hTERT-immortalized gingival fibroblasts respond to cytokines but fail to mimic primary cell responses to Porphyromonas gingivalis

Abstract

In periodontitis, gingival fibroblasts (GFs) interact with and respond to oral pathogens, significantly contributing to perpetuation of chronic inflammation and tissue destruction. The aim of this study was to determine the usefulness of the recently released hTERT-immortalized GF (TIGF) cell line for studies of host-pathogen interactions. We show that TIGFs are unable to upregulate expression and production of interleukin (IL)-6, IL-8 and prostaglandin E2 upon infection with Porphyromonas gingivalis despite being susceptible to adhesion and invasion by this oral pathogen. In contrast, induction of inflammatory mediators in TNFα- or IL-1β-stimulated TIGFs is comparable to that observed in primary GFs. The inability of TIGFs to respond directly to P. gingivalis is caused by a specific defect in Toll-like receptor-2 (TLR2) expression, which is likely driven by TLR2 promoter hypermethylation. Consistently, TIGFs fail to upregulate inflammatory genes in response to the TLR2 agonists Pam2CSK4 and Pam3CSK4. These results identify important limitations of using TIGFs to study GF interaction with oral pathogens, though these cells may be useful for studies of TLR2-independent processes. Our observations also emphasize the importance of direct comparisons between immortalized and primary cells prior to using cell lines as models in studies of any biological processes.

Figure 1

TIGFs internalize P. gingivalis but fail to mount an inflammatory response after bacterial challenge. (a) P. gingivalis internalization by PHGFs and TIGFs determined by colony-forming assay. Cells were infected with P. gingivalis (MOI 100) for 1 h and then cultured for another 1 h in medium, with or without antibiotics. Results of four independent experiments are presented as boxplots, where the line within the box denotes the median number of colony-forming units (CFU)/cell, the boxes represent the 25th and 75th percentiles, and the lines outside the box mark the minimum and maximum values. **P < 0.01; unpaired t-test (b) Secretion of IL-6 and IL-8 by PHGFs and TIGFs (n = 6–7) infected with increasing MOI of P. gingivalis (10, 50, 100) for 1 h followed by washing, then 23 h culture in fresh medium. *P < 0.05; ***P < 0.001; 1-way ANOVA followed by Bonferroni multiple comparison test. (c) Production of PGE2 by PHGFs and TIGFs (n = 8–9) infected with P. gingivalis (MOI 100) for 24 h. *P < 0.05; paired t-test. Data in (b,c) are presented as mean concentration + SEM. (d) Relative mRNA expression of IL6, IL8, COX2 and mPGES1 in PHGFs and TIGFs (n = 5–7) infected with P. gingivalis (MOI 100) for 24 h. Data are presented as mean relative expression + SEM. *P < 0.05; **P < 0.01; ***P < 0.001; paired t-test.

Figure 2

TIGFs and PHGFs respond to cytokine stimulation in a similar manner. (a) Relative mRNA expression of IL6, IL8 and COX2 in PHGFs and TIGFs that were left unstimulated in medium (med) or were stimulated with TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 24 h. Data for PHGFs from individual donors (n = 3) and TIGFs from independent experiments (n = 3) are shown on a heat map as column Z-scores calculated from ΔCt values relative to a housekeeping gene (RPLP0). (b) Secretion of IL-8 by PHGFs and TIGFs (n = 8) and stimulated as in (a). Data are presented as mean concentration + SEM. **P < 0.01; ***P < 0.001; 1-way ANOVA followed by Bonferroni multiple comparison test.

Figure 3

TNFα stimulation, but not P. gingivalis infection, activates NF-κB and MAP kinase signaling in TIGFs. Western blot analysis of p65, p38, and ERK phosphorylation in PHGFs and TIGFs (a) after stimulation with TNFα (10 ng/ml) or (b) upon infection with P. gingivalis (MOI 50) for the indicated time points. Actin was used as loading control and medium (med) indicates unstimulated/uninfected cells. Results shown are representative of 3–5 independent experiments and full-length blots are presented in Supplementary Fig. S1. (c) Densitometric analysis of p38 and p65 phosphorylation in P. gingivalis-infected cells (n = 4–5). Data are shown as mean signal intensity relative to actin. *P < 0.05; ***P < 0.001; 1-way ANOVA followed by Bonferroni multiple comparison test.

Figure 4

TIGFs lack TLR2 expression and fail to respond to TLR2 agonists. (a) Western blot analysis of TLR2 protein expression in PHGF and TIGF total cell lysates. U-251 MG cells overexpressing hTLR2 were used as a positive control and actin was used as a loading control. Results presented are representative of two independent experiments and full-length blots are presented in Supplementary Fig. S2. (b) Relative mRNA expression of TLR2 in PHGFs and TIGFs (n = 3) that were left unstimulated (med) or were stimulated with TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 24 h. (c) Relative mRNA expression of IL6, IL8 and COX2 in PHGFs and TIGFs stimulated with Pam2CSK4 (1 μg/ml) or Pam3CSK4 (1 μg/ml) for 24 h. Data for PHGFs from individual donors (n = 3) and TIGFs from independent experiments (n = 3) are shown on a heat map as column Z-scores calculated from ΔCt values relative to a housekeeping gene (RPLP0). (d) Secretion of IL-6 and IL-8 by PHGFs and TIGFs (n = 3) and stimulated as in (c). (e) TLR2 promoter methylation in PHGFs and TIGFs (n = 3) analyzed using the EpiTect methyl II PCR assay and presented as mean percentage of methylated DNA + SEM. (f) Relative TLR4 mRNA expression in PHGFs and TIGFs (n = 3). (g) Western blot analysis of TLR4 protein expression in total cell lysates of PHGFs and TIGFs. Actin was used as a loading control. (h) Secretion of IL-6 and IL-8 by PHGFs and TIGFs (n = 3–4) infected with increasing MOI of F. nucleatum (10, 50) for 24 h. (i) Secretion of IL-6 and IL-8 by PHGFs and two independent batches of TIGFs (TIGF-1 and TIGF-2) (n = 2–3) infected with increasing MOI of P. gingivalis (10, 50, 100). Data are shown as mean relative mRNA expression + SEM (b,f) or mean concentration + SEM (d,h,i). ***P < 0.001; 1-way ANOVA followed by Bonferroni multiple comparison test.