Final Report Summary - FLY FUNGAL INTERPLAY (Dissecting innate immunity to airborne opportunistic fungi through a genome-wide screen in Drosophila)
Fungi are emerging pathogens across a broad range of hosts, from plants to humans. Ubiquitous airborne filamentous fungi (molds) are major causes of life-threatening infections in an expanding population of immunocompromised individuals. Aspergillus is by far the major airborne fungal pathogen in these patients with mortality rates approaching 90% in disseminated infection. Over the past decade we have witnessed the emergence of invasive infections caused by rare airborne opportunistic molds, including Mucorales, in severely immunocompromised patients. In addition, a large epidemic of fungal meningitis caused by dematiaceous fungi in healthy individuals receiving contaminated injections of corticosteroids has been recently reported in the Unites States, illustrating the evolving epidemiology of fungal infections.
In view of the mediocre activity of current antifungal agents there is a need to better understand immunopathogenesis of fungal infections at the cellular and molecular level in robust experimental systems in order to develop targeted therapeutics. In healthy individuals, elimination of inhaled (conidia) spores of airborne fungi occurs within acidified phagolysosomal compartments of professional phagocytic cells on a daily basis Quantitative or qualitative defects in phagocyte function, as a result of chemotherapy-induced neutropenia or treatment with high doses of corticosteroids are the main predisposing factors for development of invasive mold infections. In addition, genetic defects in NADPH oxidase activity in patients with chronic granulomatous disease (CGD) lead to unique predisposition for development of invasive pulmonary aspergillosis. Despite significant achievements in understanding pathogenesis of invasive mold infections, the key molecular attributes of host-fungal interplay regulating intracellular killing of conidia within phagocytes are largely unknown.
Aim of this proposal was to identify novel evolutionarily conserved genes with important role in immunity against airborne opportunistic fungi in Drosophila melanogaster and confirm their role in antifungal immunity in humans. Because of the emerging role of autophagy in cellular homeostasis and immunity we prioritized our studies on dissection of the role of autophagy in antifungal immunity. Studies in Drosophila melanogaster flies following conditional inactivation of autophagy genes in fly hemocytes demonstrated increased susceptibility to fungal infection. In addition, induction of autophagy upon treatment with rapamycin protected fruit flies upon infection with fungi.
These preliminary results in Drosophila melanogaster model prompted us to evaluate the role of autophagy in antifungal immunity in humans. We focused our studies in human phagocytes on LC3 associated phagocytosis (LAP), a specialized form of noncanonical autophagy that links activation of pattern recognition receptors with phagosome maturation. Importantly, we found that infection of primary human monocytes with Aspergillus spores triggered selective recruitment of the autophagy hallmark protein LC3 II in phagosomes upon fungal cell wall swelling. This response was induced by surface exposure of immunostimulatory β-glucans, and was mediated by activation of the Dectin-1 receptor, as it was abolished in monocytes from patients with the homozygous early stop-codon mutation Tyr238X in Dectin-1, who display lack of surface receptor expression. LC3 recruitment in A. fumigatus-phagosomes required syk kinase-dependent production of reactive oxygen species (ROS) and was almost completely abolished in monocytes of patients with genetic defects in NADPH oxidase (chronic granulomatous disease, CGD). LAP was important for control of intracellular fungal growth, as silencing of Atg5 resulted in impaired phagosome maturation and killing of A. fumigatus. Because corticosteroid-induced immunosupression is In vivo and ex vivo administration of corticosteroids blocked LC3 recruitment in A. fumigatus phagosomes of human monocytes via rapid inhibition of syk kinase phosphorylation and downstream production of ROS. Overall, our studies link Dectin-1/syk kinase signaling with maturation of A. fumigatus phagosomes and uncover a mechanism for development of invasive fungal disease. The results of this work have been published as a featured paper in J Immunol (Kyrmizi I et al., J Immunol. 2013 191:1287-99)
In a collaborative study we also demonstrated the importance of harnessing autophagy to improve antifungal immunity in patients with CGD. Specifically, treatment with an IL1 receptor antagonist (Anakinra) restored antifungal autophagy in monocytes of CGD patients. The clinical relevance of these findings was assessed by treating CGD patients who had severe colitis with IL-1 receptor blockade using anakinra. Anakinra treatment resulted in a rapid and sustained improvement in colitis. Thus, inflammation in CGD is due to IL-1-dependent mechanisms, such as decreased autophagy and increased inflammasome activation, which are linked pathological conditions in CGD that can be restored by IL-1 receptor blockade (de Luca A et al., Proc Natl Acad Sci U S A. 2014;111:3526-31).
In the second part of our work we explored whether fungal pathogens employ virulence mechanisms that target activation of LAP. Concealing of pathogen associated molecular patterns (PAMPs) is a fundamental pathogenetic mechanism that allows fungi to avoid immune activation during phagocytosis. In line with this model, dormant conidia of Aspergillus fumigatus are immunologically inert, and fail to activate LAP, because cell wall PAMPs are covered by a layer of hydrophobic rodlet protein RodA and melanin. Upon germination of A. fumigatus conidia, β-glucan surface exposure triggers NADPH oxidase-dependent activation of LAP and facilitates fungal killing. However, in view of the major cell wall structural changes that occur during germination, it remains unclear whether masking of PAMPs is the principal mechanism of fungi to evade autophagy and killing by macrophages.
Importantly, in contrast to the prevailing model of masking of PAMPs, we found that β-glucan surface exposure on dormant conidia of RodA-deficient A. fumigatus (ΔrodA) is insufficient to activate LAP. Instead, genetic removal of cell wall melanin (ΔrodA/pksP and ΔpksP) is required to enable β-glucan-mediated LC3 recruitment in A. fumigatus phagosomes. Genetic or biochemical removal of cell wall melanin in ΔrodA and exogenous melanin complementation in melanin-deficient A. fumigatus conidia demonstrated that melanin does not interfere with signaling pathways regulating cytokine release, but selectively inhibits LAP by blocking assembly of NADPH oxidase. Importantly, the attenuated pathogenicity of melanin-deficient A. fumigatus (ΔpksP) is dependent on activation of antifungal autophagy and it is significantly restored in Atg5-deficient macrophages and in mice upon conditional inactivation of Atg5 in myeloid cells. Thus, apart from shielding PAMPs, the major role of fungal cell wall melanin is to inhibit phagosome biogenesis. This work was awarded in 6th Advances Against Aspergillosis meeting, Madrid, 27/2-1/3/2014 and the manuscript is under submission (Nature Letters).
Collectively, our work led to the discovery of a new antifungal immunity pathway that links signaling activation with intracellular elimination of conidia of airborne fungi. This pathway has major physiologic relevance in control of fungal infection by human phagocytes as evidenced by impaired autophagy protein recruitment in A. fumigatus-containing phagosomes in two distinct groups of patients with increased susceptibility for invasive aspergillosis. Furthermore, we have identified fungal cell wall melanin as a unique virulent factor that specifically targets the autophagy pathway and allows the fungus to resist killing within the phagosome. Importantly, a new model in fungal pathogenesis has emerged from our work demonstrating that two fundamental biologic events, which physiologically occur concurrently during germination of A. fumigatus, are the necessary requirements for activation of antifungal autophagy: (i) β-glucan unmasking and (ii) the removal of melanin from the fungal cell wall surface. Notably, another important finding that emerged from our study is the ability of purified synthetic melanin, which has a different chemical structure than A. fumigatus melanin, to inhibit NADPH oxidase-dependent activation of LAP. This general property of melanin to target LAP may have broad implications in pathogenesis of infections caused by other bacterial and fungal pathogens. Finally, targeting the noncanonical autophagy pathway by melanin could be relevant in regulation of inflammation and tissue homeostasis and in pathogenesis of other human diseases.
In view of the mediocre activity of current antifungal agents there is a need to better understand immunopathogenesis of fungal infections at the cellular and molecular level in robust experimental systems in order to develop targeted therapeutics. In healthy individuals, elimination of inhaled (conidia) spores of airborne fungi occurs within acidified phagolysosomal compartments of professional phagocytic cells on a daily basis Quantitative or qualitative defects in phagocyte function, as a result of chemotherapy-induced neutropenia or treatment with high doses of corticosteroids are the main predisposing factors for development of invasive mold infections. In addition, genetic defects in NADPH oxidase activity in patients with chronic granulomatous disease (CGD) lead to unique predisposition for development of invasive pulmonary aspergillosis. Despite significant achievements in understanding pathogenesis of invasive mold infections, the key molecular attributes of host-fungal interplay regulating intracellular killing of conidia within phagocytes are largely unknown.
Aim of this proposal was to identify novel evolutionarily conserved genes with important role in immunity against airborne opportunistic fungi in Drosophila melanogaster and confirm their role in antifungal immunity in humans. Because of the emerging role of autophagy in cellular homeostasis and immunity we prioritized our studies on dissection of the role of autophagy in antifungal immunity. Studies in Drosophila melanogaster flies following conditional inactivation of autophagy genes in fly hemocytes demonstrated increased susceptibility to fungal infection. In addition, induction of autophagy upon treatment with rapamycin protected fruit flies upon infection with fungi.
These preliminary results in Drosophila melanogaster model prompted us to evaluate the role of autophagy in antifungal immunity in humans. We focused our studies in human phagocytes on LC3 associated phagocytosis (LAP), a specialized form of noncanonical autophagy that links activation of pattern recognition receptors with phagosome maturation. Importantly, we found that infection of primary human monocytes with Aspergillus spores triggered selective recruitment of the autophagy hallmark protein LC3 II in phagosomes upon fungal cell wall swelling. This response was induced by surface exposure of immunostimulatory β-glucans, and was mediated by activation of the Dectin-1 receptor, as it was abolished in monocytes from patients with the homozygous early stop-codon mutation Tyr238X in Dectin-1, who display lack of surface receptor expression. LC3 recruitment in A. fumigatus-phagosomes required syk kinase-dependent production of reactive oxygen species (ROS) and was almost completely abolished in monocytes of patients with genetic defects in NADPH oxidase (chronic granulomatous disease, CGD). LAP was important for control of intracellular fungal growth, as silencing of Atg5 resulted in impaired phagosome maturation and killing of A. fumigatus. Because corticosteroid-induced immunosupression is In vivo and ex vivo administration of corticosteroids blocked LC3 recruitment in A. fumigatus phagosomes of human monocytes via rapid inhibition of syk kinase phosphorylation and downstream production of ROS. Overall, our studies link Dectin-1/syk kinase signaling with maturation of A. fumigatus phagosomes and uncover a mechanism for development of invasive fungal disease. The results of this work have been published as a featured paper in J Immunol (Kyrmizi I et al., J Immunol. 2013 191:1287-99)
In a collaborative study we also demonstrated the importance of harnessing autophagy to improve antifungal immunity in patients with CGD. Specifically, treatment with an IL1 receptor antagonist (Anakinra) restored antifungal autophagy in monocytes of CGD patients. The clinical relevance of these findings was assessed by treating CGD patients who had severe colitis with IL-1 receptor blockade using anakinra. Anakinra treatment resulted in a rapid and sustained improvement in colitis. Thus, inflammation in CGD is due to IL-1-dependent mechanisms, such as decreased autophagy and increased inflammasome activation, which are linked pathological conditions in CGD that can be restored by IL-1 receptor blockade (de Luca A et al., Proc Natl Acad Sci U S A. 2014;111:3526-31).
In the second part of our work we explored whether fungal pathogens employ virulence mechanisms that target activation of LAP. Concealing of pathogen associated molecular patterns (PAMPs) is a fundamental pathogenetic mechanism that allows fungi to avoid immune activation during phagocytosis. In line with this model, dormant conidia of Aspergillus fumigatus are immunologically inert, and fail to activate LAP, because cell wall PAMPs are covered by a layer of hydrophobic rodlet protein RodA and melanin. Upon germination of A. fumigatus conidia, β-glucan surface exposure triggers NADPH oxidase-dependent activation of LAP and facilitates fungal killing. However, in view of the major cell wall structural changes that occur during germination, it remains unclear whether masking of PAMPs is the principal mechanism of fungi to evade autophagy and killing by macrophages.
Importantly, in contrast to the prevailing model of masking of PAMPs, we found that β-glucan surface exposure on dormant conidia of RodA-deficient A. fumigatus (ΔrodA) is insufficient to activate LAP. Instead, genetic removal of cell wall melanin (ΔrodA/pksP and ΔpksP) is required to enable β-glucan-mediated LC3 recruitment in A. fumigatus phagosomes. Genetic or biochemical removal of cell wall melanin in ΔrodA and exogenous melanin complementation in melanin-deficient A. fumigatus conidia demonstrated that melanin does not interfere with signaling pathways regulating cytokine release, but selectively inhibits LAP by blocking assembly of NADPH oxidase. Importantly, the attenuated pathogenicity of melanin-deficient A. fumigatus (ΔpksP) is dependent on activation of antifungal autophagy and it is significantly restored in Atg5-deficient macrophages and in mice upon conditional inactivation of Atg5 in myeloid cells. Thus, apart from shielding PAMPs, the major role of fungal cell wall melanin is to inhibit phagosome biogenesis. This work was awarded in 6th Advances Against Aspergillosis meeting, Madrid, 27/2-1/3/2014 and the manuscript is under submission (Nature Letters).
Collectively, our work led to the discovery of a new antifungal immunity pathway that links signaling activation with intracellular elimination of conidia of airborne fungi. This pathway has major physiologic relevance in control of fungal infection by human phagocytes as evidenced by impaired autophagy protein recruitment in A. fumigatus-containing phagosomes in two distinct groups of patients with increased susceptibility for invasive aspergillosis. Furthermore, we have identified fungal cell wall melanin as a unique virulent factor that specifically targets the autophagy pathway and allows the fungus to resist killing within the phagosome. Importantly, a new model in fungal pathogenesis has emerged from our work demonstrating that two fundamental biologic events, which physiologically occur concurrently during germination of A. fumigatus, are the necessary requirements for activation of antifungal autophagy: (i) β-glucan unmasking and (ii) the removal of melanin from the fungal cell wall surface. Notably, another important finding that emerged from our study is the ability of purified synthetic melanin, which has a different chemical structure than A. fumigatus melanin, to inhibit NADPH oxidase-dependent activation of LAP. This general property of melanin to target LAP may have broad implications in pathogenesis of infections caused by other bacterial and fungal pathogens. Finally, targeting the noncanonical autophagy pathway by melanin could be relevant in regulation of inflammation and tissue homeostasis and in pathogenesis of other human diseases.