Cystic fibrosis (CF) is a monogenic disease caused by the mutation of cystic fibrosis transmembrane conductance regulator (CFTR) gene, an anion channel-protein coding sequence that plays a crucial role in the airway surface liquid homeostasis. CFTR dysfunction leads to an imbalance between Cl- secretion and Na+ uptake, resulting in airway surface dehydration and impaired mucociliary clearance (1, 2). A chronic inflammation within the lung is the hallmark of the disease, with exacerbation episodes linked to bacterial infection by opportunistic pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa, leading to progressive airflow obstruction, tissue destruction and ultimately decreased respiratory function (1, 2).

We and others have reported sex-differences in the course of CF, with females experiencing more severe inflammation and exhibiting poorer prognosis compared to males (3–6). This sex disparity in CF exists despite the progress made in the early diagnosis and in the close therapeutic monitoring over the last years (6–10). Currently, the underlying mechanisms are poorly understood. Sex hormones and X-linked sexual genetic architecture have been proposed as potential factors (11–13). The essential role of the X chromosome is supported by the finding that such sex-differences in CF outcomes are also observed in young prepubertal children. Indeed, it has been shown that prepubertal girls have a significantly higher recruitment of neutrophils within the lungs and develop more episodes of infection than boys (3).

Interestingly, genomic distribution analysis revealed a high density of micro-RNAs (miRNAs) on the X chromosome (14). miRNAs are small non-coding RNA sequences of ±22 nucleotides that regulate gene expression at post-transcriptional level by repressing translation of specific mRNA targets through complementary pairing with their 3’ untranslated regions. Growing evidence points to their potential role in chronic inflammatory diseases impacting lung tissues, such as in CF (15–21). Indeed, studies have shown that CF patients have a dysregulation in the expression of number of miRNAs, including those targeting the mutated CFTR transporter or its derivatives, such as miR-145 or miR-494 (18, 22), or those involved in inflammatory pathology, such as the miR-126, miR-17 or miR-155 (23–25). Other studies showed a significant difference in the expression of X-linked miRNAs in monocytes from CF patients compared to non-CF patients (26). However, few reports investigated whether biological sex could impact miRNAs expression profiling in CF patients. Using miRNA array analysis of the plasma from six CF male and six CF female patients, Mooney et al. reported differential expression of two circulating miRNAs, miR-885-5p and miR-193a-5p, between girls and boys (27). Both miRNAs are not localized on the X chromosome and have no apparent role in the inflammatory process. Of note, females display mosaic cells expressing two X-linked gene alleles and one of the X chromosomes is randomly inactivated early in embryogenesis to ensure the compensation dosage. Yet, about 15-23% of human X-chromosome-linked genes escape the inactivation with some variability between tissues and individuals (28, 29). This may result in differential expression of X-linked genes between males and females. In this study, we asked whether blood leukocytes from male and female CF patients exhibit a differential expression profile of X-linked miRNAs with potent impact on the inflammatory process. We focused on miRNAs that have been shown to be dysregulated in the airways of CF patients compared to healthy individuals (23) and for which there is accumulating evidence of their direct or indirect role in the regulation of inflammation.

It is well documented that the biological sex is a contributing factor in the susceptibility to CF, with a better prognosis for boys compared to girls. However, there is paucity of data related to molecular markers that could be relevant to the mechanisms at work. In this study, we examined the potential contribution of selected X-linked miRNAs that are involved in the regulation of the inflammatory process and whose aberrant expression has been described in CF patients.

Despite the overall higher production of pro-inflammatory cytokines in CF compared with controls, patients enrolled in this study exhibited a relatively stable clinical state with respect to clinical parameters routinely recorded during medical monitoring. Similar observations have been previously reported in human and experimental CF (41–45). Currently, better therapeutic care that significantly improves the outcome of the disease is progressing thanks to the newborn screening allowing an early diagnosis. While gender difference still exists in CF, significant advances in timely treatment and patient follow-up, have largely contributed to reduce the difference observed in the pathophysiological evolution and inflammatory phenotype between boys and girls. However, substantial differences between males and females could be detected at the transcript level, in particular using signal amplification methods such as qRT-PCR.

Regarding miRNAs, we observed a significant increase in the expression of miR-223-3p, miR-106a-5p, miR-502-5p and miR-221-3p in blood cells from CF patients compared with healthy controls. The miR-223-3p is highly expressed by myeloid cells and is particularly involved in the control of granulopoiesis (46). In addition, we and others have previously shown that miR-223-3p is an essential modulator of the inflammation through the regulation of neutrophil activity and macrophage polarization (47–52). The overexpression of miR-223-3p observed in blood leukocytes of CF patients is in line with previous studies describing its upregulation in CF endobronchial brush samples (23) as well as in a CFTR gene-mutated bronchial epithelial cell line (53). Further studies are needed to depict the functional role of miR-223-3p in the inflammatory pathogenesis of CF. On the other hand, the miR-106a-5p is known to be involved in the control of monocytopoiesis (54) and different studies suggested its anti-inflammatory potential (31, 55). In CF, the expression of miR-106a was shown to be downregulated in endobronchial brush samples (23). In contrast, we detected a significant expression of miR-106a in blood leukocytes in our CF cohort. This discrepancy could be explained by differences in the samples used (endobronchial brush versus blood leukocytes), in patient’s clinical status or in the experimental design related to data normalization. It is likely that the increase in the expression level of miR-106a in the blood cells of patients with cystic fibrosis can be considered as one of the compensatory mechanisms to limit excessive inflammation. On the other hand, few studies addressed the role of miR-502-5p in inflammation. Zhang G et al. reported it potential role in inhibiting the IL-1β-induced NF-κB pathway in chondrocytes by targeting TRAF2 (33). The contribution of miR-502-5p in the inflammatory signaling pathways in CF remains unclear.

Notably, this study revealed that miR-221-3p is significantly overexpressed in blood cells from CF females compared to CF males. Similar trends were obtained when comparing prepubertal CF patients, suggesting a non-prominent role of sex hormones. However, this requires a large cohort and hormone assay for a firm conclusion. Since no significant difference were observed in leukocyte count and blood cell composition between genders, the overexpression of miR-221-3p seen in females would most likely be attributed to a differential cell activation and/or gene inactivation escape occurring naturally in one of the X chromosomes. The miR-221 was mainly investigated in cancer where studies have shown its potential contribution to tumor progression through modulation of cell proliferation, invasion, and apoptosis (56). Moreover, its aberrant expression was reported in inflammatory diseases as well, like in rheumatoid arthritis, atherosclerosis and asthma (57, 58). In CF, miR-221-3p was shown to be overexpressed in CF airway epithelial cells (59). Interestingly, we found that the overexpression of miR-221-3p in CF blood cells is positively correlated with the expression of IL-1β. We also observed a trend towards lower expression in CF females of SOCS1 and PDLIM2, two mRNA targets of miR-221-3p. Of note, it would be interesting to investigate how miR-221-3p could impact the expression of its main targets at protein level as well. Although the causative link between miR-221 and IL-1β-related inflammatory profile within blood cells derived from CF patients remains unclair, this observational clinical study is in line with previous experimental studies supporting the pro-inflammatory role for miR-221-3p by promoting the activation of the NF-κB signaling pathway (32, 37–39, 60).

Our investigation addresses for the first time the potential contribution of X-linked miRNAs derived from CF blood leukocytes in the sex-bias of the inflammatory response. The study has some limitations. We cannot rule out the role of CFTR gene mutation type that may have different functional consequences, ranging from mild to a severe disease phenotype (61). As mentioned elsewhere, although our results do not appear to be affected when considering only prepubertal patients, the cohort remains too small to rule out a potential impact of sex hormones on miRNA expression (62, 63). It would be interesting to investigate, in future studies, a large homogenous cohort that includes not only low to moderate clinical phenotypes but also cases with pulmonary exacerbation to examine whether the gender difference in miRNA expression may have an important impact on the inflammatory process. Collectively, this pilot study suggests the potential contribution of X-linked pro-inflammatory miR-221-3p in the sex-bias of the inflammatory process and highlights the need to unravel its role in the pathophysiology of CF and determine how this may help optimize the personal follow-up of patients.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by the hospital ethics committee of the Brugmann University Hospital (CE 2016/162). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

MD performed experiments, analyzed data, and wrote the first draft. AP patient recruitment and reviewed the manuscript. LH patient recruitment and reviewed the manuscript. NL patient recruitment and reviewed the manuscript. LAAN performed experiments. GSO performed experiments. FC reviewed the manuscript. GC reviewed the manuscript. MC supervised the work, analyzed data, and reviewed the manuscript. All authors contributed to the article and approved the submitted version.