AhR Mediated Activation of Pro-Inflammatory Response of RAW 264.7 Cells Modulate the Epithelial-Mesenchymal Transition

Abstract

Pulmonary fibrosis, a chronic lung disease caused by progressive deterioration of lung tissue, is generated by several factors including genetic and environmental ones. In response to long-term exposure to environmental stimuli, aberrant tissue repair and epithelial cell-to- mesenchymal cell transition (EMT) trigger the subsequent progression of pulmonary fibrotic diseases. The Aryl hydrocarbon receptor (AhR) is a transcription factor that is activated by ligands providing lung dysfunction when activated by environmental toxins, such as polycyclic aromatic hydrocarbons. Our previous study demonstrated that AhR mediates α-SMA expression by directly binding to the α-SMA (fibroblast differentiation marker) promoter, suggesting the role of AhR in mediating fibrogenic progression. Here we follow the hypothesis that macrophage infiltrated microenvironments may trigger inflammation and subsequent fibrosis. We studied the expression of cytokines in RAW 264.7 cells by AhR activation through an ELISA assay. To investigate molecular events, migration, western blotting and zymography assays were carried out. We found that AhR agonists such as TCDD, IP and FICZ, promote the migration and induce inflammatory mediators such as TNF-α and G-CSF, MIP-1α, MIP-1β and MIP-2. These cytokines arbitrate EMT marker expression such as E-cadherin, fibronectin, and vimentin in pulmonary epithelial cells. Expression of proteins of MMPs in mouse macrophages was determined by zymography, showing the caseinolytic activity of MMP-1 and the gelatinolytic action of MMP-2 and MMP-9. Taken together, the present study showed that AhR activated macrophages create an inflammatory microenvironment which favours the fibrotic progression of pulmonary epithelial cells. Such production of inflammatory factors was accomplished by affecting the Wnt/β-catenin signalling pathway, thereby creating a microenvironment which enhances the epithelial-mesenchymal transition, leading to fibrosis of the lung.

Keywords: Aryl hydrocarbon receptor; MMP-9; Wnt/β-catenin; epithelial mesenchymal transition; inflammatory cytokines; macrophage.

Figure 1

Chemokine-Cytokine screening. Activation of AhR in macrophage induced inflammatory markers and fibrosis-associated genes. RAW 264.7 cells were nursed in serum-free DMEM media with TCDD (10⁻⁸ M) for 24 h. The cytokine/chemokine expression profiles in RAW 264.7 cells induced by TCDD were analyzed and compared to control cells that were treated with DMSO.

Figure 2

Activation of AhR in macrophages induces inflammatory markers and fibrosis associated cytokines. In a serum-free DMEM medium, RAW 264.7 cells were cultured for 24 h with TCDD (10⁻⁸ M), IP (10⁻⁷ M), and FICZ (10⁻⁷ M). An increase in cytokine levels is shown as folds of increase compared to the control culture. (a) TNF-α expression, (b) G-CSF expression, (c) MIP-1α expression (d) MIP-1β expression, (e) MIP-2expression Three independent experimental means ± SEM are represented in the graph. Unstimulated comparisons to control cultures were depicted by asterisks, * p < 0.05, ** p < 0.01 and *** p < 0.001 by one-way ANOVA analysis.

Figure 3

Migration of macrophage cells. In serum-free DMEM media with TCDD (10⁻⁸ M), IP (10⁻⁷ M) and FICZ (10⁻⁷ M), RAW 264.7 cells were cultured for 24 h, and images were taken. Crystal violet-stained cells take on a purple hue. The quantification of cells was performed as described above. The number of cells that migrated were totaled and presented as folds of increase compared to the control cells. Five independent experimental means ± SEM are represented in the graph. Unstimulated comparisons to control cultures were depicted by asterisks, such as. ** p < 0.01 by one-way ANOVA analysis.

Figure 4

Migration of epithelial cells. In serum-free DMEM/F12 medium that contained TCDD (10⁻⁸ M), IP (10⁻⁷ M), FICZ (10⁻⁷ M⁾, MLE-12 cells were cultured for 24 h. Images were taken after 24 h. Crystal violet-stained cells took on a purple hue. The quantification of cells was performed as described above. The number of migrating cells was totaled and presented as folds of increase compared to control cells. Five independent experimental means ± SEM are represented in the graph. Unstimulated comparisons to control cultures were depicted by asterisks, such as *** p < 0.001 and **** p < 0.0001 by one-way ANOVA analysis.

Figure 5

Effect of conditioned medium. In serum-free DMEM media with TCDD (10⁻⁸ M), IP (10⁻⁷ M), FICZ (10⁻⁷ M) for 24 h, RAW 264.7 cells were maintained. MLE-12 cells were seeded for 10–12 h before treatment for 24 h with RAW 264.7 conditioned medium. Images were taken after 24 h. Crystal violet-stained cells took on a purple hue. The quantification of cells was performed as illustrated previously. The number of cells that migrated were totaled and presented as folds of increase compared to the control cells. Five independent experimental means ± SEM were represented in the graph. Unstimulated comparisons to control cultures were depicted by asterisks, such as *** p < 0.001 and **** p < 0.0001 by one-way ANOVA analysis.

Figure 6

Activation of AhR induced MMP expression in RAW264.7 cells. In serum-free DMEM media with TCDD (10⁻⁸ M), IP (10⁻⁷ M), and FICZ (10⁻⁷ M) for 24 h, RAW 264.7 cells were incubated. The cell supernatant was collected and subjected to SDS-PAGE (containing 0.1% gelatin or 1% casein) electrophoresis. (a) Representative image of gelatin zymogram. (b) Representative image of casein zymogram. Induction of RAW 264.7 cells with AhR agonists produced (c) MMP-9, (d) MMP-2, (e) MMP-1 expressions. The levels of secreted MMPs are shown as folds of increase compared to the control. Means of five independent experiments +/-SEM were represented in the graph. Unstimulated comparisons to control cultures were depicted by asterisks, such as * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 by one-way ANOVA analysis.

Figure 7

Activation of AhR induced epithelial EMT marker expression in MLE-12 cells. MLE-12 cells were exposed for another 24 h in serum-free DMEM/F12 media with TCDD (10⁻⁸ M), IP (10⁻⁷ M), FICZ (10⁻⁷ M), TNF-α (10 ng/mL), TGF-β (10⁻⁵ M) and G-CSF (10 ng/mL). (a) Representative image of western blot. (b) e-cadherin expression, (c) fibronectin expression, (d) vimentin expression, (e) β-catenin expression. Five independent experimental means ± SEM are represented in the graph. Unstimulated comparisons to control cultures were depicted by asterisks, such as * p < 0.05 and *** p < 0.001 by one-way ANOVA analysis.