Identification of MMP9 modulation as biomarker related to clinical responses of Cystic Fibrosis patients to CFTR Modulators therapy: new development from bench to bedside

Cystic Fibrosis (CF) is a multi-organ hereditary disease caused by a mutation in the gene coding for Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein. Currently, no specific care is available for CF patients. Although various compounds have been developed to improve CFTR function, clinical responses are variable and sometimes even absent. Therefore, it is necessary to find biomarker to evaluate the effectiveness of CFTR modulators therapy related to the individual patient’s response. For this purpose, nasal epithelial cells and intestinal organoids are usually the models used; however, the discovery of CFTR expression in non-epithelial cells and the recognition of inflammation's involvement in CF have raised interest in the role of immune cells in this disease. Additionally, immune cells are easily and quickly isolable from patients, making them a useful cellular model. In this context, we firstly identified changes in the proteomic profile linked to the restoration of CFTR channel activity in CF leukocytes after ex vivo treatment with the potentiator VX770. Subsequent bioinformatic analyses revealed the downregulation of proteins within the leukocyte transendothelial migration and regulation of actin cytoskeleton pathways in response to VX770 treatment. In particular, we focused our attention on matrix metalloproteinase 9 (MMP9). Since the high expression of this protease can contribute to CF-related lung damage, its downregulation could be a positive effect of CF therapies in slowing disease progression. To confirm these data also in vivo, we used leukocytes isolated from CF patients before and following Ivacaftor therapy. We assessed MMP9 levels by Immunoblotting and CFTR activity by GST-HS-YFP assay. These data showed that the therapy promoted a decrease in leukocyte MMP9 levels together with a recovery of CFTR activity and improved of clinical parameters. Subsequently, we amplified our study and evaluated MMP9 expression in leukocytes from CF patients before and during Trikafta® therapy, the best combination of potentiator and modulators for the most CF patients. Particularly, we could observe that MMP9 was downregulated in the clinically responsive CF patients, while levels remained elevated in non-responder. Moreover, we also assessed MMP9 activity by Zymography on plasma samples from the same CF patients undergoing Trikafta® therapy. The results obtained revealed that the MMP9 levels measured in plasma reflected the same trend observed in leukocytes. Furthermore, we also analyzed the intracellular signaling pathway associated with MMP9 expression, in particular the modulation of the extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor-kB (NF-kB) pathways. The results obtained allowed us to demonstrate that the modulation of MMP9 following treatment with Trikafta® may be controlled by the NF-kB pathway. Finally, we can conclude that the downregulation of MMP9 expression could be considered a promising biomarker of therapy efficacy, useful to understand the molecular events underlying the variable clinical responses of CF patients to CFTR modulators. This knowledge, obtained with a simple blood withdraw, can be useful for future studies of personalized medicine.