Finger Stadler Amanda, Patel Mitulkumar, Pacholczyk Rafal, Cutler Christopher W, Arce Roger M
Department of Periodontics, The Dental College of Georgia at Augusta University, 1120 15th Street, Augusta, GA 30912, USA.
Department of Biochemistry and Molecular Biology, Georgia Cancer Center at Augusta University, 1120 15th Street, Augusta, GA 30912, USA.
J Immunol Methods. 2017 Oct;449:7-14. doi: 10.1016/j.jim.2017.06.007. Epub 2017 Jun 21.
Dendritic cells (DCs) are specialized antigen-presenting cells that play a pivotal role in the pathogenesis of periodontitis. The use of animal models to study the role of DCs in periodontitis has been limited by lack of a method for sustained depletion of DCs. Hence, the objectives of this study were to validate the zDC-DTR knockin mouse model of conventional DCs (cDCs) depletion, as well as to investigate whether this depletion could be sustained long enough to induce alveolar bone loss in this model. zDC-DTR mice were treated with different dose regimens of diphtheria toxin (DT) to determine survival rate. A loading DT dose of 20ng/bw, followed and maintained with doses of 10ng/bm every 3days for up to 4weeks demonstrated 80% survival. Animals were weighed weekly and peripheral blood was obtained to confirm normal neutrophil counts. Five animals per group were euthanized at baseline, 24h, 1 and 4weeks. Bone marrow (BM), spleen (SP) and gingival tissue (GT) were harvested, and cells were isolated, separated and stained for Pre-DCs precursors (CD45RMHCIICD11cFlt3CD172a) in BM, cDCs (CD11cMHCIICD209) in spleen, and DCs in GT (CD45RMHCIICD11c DC-SIGN/CD209). Pre-DCs in BM were significantly depleted at 24h and depletion maintained for up to 4weeks, as compared to blank (PBS) controls. Circulating cDCs in spleen demonstrated a non-significant trend to deplete in 1week with high variability among mice. GT also showed a similar non-significant trend to deplete in 24h. The zDC-DTR model seems to be viable for evaluating the role of DCs immune homeostasis disruption and alveolar bone loss pathogenesis in response to long-term oral infection.
树突状细胞（DCs）是特殊的抗原呈递细胞，在牙周炎的发病机制中起关键作用。利用动物模型研究DCs在牙周炎中的作用，一直受到缺乏持续清除DCs方法的限制。因此，本研究的目的是验证传统DCs（cDCs）缺失的zDC-DTR基因敲入小鼠模型，并研究这种清除是否能持续足够长的时间以在该模型中诱导牙槽骨丧失。用不同剂量方案的白喉毒素（DT）处理zDC-DTR小鼠以确定存活率。负荷DT剂量为20ng/体重，随后每3天以10ng/体重的剂量维持，持续4周，存活率达80%。每周称动物体重，并采集外周血以确认中性粒细胞计数正常。每组5只动物在基线、24小时、1周和4周时实施安乐死。采集骨髓（BM）、脾脏（SP）和牙龈组织（GT），分离细胞，对BM中的前DCs前体（CD45RMHCIICD11cFlt3CD172a）、脾脏中的cDCs（CD11cMHCIICD209）以及GT中的DCs（CD45RMHCIICD11cDC-SIGN/CD209）进行分离和染色。与空白（PBS）对照组相比，BM中的前DCs在24小时时显著减少，且减少持续长达4周。脾脏中循环的cDCs在1周时有非显著的减少趋势，小鼠之间差异较大。GT在24小时时也显示出类似的非显著减少趋势。zDC-DTR模型似乎可用于评估DCs免疫稳态破坏以及长期口腔感染后牙槽骨丧失发病机制的作用。
