Spectral flow cytometry method for immunophenotyping neutrophil activation and NET-forming capabilities in an acute dust exposure model 3649

Abstract Description The acute inflammatory response to inhaled toxicants, such as organic dust, is characterized by neutrophilia. Characterizing the recruitment and effector functions, such as neutrophil extracellular trap (NET) formation in vivo, of neutrophil populations is difficult. To address this limitation, we developed a spectral flow cytometry panel for identification of band and mature neutrophils, their precursors, and NET forming capacity in bone marrow (BM), blood (BLD), lung (LNG), and bronchoalveolar lavage fluid (BALF) of 6–7-month-old C57BL/6 mice acutely exposed to organic dust extract (ODE). We demonstrate that acute ODE increases band and mature neutrophil populations in the LNG and BALF while decreasing the number of neutrophils identified in the BM and BLD. Interestingly, the proportion of pro-neutrophils (ProNeu1 and ProNeu2) was altered following ODE in the BLD, not BM. Location-specific alterations in neutrophil activation in response to ODE were observed, and NET-forming neutrophil counts increased following ODE in LNG and BALF. Finally, we identified different forms of NET formation (lytic vs vital) in the LNG and BALF neutrophils via differential stain intensity of Hoechst 33342. This study provides a comprehensive spectral flow cytometry-based method to identify neutrophils and changes in their activation and NET forming capacity across different sites responsible for neutrophil maturation and recruitment into the lower respiratory system in response to acute ODE. Funding Sources NIH/NIMHD U54MD007601, NIH/NHLBI R01HL158926, and R01HL158926-04S1 Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)