IADR Abstract Archives
Animal Model for Conditional Dendritic Cells Depletion – Preliminary Results
Objectives: Animal models evaluating alveolar bone loss are useful to study the immunopathogenic aspects of periodontal disease. The objective of this study was to validate a murine model (conditional knockout) of conventional dendritic cells (cDCs) depletion.
Methods: B6(Cg)-Zbtb46tm1(HBEGF)Mnz/J (zDC-DTR) mice (Jackson Laboratory), 12-20 week-old, received one loading dose of diphtheria toxin (DT) of 20ng per gram of body weight (20ng/bw), and maintenance doses of 10ng/bw every 3 days for 6 weeks. Peripheral blood samples were collected at baseline, 24h, 1 week, 4 weeks and 6 weeks for neutrophil counts. Mice were also observed for weight loss or any eventual health complication. Wild-type C57BL/6J mice were also treated following the same protocol (control group). Three animals were euthanized at each time point, and spleen and bone marrow were harvested for DC phenotyping. Cells were isolated, separated by magnetic separation (Robosep, pan-DCs negative selection), and analyzed by flow cytometry. Data distribution was tested and parametric and non-parametric tests were used when appropriate.
Results: In zDC-DTR animals, the percentages of pre-DCs (CD45.R-CD11c+MHCII-Flt3+CD172a+) from bone marrow were decreased 24h after treatment, and remained lower for 6 weeks (One-Way ANOVA, p=0.024). The percentage of DC-SIGN+ cells were significantly decreased at 24 hours (p=0.025), and remained lower after 6 weeks (p=0.018). In spleen, DC-SIGN+ cells were also significantly decreased in 1 week (p=0.016), and cDCs (MHCII+CD11c+) showed a tendency to decrease, without significance (p=0.117). Neutrophils counts showed a slight decrease but remained in reported normal ranges (10-25%). Survival rate of mice was 79%.
Conclusions: Within the limitations of this preliminary study, results suggested that the cDCs can be predictably depleted for 6 weeks, and most of animals survive this period, showing that the model is viable for murine models evaluating the role of DCs in immune homeostasis and alveolar bone loss in response to oral infection.
Methods: B6(Cg)-Zbtb46tm1(HBEGF)Mnz/J (zDC-DTR) mice (Jackson Laboratory), 12-20 week-old, received one loading dose of diphtheria toxin (DT) of 20ng per gram of body weight (20ng/bw), and maintenance doses of 10ng/bw every 3 days for 6 weeks. Peripheral blood samples were collected at baseline, 24h, 1 week, 4 weeks and 6 weeks for neutrophil counts. Mice were also observed for weight loss or any eventual health complication. Wild-type C57BL/6J mice were also treated following the same protocol (control group). Three animals were euthanized at each time point, and spleen and bone marrow were harvested for DC phenotyping. Cells were isolated, separated by magnetic separation (Robosep, pan-DCs negative selection), and analyzed by flow cytometry. Data distribution was tested and parametric and non-parametric tests were used when appropriate.
Results: In zDC-DTR animals, the percentages of pre-DCs (CD45.R-CD11c+MHCII-Flt3+CD172a+) from bone marrow were decreased 24h after treatment, and remained lower for 6 weeks (One-Way ANOVA, p=0.024). The percentage of DC-SIGN+ cells were significantly decreased at 24 hours (p=0.025), and remained lower after 6 weeks (p=0.018). In spleen, DC-SIGN+ cells were also significantly decreased in 1 week (p=0.016), and cDCs (MHCII+CD11c+) showed a tendency to decrease, without significance (p=0.117). Neutrophils counts showed a slight decrease but remained in reported normal ranges (10-25%). Survival rate of mice was 79%.
Conclusions: Within the limitations of this preliminary study, results suggested that the cDCs can be predictably depleted for 6 weeks, and most of animals survive this period, showing that the model is viable for murine models evaluating the role of DCs in immune homeostasis and alveolar bone loss in response to oral infection.