Identifying Tissue-Resident Cells of Bone-Marrow Origin in Murine Oral Mucosa

Objectives: Murine models colonized with Porphyromonas gingivalis (Pg) have demonstrated the essential role of innate and adaptive immunity in periodontitis. Recruitment of bone marrow-derived cells to inflamed oral tissues is critical to the pathogenesis of alveolar bone destruction. Our objective is to develop a simple method that distinguishes tissue-resident bone marrow-derived cells from those found within blood/capillary and those present in nasal associated lymphoid tissues (NALT).
Methods: Anesthetized mice were given 1.25μg of FITC-conjugated anti-CD45 mAb in 200μl of PBS via retro-orbital intravenous injection. Mice were euthanized at 0.5, 3, 30 or 60 minutes and blood, oral mucosa and lymph node samples prepared for analysis. Single-cell suspensions were stained with rat anti-Mouse CD3, CD4, CD8, B220, CD44, CD45, CD11b, Ly6G mAbs and viability stain to identify live bone marrow-derived cell subsets by flow cytometry. For NALT analysis, mice were inoculated by oral gavage (7x) with 4x10⁹ CFUs Pg53977, and single-cell suspensions stained to identify Pg-specific antigen experienced T cells (rat anti-Mouse CD3, CD4, CD8, B220, CD44, mAbs and pR/KgpIA^b tetramer).
Results: Tissue-resident and blood/capillary bone marrow-derived cells could be separately identified within 3 minutes of anti-CD45 mAb intravenous delivery. Longer exposure resulted in poorer separation. Tissue-resident T cells were almost exclusively effector cells (CD44^hi). Twenty-eight % of bone marrow-derived cells isolated from oral mucosa resided within blood/capillary and not tissue (n=20). We also demonstrate that NALT could be a significant source of contaminating B cells and Pg-specific CD4 T cells if not avoided.
Conclusions: B cell and granulocyte frequency would be over-estimated and under-estimated, respectively, if blood/capillary-resident cells were not excluded when computing bone marrow-derived cells residing in digested oral mucosa. This simple method allows researchers to avoid analyses that are confounded by bone marrow-derived cells not recruited to inflamed murine oral mucosal tissues.