Phd in CELL AND DEVELOPMENTAL BIOLOGY

Thesis title: Analysis of the effect of long-term elexacaftor-tezacaftor-ivacaftor therapy on immune cells of people with cystic fibrosis

Background: Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene, leading to defective chloride and bicarbonate transport, airway dehydration, chronic infection and inflammation. In 2019, the triple combination of CFTR modulators elexacaftor-tezacaftor-ivacaftor (ETI) was approved for the treatment of pwCF carrying at least one F508del mutation, providing highly effective treatment for up to 90% of patients. The high clinical effectiveness of ETI has stimulated the study of the effect of therapy on other major drivers of lung disease progression in CF such as airway bacterial infections, inflammation and altered innate immune responses. Considering the importance of innate immune cells in the antimicrobial and inflammatory response e due to the lack of data on the long-term effect of ETI therapy on innate immune cells, we studied the antimicrobial activity of the ex vivo isolated mononuclear cells after 12 and up to 48 months of ETI therapy. In addition, we have investigated the expression of CFTR at the RNA and protein level in PBMCs and monocytes before and after therapy and compared the CFTR channel function between monocytes from ETI-treated pwCF and healthy donors. Finally, the effect of ETI on systemic inflammation was evaluated. Methods: The antimicrobial activity of the ex vivo isolated monocytes was evaluated by flow cytometry after infection of PBMCs with P. aeruginosa strain expressing GFP (PAO1-GFP). CFTR expression at the RNA and protein level in PBMCs and monocytes was assessed by qRT-PCR and Western blot respectively. CFTR channel function was evaluated by assessing the halide efflux using MQAE sensitive fluorescent indicator. Finally, the serum levels of IL-6, IL-8 and IL-1β was evaluated by ELISA. Results: ETI therapy confers sustained improvements in lung function, BMI, and QoL, and reduced the percentages of pwCF infected by CF pathogens after four years of therapy. Long-term ETI therapy showed that the rescue of P. aeruginosa uptake by CF monocytes is maintained over time, reaching values comparable to those of healthy donors. CFTR protein expression increased in both PBMCs and monocytes, reaching levels similar to healthy donors in PBMCs but not in monocytes. CFTR mRNA levels increased in PBMC after 12-48 months of ETI therapy while they remained unchanged in monocytes suggesting a heterogeneity of ETI responses among immune cell subsets. Although CFTR channel activity in monocytes remained lower than in cells from healthy donors, it correlated positively with improved lung function. ETI therapy significantly decreased serum levels of IL-6 and IL-1β, while IL‐8 levels didn’t change. Conclusions: Long-term ETI therapy confers sustained improvements in clinical, microbiological, and inflammatory outcomes after four years of therapy. ETI therapy further improved monocyte P. aeruginosa phagocytosis over time reaching levels similar to healthy donors while CFTR expression and channel activity remained significantly lower than those of controls. Further research is required to evaluate the effect of ETI on additional monocyte antimicrobial functions and systemic inflammation.