Treponema Denticola outer membrane extract enhances the phagocytosis of collagen coated-beads by human gingival fibroblasts

by Battikhi, Tulin

Human gingival fibroblasts (HGFs) degrade collagen by actin-dependent phagocytosis. Since Treponema denticola outer membrane extract (OM) perturbs actin, my aim was to determine whether the OM affects collagen phagocytosis by HGFs. Phagocytosis was measured by flow cytometric assessment of internalized collagen-coated fluorescent latex beads. Confluent HGFs pretreated with T. denticola ATCC 35405 OM exhibited a significant, saturable, dose-dependent increase in collagen phagocytosis index (PI; % cells with beads), and phagocytic capacity (PC; # beads per phagocytosing cell) compared with untreated controls. The enhancement was swift, and exhibited a significant, linear, timedependent increase. PI and PC of HGFs for BSA-coated beads were also stimulated following OM pretreatment in a dose- and time-dependent manner. These results were in contrast to the control OM from Veillonella atypica ATCC 17744. The T. denticola OMinduced bead uptake increase was irreversible and was eliminated by heating the OM for 30 min at 60°C, and 10 min at 100°C, and by cytochalasin D treatment of HGFs. Fluid phase accumulation of Lucifer yellow-CH (LY) was enhanced in a saturable, dose-dependent, transient manner. The findings were not due to HGF detachment or cytotoxicity in response to the T. denticola and V. atypica OM treatment as: 1) electronic particle counts indicated minimal HGF detachment; 2) propidium iodide staining revealed that the attached HGFs remained viable; and 3) flow cytometry analysis of T. denticola OM-treated cells showed that there was no significant change in their size and granularity, and that there was no development of populations containing sub-G, DNA. Thus my results collectively indicate that a heat-sensitive component(s) in T. denticola OM extract stimulates HGF collagen phagocytosis in an actin-dependent manner. The perturbation may also induce a phenotypic switch in the phagocytic capacity of HGF, an increase in nonspecific phagocytosis and an increase in pinocytosis. Increased phagocytosis of collagen and other extracellular matrix proteins may provide a mechanism by which bacteria contribute to collagenolysis and connective tissue destruction in periodontal disease.