Abstracts from the 24th Congress of the Italian Society of Cystic Fibrosis and the 14th National Congress of Cystic Fibrosis Italian Society

Abstract

s from the 24th Congress of the Italian Society of Cystic Fibrosis and the 14th National Congress of Cystic Fibrosis Italian Society Salerno, Italy. 8 -10 November 2018 Published: 16 April 2019 CFTR/CELLULAR STRESS A1 Myriocin potential as a phenotype-modifying therapeutical in Cystic Fibrosis Natalia Cirilli, Alessandra Mingione, Emeranziana Ottaviano, Fabiola Bonezzi, Michele dei Cas, Matteo Barcella, Silvia Tedesco, Tatiana Armeni, Anna Caretti, Rita Paroni, Elisa Borghi, Paola Signorelli Cystic Fibrosis Care Centre, Mother-Child Department, United Hospitals, Ancona, Italy; Italian Research Cystic Fibrosis Foundation, Verona, Italy; Biochemistry and Molecular Biology Laboratory, Health Sciences Department, University of Milan, Milan, Italy; Microbiology Laboratory, Health Sciences Department, University of Milan, Milan, Italy; Clinical Biochemistry and Mass Spectrometry Laboratory, Biochemistry and Molecular Biology Laboratory, Health Sciences Department, University of Milan, Milan, Italy; Dipartimento Scienze Cliniche, SpecialisticheDI.S.C.O., Università Politecnica delle Marche, Ancona, Italy Correspondence: Natalia Cirilli ([email protected]) Italian Journal of Pediatrics 2019, 45(Suppl 1):A1 Background Cystic Fibrosis (CF) is caused by a variety of mutations of the CFTR ion channel, with deletion of phenylalanine 508 (F508del) representing approximately 70% of patients. This mutation induces a proteinopathy, characterized by aggregates of mutated and unfolded proteins, hyper-inflammation, impaired trafficking and altered metabolism at cellular level. CFTR dysfunction has primarily a devastating effect on lungs, causing chronic inflammation and recurrent unresolved infections. CF patients develop severe comorbidities: male infertility, biliary cirrhosis, osteopenia/osteoporosis, renal failure, diabetes (50% of adult patients), malabsorption and increased synthesis of cholesterol and its accumulation in liver but also in other tissues, dyslipidemia with increased plasma triglycerides, pancreas fibrolipomatosis, hepatic lipogenesis and steatosis. Lung chronic inflammation and infection have been extensively associated to the lipotoxin ceramide, core component of membrane lipids. such as sphingomyelins and glycosphingolipids. We previously demonstrated that the sphingolipid synthesis inhibitor Myriocin is able to reduce inflammation and to ameliorate defense response against bacterial and fungal infection. Materials and methods 43 CF patients with selected genotypes were enrolled. Monocytes from peripheral blood samples were extracted and also clinical data were collected. Results We here demonstrate the mode of action of this molecule. First, we show in IB3 cell line, that Myriocin is an effective inducer of autophagy which is defective in CF proteinopathy and can sustain pathogen © The Author(s). 2019 Open Access This artic International License (http://creativecommons reproduction in any medium, provided you g the Creative Commons license, and indicate if (http://creativecommons.org/publicdomain/ze clearance (xenophagy). Second, we demonstrated that Myriocin activates key transcriptional factors, TFEB, FOXO1a and PPARgamma involved in autophagy induction, mitochondria genesis and activity, energy production and lipid mobilization and consume. We proved that Myriocin significantly increases the transcription of downstream genes regulating fatty acids entry in mitochondria (CTP1a and 1b; FATP) and their oxidation (ACAD L). Next, we showed that Myriocin drammatically reduces pathological accumulation of lipid unorganized deposits in IB3 cells. By Mass spectrometry we observed that inhibition of sphingolipid synthesis causes a reduced content of non sphingoid-base containing lipids, such as glycerol lipids and cholesterols. We then proved that Myriocin is able to change the transcriptional profile of IB3 treated cells, by gene sequencing analysis, enhancing the transcription of genes involved in lipid transport and consume and energy metabolism that resulted partially downregulated in CF. Finally, Myriocin treatment of peripheral blood monocytes from CF patients, infected with A. fumigatus conidia, significantly increases their pathogen killing ability. Conclusions Myriocin is a potent modifier of pathological gene expression profile in CF and a potential therapy for CF related infection. A2 miR-125b/NRF2/CFTR circuitry: a pilot study on oxidative stress in cystic fibrosis Maria Pelullo, Francesca Megiorni, Giuseppe Cimino , Antonio Pizzuti, Serena Quattrucci, Claudio Talora, Samantha Cialfi Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy; Department of Pediatrics, Sapienza University of Rome, Rome, Italy; Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy Correspondence: Samantha Cialfi ([email protected]) Italian Journal of Pediatrics 2019, 45(Suppl 1):A2 Background In physiopathology of Cystic Fibrosis (CF), oxidative stress implications were recognized and widely accepted. The CFTR defects in ionic transport disrupt the intracellular redox balance causing CF classical pathologic hallmarks. Therefore, oxidative stress together with detoxification genes and miRNA aberrant expression could be associated with clinical outcome. Materials and methods Patients (n=8) with CF diagnosis with the same mutation (F508del), but various clinical status, compared to healthy individuals. After nasal brushing, we extracted total RNA of patients for consecutive expression analysis of CFTR, NRF2 and its targets as well as miR-125b, by quantitative real time PCR (qPCR) using TaqMan chemistry. Written informed consent was obtained by each participant to the study. le is distributed under the terms of the Creative Commons Attribution 4.0 .org/licenses/by/4.0/), which permits unrestricted use, distribution, and ive appropriate credit to the original author(s) and the source, provide a link to changes were made. The Creative Commons Public Domain Dedication waiver ro/1.0/) applies to the data made available in this article, unless otherwise stated. Italian Journal of Pediatrics 2019, 45(Suppl 1):43 Page 2 of 26 Results In this pilot study, we analyzed the existence of a correlation among CFTR, miR-125b, NRF2 and oxidative stress genes in a representative number of CF patients. In CF patients with chronic P. aeruginosa (PA) lung infection, the mRNA levels of the NRF2 gene were significantly down-expressed and showed a direct correlation with CFTR gene expression. Interestingly, the NRF2 downmodulation was accompanied by an induced expression of miR-125b. On the contrary, PA negative CF patients showed NRF2 expression levels more similar to those of healthy individuals, with a reduction of miR-125b, even if CFTR levels remained downmodulated. These data are in line with the expression of the oxidative stress target genes NQO1 and GST-T1. Moreover, we found that PA positive patients with a FEV1>60% showed the expression of HMO1, another NRF2 target gene, higher than those with a lower FEV1. Conclusions The evidence of a CFTR, NRF2 and miR-125b impaired network as oxidative stress response in CF patients, prompt us to hypothesize that these molecular mechanisms could explain the wide CF phenotypic variability as an additional control level over the CFTR gene mutations. Furthermore, this study may allow the discovery of potential therapeutic targets in order to improve CF patient’s quality of life by screening the expression of these oxidative stress factors as prognostic markers.