RNA sequencing for ligature induced periodontitis in mice revealed important role of S100A8 and S100A9 for periodontal destruction

Abstract

Periodontitis is an inflammatory disease caused by pathogenic oral microorganisms that induce the destruction of periodontal tissue. We sought to identify the relevant differentially expressed genes (DEGs) and clarify the mechanism underlying the rapid alveolar bone loss by using ligature-induced periodontitis in mice. A silk ligature was tied around the maxillary left second molar in 9-week-old C57BL/6 J male mice. In-vivo micro-CT analysis revealed that ligation induced severe bone loss. RNA-sequencing analysis, to examine host responses at 3 days post-ligation, detected 12,853 genes with fragments per kilobase of exon per million mapped reads ≥ 1, and 78 DEGs. Gene ontology term enrichment analysis revealed the expression profiles related to neutrophil chemotaxis and inflammatory responses were significantly enriched in the ligated gingiva. The expression levels of innate immune response-related genes, including S100a8 and S100a9, were significantly higher in the ligated side. S100A8 was strongly detected by immunohistochemistry at the attached epithelium in ligated sites. Inhibition of S100A8 and S100A9 expression revealed that they regulated IL1B and CTSK expression in Ca9-22 cells. Thus, innate immune response-related molecules might be associated with the burst-destruction of periodontal tissue in ligature-induced periodontitis. Especially, S100A8 and S100A9 may play an important role in alveolar bone resorption.

Figure 1

Ligation-induced alveolar bone loss around the second molar. The 3D images of unligated side (intact (A), day 1 (C), day 3 (E), day 5 (G), day 8 (I)) and ligated side (intact (B), day 1 (D), day 3 (F), day 5 (H), day 8 (J)) are shown. The graph shows bone loss volume (mm³) at the indicated time points. Bone loss was seen to initiate from the distal root and become wide-spread at 3 days post-ligation. Bone loss volume significantly increased from 3 days post-ligation (K). Scale bar = 0.5 mm. Data are represented as the mean ± SE. Thin line: p < 0.05, thick line: p < 0.01 compared to intact.

Figure 2

Histological analysis at 8 days after ligation. Hematoxylin and eosin staining (unligated (A) and ligated (B) side) and TRAP/ALP staining (unligated (C) and ligated (D) side) are shown. Enlarged views, of the boxed area of unligated (C) and ligated (D) side, are shown in (E,F), respectively. TRAP-positive multinucleate cells were induced around the periodontal ligament in ligated side. The arrows in sections indicate osteoclasts stained with TRAP, adjacent to the alveolar bone. Black scale bar = 50 μm. HE, hematoxylin and eosin; TRAP, tartrate-resistant acid phosphatase; ALP, alkaline phosphatase; E, epithelial tissue; C, connective tissue; B, alveolar bone; T, tooth.

Figure 3

Gene profile in gingival tissue analyzed by RNA sequencing. RNA-seq detected 12,853 genes (FPKM ≥ 1.0), which were categorized into 12 patterns by hierarchical clustering approach (A; left). Cluster 1, which consists of genes down-regulated in the ligated gingiva, and Cluster 12, which consists of genes up-regulated in the ligated gingiva, were analyzed using DAVID to determine specific enriched GO terms. The figure shows the representative significantly enriched GO terms (Benjamini-Hochberg <0.05) (A; right). Statistical analysis revealed 78 (55 up-regulated and 23 down-regulated) DEGs between the ligated and unligated gingiva, using the criteria of q-value < 0.05 and fold change ≥2.0. The red and gray plots represent DEGs and non-DEGs, respectively (B). Top 10 enriched GO terms in the up-regulated DEGs in ligated gingiva were determined by DAVID. Right side of the red line designates Benjamini-Hochberg <0.05 (C). The bar plot shows fold change (on a log 2 scale) between the ligated and unligated gingiva for innate immune response-related genes. The red and gray bars represent DEGs and non-DEGs, respectively (D).

Figure 4

The mRNA expression levels of DEGs at 1, 3, and 7 days after ligation. The mRNA expression levels of DEGs were validated by qPCR after 1, 3, and 7 days of ligation. Relative expression levels of S100a8 (A), S100a9 (B), Il1b (C), Ctsk (D), Mmp9 (E), Mmp3 (F), Mmp19 (G), Timp1 (H), Ccl9 (I), Ncf1 (J), and Spp1 (K) are shown. The mean mRNA expression levels in the unligated gingiva at day 1 were set as “1”. Data are shown as the mean ± SE. *p < 0.05, **p < 0.01 compared to same-day unligated side. Thin line: p < 0.05, thick line: p < 0.01 compared to other day’s ligated side.

Figure 5

Histological analysis of periodontal tissue at 3 days and 8 days post-ligation. Hematoxylin-eosin staining (3-day unligated (A), 3-day ligated (E), 8-day unligated (I), 8-day ligated (M)), nuclear staining with DAPI (3-day unligated (B), 3-day ligated (F), 8-day unligated (J), 8-day ligated (N)), and immunohistochemistry of S100A8 (3-day unligated (C), 3-day ligated (G), 8-day unligated (K), 8-day ligated (O)) of periodontal tissue are shown. Merged images with S100A8 and DAPI (3-day unligated (D), 3-day ligated (H), 8-day unligated (L), and 8-day ligated (P)) are also shown. S100A8 expression areas were evaluated and S100A8-positive area in ligated side at 8 days post-ligation was found to be significantly increased than that of unligated side (Q). Data are shown as the mean ± SE. **p < 0.01 compared to same-day unligated side; E, epithelial tissue; C, connective tissue. Epithelium is outlined by white line.

Figure 6

S100A8 and S100A9 knockdown in Ca9-22 cells. S100A8 (A), S100A9 (B), IL1B(C), and CTSK (D) expression in Ca9-22 cells. Control cells were cultured in medium with transfection reagent. siNT, siRNA for non-target negative control; siA8-#7 and siA8-#8, siRNA for S100A8; siA9-#5 and siA9-#8, siRNA for S100A9. *p < 0.05, **p < 0.01, ***p < 0.001. ^†p < 0.05, ^††p < 0.01, ^†††p < 0.001 compared to control samples.