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. 2019 Jun 11;20(1):119.
doi: 10.1186/s12931-019-1093-z.

Pirfenidone attenuates lung fibrotic fibroblast responses to transforming growth factor-β1

Jin Jin  1   2   3 , Shinsaku Togo  4   5   6 , Kotaro Kadoya  2   3 , Miniwan Tulafu  2   3 , Yukiko Namba  2   3   7 , Moe Iwai  2   3 , Junko Watanabe  2   3 , Kumi Nagahama  2   3 , Takahiro Okabe  2   3 , Moulid Hidayat  2   3 , Yuzo Kodama  2   3 , Hideya Kitamura  7 , Takashi Ogura  7 , Norikazu Kitamura  8 , Kazuho Ikeo  8   9 , Shinichi Sasaki  10 , Shigeru Tominaga  10 , Kazuhisa Takahashi  2   3
Affiliations

Pirfenidone attenuates lung fibrotic fibroblast responses to transforming growth factor-β1

Jin Jin et al. Respir Res. .

Abstract

Background: Pirfenidone, an antifibrotic agent used for the treatment of idiopathic pulmonary fibrosis (IPF), functions by inhibiting myofibroblast differentiation, which is involved in transforming growth factor (TGF)-β1-induced IPF pathogenesis. However, unlike normal lung fibroblasts, the relationship between pirfenidone responses of TGF-β1-induced human fibrotic lung fibroblasts and lung fibrosis has not been elucidated.

Methods: The effects of pirfenidone were evaluated in lung fibroblasts isolated from fibrotic human lung tissues after TGF-β1 exposure. The ability of two new pharmacological targets of pirfenidone, collagen triple helix repeat containing protein 1(CTHRC1) and four-and-a-half LIM domain protein 2 (FHL2), to mediate contraction of collagen gels and migration toward fibronectin were assessed in vitro.

Results: Compared to control lung fibroblasts, pirfenidone significantly restored TGF-β1-stimulated fibroblast-mediated collagen gel contraction, migration, and CTHRC1 release in lung fibrotic fibroblasts. Furthermore, pirfenidone attenuated TGF-β1- and CTHRC1-induced fibroblast activity, upregulation of bone morphogenic protein-4(BMP-4)/Gremlin1, and downregulation of α-smooth muscle actin, fibronectin, and FHL2, similar to that observed post-CTHRC1 inhibition. In contrast, FHL2 inhibition suppressed migration and fibronectin expression, but did not downregulate CTHRC1.

Conclusions: Overall, pirfenidone suppressed fibrotic fibroblast-mediated fibrotic processes via inverse regulation of CTHRC1-induced lung fibroblast activity. Thus, CTHRC1 can be used for predicting pirfenidone response and developing new therapeutic targets for lung fibrosis.

Keywords: BMP-4; Collagen triple helix repeat containing protein 1(CTHRC1); Four-and-a-half LIM domain protein 2(FHL-2); Lung fibroblast; Lung fibrosis; Pirfenidone; Transforming growth factor-β1.

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Conflict of interest statement

The authors declare no competing or financial interests.

Figures

Fig. 1
Fig. 1
Effects of pirfenidone on TGF-β1-stimulated collagen gel contraction and chemotaxis in HFL-1 cells. HFL-1 cells were cultured and cast into three-dimensional collagen gels that were maintained in suspension; the gel size was measured daily. HFL-1 cells grown in a monolayer culture were trypsinized and their chemotactic activity toward fibronectin (20 μg/mL) was assessed using the Boyden blind well chamber technique. (a) Collagen gel contraction in the presence of various concentrations of pirfenidone in the presence or absence of 0.25 ng/mL (10 pM) TGF-β1. (b) Collagen gel contraction following treatment with various concentrations of TGF-β1 in the presence or absence of 100 μg/mL pirfenidone. (c) The number of migrated fibroblasts following treatment with various concentrations of pirfenidone in the presence or absence of 0.25 ng/mL TGF-β1. (d) Number of migrated fibroblasts following treatment with various concentrations of TGF-β1 in the presence or absence of 100 μg/mL pirfenidone. Collagen gel contraction vertical axis: gel size measured after 2 days of contraction expressed as a percentage of the initial value. Chemotaxis vertical axis: number of migrated cells per five high-power fields (5 HPF). Horizontal axis: conditions. Values represent means ± SEMs of three to five separate experiments, each of which included three replicates. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 2
Fig. 2
Effects of pirfenidone on collagen gel contraction and chemotaxis in primary lung fibroblasts. Fibroblasts from controls and patients with pulmonary fibrosis (PF) were cultured in the presence or absence of pirfenidone (100 μg/mL) and 0.25 ng/mL TGF-β1, and collagen gel contraction and chemotaxis were assayed. (a) Collagen gel contraction. Vertical axis: gel size measured after 2 days of contraction expressed as a percentage of the initial gel area. Horizontal axis: conditions. (b) Pirfenidone-induced suppression of collagen gel contraction in lung fibroblasts isolated from controls and patients with IP. Vertical axis: percentage of gel contracted size following pirfenidone treatment ([difference in gel size/initial gel size] × 100). Horizontal axis: conditions. (c) Fibroblast chemotaxis. Vertical axis: number of migrated fibroblasts per 5 HPF. Horizontal axis: conditions. (d) Pirfenidone-dependent suppression of chemotaxis in lung fibroblasts isolated from controls and patients with IP. Vertical axis: percentage of migrated cells following pirfenidone treatment ([difference in migrated cell number/initial migrated cells number] × 100). Horizontal axis: conditions. Each patient evaluated was expressed as an individual symbol, representing the means of two to three separate experiments, each of which included three replicates. (a, c) Lines connect the values for individual patients in the presence or absence of pirfenidone. *P < 0.05, **P < 0.01, ***P < 0.001. (b, d) Student’s paired t test and unpaired t test were used. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 3
Fig. 3
High sensitivity of TGF-β1-induced fibrotic mediators in fibrotic lung fibroblasts. Subconfluent fibroblasts from 12 controls and 12 patients with PF were cultured in serum-free (SF)-DMEM for 24 h and then incubated in the presence or absence of TGF-β1 (0.25 ng/mL) and/or pirfenidone (100 ng/mL) for 48 h. Proteins from monolayer cultured fibroblasts were extracted and subjected to western blot analysis, and media were harvested from monolayer cultures and evaluated for CTHRC1 and TGF-β1 levels using immunoassay. Expression of (a) CTHRC1 (30 kDa) and (b) FHL2 (30 kDa) from fibroblasts isolated from controls and patients with PF in the presence or absence of TGF-β1 (0.25 ng/mL). Vertical axis: Protein expression normalized to β-actin expression. Immunoassay of (c) CTHRC1 and (d) TGF-β1. Vertical axis: mediator production expressed as an amount. Symbols represent the mean values for individual patients, as assessed in two separate experiments. Horizontal axis: conditions. Student’s paired t test was used. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 4
Fig. 4
Effects of pirfenidone on TGF-β1-mediated fibrotic regulators in lung fibroblasts. Subconfluent HFL-1 cells were cultured in SF-DMEM for 24 h and then incubated in the presence or absence of TGF-β1 (0.25 ng/mL) and pirfenidone (100 ng/mL). (a) CTHRC1 and FHL2 expression in HFL-1 cells was determined using fluorescence-immunohistochemistry with primary antibodies against CTHRC1 or FHL2, followed by incubation with secondary antibodies labeled with Alexa Fluor 488 goat anti-rabbit IgG (green). The scale bar indicates 50 μm. (b) Western blot analysis to determine the effects of pirfenidone on targets related to TGF-β1-mediated fibrotic processes, including CTHRC1 (30 kDa), FHL2 (30 kDa), α-SMA (42 kDa), fibronectin (250 kDa), BMP4 (47 kDa), Gremlin1 (25 kDa), and β-actin (42 kDa). The vertical axis shows the relative intensities of (c) CTHRC1, (d) FHL2, (e) α-SMA, (f) fibronectin, (g) BMP4, and (h) Gremlin1 versus β-actin; the horizontal axis shows the conditions. Values represent means ± SEMs of three to five separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 5
Fig. 5
Effects of pirfenidone on CTHRC1-mediated regulation in lung fibroblasts. Subconfluent HFL-1 cells were cultured in SF-DMEM for 24 h and then incubated in the presence or absence of different concentrations of rhCTHRC1. Western blot analysis of the effects of different concentrations of rhCTHRC1 on targets related to fibrotic processes (a). Effects of different concentrations of rhCTHRC1 on HFL-1 cell-mediated collagen gel contraction (b) and chemotaxis (c). Effects of different concentrations of rhCTHRC1-mediated targets assayed using western blot analysis (d-h). The vertical axis shows the relative intensities of FHL2 (D), α-SMA (e), fibronectin (f), BMP4 (g), Gremlin1 (h) versus β-actin; the horizontal axis shows the conditions. Subconfluent HFL-1 cells were cultured in SF-DMEM for 24 h and then incubated in the presence or absence of rhCTHRC1 (100 ng/mL) and pirfenidone (100 ng/mL) for 48 h. Western blot analysis to analyze the effects of pirfenidone on rhCTHRC1-mediated targets related to fibrotic processes (i). Effects of pirfenidone on rhCTHRC1-mediated collagen gel contraction (j) and chemotaxis (k). Effects of pirfenidone on the expression levels of rhCTHRC1-mediated targets assayed using western blot analysis (l-p). The vertical axis shows the relative intensities of α-SMA (l), fibronectin (m), FHL2 (n), BMP4 (o), Gremlin1 (p) versus β-actin; the horizontal axis shows the conditions. Collagen gel contraction, vertical axis: gel size measured after 2 days of contraction expressed as a percentage of the initial value. Chemotaxis, vertical axis: number of migrated cells per 5 HPF. Horizontal axis: conditions. Values represent means ± SEMs of three to five separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 6
Fig. 6
Effects of CTHRC1 and FHL2 knockdown in HFL-1 cells. Collagen gel contraction and chemotaxis were assessed in CTHRC1- and FHL2-knocked down HFL-1 cells. Western blot analysis of the effects of CTHRC1 silencing on targets related to fibrotic processes (a). Collagen gel contraction (b) and chemotaxis (c) after silencing of CTHRC1. The effect of CTHRC1 knockdown on TGF-β1 (0.25 ng/mL)-induced gel contraction (d) and chemotaxis toward to fibronectin (e). Western blot analysis to analyze the effects of FHL2 silencing on targets related to fibrotic processes (f). Collagen gel contraction and (g) chemotaxis (h) after silencing of FHL2. Collagen gel contraction, vertical axis: gel size measured after 2 days of contraction expressed as a percentage of the initial value. Chemotaxis, vertical axis: number of migrated cells per 5 HPF. Horizontal axis: conditions. Values represent means ± SEMs of three separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 7
Fig. 7
Relationship between pirfenidone’s effect on TGF-β1-stimulated fibroblast bioactivity in vitro and biomarkers of lung fibrosis. Comparison of the relationships between suppression of TGF-β1-induced gel contraction by pirfenidone and serum (a) KL-6 and (b) SP-D levels, and between suppression of TGF-β1-induced migration and (c) KL-6 and (d) SP-D levels. Symbols represent individual patients. Linear regression was used. P < 0.05 indicates a positive relationship between pirfenidone response to TGF-β1-stimulated fibroblast bioactivity in vitro and biomarkers of lung fibrosis. (e) Schematic showing the mechanism via which pirfenidone controls TGF-β1-induced changes in fibrotic lung fibroblasts. Lung fibroblasts are continually exposed to TGF-β1 via airway cells-fibroblasts interaction, which is regulated by mediators released by airway cells under inflammatory conditions, resulting in distinct phenotype of fibrotic fibroblasts. This high sensitivity to TGF-β1, along with upregulation of CTHRC1 and FHL2, leads to the development of fibrosis in fibrotic fibroblasts. Treatment with pirfenidone can effectively block this process
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