The effect of histone deacetylase inhibitor Trichostatin A on IL-17A-induced transformation of MRC5 into myofibroblasts and its mechanism

Objective To elucidate potential IL-17A- and TSA-mediated regulation of fibroblasts transformation. Methods MTT assay, HDAC1 activity assay, cell immunofluorescence and Western blot were employed to detect the expression of related indicators. Results MRC5 cells expressed only a small amount of Vimentin. IL-17A treatment upregulated MRC5 cell proliferation, in a concentration-dependent manner. TSA treatment, however, suppressed MRC5 cell proliferation. IL-17A treatment also upregulated HDAC1 activity in MRC5 cells, in a concentration-dependent manner. Using immunofluorescence, we demonstrated that IL-17A-treated MRC5 cells had markedly elevated Vimentin, Collagen-I and a-SMA levels, compared to controls. However, a combined treatment of IL-17A and TSA resulted in markedly reduced levels of the Vimentin, Collagen-I and a-SMA, compared to IL-17A alone, yet the amount was higher than controls. Using western blot analysis, we also revealed that the IL-17A-treated MRC5 cells had markedly elevated levels of Vimentin, a-SMA, HDAC1, p-Smad2, and p-Smad3, and markedly reduced level of Smad7, compared to controls. In TSA intervention group, the expression effect of the above protein was opposite. Moreover, no discernible difference was observed in the levels of Smad2 and Smad3 among the treated and un-treated groups. Conclusion IL-17A stimulates proliferation of MRC5 cells and increases HDAC1 activity and protein expression. It also transforms MRC5 cells into myofibroblasts via activation of the TGF -β1/ Smads signaling network. TSA, on the other hand, strongly suppresses TGF -β1/ Smads pathway-mediated fibrosis by ceasing HDAC1 activity and protein expression.


MRC5
Human normal embryonic lung fibroblasts TSA Trichostatin A

TGF -β1
Transforming growth factor-β1    PBS-washed again three times, exposed to primary antibody (1:100) at 4 ℃ overnight (O/N), PBS-washed three times, exposed to secondary antibody (1:100) at RT for 1 h, PBS-washed three times, exposed to fluorescence reagent at RT for 1 h, PBS-washed three times, lastly, the anti fluorescence quenchant solution with DIPA was used to seal the film and images were taken under a laser confocal microscope.

Evaluation of relevant proteins by Western Blot (WB)
Amount? MRC5 cells were seeded and cultured for 48 h. Cell lysates containing phosphatase inhibitor was centrifuged at 12000 rpm for 10 minutes and the supernatant was retrieved for protein quantification by BCA method. 20 ug of total protein was subsequently used for electrophoresis (SDS-PAGE), transferred into PDVF membrane, blocked with 5% skim milk at RT for 90 min, exposed to primary antibody O/N at 4℃, rinsed 5 times with TBST, exposed to secondary antibody at RT for 60 min, TBST-rinsed 5 times, and exposed to hypersensitive ECL luminescent solution before obtaining images of proteins using a gel imaging system. GAPDH was used as internal reference. Image J software was used to calculate relative expression of target proteins.

Statistical methods
GraphPad software was employed for all statistical analyses. Data are presented as mean ± standard deviation. Pairwise comparison was done with t-test. P < 0.05 was the statistically significant threshold.

Result:
1. Identification of MRC5 cells Using microscopic evaluations, we revealed that the IL-17A-treated MRC5 cells experienced markedly elevated rate of proliferation, compared to the controls, and the proliferation density was also increased. Conversely, the growth activity of the TSA-treated MRC5 cells was decreased, and proliferation was significantly inhibited.

Discussion:
Pulmonary fibrosis is a complex process that involves inflammatory cell invasion into lung tissue, conversion of fibroblasts into interstitial cells and the production of a large quantity of interstitial protein deposition that destroys the lung structure. The production and interaction of many cytokines play a key role in this process. In recent reports, it was suggested that IL-17A participates in the occurrence and development of pulmonary fibrosis [1]. In prior experiments, we demonstrated markedly increased Th17 cell differentiation in mice with pulmonary fibrosis. Moreover, the IL-17A levels were markedly upregulated in these mice verses controls. This is indicative of IL-17A playing a principle role in lung fiber formation and development. However, not much is known about the mechanism of IL-17A-stimulated fibroblasts 20 transformation. This study was designed to fill this void.
Interleukin-17A (IL-17A) is an inflammatory cytokine produced by Th17 cell subsets, which can not only activate T lymphocytes, but also induce endothelial cells, epithelial cells and fibroblasts to secrete adhesion molecules, granulocyte macrophage stimulating factor, IL-8, IL-6, etc., thus leading to the development of acute inflammation [2]. It is the most crucial Th17 cell-secreting cytokine and was the first of its kind to be discovered. Subsequent discoveries of cytokines belonging to the same family are IL17B-F and so on. Among the various cytokines, IL-17A is essential to the early inflammatory response and participates in the development of numerous acute and chronic inflammatory diseases [3]. It stimulates the release of pro-inflammatory factor TNF-a, chemokine MCP-1 and metalloproteinase (MMP), and cause inflammatory cell infiltration and tissue destruction [4]. Multiple studies have reported that IL-17 can increase the TNF-a-induced IL-6 levels in the airway smooth muscle cells [5], and stimulate fibroblasts and airway epithelial cells to release granulocyte colony-stimulating factor and granulocyte macrophage colony-stimulating factor [6]. Moreover, in rheumatoid arthritis, IL-17 was shown to activate synovial fluid fibroblasts to secrete inflammatory factors, and stimulate colon myofibroblasts to produce inflammatory mediators [7,8]. Emerging evidences suggest that IL-17A is associated with the development of chronic inflammatory diseases in the lung, such as chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchial asthma and so on [9]. Hence, it is clear that IL-17A is intimately involved in recruiting inflammatory cells and in promoting synthesis and release of a variety of inflammatory cytokines. Simultaneously, it can cooperate with a variety of inflammatory factors to amplify the inflammatory effect, and participate in the etiology of autoimmune diseases, acute and chronic inflammation, tumor and so on.
HDACi is known to regulate various immune activities in the body. Its positive or negative effects primarily depend on the type and functional state of immune cells.
Several studies demonstrated that HDACi can suppress CD4 + T cell activity. In the Moreira [10] study, mice were administered CD4 + T cells, prior to TSA treatment.

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They discovered that TSA inhibited the translocation of NF-κB to the nucleus, resulting in reduced CD4 + T cell activity over time. Mechanistically, they proposed that TSA suppressed the expression of specific antigens on the surface of CD4 + T cells by elevating p21, an inhibitor of cyclin dependent kinase [11]. Based on these results, TSA can cease CD4 + T cell proliferation and, thereby, induce anti-tumor immune response of T cells. Furthermore, Donas et al. [12] discovered that TSA can enhance FOXP3 levels in CD4 + T cells, thereby, driving proliferation and differentiation of Treg cell subsets and augmenting immunosuppression. Given these evidences, TSA has great potential in curbing autoimmune diseases.
TSA is a highly prevalent HDACi that can effectively suppress both class I and class II HDACs. Through the inhibition of HDACs, TSA can also suppress TGF -β  [14] demonstrated that TSA can upregulate pulmonary myofibroblasts thymocyte differentiation antigen 1 (Thy-1) transcript levels and suppress fibroblasts proliferation in order to promote its anti-fibrotic role.
Moreover, studies [15] have reported that cyclooxygenase (COX) expression is downregulated in patients with pulmonary fibrosis, mainly due to the high deacetylation of the COX gene promoter, while HDACi vorinostat and pabistat can reduce the state of deacetylation of the Cox gene promoter, upregulate expression of COX2, and promote the production of the anti fibrotic factor prostaglandin E2 (PGE2).
One of the obstacles of lung fibroblast detection is that it lacks specific antigen expression. Hence, fluorescence staining exclusion method is primarily used for the identification of lung fibroblasts [16,17]. The first step, in this process, is establishing  Our protein evaluations also revealed that the levels of HDAC1, phosphorylated Smad2 and phosphorylated Smad3 in the IL-17A-stimulated MRC5 cells were markedly elevated than in control cells, whereas Smad7 protein was markedly reduced than in control cells. In cells co-treated with IL-17A and TSA, the levels of HDAC1, phosphorylated Smad2 and phosphorylated Smad3 were drastically less than in IL-17A-treated cells. Moreover, the levels of Smad7 protein were markedly more than in IL-17A-treated cells. On the contrary, Smad2 and Smad3 levels remained the same in all treatment groups. In summary, the above data revealed that in IL-17A-stimulated MRC5 cells, HDAC1 protein levels increased, HDAC1 activity increased, phosphorylated Smad2 and phosphorylated Smad3 increased, and Smad7 expression decreased, indicating that the TGF-β1/smads pathway was activated.
Conversely, the TSA-treated MRC5 cells had dramatically less HDAC1 protein levels, decreased HDAC1 activity, decreased levels of phosphorylated Smad2 and phosphorylated Smad3, and elevated Smad7 levels, which is indicative of TSA-mediated inhibition of the TGF-β1/Smads pathway. Furthermore, using co-treatments of IL-17A and TSA, we further demonstrated that TSA can effectively inhibit the IL-17A-mediated stimulation of the TGF-β1/Smads pathway and the 24 eventual transformation of MRC5 into myofibroblast cells. It was previously suggested that TSA inhibits the expression of extracellular matrix components at both the gene and protein levels during its suppression of MRC5 transformation into myofibroblast cells and fibrosis prevention. Several studies have reported [18][19][20] Smad7 localization in the nucleus and cytoplasm, using immunofluorescence, however, HDAC1 was always reported to be in the nucleus. Based on these evidences and consistent with our results, we propose that HDAC1 may directly interact with Smad7 in the nucleus. Hence, via inhibition of Smad7, the expression of the phosphorylated Smad2 and phosphorylated Smad3 may be promoted, and the subsequent TGF-β1/smads pathway may be activated.

Conclusion:
In summary, our work revealed that IL-17A promotes the proliferation and transformation of human lung fibroblasts into myofibroblast cells. IL-17A also promotes the production of interstitial protein. TSA showed obvious inhibition of human lung fibroblast proliferation and blocked the activation of key transformation pathways. The process and mechanism of pulmonary fibrosis are quite complex.
Therefore, blocking IL-17A secretion, action pathway or monoclonal antibody may play a significant role in inhibiting pulmonary fibrosis. The above research findings may provide new insight into the development and treatment of pulmonary fibrosis.