Department of Oral Biology, Yonsei University College of Dentistry, Seoul, South Korea.
Department of Applied Life Science, The Graduate School, Yonsei University, Seoul, South Korea.
J Periodontal Res. 2020 Dec;55(6):868-876. doi: 10.1111/jre.12779. Epub 2020 Jun 25.
Increased neutrophil infiltration and osteoclast formation are key characteristics of periodontitis. The effect of these neutrophils on osteoclast formation in periodontitis remains unclear. Therefore, we investigated the effects of neutrophils on osteoclast formation in a neutrophil-deficient mouse model of periodontitis.
Anti-Ly6G antibody (Ab) was used for neutrophil depletion in two mouse models: periodontitis and air pouch. In the periodontitis experiments, mice were divided into PBS-administered control (C), control Ab-administered periodontitis (P), and anti-Ly6G Ab-administered periodontitis (P + Ly6G) groups. Periodontitis was induced by ligature of mandibular first molars. In the air pouch experiments, mice were divided into PBS-administered (C), LPS and control Ab-administered (LPS), and LPS and anti-Ly6G Ab-administered (LPS + Ly6G) groups. Neutrophil migration into air pouches was induced by LPS injection. Flow cytometry was used to examine CD11b Ly6G neutrophils in the blood of periodontitis mice and CD11b Ly6G RANKL neutrophils in exudates of air pouch mice. In periodontal tissue, Ly6G neutrophil and RANKL cell numbers in periodontal ligament and alveolar bone areas were estimated using immunohistochemistry, osteoclast numbers were measured using TRAP assay, and alveolar bone loss was determined by H&E staining.
In blood, CD11b Ly6G neutrophils were found in greater percentage in the P group than in the C group on days 3 and 7. However, the percentage of neutrophils was lower in the P + Ly6G group than in the C and P groups. In periodontal tissue, the numbers of Ly6G neutrophils and RANKL cells were lower in the P + Ly6G group than in the P group on day 3. Ly6G neutrophil numbers decreased more in the P + Ly6G group than in the P group on day 7, but RANKL cell numbers did not decrease in the P + Ly6G group. In exudates, the number of CD11b Ly6G RANKL neutrophils was greater in the LPS group than in the C and LPS + Ly6G groups. On days 3 and 7, the numbers of osteoclasts and alveolar bone loss were greater in periodontal tissue in the P and P + Ly6G groups than in the C group. Interestingly, there were fewer osteoclasts in the P + Ly6G group than in the P group on day 3.
Neutrophil deficiency caused a reduction in numbers of both RANKL cells and osteoclasts in periodontitis-induced tissues only on day 3. Furthermore, in the LPS-injected air pouch model, neutrophil deficiency reduced the influx of RANKL neutrophils. These findings suggest that the presence of neutrophils induces RANKL expression and could induce osteoclast formation in the early stages of periodontitis.
中性粒细胞浸润和破骨细胞形成增加是牙周炎的关键特征。这些中性粒细胞对牙周炎中破骨细胞形成的影响尚不清楚。因此，我们研究了中性粒细胞在牙周炎中性粒细胞缺陷小鼠模型中对破骨细胞形成的影响。
抗 Ly6G 抗体（Ab）用于两种小鼠模型中的中性粒细胞耗竭：牙周炎和气囊。在牙周炎实验中，将小鼠分为 PBS 给药对照组（C）、对照 Ab 给药牙周炎组（P）和抗 Ly6G Ab 给药牙周炎组（P+Ly6G）。通过结扎下颌第一磨牙诱导牙周炎。在气囊实验中，将小鼠分为 PBS 给药组（C）、LPS 和对照 Ab 给药组（LPS）和 LPS 和抗 Ly6G Ab 给药组（LPS+Ly6G）。通过 LPS 注射诱导中性粒细胞迁移至气囊。使用流式细胞术检测牙周炎小鼠血液中的 CD11b Ly6G 中性粒细胞和气囊小鼠渗出液中的 CD11b Ly6G RANKL 中性粒细胞。在牙周组织中，使用免疫组织化学法估计牙周韧带和牙槽骨区域中的 Ly6G 中性粒细胞和 RANKL 细胞数量，使用 TRAP 测定法测量破骨细胞数量，并用 H&E 染色法测定牙槽骨丢失。
在血液中，与 C 组相比，P 组第 3 天和第 7 天的 CD11b Ly6G 中性粒细胞百分比更高。然而，与 C 组和 P 组相比，P+Ly6G 组的中性粒细胞百分比较低。在牙周组织中，与 P 组相比，P+Ly6G 组第 3 天的 Ly6G 中性粒细胞和 RANKL 细胞数量较少。与 P 组相比，P+Ly6G 组第 7 天的 Ly6G 中性粒细胞数量减少更多，但 RANKL 细胞数量并未减少。在渗出液中，LPS 组的 CD11b Ly6G RANKL 中性粒细胞数量多于 C 组和 LPS+Ly6G 组。在第 3 天和第 7 天，P 和 P+Ly6G 组的牙周组织中的破骨细胞数量和牙槽骨丢失量均大于 C 组。有趣的是，与 P 组相比，P+Ly6G 组第 3 天的破骨细胞数量更少。
中性粒细胞缺乏仅在第 3 天导致牙周炎诱导组织中 RANKL 细胞和破骨细胞数量减少。此外，在 LPS 注射气囊模型中，中性粒细胞缺乏减少了 RANKL 中性粒细胞的浸润。这些发现表明中性粒细胞的存在诱导了 RANKL 的表达，并可能在牙周炎的早期阶段诱导破骨细胞形成。
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