Classical and alternative differentiation of the mouse macrophages

Makrofagi predstavljaju stanice mijeloidne loze, nalaze se u gotovo svim tkivima našeg organizma i imaju važnu ulogu u održavanju homeostaze. U slučaju akutne infekcije makrofagi diferenciraju u proupalne makrofage (M1) koji su potrebni za učinkovito suprotstavljanje patogenima, izlučuju različite citokine, kemokine i druge upalne medijatore. Nakon aktivacije makrofaga dolazi do genske ekspresije, uključujući i gen za iNOS. U našem istraživanju smo proveli unutarstanično bojenje s monoklonskim protutijelima za iNOS, potrebnih za procjenu klasične diferencijacije u M1 tipa nakon uzgoja peritonealnih mišjih makrofaga u mediju s LPS i IFN–γ. Obje tvari potiču ekspresiju iNOS nakon 24h i održavaju ju visokom i nakon 72 sata. Zbog visoke plastičnosti, makrofagi mogu diferencirati i u M2 tip čija je glavna funkcija terminacija i rezolucija upalnog procesa. Ova alternativa diferencijacija također rezultira promjenom genske ekspresije i mogla se pratiti unutarstanična detekcija arginaze (Arg1). U našem istraživanju suplementacijom IL–4 induciranog de novo došlo je do ekspresije Arg1 nakon 24 sata, a može se detektirati i nakon 72 sata. Ovo istraživanje je potvrdilo sposobnost diferencijacije izoliranih peritonealnih mišjih makrofaga u M1 i M2 tip koji se mogu analizirati protočnim citometrom, detekcijom unutarstanične ekspresije iNOS i Arg1 te određenih površinskih markera makrofaga.Macrophages represent myeloid lineage cells, resident in many if not every tissue throughout the entire body. They patrol within tissues and participate in maintaining homeostasis. In a case of acute infection they easily differentiate in proinflammatory macrophages (M1) necessary to efficiently confront invading pathogen, secreting different cytokines, chemokines and other inflammatory mediators. Upon activation macrophages express number of genes de novo, including gene for the iNOS. In our study we used intracellular staining with monoclonal antibody for iNOS to evaluate classical differentiation into M1 type after cultivation of peritoneal mouse macrophages in medium with LPS and IFN–γ. Both compounds induced iNOS expression after 24h that maintained high after 72 hours. Due to high plasticity, macrophages can differentiate in opposite M2 type with a role in sustaining overwhelming inflammation and support resolution. This alternative differentiation also results in a gene expression change and could be monitored by intracellular Arg1 detection. In our study IL–4 supplementation induced de novo Arg1 expression after 24 hours that was upregulated after 72 hours. This study confirmed ability of isolated mouse peritoneal macrophages to differentiate into M1 and M2 type by modifying culture settings that can be analyzed by flow cytometer, detecting intracellular iNOS and Arg1 expression together with specific surface macrophage markers