Meeting Abstract 97 - 34th Annual North American Cystic Fibrosis Conference, October 7–23, 202

Castagnozzi, S

Colas, RA

Dalli, J

Gomez, EA

Isopi, E

Mattoscio, D

Plebani, R

Romano, M

Trerotola, M

Pediatric Pulmonology

Queen Mary Research Online

CFTR ABLATION BY CRISPR/CAS9 IMPAIRSTHE PRODUCTION OF PRO-RESOLVING LIPIDMEDIATORS BY HUMAN AIRWAY EPITHELIAL CELLSPlebani, R.1; Colas, R.A.2; Mattoscio, D.1; Trerotola, M.1;Gomez, E.A.2; Isopi, E.1; Castagnozzi, S.1; Dalli, J.2;Romano, M.1 1. Department of Medical Oral andBiotechnological Sciences, Gabriele d’Annunzio University ofChieti and Pescara, Chieti, Abruzzo, IT, Grottammare, AscoliPiceno, Italy; 2. Centre for Inflammation and TherapeuticInnovation, Queen Mary Univ of London, London, London,United KingdomIntroduction: Progressive lung disease, characterized by recurrentbacterial infection and unresolved inflammation is a hallmark of cysticfibrosis (CF), representing the major cause of mortality and morbidity. Ourprevious results (Mattoscio D, et al. Faseb J. 2010;26:1323-33) suggest thatimpaired formation of specialized pro-resolving lipid mediators (SPM),including lipoxins (LX), resolvins (Rv), protectins (PD), maresins (Mar)and SPM conjugates in tissue regeneration, may contribute to CF unrelentinginflammation. Moreover, we have recently uncovered pro-resolving,antimicrobial properties of RvD1 in in vivo and in vitro CF models (IsopiE, et al. Front Immunol. 2020;11:581). Thus, the understanding of the relationshipbetween CFTR loss-of-function and SPM production is of keyimportance to develop new generation drugs to combat CF inflammation.Here, we investigated the SPM profile of human airway epithelial cellsupon CFTR ablation by CRISPR/Cas9.Methods: CRISPR/Cas9 ablation of CFTR expression was carried outby targeting the exon 1 of the CFTR gene in the human airway cell line16HBE14o-. SPM profiling of parental and KO clones was carried outusing a QTrap 6500+ MS system, coupled with a Shimadzu SIL-20ACHT auto sampler, and LC-20AD LC pumps and equipped with an AgilentC18 Poroshell column (Dalli J, et al. Methods Mol Biol. 2018;1730:59-72).Multivariate OPLS-DA analysis was performed using the SIMCA software,building a model with R2X=1, R2Y=1, and Q2=1.Results: No CFTR expression and activity were detected in CRISPR/Cas9 ablated clones. The analysis of all four major fatty acid bioactivemetabolomes including arachidonic acid (AA)-derived LX and n-3 docosapentaenoic(DPA), docosahexaenoic (DHA) and eicosapentaenoic (EPA)acid-derived Rv revealed that CFTR-KO cells showed a significant reductionin overall SPM concentrations, represented by the sum of DHA-derivedD-series Rv, PD, Mar, n-3 DPA-derived Rv and PD, EPA-derivedE-series Rv and AA-derived LX (p 􀀚 0.05). We observed a significantreduction in the EPA-derived E-series Rv (RvE1, RvE2, RvE3) (p 􀀚 0.01),with RvE3 showing the greatest reduction (p 􀀚 0.001). PDn-3 DPA was alsosignificantly reduced in CFTR-KO cells (p 􀀚 0.001).Conclusions: Our present results provide the first evidence that CFTRregulates SPM generation, supporting the concept that impaired pro-resolvingmechanisms due to CFTR dysregulation may contribute to thedevelopment and progression of aberrant inflammation in CF. Thus, correctionof such resolution deficit by SPM administration may represent a more“physiologic” strategy to control CF inflammation.Acknowledgments: Supported in part by a grant from the ItalianMinistry of Health (L.548/93) to MR. JD is funded by European ResearchCouncil (ERC, grant no: 677542) and the Barts Charity (grant no:MGU0343). JD is also supported by a Sir Henry Dale Fellowship jointlyfunded by the Wellcome Trust and the Royal Society (107613/Z/15/Z).

English

CFTR ABLATION BY CRISPR/CAS9 IMPAIRS THE PRODUCTION OF PRO-RESOLVING LIPID MEDIATORS BY HUMAN AIRWAY EPITHELIAL CELLS

CFTR ABLATION BY CRISPR/CAS9 IMPAIRS THE PRODUCTION OF PRO-RESOLVING LIPID MEDIATORS BY HUMAN AIRWAY EPITHELIAL CELLS

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