WAVE1 mediates suppression of phagocytosis by phospholipid-derived DAMPs
Ulrich Matt, … , John D. Scott, Sylvia Knapp
Ulrich Matt, … , John D. Scott, Sylvia Knapp
Published June 24, 2013
Citation Information: J Clin Invest. 2013;123(7):3014-3024. https://doi.org/10.1172/JCI60681.
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Research Article Immunology

WAVE1 mediates suppression of phagocytosis by phospholipid-derived DAMPs

Abstract

Clearance of invading pathogens is essential to preventing overwhelming inflammation and sepsis that are symptomatic of bacterial peritonitis. Macrophages participate in this innate immune response by engulfing and digesting pathogens, a process called phagocytosis. Oxidized phospholipids (OxPL) are danger-associated molecular patterns (DAMPs) generated in response to infection that can prevent the phagocytic clearance of bacteria. We investigated the mechanism underlying OxPL action in macrophages. Exposure to OxPL induced alterations in actin polymerization, resulting in spreading of peritoneal macrophages and diminished uptake of E. coli. Pharmacological and cell-based studies showed that an anchored pool of PKA mediates the effects of OxPL. Gene silencing approaches identified the A-kinase anchoring protein (AKAP) WAVE1 as an effector of OxPL action in vitro. Chimeric Wave1–/– mice survived significantly longer after infection with E. coli and OxPL treatment in vivo. Moreover, we found that endogenously generated OxPL in human peritoneal dialysis fluid from end-stage renal failure patients inhibited phagocytosis via WAVE1. Collectively, these data uncover an unanticipated role for WAVE1 as a critical modulator of the innate immune response to severe bacterial infections.

Authors

Ulrich Matt, Omar Sharif, Rui Martins, Tanja Furtner, Lorene Langeberg, Riem Gawish, Immanuel Elbau, Ana Zivkovic, Karin Lakovits, Olga Oskolkova, Bianca Doninger, Andreas Vychytil, Thomas Perkmann, Gernot Schabbauer, Christoph J. Binder, Valery N. Bochkov, John D. Scott, Sylvia Knapp

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Figure 2

PKA activation mediates OxPAPC-associated cell spread and inhibition of phagocytosis.

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PKA activation mediates OxPAPC-associated cell spread and inhibition of ...
(A–C) RAW 264.7 cells were treated with carrier, DMPC, or OxPAPC (10 μg/ml, 30 minutes in A; 5 μg/m, 15 minutes in B and C) alone or following preincubation with H89 (10 μM) or PKA amide14–22 (20 μM) (30 minutes). (A) Cells were subsequently stained for F-actin (phalloidin; green) and PI (red). (B and C) Uptake of FITC-labeled E. coli was evaluated after 60 minutes and is expressed relative to carrier. (D) RAW 264.7 cells were transfected with shRNA to the α-isoform of PKAc, and silencing was verified by Western blot. (E and F) Control (vector or scrambled control) and shRNA-transfected cells were preincubated with carrier, DMPC, or OxPAPC (5 μg/ml, 15 minutes in E; 10 μg/ml, 30 minutes in F). (E) Phagocytosis of FITC-labeled E. coli was examined after 60 minutes and is expressed relative to carrier. (F) Cells were stained with phalloidin–Alexa Fluor 488 (green) and PI (red). Representative images of 3 independent experiments are shown. (G) RAW 264.7 cells were incubated with DMPC or OxPAPC at 10 μg/ml, forskolin (100 μM), or carrier for 15 minutes. PKA activity was measured as described in Methods. Arrow indicates activated PKA (lower band); “positive” and “negative” indicate assay control. Data are mean ± SEM of triplicates and representative of 3 independent experiments; *P < 0.05; **P < 0.01; ***P < 0.001 versus corresponding carrier. Original magnification, ×800.