71 <i>In vitro</i> human alveolar macrophage (ImmuPHAGE™) responses to particulate matter exposure

Abstract Air pollution is linked to approximately 4.2 million deaths annually, with particulate matter (PM) playing a major role in respiratory diseases. PM2.5, comprising fine particles capable of penetrating deep into the lungs, induces oxidative stress and inflammation. Alveolar macrophages are central to the lung’s immune response, making their interaction with PM critical for understanding toxicity and inflammation. This study evaluates the in vitro respiratory toxicity of PM2.5 collected from Prishtina and Kastriot, Kosovo. Human alveolar macrophages (ImmuPHAGE™) were exposed to PM2.5 concentrations (25 to 100 µg/mL) for 24 and 48 h. Cell viability, cytotoxicity, and phagocytic function were assessed using standard assays and fluorescent microspheres. High-content imaging (In Cell Analyzer 600) enabled single-cell profiling of morphology and stress markers. Results showed significant (P &amp;lt; 0.05) reductions in mitochondrial activity across all exposure levels. Phagocytic activity decreased (P &amp;lt; 0.05) after 24 h but increased (P &amp;lt; 0.05) after 48 h. High-content imaging revealed distinct phenotypic changes: early responses (24 h) were marked by elevated cellular stress, while later responses (48 h) showed enlarged cells and increased vacuolation. These findings suggest a time-dependent evolution of macrophage response to PM2.5, indicating a pathophysiological shift toward airway inflammation. This study highlights the utility of in vitro tools and high-content imaging in characterizing individual cell responses to environmental PM. Such approaches enhance our understanding of acute and chronic lung responses to air pollution and support efforts to mitigate its health impacts.