Abstract
Suppressor of cytokine signaling (SOCS) proteins are recognized as key feedback inhibitors modulating the inflammatory activities of macrophages, but comparatively little is known about whether and how they affect phagocytosis. Here, we evaluated the role of SOCS3 in driving the inflammatory phenotype and phagocytic uptake of apoptotic cells by human macrophages and the signaling pathways that are necessary for efficient phagocytosis. In M1-activated human monocyte-derived macrophages, SOCS3 silencing, using short interfering RNA technology, resulted in a decreased expression of proinflammatory markers and an increased expression of M2 macrophage markers. Strikingly, we demonstrated for the first time that SOCS3 knockdown significantly enhances the phagocytic capacity of M1 macrophages for carboxylate-modified beads and apoptotic neutrophils. With the use of live-cell video microscopy, we showed that SOCS3 knockdown radically affects the temporal dynamics of particle engulfment, enabling more rapid uptake of a second target and delaying postengulfment processing, as evidenced by deferred acquisition of phagosome maturation markers. SOCS3 knockdown impacts on phagocytosis through increased PI3K and Ras-related C3 botulinum toxin substrate 1 (Rac1) activity, pathways essential for engulfment and clearance of apoptotic cells. Enhanced phagocytosis in SOCS3-silenced cells was reversed by pharmacological PI3K inhibition. Furthermore, we revealed that actin polymerization, downstream of PI3K/Rac1 activation, was significantly altered in SOCS3-silenced cells, providing a mechanism for their greater phagocytic activity. The findings support a new model, whereby SOCS3 not only plays an important role in driving macrophage inflammatory responses but modulates key signaling pathways organizing the actin cytoskeleton to regulate the efficiency of phagocytic processes.
Highlights
Macrophages are highly heterogeneous cells that can adopt many phenotypes and functions to shape immune responses and maintain and restore tissue homeostasis
SOCS3 knockdown results in a decrease in expression of proinflammatory genes (TNF, IL-6, HLA-DR), while enhancing those markers associated with M2 macrophages; by contrast, MARCO showed a minimal change in expression levels (Fig. 1 and Supplemental Fig. 1A–D)
We demonstrate that SOCS3 is essential for controlling the proinflammatory phenotype of human monocytederived macrophages but is a previously unidentified determinant regulating the efficiency with which they phagocytose apoptotic cells
Summary
Macrophages are highly heterogeneous cells that can adopt many phenotypes and functions to shape immune responses and maintain and restore tissue homeostasis. Microbial products and IFN-g (M1 activation), for example, can drive proinflammatory, microbicidal, and tumoricidal macrophages, whereas IL-4 (M2 activation) results in anti-inflammatory, tissue-healing macrophages [1, 2]. These are, extremes on a continuum of the phenotypes found in vivo. A fundamental function of macrophages for conserving homeostasis and resolving inflammation is engulfment and clearance of apoptotic cells [4,5,6,7] This uptake results in production of anti-inflammatory cytokines, including TGF-b and IL-10 [8], which inhibits inflammatory mediator production, further dampening the inflammation
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