**3. Regulation of key elements of inflammation and innate immune system**

miRNAs are crucial regulators of innate and adaptive immune responses and their abnormal expression or function or both have been linked to multiple human inflammatory disorders [12]. The link between *CFTR* mutations and the mechanisms underlying disproportionate proinflammatory responses remains poorly understood. Aberrant release of proinflammatory mediators by epithelial and immune cells in CF includes hypersecretion of interleukin-8 (IL-8). The fact that this can be detected in lungs of children with CF [13] even during fetal life [14, 15] suggests that it is a constitutive process of CF. miRNA-based post-transcriptional regula‐ tions, known to be highly sensitive to a range of homeostasis signals, such as changes in hypoxia, pH, ion concentration and osmolarity, have recently been considered as potentially regulating immune responses in CF, and more specifically IL-8 hyperproduction.

The first published contribution of miRNA involvement in CF [8] reported that miR-126 was found to be abundantly expressed in normal lungs and that it was reduced in lungs of patients with CF. This process cannot account for the pathophysiological mechanism of changes observed in patients with CF, based on the observation of high concentrations of IL-8 depend‐ ing on constitutive activation of nuclear factor kappa B (NF-κB) [15-17]. Indeed, Oglesby et al. [8] found that in patients with CF, miR-126 was strongly downregulated and expression of TOM1, which is itself a negative regulator of the NF-κB signaling pathway, was upregulated. On this basis, a decreased expression of IL-8 would be expected, in contradiction to the wellestablished increased IL-8 production. These observations indicate that miR-126 alone does not play a role via the TOM1 cascade and that other factors may also be involved. Other miRNAs originated from host or part of this microbioma could also be involved. For example, Rao et al. [18] reported their finding of bacterial miR-146, which binds to a receptor of the TLR family, in sputum of CF patients infected with *Pseudomonas aeruginosa*.

As for other miRNAs, it has been suggested that expression of miR-155 might contribute to the activation of IL-8--dependent inflammation in patients with CF. Clinically, overexpression of miR-155 has been observed in lung epithelial cells and in neutrophils from patients with CF [18]. Interestingly, miR-155 expression was also elevated in primary CF bronchial epithelial cells and in IB-3 and CuFi-1 CF cell lines [16, 19]. Upregulating miR-155 expression in these cells increased production of IL-8 through activation of the PI3K/Akt signaling pathway [16]. Another study confirmed these results [17] by showing that high levels of miR-155 in sterile CF cells, reduced after exposure to the anti-inflammatory cytokine IL-10 or following inhibition 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 such a dangerous potential when deregulated.

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

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 other lung diseases.

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 imbalance by increasing production of proinflammatory mediators or directly repress CFTR protein expression, forming a vicious circle.
