Myriocin ameliorates defective killing of pathogens in cystic fibrosis patients

Introduction- Chronic inflammatory diseases affecting airway mucosa, such as Cystic Fibrosis (CF), are characterized by defective immunity and recurrent infections of pathogens organized in drug resistant microbial communities, named biofilms. Aspergillus fumigatus is the most prevalent filamentous fungus in the respiratory tract of CF patients, contributing to lung deterioration. Myriocin, an orphan drug with pleiotropic action, reduces pro-inflammatory lipids accumulation, promoting anti-oxidant response to stress. This compound, administered in vivo by nanocarriers, has been demonstrated to reduce inflammation and to ameliorate, in a mice model of CF, host response to pathogens. Moreover, myriocin exerts a direct fungistatic activity against in vitro preformed biofilms of A. fumigatus. Materials and Methods – The effect of myriocin on A. fumigatus phagocytosis and killing has been investigated in: i) IB3-1 cells, an adeno-associated virus-transformed human bronchial epithelial cell line derived from a CF patient (ΔF508/W1282X) and its isogenic C38 cells corrected by insertion of CFTR; ii) CF patient monocytes, isolated from peripheral blood. We infected cells, either pre-treated or not with myriocin (10 μM, 2 h), with A. fumigatus (Af293, MOI 1:1). Non-internalized conidia were removed after 1 hour and cells allowed killing the internalized fungus for 4 hours. Still alive conidia were counted after osmotic cell lysis by colony forming unit (CFU count). For IB3-1 and C38 cells, the expression of pathogens receptors TLR2 and NOD2 were investigated by real-time PCR, as well as of NRF2 and HO1, involved in anti-oxidant response. Results –CF epithelial cells displayed a markedly reduced ability to kill internalized fungi compared with controls. Myriocin corrected the defective IB3-1 killing ability, decreasing more than two folds the number of CFU/cell. Preliminary results on CF monocytes seem to confirm this data. A basal significantly reduced expression of HO1, NOD2 and TLR2 in IB3-1 versus C38 was observed, suggesting that these proteins may account for IB3-1 defective fungal killing. Myriocin treatment significantly restored the TLR2 and NOD2 expression and HO1 via NRF2 activation. Discussion and Conclusions - Internalized A. fumigatus conidia remain alive in CF epithelial cells whereas are promptly killed by control cells. We demonstrated that myriocin, by reducing the inflammatory and oxidative state of CF cells, it is also able to correct CF defective killing of internalized fungal conidia, possibly by enhancing microbe-targeted autophagy.