Single-Cell Analysis of Fungal Uptake in Cultured Airway Epithelial Cells Using Differential Fluorescent Staining and Imaging Flow Cytometry
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Updated: Nov 22, 2025
Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi
Published on: November 28, 2019
Margherita Bertuzzi1,2, Gareth J Howell3
1Manchester Fungal Infection Group, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, Core Technology Facility Building, The University of Manchester, Manchester, UK. margherita.bertuzzi@manchester.ac.uk.
Abstract:
The respiratory epithelium is the initial point of host contact for inhaled particles, leading to orchestrated, but highly heterogeneous, responses. Human airway epithelial cells (AECs) play a crucial role in host defense by promoting uptake and killing of inhaled microorganisms and concomitant cytokine production in order to recruit professional phagocytes to the site of infection. However, inhaled pathogens can also reside and replicate intracellularly to evade host immune defenses or circulating antimicrobial drugs, ultimately causing apoptosis or cell death of the infected AECs. Imaging flow cytometry (IFC) combines flow cytometry, fluorescent microscopy, and advanced data-processing algorithms to dissect the heterogeneity of the interaction of AECs and inhaled microorganisms and its outcomes at the single-cell level. Here, we describe a novel single-cell approach based on differential fluorescent staining and state-of-the-art IFC to identify, quantify, and analyze individual host-pathogen complexes from cultured AECs infected with spores of the major human fungal pathogen Aspergillus fumigatus.
Insights
This study introduces a new single-cell method using imaging flow cytometry to analyze how airway epithelial cells interact with Aspergillus fumigatus. This technique helps understand host-pathogen dynamics in respiratory infections.
Area of Science:
- Immunology
- Cell Biology
- Microbiology
Background:
- The respiratory epithelium is the first line of defense against inhaled particles and pathogens.
- Airway epithelial cells (AECs) are critical for host defense, engulfing and eliminating microbes, but can also be infected.
- Intracellular pathogen replication within AECs can lead to cell death and immune evasion.
Purpose of the Study:
- To develop and validate a novel single-cell approach for analyzing host-pathogen interactions.
- To quantify and characterize the interaction between human airway epithelial cells and Aspergillus fumigatus spores.
- To dissect the heterogeneity of cellular responses to fungal infection at the single-cell level.
Main Methods:
- Utilized imaging flow cytometry (IFC), a technique combining microscopy and flow cytometry.
- Employed differential fluorescent staining for precise cell and pathogen identification.
- Applied advanced data-processing algorithms to analyze single-cell data from cultured AECs infected with Aspergillus fumigatus.
Main Results:
- Successfully identified and quantified individual host-pathogen complexes.
- Dissected the heterogeneity of AEC responses to Aspergillus fumigatus infection.
- Provided a detailed single-cell analysis of the host-pathogen interaction dynamics.
Conclusions:
- The novel IFC-based method enables detailed, single-cell level analysis of host-pathogen interactions in respiratory infections.
- This approach offers a powerful tool for studying fungal pathogenesis and host defense mechanisms.
- Understanding these interactions is crucial for developing effective treatments against inhaled fungal pathogens like Aspergillus fumigatus.
