Antisynthetase syndrome (ASSD) is an autoimmune disease characterized by the positivity of autoantibodies against different aminoacyl transfer RNA (tRNA) synthetases. Morbidity and mortality of this disease are highly affected by interstitial lung disease (ILD) which is present in about 80% of patients. In this study, we investigated possible differences in 84 immune-related circulating miRNAs between ASSD patients with and without ILD; we enrolled 15 ASSD patients, 11 with ILD (ILD+) and 4 without ILD (ILD-), and 5 patients with idiopathic pulmonary fibrosis (IPF) as an additional control group. All patients were at disease onset and not on therapy at the time of inclusion. Differentially expressed miRNAs were identified in plasma-derived exosomes, using an miRNA PCR array (MIHS-111ZG, Qiagen, Hilden, Germany); miR-30a-5p and miR-29c-3p were upregulated in ASSD-ILD patients compared to patients without lung involvement (adjusted p-value < 0.05). IPF patients showed higher miR-29c-3p expression levels with respect to both ASSD and ASSD-ILD (p = 0.0005), whereas levels of miR-30a-5p were not different. miR-29c-3p and miR-30a-5p are overexpressed in ASSD-ILD+ patients compared with ILD−. These miRNAs are involved in the regulation of inflammation and fibrosis through their action on NF-κB and TGF-β1. Although the mechanistic role of these miRNAs in ASSD-ILD development has to be elucidated, we suggest that their exosome levels could be useful in identifying patients at risk of ILD.

Recent studies have emphasized the critical role of alteration in cellular plasticity in the development of fibrotic disorders, particularly pulmonary fibrosis, prompting further investigation into molecular mechanisms and therapeutic approaches. In this context, Precision Cut Lung Slices (PCLSs) emerge as a valuable ex vivo research tool. The process of PCLSs generation preserves most features of the naïve lung tissue, such as its architecture and complex cellular composition. We previously stimulated normal lung PCLSs with two different stimuli (fibrotic cocktail, composed by platelet lysate and TGFβ, or neutrophil extracellular traps) and we observed a significant elevation of Epithelial–Mesenchymal Transition (EMT) markers from 24 h to 72 h of culture. The aim of our work was to exploit this PCLSs based ex vivo model of EMT, to evaluate the effect of imatinib, an old tyrosine kinase inhibitor with reported anti-remodeling activities in vitro and in animal models. Imatinib treatment significantly decreased α-SMA and collagen expression already starting from 24 h on stimulated PCLS. Imatinib showed a significant toxicity on unstimulated cells (3-fold increase in ACTA2 expression levels at 24 h, 1.5-fold increase in COL1A1 expression levels at 24 h, 2-fold increase in COL3A1 expression levels at 72 h). Further evaluations on specific cell lines pointed out that drug effects were mainly directed towards A549 and LFs. In conclusion, our model confirms the anti-remodeling activity of imatinib but suggests that its direct delivery to alveolar epithelial cells as recently attempted by inhalatory preparation of the drug might be associated with a non-negligible epithelial cell toxicity.

Background: Pulmonary fibrosis (PF) is a form of progressive lung disease characterized by epithelial-endothelial mesenchymal transition (EMT) and extracellular matrix deposition. Aim: We aimed to test Imatinib (an anti-remodelling Tyrosine-Kinase inhibitors) activity in an innovative “in vitro” model of EMT, which is obtained in human precision cut lung slices (PCLS). Methods: PCLS (obtained by normal lung tissue) within 4 hrs from retrieval, were cultured for up to 72 hrs in presence of: a) Platelet Lysate (LP) (3%) + TGF-β (10ng/mL); b) Neutrophil Extracellular Traps (NETs) (from 100 nM PMA stimulation of 5x106 Neutrophils). 15 µM Imatinib was added to certain PCLS. Markers of EMT were detected at 24-48-72 hrs of culture: expression of α-SMA (ACTA2), Collagen1a1 (COL1a1) and Collagen3a1 (COL3a1) by RT-PCR and protein levels at 48-72 hrs by WB analysis. Results: Expression profiles with both stimulations showed an increase of all markers by 24 to 72 hrs. Surprisingly, Imatinib alone caused an increase in the expression levels of COL1a, COL3a1 and α-SMA compared to untreated slices. This drug, when added to LP-TGFβ or to NETs significantly down regulated or abolished the expression α-SMA, COL1a1, COL3a1. All data were confirmed by WB analysis at 72 hrs. Conclusion: PCLS allow the generation of a “whole lung” EMT model useful to assess the activity of anti-remodeling therapeutic strategies.

Precision-cut lung slices (PCLS) derived from lung resections or explants are attracting growing interest as human lung diseases models. Based on our previous results, we patented hyaluronic acid functionalized liposomes (xhalip) loaded with Imatinib, designed for inhalation therapy of Chronic Lung Allograft Dysfunction (CLAD) and other forms of inflammatory/immune driven lung fibrosis. To evaluate the anti-remodeling effects of xhalip, we devised an Epithelial Mesenchymal Transition (EMT) model using PCLS. PCLS were subjected to treatment with either a "fibrogenic cocktail (FC)" consisting of platelet lysate and TGFbeta (10ng/ml), or purified NETs (from 2.5 x 106 neutrophils activated with PMA). Gene expression levels of a-SMA (ACTA2), type 1a1 collagen (COL1A1) and type 3a1 collagen (COL3A1) were monitored up to 96 hours. Moreover, cellular observations were conducted using confocal laser microscopy to assess xhalip uptake and distribution. Fibrosis markers expression levels were significantly upregulated by NETs or FC with respect to control up to 96 hrs of culture. Treatment of stimulated PCLS with xhalip, or with free Imatinib, led to a significant reduction in ACTA2 expression (70-50% decrease at 48 hrs with xhalip and 50-30% decrease with Imatinib at 48 and 72 hrs, respectively), as well as COL1A1 (60%-10% reduction with both treatments at 48 and 72 hrs) and COL3A1 (reduced by 40% and 5% with both treatments at 48 and 72 hrs, respectively). Moreover, with confocal microscopy we observed a significant uptake of xhalip in PCLS up to 48 hours. Imatinib-loaded xhalip presents a promising avenue for inhalatory treatment of CLAD, especially targeting the obstructive phenotype.