Defining the calcineurin-dependent innate immune response of human alveolar macrophages to Aspergillus fumigatus

Abstract

Pulmonary aspergillosis is a lethal mould infection in the immunocompromised host. Understanding initial control of infection, and how this is altered in the immunocompromised host, is a key goal for understanding the pathogenesis of pulmonary aspergillosis. My thesis aims to characterise the outcome of human macrophage infection with Aspergillus fumigatus, and understand how this is altered in transplant recipients on calcineurin inhibitor immunosuppressants. I initially quantified the impact of calcineurin inhibitors on fungal killing following in vitro human alveolar macrophage infection with A. fumigatus. A human monocyte-derived macrophage model was then used to systematically analyse calcineurin-dependent effects on phagocytosis, phagosomal maturation, reactive oxygen species and cytokine production. Calcineurin-dependent mechanisms of fungal killing were then further characterised by a combination of single cell fluorescence imaging, proteomics, and in vivo studies. Human alveolar macrophage killing of A. fumigatus is calcineurin-dependent. Macrophage phagocytosis of A. fumigatus enabled control of 90% of fungal germination. However fungal germination in the late phagosome led to macrophage necrosis. During programmed necrosis, I observed frequent cell-cell transfer of A. fumigatus between macrophages which assists subsequent control of germination in recipient macrophages. Lateral transfer occurred through actin-dependent exocytosis of the late endosome in a vasodilator-stimulated phosphoprotein (VASP) envelope. Its relevance to the control of fungal germination was also shown by direct visualisation in a zebrafish aspergillosis model in vivo. The calcineurin inhibitor FK506/tacrolimus reduced cell death and lateral transfer in vitro by 50%. This resulted in uncontrolled fungal germination in macrophages and hyphal escape. This thesis identifies programmed necrosis-dependent lateral transfer of A. fumigatus between macrophages as an important host strategy for controlling fungal germination. This process is critically dependent on calcineurin. This provides fundamental insights into the pathogenesis of pulmonary aspergillosis in the immunocompromised host.