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Phagocytosis-dependent activation of a TLR9-BTK-calcineurin-NFAT pathway co-ordinates innate immunity to Aspergillus fumigatus.

ABSTRACT: Transplant recipients on calcineurin inhibitors are at high risk of invasive fungal infection. Understanding how calcineurin inhibitors impair fungal immunity is a key priority for defining risk of infection. Here, we show that the calcineurin inhibitor tacrolimus impairs clearance of the major mould pathogen Aspergillus fumigatus from the airway, by inhibiting macrophage inflammatory responses. This leads to defective early neutrophil recruitment and fungal clearance. We confirm these findings in zebrafish, showing an evolutionarily conserved role for calcineurin signalling in neutrophil recruitment during inflammation. We find that calcineurin-NFAT activation is phagocytosis dependent and collaborates with NF-κB for TNF-α production. For yeast zymosan particles, activation of macrophage calcineurin-NFAT occurs via the phagocytic Dectin-1-spleen tyrosine kinase pathway, but for A. fumigatus, activation occurs via a phagosomal TLR9-dependent and Bruton's tyrosine kinase-dependent signalling pathway that is independent of MyD88. We confirm the collaboration between NFAT and NF-κB for TNF-α production in primary alveolar macrophages. These observations identify inhibition of a newly discovered macrophage TLR9-BTK-calcineurin-NFAT signalling pathway as a key immune defect that leads to organ transplant-related invasive aspergillosis.

SUBMITTER: Herbst S

PROVIDER: S-EPMC4364943 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Calcineurin plays an important role in the control of cell morphology and virulence in fungi. Calcineurin is a serine/threonine-specific protein phosphatase heterodimer consisting of a catalytic subunit A and a regulatory Ca2+/Calmodulin binding subunit. A mutant of A. fumigatus lacking the calcineurin A (calA) catalytic subunit exhibited defective hyphal morphology related to apical extension and branching growth, which resulted in drastically decreased filamentation. Here, we investigated which pathways are influenced by A. fumigatus calcineurin during proliferation by comparatively determining the transcriptional profile of A. fumigatus wild type and delta calA mutant strains. Our results showed that although the mitochondrial function is reduced in the delta calA mutant strain, its respiratory chain is functional and the mutant has increased alternative oxidase (aoxA) mRNA accumulation and activity. Furthermore, we identified several genes that encode transcription factors that have increased mRNA expression in the delta calA mutant and that could be involved in the Cal-CrzA pathway. Deletion mutants for these transcription factors had also reduced susceptibility to itraconazole, caspofungin, and sodium dodcyl sulfate. For the time course microarray experiments, 1.0 x 109 conidia of both A. fumigatus wild type [CEA17 delta akuB(KU80)] and mutant strain (delta calA) were used to inoculate 400 ml of pre-warmed liquid cultures in complete medium (YG) in 1 L erlenmeyer flasks. Cultures were allowed to grow for 6, 8, 12, 18 and 24 hours in a reciprocal shaker (250 rpm) at 37°C. At each time point, germilings were harvested by centrifugation or filtration and total RNA was extracted. Hybridization experiments were competitive using Cy3 or Cy5-labeled probes derived from both wild type and delta calA strain grown for 6, 8, 12, 18 and 24 hours. Hybridizations were performed using the wild type RNA as reference in comparison to the mutant strain (test RNA) for the same time point. Normalized signal intensities were used to generate relative hybridization ratios (query/reference). Following normalization, the values for each gene in-slide replicates were condensed (median variance <0.01), and corresponding flip-dye replicates were averaged to compensate for dye-specific effects (see Supplementary files linked below).

Project description:BackgroundAspergillosis is a common cause of morbidity and mortality in immunocompromised populations. PU.1 is critical for innate immunity against Aspergillus fumigatus (AF) in macrophages. However, the molecular mechanism underlying PU.1 mediating immunity against AF infection in human alveolar macrophages (AMs) is still unclear.MethodsIn this study, we detected the expressions of PU.1, CD23, p-ERK, CCL20 and IL-8 and key inflammatory markers IL-1β, IL-6, TNF-α and IL-12 in human THP-1-derived macrophages (HTMs) or PU.1/CD23-overexpressed immunodeficient mice with AF infection. Moreover, we examined these expressions in PU.1-overexpressed/interfered HTMs. Additionally, we detected the phagocytosis of macrophages against AF infection with altered PU.1 expression. Dual luciferase, ChIP and EMSAs were performed to detect the interaction of PU.1 and CD23. And we invested the histological changes in mouse lung tissues transfected with PU.1/CD23-expressing adenoviruses in AF infection.ResultsThe results showed that the expressions of PU.1, CD23, p-ERK, CCL20, IL-8, IL-1β, IL-6, TNF-α and IL-12 increased significantly with AF infection, and PU.1 regulated the later 8 gene expressions in HTMs. Moreover, CD23 was directly activated by PU.1, and overexpression of CD23 in PU.1-interfered HTMs upregulated IL-1β, IL-6, TNF-α and IL-12 levels which were downregulated by PU.1 interference. PU.1 overexpression strengthened the phagocytosis of the HTMs against AF. And injection of PU.1/CD23-expressing adenoviruses attenuated pathological defects in immunodeficient mouse lung tissues with AF infection. Adenovirus (Ad)-PU.1 increased the CD23, p-ERK, CCL20, IL-8 levels.ConclusionsOur study concluded that PU.1-CD23 signaling mediates innate immunity against AF in lungs through regulating inflammatory response. Therefore, PU.1-CD23 may be a new anti-aspergillosis therapeutic for the treatment of invasive aspergillosis with the deepening of gene therapy and its wide application in the clinic.

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Phagocytosis-dependent activation of a TLR9-BTK-calcineurin-NFAT pathway co-ordinates innate immunity to Aspergillus fumigatus.

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