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Phospholipase D (PLD) produces phosphatidic acid (PA), an established intracellular signalling lipid that has been also implicated in vesicular trafficking, and as such, PLD could play multiple roles during phagocytosis. Using an RNA interference strategy, we show that endogenous PLD1 and PLD2 are necessary for efficient phagocytosis in murine macrophages, in line with results obtained with wild-type constructs and catalytically inactive PLD mutants which, respectively, enhance and inhibit phagocytosis. Furthermore, we found that PA is transiently produced at sites of phagosome formation. Macrophage PLD1 and PLD2 differ in their subcellular distributions. PLD1 is associated with cytoplasmic vesicles, identified as a late endosomal/lysosomal compartment, whereas PLD2 localizes at the plasma membrane. In living cells undergoing phagocytosis, PLD1 vesicles are recruited to nascent and internalized phagosomes, whereas PLD2 is only observed on nascent phagosomes. These results provide evidence that both PLD isoforms are required for phagosome formation, but only PLD1 seems to be implicated in later stages of phagocytosis occurring after phagosomal internalization. Phagocytosis is an essential process of the innate immune response, enabling immune cells, such as macrophages, to eliminate large extracellular particles, invasive pathogens, cellular debris and apoptotic cells, by internalizing them in membrane-bound vacuoles, the phagosomes [reviewed in (1,2)]. Binding of the particle to diverse cell-surface receptors triggers phagocytosis. Certain phagocytic receptors recognize particle ligands, for example mannose and phosphatidylserine residues, whereas opsonin receptors, like the Fc receptors and complement receptors bind, respectively, the constant domain of immunoglobulins and complement proteins that coat particles. Clustering of phagocytic receptors activates complex intracellular signalling networks that have been extensively studied in the case of the Fc receptor [reviewed by (3)], and this initiates the extension of pseudopods which engulf the particle. Following internalization, phagosomes mature into phagolysosomes where the ingested material is degraded or processed for antigen presentation.During phagosome formation, pseudopod extension around the particle is driven by transient localized actin polymerization (4,5). This surface expansion also requires focal exocytosis of endomembranes, as demonstrated by capacitance and spectroscopic measurements (6,7). Hence, phagosome formation, as well as maturation, involves membrane trafficking between multiple intracellular membrane compartments [reviewed by (2)]. Classical actors of the exocytotic process such as the SNARE complex proteins (8) and specific phospholipids (9,10) participate in these trafficking events, although details of how these elements are interlinked to ensure an efficient machinery for phagocytosis remain to be established.Phospholipase D (PLD) has been recently described as a critical factor for exocytosis in neurons (11) and endocrine cells ...

Omega-3 fatty acids (n-3 PUFAs) are essential for the functional maturation of the brain. Westernization of dietary habits in both developed and developing countries is accompanied by a progressive reduction in dietary intake of n-3 PUFAs. Low maternal intake of n-3 PUFAs has been linked to neurodevelopmental diseases in Humans. However, the n-3 PUFAs deficiency-mediated mechanisms affecting the development of the central nervous system are poorly understood. Active microglial engulfment of synapses regulates brain development. Impaired synaptic pruning is associated with several neurodevelopmental disorders. Here, we identify a molecular mechanism for detrimental effects of low maternal n-3 PUFA intake on hippocampal development in mice. Our results show that maternal dietary n-3 PUFA deficiency increases microglia-mediated phagocytosis of synaptic elements in the rodent developing hippocampus, partly through the activation of 12/15-lipoxygenase (LOX)/12-HETE signaling, altering neuronal morphology and affecting cognitive performance of the offspring. These findings provide a mechanistic insight into neurodevelopmental defects caused by maternal n-3 PUFAs dietary deficiency.