Invasive fungal infections are a major cause of mortality in solid-organ transplantation where steroids and calcineurin inhibitors form the core of immunosuppression. Our group has previously shown in established hydrocortisone-based mouse models of invasive aspergillosis that calcineurin inhibitors increase mortality through effects on the innate immune response.1 As alveolar macrophages present the primary resident innate immune cell in the airways responsible for fungal clearance, we perform a detailed study of the role of the calcineurin pathway in the human macrophage response to A. fumigatus (AF).
We show that the calcineurin-NFAT pathway is highly activated in the human lung transplant alveolar macrophage response to AF with inhibition resulting in impaired fungal clearance. Calcineurin inhibition leads to delayed phagocytosis, reduction in reactive-oxygen species production and an impairment of a novel actin-dependent process of lateral transfer of swollen AF conidia between human macrophages. Further characterisation reveals that transfer of AF occurs during macrophage necroptosis with subsequently around 50% control of germination in the receiving macrophage. To understand the calcineurin-dependent mechanism, next generation RNA sequencing was performed which confirms that calcineurin inhibition impairs the macrophage programmed cell death immune response. Utilising phosphoproteomics we additionally show that calcineurin inhibition impairs dephosphorylation of vasodilator-stimulated phosphoprotein (VASP), an important actin regulatory protein which promotes actin filament formation. High-resolution confocal microscopy confirms that VASP strongly co-localises to AF conidial phagocytosis and facilitates lateral transfer through tunnel-like structures. Lastly, we utilise a zebrafish model of invasive aspergillosis to confirm the in-vivo relevance of AF macrophage lateral transfer.
In conclusion our data shows the importance of the calcineurin pathway in the macrophage innate immune response to AF and highlights a novel calcineurin-actin dependent host defense mechanism which may have significant implications on persistence and dissemination within solid organ transplantation. To our knowledge this is the first report of a host-mediated cell-cell transfer mechanism for any pathogen.
Reference 1 Herbst S, Shah A, Mazon Moya M, Marzola V, Jensen B, Reed A, et al. Phagocytosis-dependent activation of a TLR9-BTK-calcineurin-NFAT pathway co-ordinates innate immunity to Aspergillus fumigatus. EMBO Mol Med. 2015;7(3):240–58