Efficacy of probiotic Streptococcus thermophilus in counteracting TGF-β1-induced fibrotic response in normal human dermal fibroblasts

Abstract

Background: Abnormal and deregulated skin wound healing associated with prolonged inflammation may result in dermal fibrosis. Since the current therapeutic strategies revealed unsatisfactory, the investigation of alternative approaches such as those based on the use of specific probiotic strains could provide promising therapeutic options. In this study, we aimed to evaluate whether the lysate from S. thermophilus could antagonize the fibrogenic effects of TGF-β1 in normal human dermal fibroblasts (NHDF).

Methods: NHDF were exposed to TGF-β1 to establish a fibrotic phenotype. Proliferation rate and cell number were measured using the IncuCyte® Live Cell Imager system and the trypan blue dye exclusion test. Phenoconversion markers (α-SMA and fibronectin) and collagen I levels were assessed by western blot and immunofluorescence. The mRNA levels of TGF-β1 were evaluated by RT-PCR. The Smad2/3 phosphorylation level as well as β-catenin and PPARγ expression, were assessed by western blot. The cell contractility function and migration of NHDF were studied using collagen gel retraction assay, and scratch wound healing assay, respectively. The effects of S. thermophilus lysate, alone or combined with TGF-β1, were evaluated on all of the above-listed parameters and markers associated with TGF-β1-induced fibrotic phenotype.

Results: Exposure to the S. thermophilus lysate significantly reduced the key mediators and events involved in the abnormal activation of myofibroblasts by TGF-β1 within the fibrotic profile. The S. thermophilus treatment significantly reduced cell proliferation, migration, and myo-differentiation. In addition, the treatment with probiotic lysate reduced the α-SMA, fibronectin, collagen-I expression levels, and affected the collagen contraction ability of activated dermal fibroblasts. Moreover, the probiotic targeted the TGF-β1 signaling, reducing Smad2/3 activation, TGF-β1 mRNA level, and β-catenin expression through the upregulation of PPARγ.

Conclusion: This is the first report showing that S. thermophilus lysate had a remarkable anti-fibrotic effect in TGF-β1-activated NHDF by inhibiting Smad signaling. Notably, the probiotic was able to reduce β-catenin and increase PPARγ levels. The findings support our point that S. thermophilus may help prevent or treat hypertrophic scarring and keloids.

Keywords: Fibrotic markers; PPARγ; S. thermophilus; Skin fibrosis; Smad signaling; TGF-β1; β-catenin.

Fig. 1

Effect of S. thermophilus lysate on TGF-β1-induced proliferation and migration of NHDF. A Growth curves of NHDF were analyzed as cell confluence percentage through IncuCyte® Live Cell Imager and monitored dynamically from 0 to 72 h. NHDF were starved for 24 h and then incubated until 3 days with TGF-β1 (10 ng/ml) in the presence or absence of S. thermophilus lysate (25, 50 or 100 µg/ml), in 0.5% FBS medium. Results relating to one representative out of three experiments performed in triplicate, are expressed as mean ± SD. B NHDF were treated for 48 h as reported in A and cell number was counted by trypan blue staining. The results, derived from three experiments performed in duplicate, are expressed as mean ± SEM. C Quantitative analysis of wound healing assay in NHDF treated as above in A. The wound closure was captured at 0, 24, and 48 h after scratch generation and expressed as the wound closure rate (% vs. relative T0) of scratched monolayers. Values are expressed as mean ± SEM of two independent experiments in duplicate. In each case, the comparative analysis of groups of data has been performed by the two-way analysis of variance (ANOVA) followed by post hoc Tukey’s test (** P < 0.01, **** P < 0.0001 vs. CNTR; § P < 0.05, §§ P < 0.01, §§§ P < 0.001, §§§§ P < 0.0001 vs. TGF-β1). Representative images of cell confluence, cell number and NHDF monolayer re-epithelialization (the yellow dashed lines indicate wound edges at T0) are inserted in A, B and C respectively

Fig. 2

Effects of S. thermophilus lysate on TGF-β1-induced myofibroblast phenoconversion. Immunoblotting assays for A α-SMA and C fibronectin were performed on NHDF treated as previously reported. Densitometric analysis was performed by normalizing vs. GAPDH. Values are expressed as mean ± SEM of three independent experiments. For comparative analysis of data, a one-way analysis of variance (ANOVA) with post hoc Tukey’s test was used. (* P < 0.05, ** P < 0.01 vs. CNTR; § P < 0.05, §§ P < 0.01, §§§ P < 0.001 vs. TGF-β1). Representative images of each immunoblotting are shown. Representative immunofluorescence images of NHDF stained with anti-α-SMA antibody (B) (green) or anti-fibronectin antibody (D) (green) and with TRITC-phalloidin (red) to reveal F-actin. Nuclei were counterstained with DAPI (blue) (magnification 100 x). The images are representative of three independent experiments in duplicate

Fig. 3

Effects of S. thermophilus lysate on collagen I production in TGF-β1-activated NHDF and ECM remodeling. A Immunoblotting assay for collagen I was performed on NHDF treated as previously described. Following densitometric analysis, obtained values were normalized vs. GAPDH. Values are expressed as mean ± SEM of three independent experiments performed in duplicate. Images from one representative out of three independent experiments are presented. B Representative immunofluorescence images of NHDF stained with anti-collagen I antibody (green) and with TRITC-phalloidin (red) to reveal F-actin. Nuclei were counterstained with DAPI (blue) (magnification 100 x). The images are representative of three independent experiments in duplicate. C Collagen gel retraction assay of NHDF-populated collagen lattices. The gel contraction was digitally photo-documented, and the gel area was measured with ImageJ and normalized to pre-release area (T0). Values of normalized area are expressed as mean ± SEM of two independent experiments in duplicate. D Representative images of collagen gel pre-release (T0) and taken 48 h after treatments are shown. In all cases, the comparative analysis of data has been carried out by using one-way analysis of variance (ANOVA) followed by post hoc Tukey’s test (** P < 0.01 vs. CNTR; *** P < 0.001 vs. CNTR; § P < 0.05, §§ P < 0.01 vs. TGF-β1)

Fig. 4

Effects of S. thermophilus lysate on TGF-β1-induced Smad signaling. A Immunoblotting assay for pSmad2/3 protein expression was performed on NHDF treated as described above. Following densitometric analysis, obtained values were normalized vs. GAPDH. Values are expressed as mean ± SEM of two independent experiments performed in duplicate. Representative images of immunoblotting for pSmad2/3 and GAPDH are shown. B The SYBRGreen Real-Time PCR analysis of the TGF-β1 gene was performed on NHDF. mRNA levels were relative to the amount of GAPDH mRNA. Data from one of two independent experiments in duplicate are shown as mean ± SD. In all cases, the comparative analysis of data has been carried out by using one-way analysis of variance (ANOVA) followed by post hoc Tukey’s test (** P < 0.01 vs. CNTR; **** P < 0.0001 vs. CNTR; §§ P < 0.01 vs. TGF-β1, §§§ P < 0.001 vs. TGF-β1)

Fig. 5

Effect of S. thermophilus lysate on β-catenin and PPARγ expression in TGF-β1-activated NHDF. A β-catenin and B PPARγ protein expressions were evaluated on NHDF incubated as above described, by western blot analysis. Following densitometric analysis, obtained values were normalized vs. GAPDH. Values are expressed as mean ± SEM of two independent experiments in duplicate. For comparative analysis of data, a one-way analysis of variance (ANOVA) with post hoc Tukey test was used (*P < 0.05, **P < 0.01 vs. CNTR; §§ P < 0.01, §§§ P < 0.001 vs. TGF-β1). Representative images of immunoblotting for β-catenin, PPARγ, and GAPDH are shown