Phagocytosis of Aspergillus fumigatus by Human Bronchial Epithelial Cells Is Mediated by the Arp2/3 Complex and WIPF2

Aspergillus fumigatus is an opportunistic fungal pathogen capable of causing severe infection in humans. One of the limitations in our understanding of how A. fumigatus causes infection concerns the initial stages of infection, notably the initial interaction between inhaled spores or conidia and the human airway. Using publicly-available datasets, we identified the Arp2/3 complex and the WAS-Interacting Protein Family Member 2 WIPF2 as being potentially responsible for internalization of conidia by airway epithelial cells. Using a cell culture model, we demonstrate that RNAi-mediated knockdown of WIPF2 significantly reduces internalization of conidia into airway epithelial cells. Furthermore, we demonstrate that inhibition of Arp2/3 by a small molecule inhibitor causes similar effects. Using super-resolution fluorescence microscopy, we demonstrate that WIPF2 is transiently localized to the site of bound conidia. Overall, we demonstrate the active role of the Arp2/3 complex and WIPF2 in mediating the internalization of A. fumigatus conidia into human airway epithelial cells.


RNA interference of WIPF2
Transfections were performed using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, 13778100) with the TriFECTa RNAi kit (Integrated DNA Technologies, hs.Ri.WIPF2.13). The TriFECTa RNAi kit contained three species of siRNA that targeted the transcript encoding WIPF2. The kit also contained NC-1 siRNA, which does not target any known sequence in the human or A. fumigatus transcriptome. A 1:50 solution of Lipofectamine RNAiMAX in DMEM was combined with an siRNA solution that contained the three WIPF2 siRNA species in equal molar proportions of 39.6µL in DMEM. After 5 minutes of incubation at room temperature, 100µL of transfection solution was added to each well of a 24-well plate. 1HAEo-cells (30,000) were added to each well in DMEM supplemented with 10% FBS yielding a final volume of 600µL with siRNA concentrations of 3.3 nM each. NC-1 siRNA was transfected with a final concentration of 10 nM. Transfected cells were grown for 72 h until experimental usage.

Chemical Inhibition of Arp2/3
For chemical inhibition experiments, 200µM CK-666 (Sigma-aldrich, SML0006) in DMSO or equivalent volume of DMSO alone in media was added to cells 30 minutes before infection. Cells were infected for three hours as in the Nystatin Protection Assay experiment (above). Two-color immunofluorescence was used to assess internalization of A. fumigatus, and was performed as described previously (Wasylnka and Moore, 2002), with five fields of view quantified per sample.

Nystatin Protection Assay
This assay has been previously validated in studies investigating the internalization of conidia into airway epithelial cells (Wasylnka and Moore, 2002;Gomez et al., 2010;Chen et al., 2015). Briefly, 1HAEo-cells were transfected for 72 h as described above with siRNA targeting WIPF2, NC-1 siRNA, or left untreated as control. Cells were then infected for three hours with 10 6 conidia in DMEM at 37°C. Conidia solutions were then aspirated, cultures were washed once with PBS-T and treated with 25µg/mL Nystatin and 0.5% DMSO in DMEM for three hours at 37°C. Cells were washed three times with PBS-T and lysed for 15 minutes at room temperature with 0.05% Triton X-100 in sterile dH2O. Lysates were diluted with sterile dH2O, plated in duplicate onto YAG and grown for 24 h at 37°C. Colonies were counted manually.

Immunofluorescence of WIPF2
1HAEo-cells were grown as described above in 8-well tissue culture treated chamber slides (Thermo Fisher Scientific, 154534PK). Cells were infected with conidia at a MOI of 10 in DMEM for the indicated periods of time. After infection, cells were washed three times with PBS-T and fixed with 4% paraformaldehyde in PBS for 20 minutes. Cells were washed three times with PBS and permeabilized for five minutes using cytoskeleton solution (10mM MES, 138mM KCl, 3mM MgCl, 2mM EGTA, 320mM sucrose, pH 6.1) supplemented with 0.5% Triton X-100. Cells were washed three times with PBS and blocked for one hour at 4°C in PBS with 5% BSA. Samples were then immunolabeled with 1:200 rabbit anti-WIPF2 antibody in blocking solution for one hour, washed three times with PBS, and incubated with 1:400 goat-anti rabbit Alexa-fluor 596 antibody (Jackson laboratories, 111-585-045) in blocking solution for one hour. Samples were washed three times with PBS and treated with 0.1µg/mL of DAPI for five minutes to stain nuclei. Cells were washed three times with PBS, and cover slipped with Fluoromount medium (Sigma-Aldrich, F4680) after removing the chambers. Finally, the slides were sealed with nail polish.

Airyscan confocal microscopy
Slides were imaged using a Zeiss LSM-880 inverted confocal microscope with Airyscan technology and ZEN black software (Zeiss). The microscope settings were: frame size 1488x1488px, pixel size 0.05µm, pixel dwell 1.41µs with 4x averaging in the left-right direction with the mean method and 12-bit colour depth. For airyscan processing, "auto" was used. Z-stack images were obtained as 11 Z-slices with 0.6µm spacing. Pinhole size/gain for each channel in the 15 minute post-infection sample was 52.5/900R, 42.8/850G, and 35.9/650B (R = red, G = green, B = blue). For the 60-minute sample, the pinhole size and gain were 85.1/900R, 79.3/850G, and 48.0/650B.