Abstract
Periodontitis is a chronic inflammatory response to a microbial biofilm that destroys bone and soft tissues supporting the teeth. Murine models of periodontitis based on Porphyromonas gingivalis (Pg) colonization have shown that extravasation of leukocytes into oral tissue is critical to driving alveolar bone destruction. Identifying interstitial leukocytes is key to understanding the immunopathogenesis of periodontitis. Here, we describe a robust flow cytometry assay based on intravenous FITC-conjugated anti-mouse CD45 mAb that distinguishes interstitial leukocytes in the oral mucosa of mice from those circulating within the vasculature or in post-dissection contaminating blood. Unaccounted circulating leukocytes skewed the relative frequency of B cells and granulocytes and inflated the numbers of all leukocyte cell types. We also describe a dissection technique that avoids contamination of oral mucosal tissues with nasal-associated lymphoid tissues (NALT), a B cell rich organ that can inflate leukocyte numbers at least 10-fold and skew the assessment of interstitial CD4 T cell phenotypes. Unlike circulating CD4 T cells, interstitial CD4 T cells were almost exclusively antigen-experienced cells (CD44hi). We report for the first time the presence of antigen-experienced Pg-specific CD4 T cells in NALT following oral feeding of mice with Pg. This new combined flow cytometry and dissection approach allows identification of leukocytes infiltrating the connective tissues of the murine oral mucosa and avoids confounding analyses of leukocytes not recruited to inflamed oral mucosal tissues in disease conditions like periodontitis, candidiasis, or sialadenitis.
Highlights
The mouth harbors a species-rich microbiome that includes microorganisms with pathogenic and invasive potentials and the ability to form biofilms on hard surfaces like teeth [1, 2]
We describe a simple robust method that combines intravenous delivery of a pan-leukocyte (CD45) monoclonal antibody with a flow cytometry approach to discriminate interstitial leukocytes in the oral mucosa from those found within blood; either residing within the tissue vasculature itself or in blood dispersed during dissection that could contaminate the surface of the mucosal tissue
Histological techniques have been useful in identifying a number of cell types within the oral mucosa of mice and its vasculature system [45, 46], histological techniques do not have the same quantitative and qualitative accuracy offered by flow cytometry-based methods [10, 47]
Summary
The mouth harbors a species-rich microbiome that includes microorganisms with pathogenic and invasive potentials and the ability to form biofilms on hard surfaces like teeth [1, 2]. Destructive inflammatory diseases can occur when microorganisms breach the sulcular epithelium around teeth As one such disease, periodontitis is triggered by a community of symbiont microorganisms whose relative proportions are altered by a handful of microorganisms, so called “keystone pathogens,” which are capable of altering the nutrient foundation of the community itself [5,6,7]. Periodontitis is triggered by a community of symbiont microorganisms whose relative proportions are altered by a handful of microorganisms, so called “keystone pathogens,” which are capable of altering the nutrient foundation of the community itself [5,6,7] This changed nutritional environment fosters the growth of pathobionts that stimulate the extravasation and infiltration of the leukocytes into the interstitium of the gingival tissues. Though recruited to fight the invading pathobionts, the cellular infiltrate triggers osteoclastogenesis leading to alveolar bone destruction as well as irreversible loss of the connective tissue attachment that holds teeth into their socket [12, 13]
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