Photobiomodulation of oral fibroblasts stimulated with periodontal pathogens

Abstract

Photobiomodulation (PBM) utilises light energy to treat oral disease, periodontitis. However, there remains inconsistency in the reporting of treatment parameters and a lack of knowledge as to how PBM elicits its molecular effects in vitro. Therefore, this study aimed to establish the potential immunomodulatory effects of blue and near infra-red light irradiation on gingival fibroblasts (GFs), a key cell involved in the pathogenesis of periodontitis. GFs were seeded in 96-well plates in media + / - Escherichia coli lipopolysaccharide (LPS 1 μg/ml), or heat-killed Fusobacterium nucleatum (F. nucleatum, 100:1MOI) or Porphyromonas gingivalis (P. gingivalis, 500:1MOI). Cultures were incubated overnight and subsequently irradiated using a bespoke radiometrically calibrated LED array (400-830 nm, irradiance: 24 mW/cm² dose: 5.76 J/cm²). Effects of PBM on mitochondrial activity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and adenosine triphosphate (ATP) assays, total reactive oxygen species production (ROS assay) and pro-inflammatory/cytokine response (interleukin-8 (IL-8) and tumour growth factor-β1 (TGFβ1)) were assessed 24 h post-irradiation. Data were analysed using one-way ANOVA followed by the Tukey test. Irradiation of untreated (no inflammatory stimulus) cultures at 400 nm induced 15%, 27% and 13% increases in MTT, ROS and IL-8 levels, respectively (p < 0.05). Exposure with 450 nm light following application of P. gingivalis, F. nucleatum or LPS induced significant decreases in TGFβ1 secretion relative to their bacterially stimulated controls (p < 0.001). Following stimulation with P. gingivalis, 400 nm irradiation induced 14% increases in MTT, respectively, relative to bacteria-stimulated controls (p < 0.05). These findings could identify important irradiation parameters to enable management of the hyper-inflammatory response characteristic of periodontitis.

Keywords: Fibroblast; Mitochondria; PBM; Periodontitis; Photobiomodulation.

Fig. 1

Blue light modulates markers for mitochondrial activity and inflammation. a High-throughput analysis of various wavelengths (400–830 nm) and irradiation periods (30–240 s) on cell metabolic activity of pooled primary human gingival fibroblasts (B19, B16, B15 samples, 24mW/cm², 0.72–5.76 J/cm², 30–240 s) 24 h post-irradiation was assessed via MTT assay. The effects of PBM (400 nm, 450 nm or 810 nm, 5.76 J/cm², 24mW/cm²) on markers for mitochondrial activity and inflammation were measured 24 h post-irradiation. PBM-induced changes in mitochondrial activity were assessed via ROS (b) and ATP (c) assays. Cell lysates were collected, RNA extracted and RT-PCR employed to elucidate PBM-induced changes in IL-8 (d) and TGFβR1 (e) gene expression. Evaluation of PBM on IL-8 (f) and TGFβ1 (g) secretion was undergone via collection of supernatants and analysis by ELISA. All experiments were performed in triplicate and presented as mean ± SD. Significance was assessed using one-way ANOVA followed by Tukey test and is indicated by ***p < 0.001, **p < 0.01 and *p < 0.05 relative to the non-irradiated control, where all data is shown as a percentage of the non-irradiated control, where the non-irradiated control was normalised to 0%

Fig. 2

LPS, F. nucleatum and P. gingivalis modulate markers for inflammation in a dose-dependent manner. Following 24 h incubation, pHGFs (B15, B16 and B19, p5-8) ± LPS (1-5 μg/ml)/heat-inactivated F. nucleatum (50–500: 1 MOI)/heat-inactivated P. gingivalis (50–500:1 MOI) were assayed for ROS production (ROS assay a, c) and IL-8 secretion (ELISA, b, d). All experiments were performed in triplicate and presented as mean ± SD. Significance was assessed using one-way ANOVA followed by the Tukey test and is indicated by ***p < 0.001, **p < 0.01 and *p < 0.05 relative to the non-irradiated control, where all data is shown as a percentage of the corresponding untreated control, where the untreated control was normalised to 0%

Fig. 3

PBM modulates bacterially induced changes in markers for cell metabolic activity and inflammation. ppHGFs (B15, B16 and B19, p5-8) ± LPS (1 μg/ml, i)/heat-inactivated P. gingivalis (500:1 MOI, ii)/heat-inactivated F. nucleatum (100:1 MOI, iii) were subsequently treated with PBM (400–810 nm, 5.76 J/cm², 24mW/cm²). PBM-induced changes in bacterially stimulated changes in a cell metabolic activity (MTT assay), b ROS production (marker for mitochondrial activity, ROS assay) and c TGFβ1 secretion (marker for inflammation, ELISA on collected supernatants) were then assessed 24 h post-irradiation. All experiments were performed in triplicate and presented as mean ± SD. Significance was assessed using one-way ANOVA followed by Tukey’s test and results are indicated by ****p < 0.0001, ***p < 0.001, **p < 0.01 and *p < 0.05 relative to the respective non-irradiated controls. All data is shown as a percentage of the non-irradiated control, where the non-irradiated control in each experimental group (LPS, F. nucleatum or P. gingivalis) was normalised to 0%

Fig. 4

The effects of PBM on IL-8 secretion are bacterial stimulus–dependent. pHGFs (B15, B16 and B19, p5-8) ± LPS (1 μg/ml, a)/heat-inactivated F. nucleatum (100:1 MOI, b)/heat-inactivated P. gingivalis (500:1 MOI, c) were subsequently treated with ± (400–810 nm, 5.76 J/cm², 24mW/cm²). PBM-induced changes in bacterially stimulated changes in IL-8 secretion were then assessed 24 h post-irradiation. All experiments were performed in triplicate and presented as mean ± SD. Significance was assessed using one-way ANOVA followed by Tukey’s test and is indicated by ****p < 0.0001 ***p < 0.001, **p < 0.01 and *p < 0.05 relative to the respective non-irradiated controls. All data is shown as a percentage of the corresponding stimulated control, where the stimulated control in each experimental group (LPS, F. nucleatum or P. gingivalis) was normalised to 100%