Diphtheria Toxin-induced Ablation of Dendritic Cells Leads to Neutrophilia I. Mohammad*, K. Thieu, L. Fischer, P. Bittner-Eddy, M. Costalonga University of Minnesota, School of Dentistry, Minneapolis, MN - USA Poster # 902 Background Objective Objective Results Neutrophils numbers in the blood of CD11c-DTR transgenic mice are significantly higher 2 to 6 days after administration of DT when compared to CD11c-DTR BM chimera mice (p<0.05). Chimera mice show only mild neutrophilia. Periodontal disease is a multifactorial disorder involving oral bacteria and immune cell interactions. Activation of T cells by dendritic cells in response to bacteria, lead to multiple cellular responses which cause the release of cytokines that promote osteoclastogenesis. The net result is the destruction of alveolar bone by osteoclasts. Compare neutrophilia levels between CD11c-DTR transgenic mice and CD11c-DTR bone marrow chimera mice after treatment with diphtheria toxin. Flow cytometry scatter plots of blood comparing transgenic and BM chimera mice 4 days after administration of diphtheria toxin (Fig. 1-3) or PBS (data not shown). Figure 1 displays Forward Scatter (FSC) vs. Side Scatter (SSC) analysis of live gated cells, which represents all of the blood cells in the sample. From these population of cells we selected PMNs (Gr1+/I-Ab-) and antigen presenting cells [APCs] (Gr1-/I-Ab+) as shown in figure 2. Furthermore, we gated primarily B cells (B220+/I-Ab+) from the population of APCs (Gr1-/I-Ab+) shown in figure 3. The ratio of PMNs (Gr1+/I-Ab-) to B cells (B220+/I-Ab+) was calculated to normalize the population of neutrophils against a stable population of B cells in blood. The comparison of neutrophil levels between chimera and transgenic mice treated with DT or PBS is shown in figure 4. Methods Ratio of Gr-1+ I-Ab- to B220+ CD3 cells Days after DT injection Transgenic BM chimera Dying mice DT injections DT injection Fig. 4 Ten CD11c-DTR transgenic and 10 CD11c-DTR bone marrow chimera were injected with PBS or 8 ng diphtheria toxin (DT) per gram of body weight on day 0 and/or day 7. After the first DT injection, blood cells (B) were collected from submandibular vein every 2-3 days for 15 days as outlined below. Tissue Inflammation Antibody Synthesis Cyokine Secretion bacteria Dendritic cell T Cell Osteoclasts Cytokines Alveolar Bone Resorption Root Gingiva Crown CD11c-DTR Transgenic 0 2 4 6 7 8 9 11 13 15 Days DT B A mouse model lacking dendritic cells provides a useful tool in determining the role of dendritic cells in T cell activation during periodontal disease. CD11c+ dendritic cells in mice can be depleted by using the diphtheria toxin (DT)-based system. In this system, injection of DT induces rapid apoptosis in cells carrying a transgene encoding DT receptor (DTR) under the control of CD11c promoter. CD11c-DTR Bone Marrow Chimera Day 4 after DT injection Summary Transgenic BM chimera 0 2 4 6 7 8 9 11 13 15 DT B Fig. 1 Neutrophil levels were significantly higher in transgenic compared to BM chimera mice from day 2 to day 6 (p<0.05), and returned to normal levels by day 8. Unlike BM chimeras, all transgenic mice died by day 8. In BM chimeras the second dose of DT on day 7 did not repeat the induction of neutrophilia. CD11c promoter DTR GFP Blood cells were collected in FACS tubes containing heparin on ice, lysed with ACK lysing buffer, blocked using Fc block, and stained by direct immunofluorescence with appropriate antibodies and analyzed by flow cytometry. Depletion of dendritic cells in transgenic CD11c-DTR mice can be problematic for periodontitis studies as it can lead to neutrophilia, an abnormally high number of neutrophils in the blood (Tittel et al, 2012). Therefore, in this study we compared the neutrophilia levels in CD11c-DTR Transgenic vs. CD11c-DTR Bone Marrow Chimera mice in order to identify the best mouse model to study periodontitis after oral infection with Porphyromonas gingivalis. SSC FSC Fig. 2 Conclusion Flow Cytometry Staining Strategy Injection of DT leads to neutrophilia and eventually death of CD11c-DTR transgenic mice. CD11c-DTR bone marrow chimeras survive multiple DT doses and have only mild transient neutrophilia. Fluoro-chrome Cell Marker Target Cell APC Gr1 PMN FITC CD11b PMN, DC, Mf PE I-Ab Antigen presenting cell (APC) PerCP B220 B cell CD3 T cell Cell Receptor Antibody Fluorescent label Gr1 Mouse Models Being Tested for Neutrophilia Significance CD11c promoter DTR GFP Mouse embryo injected with transgene Females implanted with embryo Offspring carrying the transgene CD11c-DTR Transgenic CD11c-DTR Bone Marrow Chimera Donor Normal Recipient Extract bone marrow cells Whole body irradiation Inject cells into recipient mouse Bone Marrow Chimera Mouse Fig. 3 BM chimera mouse model system is better suited for use in studies of periodontal disease compared to transgenic because the negligible neutrophilia is unlikely to affect the oral colonization with Porphyromonas gingivalis. B220/CD3 Reference Tittel A, Heuser C, Ohliger C, Llanto C, Yona S, Hammerling G, Engel D, Garbi N, and Kurts C. Functionally relevant neutrophilia in CD11c diphtheria toxin receptor transgenic mice. Nature Methods. Vol 9 No. 4: 385-391, 2012. I-Ab