Anti-fibrotic effect of ciglitazone in HRV-induced airway remodelling cell model

Abstract

Fibrosis is an important phenomenon as it can occur early in the pathogenesis of asthma; it may be associated with disease severity and resistance to therapy. There is a strong evidence that infection caused by human rhinovirus (HRV) contributes to remodelling process, but there is lack of studies clearly explaining this pathway. Synthetic peroxisome proliferator-activated receptor (PPAR) γ presents immunomodulatory and anti-inflammatory features. In this study, we examined immunomodulatory properties of ciglitazone - PPAR-γ agonist, in development and modulation of airway remodelling. Epithelial cells (NHBE) and two lines of fibroblasts (WI-38, HFL1) were stimulated with ciglitazone and rhinovirus. The expression of genes related to airway remodelling process were analysed in the cells; moreover NF-κB, c-Myc and STAT3 were silenced in order to estimate potential pathways involved. Ciglitazone decreased mRNA expression of MMP-9 and TGF-β. It also modified the expression of α-SMA and collagen after rhinovirus infection. Transcription factors knockdown altered the levels of expression. The results suggest possible anti-fibrotic activity of PPAR-γ agonist in human airway cells. Ciglitazone has been shown to be dependent on NF-κB- and STAT3-related pathways, thus, the PPAR-γ agonist may have therapeutic potential for the treatment of airway remodelling in asthma.

Keywords: MMP-9; PPAR-γ; TGF-β; airway remodelling; asthma; ciglitazone; collagen I.

FIGURE 1

Viability of WI‐38, HFL1 and NHBE cells after exposure to ciglitazone in concentration of 1 μM (A), 3 μM (B) and 5 μM (C). Cell viability was assessed after 24 h. The values represent mean viability.

FIGURE 2

The result of ciglitazone stimulation on rhinovirus‐triggered mRNA expression of airway remodelling‐involved genes. Two serotypes of Rhinovirus 2 (minor serotype) and 16 (major serotype) were used. Ciglitazone (Ci) has been added before rhinovirus infection (Ci+H16, Ci+H2) or after rhinovirus infection (H16+Ci, H2+Ci). Ciglitazone decreased the expression of MMP‐9 (A) in fibroblasts, and TGF‐β (B) in fibroblasts and epithelial cells (p < 0.05). Rhinoviruses increased the expression of all genes analysed in fibroblasts (p < 0.05). Ciglitazone reversed the effect of rhinoviruses by decreasing the mRNA expression of MMP‐9 (A), TGF‐β (B), ADAM33 (D), and α‐SMA (E) in fibroblasts, whereas in the case of epithelial cells, its action was observed in the COL I expression only (C, p < 0.05). *p < 0.05 in comparison to the control sample, +p < 0.05 in comparison to the rhinovirus sample (HRV‐2 or HRV‐16, respectively); C – control sample with medium only.

FIGURE 3

Effect of ciglitazone to reverse HRV‐stimulated proteins. Fibroblasts (A, C – HFL1 cell line and B – WI‐38 cell line) were infected with human rhinovirus and stimulated with PPAR‐γ agonist – ciglitazone (Ci). The cells were chosen to be presented, as no changes were observed in epithelial cells. Ciglitazone (Ci) has been added before rhinovirus infection (Ci+H16, Ci+H2) or after rhinovirus infection (H16+Ci, H2+Ci). Concentrations of MMP‐9 (A) and TGF‐β (B,C), were measured in supernatants utilizing immunoassay in triplicate. Data presented as % of change, normalizing to control sample ± SD. +<0.05. *p < 0.05 in comparison to the control sample, +p < 0.05 in comparison to the rhinovirus sample (HRV‐2 or HRV‐16, respectively).

FIGURE 4

The effect of ciglitazone treatment on remodelling process induced by rhinovirus, under conditions of siRNA knock down of NF‐κB transcription factor. Rhinovirus 2 (H2, minor serotype) and 16 (H16, major serotype) were used. Ciglitazone (Ci) has been added before rhinovirus infection (Ci+H16, Ci+H2) or after rhinovirus infection (H16+Ci, H2+Ci). NF‐κB silencing eliminated effects of HRV and ciglitazone in MMP‐9 (A) and TGF‐β (B), (p > 0.05). The knockdown reversed changes introduced by ciglitazone in the expression of MMP‐9 (A), COL I (C), α‐SMA (E), and partly in TGF‐β (B) and ADAM 33 (D), in comparison to cells expressing NF‐κB (p < 0.05). *p < 0.05 in comparison to the control sample, +p < 0.05 in comparison to the rhinovirus sample (HRV‐2 or HRV‐16, respectively); C – control sample with medium only.

FIGURE 5

Action of ciglitazone (Ci) in c‐Myc knockdown cells under Rhinovirus infection conditions. Rhinovirus 2 (minor serotype, H2) and 16 (major serotype, H16) were used in the experiments. siRNA silencing of c‐Myc transcription factor reversed the changes in the expression of MMP‐9 (A), TGF‐β (B), COL I (C) ADAM33 (D) and α‐SMA (E). Interestingly, under condition of c‐Myc silencing, ciglitazone effect on COL I was stronger and it significantly decreased HRV‐induced expression of this gene. *p < 0.05 in comparison to the control sample, +p < 0.05 in comparison to the Rhinovirus sample (HRV‐2 or HRV‐16, respectively). Ciglitazone (Ci) has been added before rhinovirus infection (Ci+H16, Ci+H2) or after rhinovirus infection (H16+Ci, H2+Ci), C – control sample with medium only.

FIGURE 6

Collagen I protein expression in experimental set with c‐Myc siRNA knockdown. In each cell type – fibroblasts (A, B) and epithelial cells (C), collagen I protein expression was induced by Rhinovirus (both serotypes) and decreased by ciglitazone (Ci) after 24 h incubation. No differences were observed in the order of HRV and CI stimulation. Collagen I concentrations were measured in supernatants utilizing immunoassay in triplicate. Ciglitazone (Ci) has been added before rhinovirus infection (Ci+H16, Ci+H2) or after rhinovirus infection (H16+Ci, H2+Ci). Data presented as % of change, normalizing to control sample ± SD +<0.05.

FIGURE 7

Effects of STAT3 knockdown on ciglitazone (Ci) abilities under Rhinovirus infection conditions. Rhinovirus 16 (H16 in the Figure, major serotype) and 2 (H2 in the Figure, minor serotype) and were used. In comparison to the conditions without the knockdown, the MMP‐9 (A) and TGF‐β (B) mRNA expressions were decreased. Ciglitazone effect were observed in COL mRNA expression, especially its decreasing effect on HRV‐induced COL and a‐SMA expression (C, E). No effect of ciglitazone was observed in ADAM33 (D). *p < 0.05 in comparison to the control sample, +p < 0.05 in comparison to the Rhinovirus sample (HRV‐2 or HRV‐16, respectively). Ciglitazone (Ci) has been added before rhinovirus infection (Ci+H16, Ci+H2) or after rhinovirus infection (H16+Ci, H2+Ci). C – control sample with medium only.

FIGURE 8

The result of siRNA knockdown of STAT3 on collagen I and α‐SMA protein expression. Ciglitazone (Ci) has been added before rhinovirus infection (Ci+H16, Ci+H2) or after rhinovirus infection (H16+Ci, H2+Ci). The confirmation of mRNA expression on protein level was observed in COL I (A, B) and α‐SMA (C, D). Ciglitazone (Ci) reversed the HRV‐induced augmentation, regardless the virus serotype and the order of stimulation. α‐SMA protein expression has been decreased significantly only after HRV‐16 (H16) stimulation (p < 0.05). The bands presented were obtained from Western Blot analysis. The concentrations were measured in supernatants utilizing immunoassay in triplicate. Data presented as % of change, normalizing to control sample ± SD. +<0.05.

FIGURE 9

Effects of ciglitazone and two Rhinovirus serotypes on PPAR‐ levels in fibroblast and epithelial cells. Ciglitazone caused an increase in PPAR‐γ while both rhinovirus serotypes decreased it. Ciglitazone added after HRV infection increased PPAR‐γ concentrations in HFL1 fibroblasts and epithelial cells. Ciglitazone (Ci) has been added before rhinovirus infection (Ci+H16, Ci+H2) or after rhinovirus infection (H16+Ci, H2+Ci). Data presented as mean ± SEM, *p < 0.05 in comparison to control, #p < 0.05 in comparison to HRV16 or HRV2.