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Bifunctional Small Molecules Enhance Neutrophil Activities Against Aspergillus fumigatus in vivo and in vitro.

ABSTRACT: Aspergillosis is difficult to treat and carries a high mortality rate in immunocompromised patients. Neutrophils play a critical role in control of infection but may be diminished in number and function during immunosuppressive therapies. Here, we measure the effect of three bifunctional small molecules that target Aspergillus fumigatus and prime neutrophils to generate a more effective response against the pathogen. The molecules combine two moieties joined by a chemical linker: a targeting moiety (TM) that binds to the surface of the microbial target, and an effector moiety (EM) that interacts with chemoattractant receptors on human neutrophils. We report that the bifunctional compounds enhance the interactions between primary human neutrophils and A. fumigatus in vitro, using three microfluidic assay platforms. The bifunctional compounds significantly enhance the recruitment of neutrophils, increase hyphae killing by neutrophils in a uniform concentration of drug, and decrease hyphal tip growth velocity in the presence of neutrophils compared to the antifungal targeting moiety alone. We validated that the bifunctional compounds are also effective in vivo, using a zebrafish infection model with neutrophils expressing the appropriate EM receptor. We measured significantly increased phagocytosis of A. fumigatus conidia by neutrophils expressing the EM receptor in the presence of the compounds compared to receptor-negative cells. Finally, we demonstrate that treatment with our lead compound significantly improved the antifungal activity of neutrophils from immunosuppressed patients ex vivo. This type of bifunctional compounds strategy may be utilized to redirect the immune system to destroy fungal, bacterial, and viral pathogens.

SUBMITTER: Jones CN

PROVIDER: S-EPMC6465576 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Bifunctional Small Molecules Enhance Neutrophil Activities Against Aspergillus fumigatus in vivo and in vitro.

Jones Caroline N CN Ellett Felix F Robertson Anne L AL Forrest Kevin M KM Judice Kevin K Balkovec James M JM Springer Martin M Markmann James F JF Vyas Jatin M JM Warren H Shaw HS Irimia Daniel D

Frontiers in immunology 20190409

Aspergillosis is difficult to treat and carries a high mortality rate in immunocompromised patients. Neutrophils play a critical role in control of infection but may be diminished in number and function during immunosuppressive therapies. Here, we measure the effect of three bifunctional small molecules that target Aspergillus fumigatus and prime neutrophils to generate a more effective response against the pathogen. The molecules combine two moieties joined by a chemical linker: a target ...[more]

PMID: 31024528

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Project description:AimTo determine whether lectin-like ox-LDL receptor (LOX-1) regulates adhesion molecules expression and neutrophil infiltration in Aspergillus fumigatus (A. fumigatus) keratitis of C57BL/6 mice.MethodsC57BL/6 mice were pretreated with a neutralizing antibody to LOX-1 (5 µg/5 µL) or control nonspecific IgG (5 µg/5 µL), LOX-1 inhibitor Poly-I (2 µg/5 µL) or PBS by subconjunctival injection. Fungal keratitis (FK) mouse models of C57BL/6 mice were established by scraping corneal central epithelium, smearing A. fumigatus on the corneal surface and covering the eye with contact lenses. The corneal response to infection was assessed via clinical score. The mRNA levels of the adhesion molecules intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), P-selectin and E-selectin were tested in control and infected corneas by reverse transcription-polymerase chain reaction (RT-PCR). The protein levels of ICAM-1 were evaluated by immunofluorescence (IF) and Western blot. Neutrophils were extracted from the abdominal cavity of C57BL/6 mice followed by pretreatment using antibody to LOX-1 (10 µg/mL) or control nonspecific IgG (10 µg/mL), the Poly-I (4 µg/mL) or PBS. The cells were then stimulated with A. fumigatus and tested mRNA and protein levels of lymphocyte function-associated antigen-1 (LFA-1) using RT-PCR and Western blot. IF and myeloperoxidase (MPO) assays were used to assess neutrophil infiltration in mice corneas.ResultsPretreatment of LOX-1 antibody or the Poly-I reduced the degree of inflammation of cornea and decreased the clinical FK score compared with pretreatment of IgG or PBS (both P<0.01). And these pretreatment also displayed an obvious decline in the mRNA levels of ICAM-1, VCAM-1, P-selectin, E-selectin and LFA-1 expression compared with control groups (all P<0.01). Furthermore, pretreated with LOX-1 antibody or Poly-I, the protein levels of ICAM-1 and LFA-1 also decreased compared with control groups (all P<0.05). Neutrophil infiltration in the cornea was significantly reduced after pretreatment of LOX-1 antibody or Poly-I compared with control groups by IF and MPO assays (both P<0.01).ConclusionInhibition of LOX-1 can decrease the expression of adhesion molecules and reduce neutrophil infiltration in A. fumigatus infected corneas of C57BL/6 mice.

Project description:Fungal infections remain a major global concern. Emerging fungal pathogens and increasing rates of resistance mean that additional research efforts and resources must be allocated to advancing our understanding of fungal pathogenesis and developing new therapeutic interventions. Neutrophilic granulocytes are a major cell type involved in protection against the important fungal pathogen Aspergillus fumigatus, where they employ numerous defense mechanisms, including production of antimicrobial extracellular vesicles. A major drawback to work with neutrophils is the lack of a suitable cell line system for the study of fungal pathogenesis. To address this problem, we assessed the feasibility of using differentiated PLB-985 neutrophil-like cells as an in vitro model to study A. fumigatus infection. We find that dimethylformamide-differentiated PLB-985 cells provide a useful recapitulation of many aspects of A. fumigatus interactions with primary human polymorphonuclear leukocytes. We show that differentiated PLB-985 cells phagocytose fungal conidia and acidify conidia-containing phagolysosomes similar to primary neutrophils, release neutrophil extracellular traps, and also produce antifungal extracellular vesicles in response to infection. In addition, we provide an improved method for the isolation of extracellular vesicles produced during infection by employing a size exclusion chromatography-based approach. Advanced liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics revealed an enrichment of extracellular vesicle marker proteins and a decrease of cytoplasmic proteins in extracellular vesicles isolated using this improved method. Ultimately, we find that differentiated PLB-985 cells can serve as a genetically tractable model to study many aspects of A. fumigatus pathogenesis. IMPORTANCE Polymorphonuclear leukocytes are an important defense against human fungal pathogens, yet our model systems to study this group of cells remain very limited in scope. In this study, we established that differentiated PLB-985 cells can serve as a model to recapitulate several important aspects of human polymorphonuclear leukocyte interactions with the important human fungal pathogen Aspergillus fumigatus. The proposed addition of a cultured neutrophil-like cell line to the experimental toolbox to study fungal pathogenesis will allow for a more mechanistic description of neutrophil antifungal biology. In addition, the easier handling of the cell line compared to primary human neutrophils allowed us to use PLB-985 cells to provide an improved method for isolation of neutrophil-derived extracellular vesicles using size exclusion chromatography. Together, these results provide significant tools and a baseline knowledge for the future study of neutrophil-derived extracellular vesicles in the laboratory.

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Bifunctional Small Molecules Enhance Neutrophil Activities Against Aspergillus fumigatus in vivo and in vitro.

Publications

Bifunctional Small Molecules Enhance Neutrophil Activities Against <i>Aspergillus fumigatus in vivo</i> and <i>in vitro</i>.

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