Aspartyl-aspartic acid

Alveolar macrophages or RAW-dectin macrophages [23 (link)] (3 × 10⁵) were pre-treated with the anti–dectin-1 antibody 2A11 (5 μg/ml) or isotype for 30 min [26 (link)] and thereafter co-cultured with A. fumigatus conidia at a ratio of 1:1 for various times in a 96-well plate at 37 °C, 5% CO₂. Controls included alveolar or RAW macrophages cultured in medium alone. Thereafter, the contents of each well were collected and the supernatants analyzed for IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12p40, IL-12p70, IL-17, IFN-γ, GM-CSF, G-CSF, TNF-α, MIP-1α, RANTES, and KC levels using the Bio-Plex Protein Array System (Bio-Rad, Hercules, California, United States) as per manufacturer's instructions. MIP-2 concentrations were determined using a commercially available ELISA kit (R&D Systems, Minneapolis, Minnesota, United States) as per manufacturer's instructions. To determine the response to SC, A. fumigatus RC were cultured for 6 h at 37 °C, 5% CO₂ and allowed to swell [10 (link),31 (link),42 (link)]. Thereafter, macrophages were added for an additional 4 h. Controls for these studies included alveolar macrophages added to RC for 4 h. To determine the response to A. fumigatus hyphae, A. fumigatus conidia were cultured for 24 h at 37 °C, 5% CO₂ prior to the addition of macrophages for 4 h, 8 h, or 24 h. Spontaneous cytokine and chemokine production in unstimulated cultures was subtracted from stimulated cultures in order to calculate the net concentration induced by A. fumigatus. To determine whether macrophage cell death occurred in co-cultures, supernatants were analyzed for LDH levels using an LDH kit (Sigma, St. Louis, Missouri, United States) as per manufacturer's instructions. Caspase 3 levels in cell lysates were also analyzed using the EnzChek Caspase 3 Assay Kit containing the rhodamine 110 bis-(N-CBZ-L-aspartyl-Lglutamyl-L-valyl-L-aspartic acid amide) (Z-DEVD–R110) substrate (Molecular Probes, Eugene, Oregon, United States) as per manufacturer's instructions. In specific experiments, macrophages were pretreated with cytochalasin D (10 μM), 250 μg/ml mannan (both from Sigma, St. Louis, Missouri, United States), or 100 μg/ml glucan phosphate [23 (link),26 (link)] for 30 min at room temperature prior to addition to RC or SC. For analysis of responses to heat-killed A. fumigatus, A. fumigatus conidia were cultured for 3 h, 6 h, or 9 h at 37 °C, 5% CO₂ followed by heat-killing at 100 °C for 10 min [10 (link)]. Thereafter, heat-killed A. fumigatus were co-cultured with alveolar macrophages at a ratio of 1:1 for 6 h at 37 °C, 5% CO₂ in a 96-well plate. Some experiments employed A. fumigatus organisms killed by 70% ethanol treatment for 30 min at room temperature. All killed A. fumigatus were plated onto potato dextrose agar at 37 °C for 48 h to confirm negative growth. In specific experiments, RAW 264.7 macrophages were cultured with LPS (100 ng/ml) or Pam(3)Cys (10 μg/ml) (both from InvivoGen, San Diego, California, United States) in the presence or absence of 2A11 for 16 h, followed by analysis of cytokine and chemokine levels in supernatants.

Structural and biochemical markers of apoptosis like percent apoptotic nulcei and caspase 3/7 activity, respectively were assessed. Percent apoptotic nuclei was quantified by characteristic nuclear morphological changes and visualized by treatment with the fluorescent DNA-binding dye, DAPI (4’, 6-diamidine-2-phenylindole dihydrochloride) as described [25 (link)]. Briefly, cells were stained with 5 μg/ml of DAPI for 20–30 minutes at 37°C. Apoptotic nuclei (condensed, fragmented) were counted and presented as a percent of total nuclei. At least 200 cells were counted per well and experiments were performed in triplicate. Caspase 3/7 activity was measured using rhodamine 110 bis-(N-CBZ-I-aspartyl-I-glutamyl-I-Valyl-aspartic acid amide (Z-DEVD-R110) substrate. The caspase 3 and 7 enzyme activity in the cells will cleave the DEVD peptide in the substrate and release the rhodamine 110 fluorophore which can be measured spectrofluorometrically (Biotek Synergy) with 498nm wavelength of excitation and 521nm emission. The activity is measured by net fluorescence (assay rate of fluorescence (RFU)—negative control RFU) according to the manufacturer’s instructions (APO-One, Promega, Madison, WI #G7791). The data were reported as fold-change of net fluorescence compared to vehicle treated cells, with experiments performed in quadruplicate.

The Apo-ONE^® Homogeneous Caspase-3/7 Assay (Promega, Madison, WI, USA) is a fluorescent assay that measures caspase-3 and -7 activities homogeneously and includes a profluorescent substrate with an enhanced bifunctional cell lysis buffer for caspase-3/7 activity assays. Caspase-3/7 rhodamine 110 substrate, bis-(N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide) (Z-DEVD-R110) is the fluorescent substrate used in this study. To perform the Apo-ONE assay, the buffer and substrate were mixed and added to each sample. After cleavage and removal of DEVD peptides due to caspase-3/7 activity (excitation wavelength 499 nm), rhodamine 110 becomes highly fluorescent (emission wavelength 521 nm). The magnitude of fluorescence generated is proportional to the caspase-3/7 cleavage of the sample.
The SH-SY5Y cell line (15 × 10³ cells/well) was grown in black 96-well plates and exposed to flumethrin (1–1000 μM) for 24 h. After treatment, the Apo-ONE^® Caspase-3/7 Assay was prepared according to the manufacturer’s instructions. We removed the 96-well plates containing the treated cells from the incubator, and 100 µL of homogeneous Caspase-3/7 reagent was added to 100 µL of the culture medium containing the previously treated cells in each well and incubated at room temperature for 60 min in the dark. Fluorescence (excitation/emission wavelength 485/528 nm) was measured using the plate reader (FLx800, BioTek, Winooski, VT, USA). Data were normalized as % control.

Apoptosis was analyzed via assessing percent apoptotic nuclei and caspase 3/7 activity, which represent structural and biochemical markers of apoptosis, respectively. Percent apoptotic nuclei was quantified by characteristic nuclear morphology and visualized with the treatment of DNA binding fluorescent dye, DAPI (4’, 6-diamidine-2-phenylindole dihydrochloride) [14 (link)]. Briefly, cells were stained with DAPI (5 µg/ml) for 10–15 min at 37 °C. Apoptotic cells, characterized by condensed and fragmented nuclei were counted and presented as percent of total nuclei. Experiments were performed in triplicates and at least 200 cells were counted per well. Caspase 3 and 7 activity was analyzed using rhodamine 110 bis-(N-CBZ-l-aspartyl-l-glutamyl-l-valyl-aspartic acid amide) (Z-DEVD-R110) caspase substrate according to manufacturer’s instructions (Promega, Madison, WI #G7791). Briefly, cells with active caspase 3/7 enzyme cleaves the substrate Z-DEVD releasing rhodamine which was quantified spectrofluorometrically using Biotek Synergy plate reader [14 (link)]. The experiment was performed in quadruplicate and were reported as fold change compared to vehicle-treated cells.

The metabolic capacity of cells was determined by measuring cellular ATP levels with a luciferase based CellTiterGlo 3D (Promega) according to the manufacturer’s protocol and luminescence measurement (Synergy HT, Biotek, Winooski, VT, USA⁾. Caspase3/7 activity in the cells was determined using the Apo-ONE Homogeneous Caspase-3/7 Assay (Promega) using an a profluorescent consensus substrate, rhodamine 110 bis-(N-CBZ-l-aspartyl-l-glutamyl-l-valyl-aspartic acid amide; Z-DEVD-R110) according to the instructions from the manufacturer.