Oral fibroblasts modulate the macrophage response to bacterial challenge

Abstract

Tissue damage in chronic periodontal disease is driven by the host response to a dysbiotic microbiota, and not by bacteria directly. Among chronic inflammatory diseases of the oral cavity, inflammation and tissue damage around dental implants (peri-implantitis) is emerging as a major clinical challenge, since it is more severe and less responsive to treatment compared to inflammation around natural teeth. We tested whether oral fibroblasts from the periodontal ligament (PDLF), which are present around natural teeth but not around dental implants, actively regulate inflammatory responses to bacterial stimulation. We show that human PDLF down-regulate TNF-α post-transcriptionally in macrophages stimulated with the oral pathogen Porphyromonas gingivalis. Cell contact and secretion of IL-6 and IL-10 contribute to the modulation of inflammatory cytokine production. Although fibroblasts decreased TNF-α secretion, they enhanced the ability of macrophages to phagocytose bacteria. Surprisingly, donor matched oral fibroblasts from gingival tissues, or fibroblasts from peri-implant inflamed tissues were at least as active as PDLF in regulating macrophage responses to bacteria. In addition, priming fibroblasts with inflammatory mediators enhanced PDLF regulatory activity. A further understanding of the spectrum of fibroblast activities in inflammatory lesions is important in order to design ways to control inflammatory tissue damage.

Figure 1

PDLF down-regulate macrophage cytokine production in response to P. gingivalis. Human macrophages (Mφ) were mono or co-cultured with PDLF prior to stimulation with increasing MOIs of P. gingivalis. TNF-α (a) and IL1-β (b) production were measured in the supernatant 5 hours after bacterial stimulation. Contact dependency of the regulatory effect of PDLF over the TNF-α production by stimulated Mφ was tested using PDLF pc-media (c) or a 0.4 μm transwell chamber (d) separating the two cell populations. Data represent the means + SEM of at least 3 separate experiments. *P value < 0.05 compared to the cytokine production by Mφ in mono-culture stimulated with the same MOI. ***P < 0.001 compared to the cytokine production by Mφ in mono-culture stimulated with the same MOI.

Figure 2

TNF-α regulation by PDLF is post-transcriptional. Human macrophages (Mφ) were mono or co-cultured with PDLF prior to stimulation with increasing MOIs of P. gingivalis. TNFA (a), IL1B (b), TIA-1 (c) and TTP (d) levels were measured by qRT-PCR 5 hours after bacterial stimulation and normalized to the level of HBA. Relative quantity of each transcript at the indicated MOI was compared to MOI 0 in the same culture type. Data represent means + SEM of at least 3 separate experiments. Transcript levels were compared to the level in MOI 0 of the same culture, and between culture types at the same MOI. *P < 0.05; ***P < 0.001; n.s = not significant.

Figure 3

IL-6 and IL-10 contribute to the modulation of TNF-α secretion by stimulated Mφ. Human macrophages (Mφ) were mono or co-cultured with PDLF prior to stimulation with increasing MOIs of P. gingivalis. IL-6 (a), IL-10 (b) and TGF-β (c) production were measured in the supernatant 5 hours after bacterial stimulation. Dashed line represents cytokine secretion by mono-cultured fibroblasts. Blocking of IL-10 or IL-6 in co-cultures of Mφ and PDLF decreased the modulatory effect over TNF-α production following stimulation with P. gingivalis (d). Data represent the means + SEM of at least 3 separate experiments. **P value < 0.01 compared to the cytokine production by Mφ in mono-culture stimulated with the same MOI (b) or to co-cultures of Mφ and PDLF treated with isotype control and the same MOI (d). *P < 0.05; **P < 0.01; ***P < 0.001 compared to the cytokine production by macrophages in mono-culture stimulated with the same MOI (a,b,c), or to co-cultures of Mφ and PDLF treated with isotype control and the same MOI (d).

Figure 4

PDLF increase Mφ phagocytosis of P. gingivalis. Mono-cultures or co-cultures of Mφ and PDLF were infected with FITC-labeled P. gingivalis for up to 105 minutes. Following incubation, cells were thoroughly washed and quenched with trypan blue. Phagocytosis was assessed using a fluorescence reader, and data are shown as relative fluorescence units (RFU). Data represent the means + SEM of at least 5 separate experiments. ***P < 0.001 between Mφ in mono-culture and Mφ in co-culture with PDLF at the tested time point.

Figure 5

Fibroblasts down regulate SDF-1α mediated monocyte migration. CFSE labeled human monocytes were migrating in a 5 mm transwell chamber towards a gradient of SDF-1α, either with or without PDLF in the bottom chamber. After 3 hours of migration, CFSE positive cells in the upper and the lower chambers were counted by FACS. Fold induction of migration was calculated in comparison to spontaneous migration of monocytes in mono-culture without the presence of an SDF-1α gradient in the chamber. Data represent means + SEM of 3 different experiments. **P < 0.01; ***P < 0.001; n.s = not significant.

Figure 6

HGF cell line, but not donor matched primary GFs, is less modulatory than PDLF. Human Mφ were cultured alone or together with PDLF, or an HGF-1 cell line (a). Separately, Mφ were cultured alone or together with primary GF vs. PDLF from the same or different donors (b). Cells were stimulated with increasing MOIs of P. gingivalis and TNF-α production was tested in the supernatant after 5 hours. Data represent the means + SEM of at least 3 separate experiments. *P < 0.05; **P < 0.01 and ***P < 0.001 in comparison to the cytokine production by Mφ in mono-culture stimulated with the same MOI.

Figure 7

Inflammation increases the modulatory activity of oral fibroblasts. Mφ were cultured alone or together with primary PDLF, GF and PIF (a) or with primary PDLF or GF primed with IL1-β or TNF-α for 3 days. TNF-α production was tested following stimulation with P. gingivalis for 5 hours. Data represent the means + SEM of at least 3 separate experiments. *P < 0.05; **P < 0.01 and ***P < 0.001 in comparison to the cytokine production by Mφ in mono-culture stimulated with the same MOI (a), or to cytokine production by Mφ co-cultured with un-primed fibroblasts and stimulated with the same MOI.