Abstract
Phagocytosis is a complex process that involves membranelipid remodeling and the attraction and retention of key effector proteins. Phagosome phenotype depends on the type of receptor engaged and can be influenced by extracellular signals. Interleukin 4 (IL-4) is a cytokine that induces the alternative activation of macrophages (MΦs) upon prolonged exposure, triggering a different cell phenotype that has an altered phagocytic capacity. In contrast, the direct effects of IL-4 during phagocytosis remain unknown. Here, we investigate the impact of short-term IL-4 exposure (1 hour) during phagocytosis of IgG-opsonized yeast particles by MΦs. By time-lapse confocal microscopy of GFP-tagged lipid-sensing probes, we show that IL-4 increases the negative charge of the phagosomal membrane by prolonging the presence of the negatively charged second messenger PI(3,4,5)P3. Biochemical assays reveal an enhanced PI3K/Akt activity upon phagocytosis in the presence of IL-4. Blocking the specific class I PI3K after the onset of phagocytosis completely abrogates the IL-4-induced changes in lipid remodeling and concomitant membrane charge. Finally, we show that IL-4 direct signaling leads to a significantly prolonged retention profile of the signaling molecules Rac1 and Rab5 to the phagosomal membrane in a PI3K-dependent manner. This protracted early phagosome phenotype suggests an altered maturation, which is supported by the delayed phagosome acidification measured in the presence of IL-4. Our findings reveal that molecular differences in IL-4 levels, in the extracellular microenvironment, influence the coordination of lipid remodeling and protein recruitment, which determine phagosome phenotype and, eventually, fate. Endosomal and phagosomal membranes provide topological constraints to signaling molecules. Therefore, changes in the phagosome phenotype modulated by extracellular factors may represent an additional mechanism that regulates the outcome of phagocytosis and could have significant impact on the net biochemical output of a cell.
Highlights
The constant threat posed by pathogens and cell debris is tackled by phagocytosis, the process through which cells engulf and destroy dangerous material [1]
In the presence of Interleukin 4 (IL-4), Kmyr did eventually subside from the phagosomal membrane, but on a much longer timescale (5–10 min, data not shown) as compared to the phagosomes formed in the absence of IL-4
Our results show a prolonged negative charge at the membrane of early phagosomes in the presence of IL-4 and we demonstrate this is due to an extended association of the negatively charged lipid second messenger PI(3,4,5)P3, as a result of an increased phosphatidylinositol 39kinase (PI3K) activity
Summary
The constant threat posed by pathogens and cell debris is tackled by phagocytosis, the process through which cells engulf and destroy dangerous material [1]. A variety of self and pathogen derived signals can influence phagocytosis and the microbicidal activity of specialized phagocytes such as macrophages (MWs) and neutrophils [2,3,4]. Among these signals is the type I cytokine Interleukin 4 (IL-4), mainly produced by T helper 2 cells and mast cells and involved in a variety of (patho)physiological events ranging from tissue adhesion and inflammation, to specificity of immunoglobulin class switching and the regulation of immune responses to allergens and parasites [5]. New insight into the effects of a direct IL-4 signaling on phagocytosis is needed
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