**4. Regulation of lung cell development and differentiation**

of IL-1β signaling, was accompanied by a reduction of IL-8 production. These observations suggest a general role of miR-155 as an IL-8 expression regulator and consequently of the NFκB pathway [16, 17, 19, 20]. The expression of miR-155 is also increased in patients with asthma, idiopathic pulmonary disease and acute lung injury [21]. It has been underlined that a tight control is required for the expression of a molecule such as miR-155 which is overexpressed in cancers of B-cell origin. It is noteworthy that a signaling molecule as small as miR-155 has

Although so far, few studies have focused on the role of miRNA as regulator of the immune response specifically in the CF context, it is of prime importance to consider other miRNAs, the expression of which is deregulated in inflammatory lungs, and which could be nonspecifically modulated in CF. CF cells display a new profile of miRNAs, including high expression of miR-215 which is a strong modulator of cell cycle through the p53-signaling pathway [17]. As an example of unspecific modulation, miR-509-3p and miR-494 (which directly target CFTR expression, as described above) are overexpressed in CF bronchial epithelial cells [9]. In addition, bacterial infection and tumor necrosis factor-alpha and IL-1β exposure increase miR-509–3p and miR-494 concentrations in part via the action of the NF-kB transcriptional activator complex. These findings, together with those showing that miR-494 is upregulated in CF cells [9, 10], support the idea of a role of miRNAs in inflammatory responses in CF respiratory epithelia, either directly by activation of transcription factors such

Another crucial aspect of the immune response in lungs of patients with CF is the dysregulation of the protease-antiprotease balance which eventually leads to bronchiectasis [22]. Much attention has been given to serine proteases, in particular elastase secreted by neutrophils massively recruited during lung inflammation in patients with CF. However, other proteases secreted by epithelial cells themselves impact airway function. Recently, involvement of cysteine proteases cathepsin (CTS) B and S, overexpressed in lungs of patients with CF, has been described [23]. CTSS is constitutively released at high levels by airways of patients with CF. Weldon et al. showed that the overproduction of CTSS in lungs of patients with CF was indirectly modulated by miR-31 in HBEs via repression of the transcription factor, interferon regulatory factor 1 (IRF1), which directly controls the *CTSS* gene [24]. It could be predicted that evidence will be brought up, showing that other proteases, such as metalloproteinases, often detected at high levels in CF patients sputum, are also regulated by miRNA-dependent mechanisms. Similarly, miRNAs could be involved in the expression of anti-proteases (e.g., α-1 antitrypsin, secretory leucocyte protease inhibitor (SLPI), tissue inhibitors of metallopro‐ teinase 1 (TIM-1)), opening new perspectives in development of novel therapies for CF and

It is quite likely that a number of miRNAs could find several target points in the network of cellular and molecular players of the inflammation, imbalanced in CF. In turn, inflammatory responses themselves drive the expression of several miRNA species that either worsen the

such a dangerous potential when deregulated.

238 Cystic Fibrosis in the Light of New Research

as NF-κB, or indirectly by inhibition of CFTR expression.

other lung diseases.

Studies of miRNAs expression patterns have revealed that 27 miRNAs are differentially expressed during lung embryogenesis [25] following a characteristic pattern. miR-29a is highly expressed in late stages of lung development and in adult life [26]. In contrast, the miR-17-92 locus is highly expressed in undifferentiated lung epithelial cells and in lung cancer cells [27], and its expression progressively declines as differentiation progresses. Interestingly, miR-127 and miR-351 are transiently expressed during late phases of lung embryogenesis, first in the mesenchymal network, and then in epithelial cells. These observations suggest that miRNAs play distinct roles in the differentiation processes during the mesenchymal-to-epithelial transition.

The chronic inflammation in CF leads to irreversible airway tissue remodeling characterized by loss of multiciliated cells, gain in mesenchymal cells, goblet cells hyperplasia, and squamous metaplasia. Two miRNAs involved in multiciliogenesis have particularly attracted attention. miR-449a accumulates in bronchial epithelial cells during their transition to full differentiation [28]. Involvement of this particular miRNA in multiciliogenesis has been previously described [29]. Expression of miR-449a remains high in differentiated cells, indicating that it also plays a role in maintaining the multiciliated phenotype [29]. Conversely, expression of miR-455-3p, which negatively regulates the mucin 1 gene, is lost during the differentiation process of HBEs, reinforcing the control of epithelial (de)differentiation by miRNAs. Consequently, high expression of miR-449a and low expression of miR-455-3p can serve as biomarkers for the differentiation of bronchial epithelia. Further studies would be welcome to determine whether these miRNAs are deregulated in CF cells.

Airway epithelium dedifferentiation is part of tissue remodeling through induction of epithelial-mesenchymal transition (EMT). EMT is a process during which epithelial cells lose their phenotype, including loss of cell polarity and dissolution of cell-cell junctions, and acquire mesenchymal characteristics. Transforming growth factor (TGF)-β is a key mediator of EMT and epithelial remodeling. TGF-β signaling was shown to be increased in CF lungs [30] and several miRNAs are regulated via the TGF-β pathway [31]. Among them, miR-155 expression is reduced in human fibroblast cells upon exposure to TGF-β [32]. Although it may contribute to reduce inflammation (through its effect of IL-8 secretion in HBEs described earlier), reduction of miR-155 expression at the airway level might contribute to loss of epithelial polarity. Moreover, another study utilizing epithelial cells indicated that miR-155 plays an important role in TGF-β-induced EMT as it facilitates tight junction dissolution [33]. Because CFTR expression is important to maintain epithelial differentiation and polarity [34], in particular in CF cells [35], it could be expected that at least some of the other miRNAs controlling CFTR expression might be involved in airway remodeling as well. Likewise, other miRNAs might non-specifically regulate epithelial differentiation in CF cells and could be involved in loss of epithelial polarity.

**Figure 2.** Main miRNAs involved in cystic fibrosis pathogenesis The left part depicts miRNA-dependent regulation of cystic fibrosis transmembrane conductance regulator (CFTR) biogenesis and trafficking. The right part zooms into the regulation of interleukin (IL)-8 production by miRNAs. miRNAs which may drive phenotypic changes on multicilio‐ genesis, airway remodeling and disorganization of tight junction are also represented. Red arrows depict changes re‐ ported in CF; up arrows indicate upregulated miRNAs and phenotype; and down arrows indicate miRNA and phenotype downregulation. CSE: cigarette smoke exposure; CTS: cathepsin.

MicroRNAs potentially altered and phenotypic consequences in CF are summarized in Table 1.



**Table 1.** microRNAs potentially altered and phenotypic consequences in cystic fibrosis (CF)
