Responsiveness of human bronchial fibroblasts and epithelial cells from asthmatic and 1 non-asthmatic donors to the transforming growth factor- β 1 in epithelial-mesenchymal 2 trophic unit model 3

16 Background: Asthma-related airway wall remodeling is associated with i.a. damage of 17 bronchial epithelium and subepithelial fibrosis. Functional interactions between human 18 bronchial epithelial cells and human bronchial fibroblasts are known as the epithelial- 19 mesenchymal trophic unit (EMTU) and are necessary for a proper functioning of lung tissue. 20 However, a high concentration of the transforming growth factor- β 1 (TGF- β 1 ) in the asthmatic 21 bronchi drives the structural disintegrity of epithelium with the epithelial-to-mesenchymal 22 transition (EMT) of the bronchial epithelial cells, and of subepitheial fibrosis with the 23 fibroblast-to-myofibroblast transition (FMT) of the bronchial fibroblasts. Since previous 24 reports indicate different intrinsic properties of the human bronchial epithelial cells and the human bronchial fibroblasts which affect their EMT/FMT potential beetween cells derived 26 from asthmatic and non-asthmatic patients, cultured separatelly in vitro , we were interested to 27 see whether corresponding effects could be obtained in co-cultures of bronchial epithelial cells 28 and bronchial fibroblasts. In this study, we investigate the effects of the TGF- β 1 on the EMT 29 markers of the bronchial epithelial cells cultured in the air-liquid-interface and effectiveness of 30 FMT in the bronchial fibroblast populations in the EMTU models. 31 Results: Our results show that the asthmatic co-cultures are more sensitive to the TGF- β 1 than 32 the non-asthmatic ones, which is associated with a higher potential of asthmatic bronchial cells 33 for a profibrotic response, analogously to be observed in “2D” cultures. Moreover, our results 34 indicate a noticeable impact of human bronchial epithelial cells on the TGF- β 1 -induced FMT, 35 stronger in the asthmatic bronchial fibroblast populations in comparison to the non-asthmatic 36 ones. 37 Conclusions: Our data are the first to demonstrate a protective effect of human bronchial 38 fibroblasts on the properties of human bronchial epithelial cells, which suggests that intrinsic 39 properties of not only epithelium but also subepithelial fibroblasts affect a proper condition and 40 function of the EMTU in both normal and asthmatic individuals. Normality of distribution was estimated using the Shapiro-Wilk test and then appropriate parametric or non-parametric test was used. Data are presented as a mean ± SEM. Statistical significances were tested using non-parametric Mann-Whitney (the comparison between AS and NA group; # p < 0.05, ## p < 0.01, ###< 0.001), the Student’s t-test; * p < 0.05, ** p < 0.01, *** p < 0.001 (the comparison in one group between only two different experimental conditions) or one-way ANOVA with the Bonferroni multiple comparison post hoc test; (the comparison in one group between more than two different experimental conditions); * p < 0.05, 472 ** p < 0.01, *** p < 0.001. The statistical analyses were performed using GraphPad Prism 5.0 473 software. muscle actin), TAGLN (transgelin), FN1 (fibronectin); (F- 716 G) markers of mucociliary differentiated HBECs: DNAH9 (dynein axonemal heavy chain 9), MUC5AC 717 (mucin 5AC) and (H-K) EMT-related genes: ACTA2 , CDH2 (N-cadherin), SNAI1 (Snail) and SNAI2 718 (Slug) was measured by RT-qPCR and presented as 2 - ΔCt value in relation to GAPDH as a reference 719 gene. Data from all experiments represent the mean ± SEM carried out on HBFs AS (n = 4) and NA (n 720 = 4). Statistical significances were tested using non-parametric Mann-Whitney (the comparison between 721 AS and NA group; ## p < 0.01, ###< 0.001) or the Student’s t-test; * p < 0.05, ** p < 0.01,*** p < 0.001 722 (the comparison in one group between only two different experimental conditions).