The oral mucosa is a barrier site constantly exposed to rich and diverse commensal microbial communities, yet little is known of the immune cell network maintaining immune homeostasis at this interface. Characterization of the immune cell subsets at the gingival interface in health will provide important new insights into the cellular mechanisms operating to establish physiologic oral/periodontal immunity. We have performed a detailed characterization of the immune cell subsets of the oral cavity in a cohort of healthy/young adult subjects. For these studies, we evaluated more than 100 healthy volunteers in order to obtain biopsies from 50 healthy/young individuals to study the oral/gingival immunological network of oral health. We established protocols for isolation of mononuclear cells from tissue and employed multi-color flow cytometry to characterize in depth the immune cell subtypes in oral tissues. We focused our characterization on the gingival interface, a particularly vulnerable mucosal site, with a thin epithelial lining and constant exposure to the tooth-adherent biofilm. In health, we find a predominance of T cells, low numbers of B cells, a large presence of granulocytes/neutrophils, a sophisticated network of professional antigen-presenting cells (APCs), and a small population of innate lymphoid cells (ILCs) policing the gingival barrier. For comparison, we interrogated shifts in immune cell populations in periodontitis. In periodontitis, we observe increased inflammatory cells (particularly T cells and neutrophils) as well as a significant increase in Th17 cells (CD4 T cells secreting IL-17A). Collectively, our studies provide a view of the landscape of physiologic oral immunity and serve as a baseline for the characterization of local immunopathology. We have also developed techniques tailored to the study of oral tissue immunity in murine models. Specifically, we have developed protocols for the isolation and study of immune cells from murine gingival/oral tissues. Our protocol allows for the isolation of viable cells from gingiva, buccal mucosa and tongue and yields sufficient cells for a detailed phenotypic characterization of the immune cell populations resident in the oral cavity and gingiva, even at steady state (health). Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network will aid our future investigation of the mechanisms involved in oral immunity and periodontal homeostasis and advance our understanding of the mechanisms involved in local immunopathology.
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