Observation of autophagosome maturation in the interferon-γ-primed and lipopolysaccharide-activated macrophages using a tandem fluorescent tagged LC3

Macrophages are engaged in many essential host functions, and their activation is a dynamic process that results in diverse functional outcomes such as the potentiation of bactericidal activity and production of chemokines, cytokines, and mediators that coordinate the inflammatory response. This pro-inflammatory response is bimodal, comprising a “prime” event, classically through interferon-γ (IFN-γ), and a “trigger,” such as lipopolysaccharide (LPS). Recently, autophagy, which is one of the major degradative pathways in eukaryotic cells, has been shown to play an important role in both IFN-γ-primed and LPS-activated macrophages. In this study, we sought to characterize the mechanisms of autophagy activation in primed and activated macrophages. To this end, we established a macrophage RAW 264.7 cell line that expressed high levels of a tandem fluorescently tagged LC3 (tfLC3) autophagy marker. By using this macrophage cell line, autophagosome formation was observed in both IFN-γ- and LPS-stimulated cells. Moreover, our data demonstrated that IFN-γ, but not LPS, facilitated autophagosome maturation to autophagolysosomes, suggesting that 2 distinct mechanisms regulating autophagy exist in IFN-γ-primed and LPS-activated macrophages.