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Background Neutrophil count and neutrophil extracellular trap (NET) markers are elevated within bronchoalveolar lavage fluid (BALF) of patients with Idiopathic Pulmonary Fibrosis (IPF). 1 2 However, the activity of neutrophils within the alveolar spaces of IPF lung tissue is undetermined, as is its contribution to fibrosis. We hypothesised that the fibrotic alveolar microenvironment promotes neutrophil survival and activation, with accompanying enhanced respiratory burst and NET formation. This was investigated using a novel in vitro model of IPF.Methods Neutrophils, isolated from venous blood of healthy donors using Percoll density-gradient centrifugation, were incubated in pooled BALF collected either from IPF patients (n=5) or age matched control participants (n=3). Flow cytometric analyses of neutrophil viability (PI/Annexin V staining) and cell surface activation marker (CD11b, CD66b, CD62L) expression were performed. Reactive oxygen species (ROS) generation and NET formation were assessed using luminol-amplified chemiluminescence and fluorometric analysis of SYTOX TM green stained DNA, respectively.Results Fewer neutrophils entered early apoptosis during 3 hours of incubation with IPF BALF compared to control BALF (p = 0.018). IPF BALF exposure increased cell surface expression of the activation marker CD11b (p = 0.011) and degranulation marker CD66b (p < 0.001), compared to control BALF. CD62L expression was equivalent following exposure to control and IPF BALF (p > 0.05). IPF BALF exposure increased total neutrophil ROS generation over 3 hours, compared to control BALF (p = 0.018). Despite protein-normalised IPF BALF samples containing higher concentrations of cell free DNA (a NET marker) compared to protein-normalised control BALF samples (p = 0.036), NET formation by neutrophils was equivalent following exposure to pooled IPF and control BALF (p > 0.05).Abstract S168 Figure 1(A) Flow cytometric analysis of neutrophil activation marker (CD11b and CD66b) expression following exposure to pooled IPF and control BALF. (B) Summary histogram and (C) representative trace of luminol-amplified chemiluminescence experiments assessing neutrophil ROS generation (AUC – area under the curve)Conclusions This study demonstrates that IPF BALF promotes a phenotypic shift in neutrophils towards an activated state. This corresponds with a decrease in neutrophil apoptosis and an augmented respiratory burst. The implications of these findings in IPF’s pathogenesis and progression warrant further investigation. Namely, exploration of the impact of enhanced neutrophil ROS generation on TGF-β activation within the fibrotic lung is vital. Identification of neutrophil-activating components of IPF BALF may enable therapeutic modulation of neutrophil function in IPF.References https://doi.org/10.1378/chest.07-1948https://doi.org/10.3389/fimmu.2020.02190