RESUMEN
Airway inflammation, mucus hyperproduction and epithelial remodelling are hallmarks of many chronic airway diseases, including asthma, COPD and cystic fibrosis. While several cytokines are dysregulated in these diseases, most studies focus on the response of airways to interleukin (IL)-4 and IL-13, which have been shown to induce mucus hyperproduction and shift the airway epithelium towards a hypersecretory phenotype. We hypothesised that other cytokines might induce the expression of chloride (Cl-) channels/transporters, and regulate epithelial differentiation and mucus production. To this end, fully differentiated human airway basal cells (BCi-NS1.1) were treated with cytokines identified as dysregulated in those diseases, namely IL-8, IL-1ß, IL-4, IL-17A, IL-10 and IL-22, and tumour necrosis factor-α. Our results show that the cystic fibrosis transmembrane conductance regulator (CFTR) is the main Cl- channel modulated by inflammation, in contrast to transmembrane protein 16A (TMEM16A), whose levels only changed with IL-4. Furthermore, we identified novel roles for IL-10 and IL-22 by influencing epithelial differentiation towards ciliated cells and away from pulmonary ionocytes. In contrast, IL-1ß and IL-4 reduced the number of ciliated cells while increasing club cells. Interestingly, while IL-1ß, IL-4 and IL-10 upregulated CFTR expression, IL-4 was the only cytokine that increased both its function and the number of CFTR-expressing club cells, suggesting that this cell type may be the main contributor for CFTR function. Additionally, all cytokines assessed increased mucus production through a differential upregulation of MUC5AC and MUC5B transcript levels. This study reveals a novel insight into differentiation resulting from the cross-talk of inflammatory mediators and airway epithelial cells, which is particularly relevant for chronic airway diseases.
RESUMEN
Cystic fibrosis (CF) cells display a more cancer-like phenotype vs. non-CF cells. KLF4 overexpression has been described in CF and this transcriptional factor acts as a negative regulator of wt-CFTR. KLF4 is described as exerting its effects in a cell-context-dependent fashion, but it is generally considered a major regulator of proliferation, differentiation, and wound healing, all the processes that are also altered in CF. Therefore, it is relevant to characterize the differential role of KLF4 in these processes in CF vs. non-CF cells. To this end, we used wt- and F508del-CFTR CFBE cells and their respective KLF4 knockout (KO) counterparts to evaluate processes like cell proliferation, polarization, and wound healing, as well as to compare the expression of several epithelial differentiation markers. Our data indicate no major impact of KLF4 KO in proliferation and a differential impact of KLF4 KO in transepithelial electrical resistance (TEER) acquisition and wound healing in wt- vs. F508del-CFTR cells. In parallel, we also observed a differential impact on the levels of some differentiation markers and epithelial-mesencymal transition (EMT)-associated transcription factors. In conclusion, KLF4 impacts TEER acquisition, wound healing, and the expression of differentiation markers in a way that is partially dependent on the CFTR-status of the cell.
Asunto(s)
Diferenciación Celular/genética , Proliferación Celular/genética , Fibrosis Quística/genética , Fibrosis Quística/patología , Células Epiteliales/patología , Factores de Transcripción de Tipo Kruppel/genética , Biomarcadores/metabolismo , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Células Cultivadas , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Transición Epitelial-Mesenquimal/genética , Transición Epitelial-Mesenquimal/fisiología , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción/genética , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiologíaRESUMEN
Airway mucus obstruction is the main cause of morbidity in cystic fibrosis, a disease caused by mutations in the CFTR Cl- channel. Activation of non-CFTR Cl- channels such as TMEM16A can likely compensate for defective CFTR. However, TMEM16A was recently described as a key driver in mucus production/secretion. Here, we have examined whether indeed there is a causal relationship between TMEM16A and MUC5AC production, the main component of respiratory mucus. Our data show that TMEM16A and MUC5AC are inversely correlated during differentiation of human airway cells. Furthermore, we show for the first time that the IL-4-induced TMEM16A up-regulation is proliferation-dependent, which is supported by the correlation found between TMEM16A and Ki-67 proliferation marker during wound healing. Consistently, the notch signaling activator DLL4 increases MUC5AC levels without inducing changes neither in TMEM16A nor in Ki-67 expression. Moreover, TMEM16A inhibition decreased airway surface liquid height. Altogether, our findings demonstrate that up-regulation of TMEM16A and MUC5AC is only circumstantial under cell proliferation, but with no causal relationship between them. Thus, although essential for airway hydration, TMEM16A is not required for MUC5AC production.