Spectral Flow Cytometry Method for Immunophenotyping Neutrophil Activation and NETs in an Acute Dust Exposure Model

Although the contributions of neutrophils and neutrophil extracellular trap (NET) in chronic respiratory diseases associated with environmental dust inhalation has advanced, changes in neutrophil populations and their response following acute dust inhalation are relatively limited. Our understanding of neutrophil response during acute lung injury relies on blood and lung samples, which limit investigations on neutrophil dynamics such as neutrophil production, release, trafficking, and NET formation in response to acute exposures.

To address this limitation, we designed a spectral flow cytometry panel to identify neutrophil progenitors, banded, and mature neutrophils across different sites; bone marrow, blood, lung, and bronchoalveolar lavage fluid (BALF) following acute organic dust extract (ODE) exposure.

We demonstrate that acute ODE exposure increases band and mature neutrophils in the lung and BALF while decreasing band neutrophils in the bone marrow and blood. Interestingly, the proportion of pro-neutrophils (ProNeu1 and ProNeu2) was altered following ODE in the blood but not bone marrow. We also analyzed the expression of surface markers associated with neutrophil recruitment and activation, as well as their size and granularity after ODE exposure. Regardless of their maturation status, neutrophils in BALF and lung exhibited significant changes in CD11b, CXCR2, and CXCR4 levels post-ODE, while CD62L levels were specifically elevated in the blood. Finally, we identified lytic and vital NET forming neutrophils via Hoechst 33342 intensity in the lung and BALF, demonstrating increases in NET-forming neutrophil counts following ODE.

Our spectral flow cytometry method provides valuable insight into neutrophil response, activation, and NET-forming capacity in response to acute ODE exposure.