Serum opsonin ficolin-A enhances host-fungal interactions and modulates cytokine expression from human monocyte-derived macrophages and neutrophils following Aspergillus fumigatus challenge.

Invasive aspergillosis is a devastating invasive fungal disease associated with a high mortality rate in the immunocompromised, such as leukaemia patients, transplant patients and those with HIV/AIDS. The rodent serum orthologue of human L-ficolin, ficolin-A, can bind to and opsonize Aspergillus fumigatus, the pathogen that causes invasive aspergillosis, and may participate in fungal defence. Using human monocyte-derived macrophages and neutrophils isolated from healthy donors, we investigated conidial association and fungal viability by flow cytometry and microscopy. Additionally, cytokine production was measured via cytometric bead arrays. Ficolin-A opsonization was observed to significantly enhance association of conidia, while also inhibiting hyphal growth and contributing to increased fungal killing following incubation with monocyte-derived macrophages and neutrophils. Additionally, ficolin-A opsonization was capable of manifesting a decrease in IL-8, IL-1β, IL-6, IL-10 and TNF-α production from MDM and IL-1β, IL-6 and TNF-α from neutrophils 24 h post-infection. In conclusion, rodent ficolin-A is functionally comparable to human L-ficolin and is capable of modulating the innate immune response to A. fumigatus, down-regulating cytokine production and could play an important role in airway immunity

Ficolins and the Recognition of Pathogenic Microorganisms: An Overview of the Innate Immune Response and Contribution of Single Nucleotide Polymorphisms

Ficolins are innate pattern recognition receptors (PRR) and play integral roles within the innate immune response to numerous pathogens throughout the circulation, as well as within organs. Pathogens are primarily removed by direct opsonisation following the recognition of cell surface carbohydrates and other immunostimulatory molecules or via the activation of the lectin complement pathway, which results in the deposition of C3b and the recruitment of phagocytes. In recent years, there have been a number of studies implicating ficolins in the recognition and removal of numerous bacterial, viral, fungal, and parasitic pathogens. Moreover, there has been expanding evidence highlighting that mutations within these key immune proteins, or the possession of particular haplotypes, enhance susceptibility to colonization by pathogens and dysfunctional immune responses. This review will therefore encompass previous knowledge on the role of ficolins in the recognition of bacterial and viral pathogens, while acknowledging the recent advances in the immune response to fungal and parasitic infections. Additionally, we will explore the various genetic susceptibility factors that predispose individuals to infection

The serum opsonin L-ficolin is detected in lungs of human transplant recipients following fungal infections and modulates inflammation and killing of Aspergillus fumigatus.

BACKGROUND: Invasive aspergillosis (IA) is a life-threatening systemic fungal infection in immunocompromised individuals that is caused by Aspergillus fumigatus. The human serum opsonin, L-ficolin, has been observed to recognize A. fumigatus and could participate in fungal defense. METHODS: Using lung epithelial cells, primary human monocyte-derived macrophages (MDMs), and neutrophils from healthy donors, we assessed phagocytosis and killing of L-ficolin-opsonized live A. fumigatus conidia by flow cytometry and microscopy. Additionally, cytokines were measured by cytometric bead array, and L-ficolin was measured in bronchoalveolar lavage (BAL) fluid from lung transplant recipients by enzyme-linked immunosorbent assay. RESULTS: L-ficolin opsonization increased conidial uptake and enhanced killing of A. fumigatus by MDMs and neutrophils. Opsonization was also shown to manifest an increase in interleukin 8 release from A549 lung epithelial cells but decreased interleukin 1β, interleukin 6, interleukin 8, interleukin 10, and tumor necrosis factor α release from MDMs and neutrophils 24 hours after infection. The concentration of L-ficolin in BAL fluid from patients with fungal infection was significantly higher than that for control subjects (P = .00087), and receiving operating characteristic curve analysis highlighted the diagnostic potential of L-ficolin for lung infection (area under the curve, 0.842; P < .0001). CONCLUSIONS: L-ficolin modulates the immune response to A. fumigatus. Additionally, for the first time, L-ficolin has been demonstrated to be present in human lungs

Bis(N-picolinamido)cobalt(II) Complexes Display Antifungal Activity toward Candida albicans and Aspergillus fumigatus

This report highlights the synthesis and characterization of ten new bis(N-picolinamido)cobalt(II) complexes of the type [(L)2CoX2]0/2+, whereby L=N-picolinamide ligand and X=diisothiocyanato (−NCS), dichlorido (−Cl) or diaqua (−OH2) ligands. Single crystal X-ray (SC-XRD) analysis for nine of the structures are reported and confirm the picolinamide ligand is bound to the Co(II) center through a neutral N,O binding mode. With the addition of powder X-ray diffraction (PXRD), we have confirmed the cis and trans ligand arrangements of each complex. All complexes were screened against several fungal species and show increased antifungal activity. Notably, these complexes had significant activity against strains of Candida albicans and Aspergillus fumigatus, with several compounds exhibiting growth inhibition of >80 %, and onecompound inhibiting Aspergillus fumigatus hyphal growth by >90 %. Conversely, no antifungal activity was exhibited toward Cryptococcus neoformans and no cytotoxicity towards mammalian cell lines

Host genetic signatures of susceptibility to fungal disease

Our relative inability to predict the development of fungal disease and its clinical outcome raises fundamental questions about its actual pathogenesis. Several clinical risk factors are described to predispose to fungal disease, particularly in immunocompromised and severely ill patients. However, these alone do not entirely explain why, under comparable clinical conditions, only some patients develop infection. Recent clinical and epidemiological studies have reported an expanding number of monogenic defects and common polymorphisms associated with fungal disease. By directly implicating genetic variation in the functional regulation of immune mediators and interacting pathways, these studies have provided critical insights into the human immunobiology of fungal disease. Most of the common genetic defects reported were described or suggested to impair fungal recognition by the innate immune system. Here, we review common genetic variation in pattern recognition receptors and its impact on the immune response against the two major fungal pathogens Candida albicans and Aspergillus fumigatus. In addition, we discuss potential strategies and opportunities for the clinical translation of genetic information in the field of medical mycology. These approaches are expected to transfigure current clinical practice by unleashing an unprecedented ability to personalize prophylaxis, therapy and monitoring for fungal disease.This work was supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000013), the Fundação para a Ciência e Tecnologia (FCT) (IF/00735/2014 to AC, and SFRH/BPD/96176/2013 to CC), the Institut Mérieux (Mérieux Research Grant 2017 to CC), and the European Society of Clinical Microbiology and Infectious Diseases (ESCMID Research Grant 2017 to AC)

Positive allosteric modulation of P2X7 promotes apoptotic cell death over lytic cell death responses in macrophages

P2X7 is an ATP-gated ion channel that is highly expressed by leukocytes, such as macrophages. Here, it has been demonstrated to be involved in the regulation of various cell death pathways; including apoptosis, pyroptosis, necrosis, and autophagy. However, cell death induction via P2X7 is complex and is reliant upon the nature of the stimulus, the duration of the stimulus, and the cell type investigated. Previous reports state that high extracellular ATP concentrations promote osmotic lysis, but whether positive allosteric modulation of P2X7 in the presence of lower concentrations of ATP condemns cells to the same fate is unknown. In this study, we compared cell death induced by high ATP concentrations, to cell death induced by compound K, a recently identified and potent positive allosteric modulator of P2X7. Based on our observations, we propose that high ATP concentrations induce early cell swelling, loss of mitochondrial membrane potential, plasma membrane rupture, and LDH release. Conversely, positive allosteric modulation of P2X7 primarily promotes an intrinsic apoptosis pathway. This was characterised by an increase in mitochondrial Ca2+, accelerated production of mitochondrial ROS, loss of mitochondrial membrane permeability in a Bax-dependent manner, the potential involvement of caspase-1, and caspase-3, and significantly accelerated kinetics of caspase-3 activation. This study highlights the ability of positive allosteric modulators to calibrate P2X7-dependent cell death pathways and may have important implications in modulating the antimicrobial immune response and in the resolution of inflammation

Mapping a novel positive allosteric modulator binding site in the central vestibule region of human P2X7

P2X7 receptors are important in the regulation of inflammatory responses and immune responses to intracellular pathogens such as Mycobacterium tuberculosis and Toxoplasma gondii. Enhancement of P2X7 receptor responses may be useful in pathogen clearance particularly in individuals with defective microbial killing mechanisms. Ginsenosides from Panax ginseng have been discovered to act as positive allosteric modulators of P2X7. Here we describe a novel modulator binding site identified by computational docking located in the central vestibule of P2X7 involving S60, D318, and L320 in the lower body β-sheets lining the lateral portals. Potentiation of ATP-mediated responses by ginsenosides CK and Rd caused enhanced ionic currents, Ca2+ influx and YOPRO-1 uptake in stably transfected HEK-293 cells (HEK-hP2X7) plus enhanced cell death responses. Potentiation of ATP responses by CK and Rd was markedly reduced by mutations S59A, S60A, D318L and L320A supporting the proposed allosteric modulator binding site. Furthermore, mutation of the conserved residues S60 and D318 led to alterations in P2X7 response and a higher sensitivity to ATP in the absence of modulators suggesting residues in the connecting rods play an important role in regulating P2X7 gating. Identification of this novel binding site location in the central vestibule may also be relevant for structurally similar channels