Quantifying Intracellular Viral Pathogen: Specimen Preparation, Visualization and Quantification of Multiple Immunofluorescent Signals in Fixed Human Airway Epithelium Cultured at Air-Liquid Interface

Abstract

Infection control and aggressive antibiotic therapy play an important role in the management of airway infections in individuals with cystic fibrosis (CF). The responses of airway epithelial cells to pathogens are likely to contribute to the pathobiology of CF lung disease. Primary airway epithelial cells obtained from individuals with CF, cultured and differentiated at air-liquid interface (ALI), effectively mimic the structure and function of the in vivo airway epithelium. With the recent respiratory viral pandemics, ALI cultures were extensively used to model respiratory infections in vitro to facilitate physiologically relevant respiratory research. Immunofluorescence staining and imaging were used as an effective tool to provide a fundamental understanding of host-pathogen interactions and for exploring the therapeutic potential of novel or repurposed drugs. Therefore, we described an optimized quantitative fluorescence microscopy assay for the wholemount staining and imaging of epithelial cell markers to identify distinct cell populations and pathogen-specific targets in ALI cultures of human airway epithelial cells grown on permeable support insert membranes. We present a detailed methodology using a graphical user interface (GUI) package to quantify the detected signals on a tiled whole membrane. Our method provided an imaging strategy of the entire membrane, overcoming the common issue of undersampling and enabling unbiased quantitative analysis.

Keywords: SARS-CoV-2; air-liquid interface; airway infection; cystic fibrosis; quantification; tiled imaging; wholemount immunofluorescence.

Figure 4

Quantification of SARS nucleocapsid protein signal in air-liquid interface cultures. (A) Graphical User Interface (GUI) ‘ALIIFAnalysis’ interface in MATLAB software. (B) Enclosed polygon outlining the region of the air-liquid interface (ALI) membrane for the analysis of the nucleocapsid protein (NP) signal. (C) Binary image of the NP signal. (D,E) Magnified inset of the NP signal (D) before and (E) after size exclusion.

Figure 5

Representative graphical user interface analysis of data from CF air-liquid interface cultures. (A) Binary (left) and background corrected images (right) of SARS nucleocapsid protein (NP, green) and acetylated tubulin (Actub, yellow). Scale bars: 1 mm. (B) Area coverage (%) of NP signal. (C) Intensity statistics of the NP signal. (D) Statistics of the clusters after segmentation and filtering, for the NP and Actub signal.

Figure 6

Representative stitched tiled images (25 × 25) of air-liquid interface cultures. (A) Acetylated tubulin (Actub) and MUC5AC with magnified insets (3 × 3). (B) SARS nucleocapsid protein (NP, grey) in SARS-CoV-2-infected air-liquid interface (ALI) cultures. A 20×/0.5 objective. Scale bars: 1 mm.

Figure 1

The characterization of airway epithelial cells differentiated at the air-liquid interface. Representative confocal images of acetylated tubulin (actub, ciliated cells), MUC5AC (secretory goblet cells), p63 (basal cells), ZO-1 (tight junction) and phalloidin (actin). A 63x/1.4 objective. Scale bars: 20 µm.

Figure 2

Visualization of pathogen infection in airway epithelial cell air-liquid interface cultures derived from CF and non-CF participants. (A) Representative confocal images of double-stranded RNA (dsRNA) in HCoV-OC43-infected air-liquid interface (ALI) cultures. (B) Representative confocal images of SARS nucleocapsid protein (NP) in SARS-CoV-2-infected ALI cultures. (C) NP signal in SARS-CoV-2-infected ALI cultures, with or without treatment with experimental compound, and in non-infected ALI cultures. A 63×/1.4 objective. Scale bars: 20 µm.

Figure 3

Visualization of pathogen tropism in air-liquid interface cultures. Representative confocal images of SARS nucleocapsid protein (NP) in SARS-CoV-2-infected air-liquid interface (ALI) cultures co-stained with (A) acetylated tubulin (actub, ciliated cells) and (B) MUC5AC (secretory goblet cells). Magnified insets show NP-positive ciliated and goblet cells (orange arrows). A 63×/1.4 objective. Scale bars: 20 µm.