Human dental stem cells suppress PMN activity after infection with the periodontopathogens Prevotella intermedia and Tannerella forsythia

Abstract

Periodontitis is characterized by inflammation associated with the colonization of different oral pathogens. We here aimed to investigate how bacteria and host cells shape their environment in order to limit inflammation and tissue damage in the presence of the pathogen. Human dental follicle stem cells (hDFSCs) were co-cultured with gram-negative P. intermedia and T. forsythia and were quantified for adherence and internalization as well as migration and interleukin secretion. To delineate hDFSC-specific effects, gingival epithelial cells (Ca9-22) were used as controls. Direct effects of hDFSCs on neutrophils (PMN) after interaction with bacteria were analyzed via chemotactic attraction, phagocytic activity and NET formation. We show that P. intermedia and T. forsythia adhere to and internalize into hDFSCs. This infection decreased the migratory capacity of the hDFSCs by 50%, did not disturb hDFSC differentiation potential and provoked an increase in IL-6 and IL-8 secretion while leaving IL-10 levels unaltered. These environmental modulations correlated with reduced PMN chemotaxis, phagocytic activity and NET formation. Our results suggest that P. intermedia and T. forsythia infected hDFSCs maintain their stem cell functionality, reduce PMN-induced tissue and bone degradation via suppression of PMN-activity, and at the same time allow for the survival of the oral pathogens.

Figure 1. Surface marker expression of hDFSCs.

Representative FACS analysis of the stem cell surface marker on hDFSCs after incubation under aerobic and anaerobic conditions. The black line refers to the isotype control, green describes the distinct antibody binding on the stem cell surface marker.

Figure 2. Direct interaction of P. intermedia and T. forsythia with human cells.

HDFSCs and Ca9-22 were infected with P. intermedia or T. forsythia under anaerobic conditions for 2 h. Adherent and internalized bacteria were quantified. Adherent bacteria were related to reference bacteria present in the inoculum, and internalized bacteria were related to adherent bacteria. Results are displayed as median ± interquartile range, *p < 0.05, **p < 0.01 (Mann-Whitney U test), n ≥ 4.

Figure 3. Differentiation potential of infected hDFSCs.

Representative microscopic photograph of infected hDFSCs after 21 days of differentiation stimulation. Adipogenic (A), chondrogenic (B) and osteogenic (C) differentiation stain. (D) to (F) are infected control cells with cell culture medium.

Figure 4. Migration of hDFSCs after 24 h.

HDFSCs were incubated under aerobic or anaerobic conditions and in parallel infected with P. intermedia or T. forsythia. Uninfected hDFSCs were used as control. Migration was analyzed in a scratch assay, while the scratch diameter was measured each 4 h up to 24 h. The initial scratch diameter was defined as 100%. The migration dynamic of infected and uninfected hDFSCs is observed over 24 h, results are displayed as median ± standard deviation (A) and final results are presented after 24 h of incubation. Results are displayed as median ± interquartile range, *p < 0.05 (Mann-Whitney U test), n = 4 (B). Representative microscopic photograph of the migrating HDFSCs after 24 h of aerobic (C) and anaerobic (D) incubation.

Figure 5. Secretion of IL-6, IL-8 and IL-10 after infection of human cells.

HDFSCs and Ca9-22 were infected with P. intermedia or T. forsythia under anaerobic conditions. IL-6 (A), IL-8 (B) and IL-10 (C) levels were quantified via ELISA after 2 h, 4 h and 24 h from the supernatant of infected cells with P. intermedia (left) and T. forsythia (right). Values from uninfected hDFSC controls matching the experimental time points were subtracted beforehand. Results are displayed as median ± interquartile range, *p < 0.05 (Mann-Whitney U test), n = 4.

Figure 6. Chemotactic behavior of PMNs towards infected hDFSCs.

Migration of PMNs was assessed via transwell assay. After 24 h sterile filtered supernatants of P. intermedia and T. forsythia in cell culture medium with/without hDFSCs were used as chemo attractants in the lower wells. PMNs in fresh cell culture medium without serum were added to the insert. After 2 h of aerobic incubation the count of PMNs migrated into the lower compartment was determined. Migration of PMNs in the presence of non-infected hDFSCs was set to 100% (dashed line). Results are displayed with median ± interquartile range. *p < 0.05, **p < 0.01 (Mann-Whitney U test), n ≥ 4.

Figure 7. Phagocytic activity of PMNs against oral bacteria.

Phagocytic activity is shown as bacterial clearance from the supernatant of P. intermedia or T. forsythia with hDFSCs relative to the control of bacteria only. Results are displayed with median ± interquartile range. **p < 0.01, ***p < 0.001 (Mann-Whitney U test), n ≥ 4.

Figure 8. NET formation of PMNs against oral bacteria.

NETs were quantified via quantification of extracellular DNA. HDFSCs and P. intermedia or T. forsythia were used as stimuli for PMNs. After 180 min, extracellular DNA was quantified and uninfected hDFSCs were set to 100%. Results are displayed with median ± interquartile range. *p < 0.05 (Mann-Whitney U test) significance to hDFSC control, n = 4.