Deficiencies of the Lipid-Signaling Enzymes Phospholipase D1 and D2 Alter Cytoskeletal Organization, Macrophage Phagocytosis, and Cytokine-Stimulated Neutrophil Recruitment

Abstract

Cell migration and phagocytosis ensue from extracellular-initiated signaling cascades that orchestrate dynamic reorganization of the actin cytoskeleton. The reorganization is mediated by effector proteins recruited to the site of activity by locally-generated lipid second messengers. Phosphatidic acid (PA), a membrane phospholipid generated by multiple enzyme families including Phospholipase D (PLD), has been proposed to function in this role. Here, we show that macrophages prepared from mice lacking either of the classical PLD isoforms PLD1 or PLD2, or wild-type macrophages whose PLD activity has been pharmacologically inhibited, display isoform-specific actin cytoskeleton abnormalities that likely underlie decreases observed in phagocytic capacity. Unexpectedly, PA continued to be detected on the phagosome in the absence of either isoform and even when all PLD activity was eliminated. However, a disorganized phagocytic cup was observed as visualized by imaging PA, F-actin, Rac1, an organizer of the F-actin network, and DOCK2, a Rac1 activator, suggesting that PLD-mediated PA production during phagocytosis is specifically critical for the integrity of the process. The abnormal F-actin reorganization additionally impacted neutrophil migration and extravasation from the vasculature into interstitial tissues. Although both PLD1 and PLD2 were important in these processes, we also observed isoform-specific functions. PLD1-driven processes in particular were observed to be critical in transmigration of macrophages exiting the vasculature during immune responses such as those seen in acute pancreatitis or irritant-induced skin vascularization.

Pharmacological Inhibition of Phospholipase D Protects Mice From Occlusive Thrombus Formation and Ischemic Stroke—Brief Report

“…14,17 Further studies confirmed its specificity and effectiveness for inhibiting PLD through observation of PLD-dependent phenotypes in FIPItreated cells. 14,[18][19][20] Moreover, FIPI has become an essential tool for studying the physiological relevance of PLD in therapeutic ; nonetheless, halopemide is used clinically at levels that should accomplish full PLD inhibition, 22 suggesting that PLD inhibition in humans can be achieved without overt toxicity. We recently demonstrated that FIPI treatment prevents tumor growth and metastasis in mice to the same extent as genetic ablation of Pld1.…”

Thematic Review Series: Phospholipases: Central Role in Lipid Signaling and Disease

“…Roles for PLD2 in thrombotic disease ( 37,66 ), cancer ( 67, 68 ), Alzheimer's disease (AD) ( 69 ), and immune function ( 65 ), based on animal model studies, have recently been summarized ( 5 ). Other potential functions have been raised by tissue culture studies, some of which will be reviewed here.…”

“…As PLD2 knockout mice are grossly normal to inspection ( 69 ), PLD2 would appear to fi t the category of a "temporarily dispensable host gene" that could be acutely targeted to suppress viral replication. One caution for this approach would entail potential effects of PLD2 inhibition on the immune system, which were previously reported to decrease macrophage phagocytosis and neutrophil migration ( 65 ). However, this might not be a substantive issue if the effects on the immune response to infl uenza were limited, whereas the effects on viral replication are profound.…”

Phospholipase D in Cell Signaling: From a Myriad of Cell Functions to Cancer Growth and Metastasis

“…ARF6 activation by ARNO stimulates epithelial cell migration through Rac1 and PLD (44), and PLD is necessary for actin localization and actin-based motility in Dictyostelium via phosphatidylinositol 4,5-bisphosphate (45). PLD2 mediates adhesion via regulation of cell surface integrins (46) and is involved in cytoskeletal organization, macrophage phagocytosis and neutrophil recruitment (43,47,48). In leukocytes, PA is a chemoattractant that acts via ribosomal S6 kinase (S6K) (49) and Fer (17), and 5-fluoro-2-indolyl des-chlorohalopemide (FIPI) is a PLD inhibitor that alters cell spreading and inhibits chemotaxis (50).…”