 positive relative frequency of gated populations from PAPT. (B (ii)) Quantitative analysis of F4/80+ flow cytometry data (n = 5). Relative mRNA expression of (C) M1, and (D) M2 macrophage polarization related genes analyzed by RNA sequencing (n = 3). (E) Protein level expression of CD16/32 in PAPT (n = 6). Protein level expression of, (F) CD16/32 in cell lysates of LPS-treated BMMs. Protein expression of (G) IL-6, (H) IL-1β, and (I) TNF-α in conditioned medium of LPS-treated BMMs (n = 4). Data are presented as mean ± SD. The significant difference among the groups, *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 6

SLIT2/ROBO1 signaling possibly regulates the SLIT2-induced inflammation in periodontitis. (A (i)) Heat map the differential expression of Slit2 receptors Robo1-4, and (A (ii)) Relative mRNA expression of Robo1, Robo2, Robo3, and Robo4 in PAPT analyzed by RNA sequencing (n = 3). Relative mRNA expression of, (B) Robo1, (C) Robo2, and (D) Robo4 in PAPT analyzed by RT-qPCR (n = 6). (E) Heat map of differentially expressed 134 mRNAs in PAPT of Slit2-Tg mice to wild-type mice (fdr < 0.05,| FC| > 2). (F) GO pathway enrichment analysis. Data are presented as mean ± SD. The significant difference among the groups, *P < 0.05, **P < 0.01.

FIGURE 7

SLIT2 overexpression activates MAPK signaling pathway in periodontitis milieu. (A (i)) Relative mRNA expression (n = 3), and (A (ii)) Heat map of the differential expression pattern of MAPK signaling pathway-related genes in PAPT analyzed by RNA sequencing. Relative mRNA expression of (B) Traf6, and (C) p38 in PAPT analyzed by RT-qPCR (n = 6). (D (i)) Representative western blot images of MAPK signaling related proteins. (D (ii), (iii)) Quantitative analysis of TRAP and P-P38 (n = 4). Data are presented as mean ± SD. The significant difference among the groups, *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 8

Scheme showing the role of SLIT2 overexpression in pathophysiology of periodontitis.