The utility of sphingolipid pathway inhibitors in the treatment of aspergillosis

Abstract

A. fumigatus is a ubiquitous fungus that causes invasive pulmonary aspergillosis (IPA). IPA is one of the most life-threatening infections in immune compromised patients. Corticosteroids are commonly prescribed as immunosuppressive agents for transplant patients, and IPA mostly occurs in the setting of potent transplant maintaining immune suppressive therapy. Corticosteroid immune suppression represents a major risk factor for IPA, as it inhibits the killing capacity of alveolar macrophages and results in fungal growth that recruits non-sterilizing PMNs to the site of infection that leads to tissue damage. This PhD aimed to characterize effects of the sphingosine-1-phosphate (S1P) pathway inhibitors SKI-II and FTY720 for their utility in attenuating the characteristics of IPA immunopathology, such as inflammation and fungal development. Firstly, it was demonstrated that A. fumigatus-mediated cytokines and chemokines were down regulated in vitro in the presence of inhibitors, and direct sphingosine kinase (SphK) inhibition resulted in a phagocytic defect but not S1P1 receptor antagonism. Inflammatory responses and fungal burden were then evaluated in immunocompetent mice with FTY720, and it was shown that cytokine and chemokine responses were inhibited without compromising fungal clearance. Further to this, FTY720 was evaluated for its efficacy in the hydrocortisone model of aspergillosis. Both pro-inflammatory mediators and fungal burden were reduced, and increased survival was observed. Secondly, the antifungal properties of both SKI-II and FTY720 were investigated. SKI-II was shown to have limited activity with germination kinetics but inhibited hyphal extension, whereas FTY720 was demonstrated to inhibit fungal germination and hyphal growth significantly. Genetic deletions in A. fumigatus of SphK and gprD putative genes were generated, corresponding to mammalian sphingosine kinase 1 and S1P1. Phenotypic examination showed that mutants were sensitive to cell wall stress and had altered germination kinetics, and ∆gprD was less immunogenic. Overall these findings in this thesis indicate the S1P1 inhibitor FTY720 has antifungal activity in vitro against A. fumigatus with therapeutic effects in vivo in the HC model of IPA and could be developed as an antifungal therapeutic.