Impact of specialized pro-resolving lipid mediators on the colonization of CF respiratory epithelia by aspergillus fumigatus

Introduction

Inflammation is normally self-regulated by an active resolution phase, orchestrated by specialized pro-resolving mediators (SPMs) such as lipoxins (LX), resolvins (Rv), protectins (PD) and maresins. We recently showed that SPMs biosynthesis by airway epithelial cells is altered in cystic fibrosis (CF). CF is a genetic disease mainly characterized by airway chronic infection and excessive inflammation. Aspergillus fumigatus, a predominant filamentous fungus in CF is associated with lung injury and function decline. The consequences of the dysregulated SPMs biosynthesis on the colonization of the CF respiratory track by the filamentous fungi, A. fumigatus has not been investigated before this study.

Methods

We used human nasal epithelial primary cultures (hNEC) from CF and non-CF patients and the bronchial epithelial cell line (CFBE). We inoculated A. fumigatus (Af Dsred) conidia (MOI 1: 6.24 h) on epithelial cells grown on plastic or at air-liquid interface, treated or not with SPMs (10 nM). SPM receptors expression was studied by immunofluorescence. Fungal load was quantified by optical density using a microplate reader, by qPCR of gDNA, and using a galactomannan assay on the culture medium. Tight junctions were visualized using ZO-1 protein immunostaining. Antimicrobial peptides were explored using RTqPCR. The role of SPM receptors was evaluated using selective agonists and/or antagonists.

Results

On one hand, A. fumigatus infection for 24 h disrupted most of the airway epithelial tight junctions. When cells were treated with SPMs (LXA4, LXB4, RvE1, RvD₅and PD₁) simultaneously with A. fumigatus, the epithelial tight junction integrity was significantly protected. The SPMs’ protecting effect on tight junctions was observed up to 12 h after A. fumigatus exposure. The SPM protecting effect on tight junctions involved the G-protein coupled receptors, FPR2 (LXA4 and LXB4), GPR32 (RvD₅and LXB4), ChemR23 (RvE1) and GPR37 (PD₁) that we found to be expressed airway epithelial models. On the other hand, airway epithelium reduced A. fumigatus growth and secretion of one of its virulence factor galactomannan. The treatment of the airway epithelial cells with SPMs (LXA4, LXB4, RvE1, RvD₂, RvD₅, PD₁) enhanced this latter effect with a further decrease of A. fumigatus galactomannan. Some SPMs as RvE1 stimulated the transcription of HBD₂antimicrobial peptides.

Conclusion

Our results show that several SPMs stimulate the protecting role of epithelial cells in enhancing its barrier function. SPMs also reduce the secretion of galactomannan, which is an A. fumigatus virulence factor playing a critical role in biofilm formation. This suggests that the anomaly of SPMs biosynthesis in CF could significantly contribute to the reduced capacity of respiratory epithelial cells to protect themselves and to fight A. fumigatus. This study opens new therapeutic perspectives.