WAVE1 mediates suppression of phagocytosis by phospholipid-derived DAMPs - PubMed (original) (raw)

. 2013 Jul;123(7):3014-24.

doi: 10.1172/JCI60681. Epub 2013 Jun 24.

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

Affiliations

WAVE1 mediates suppression of phagocytosis by phospholipid-derived DAMPs

Ulrich Matt et al. J Clin Invest. 2013 Jul.

Erratum in

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.

PubMed Disclaimer

Figures

Figure 1

Figure 1. Oxidation of lipids occurs in E. coli peritonitis in vivo and leads to an actin-dependent change in cell shape in vitro.

(A) Endogenous levels of oxidized phosphatidylcholine were measured in PLF of mice infected with E. coli after 8 or 16 hours, respectively, compared with supernatants of RAW 264.7 cells after adding 5 or 10 μg/ml of OxPAPC, respectively. Co, control. (B) RAW 267.4 cells were incubated with indicated doses of OxPAPC or DMPC for 15 minutes, and phagocytosis of E. coli was assessed after 60 and 120 minutes (triplicates, representative of 3 independent experiments). (C) FACS histogram showing uptake of FITC-labeled E. coli by resident peritoneal macrophages pretreated with 10 μg/ml of OxPAPC or DMPC after 60 minutes. (D) Mice (n = 8/group) were infected with 104 CFU E. coli i.p. and treated with 2.5 mg/kg DMPC or OxPAPC i.p. Peritoneal CFU counts were enumerated 10 hours after infection. Data (A–D) are presented as mean ± SEM; *P < 0.05; **P < 0.01 versus controls. ***P < 0.001. (E) RAW 264.7 cells were incubated with carrier, DMPC, or OxPAPC (10 μg/ml; 30 minutes) alone or following incubation with 2 μM cytochalasin D (30 minutes). Cells were subsequently stained for F-actin using phalloidin (green) and PI for nuclei (red). Scale bar: 30 μm.

Figure 2

Figure 2. PKA activation mediates OxPAPC-associated cell spread and inhibition of phagocytosis.

(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.

Figure 3

Figure 3. OxPL-induced inhibition of phagocytosis requires anchoring of PKA in vivo and in vitro.

(A) RAW 264.7 cells were incubated with carrier, DMPC, or OxPAPC (10 μg/ml) alone or after treatment with 100 μM Ht-31 (30 minutes) and stained with phalloidin (green) and PI (red). Original magnification, ×800. (B) RAW 264.7 cells were treated with carrier or phospholipids (5 μg/ml; 15 minutes) alone or after preincubation with Ht-31 (100 μM) for 30 minutes. Phagocytosis of FITC-labeled E. coli was analyzed using FACS after 60 minutes. Uptake is expressed relative to carrier. **P < 0.01. Data show mean ± SEM of triplicates and are representative of 3 independent experiments. (C) Mice received carrier or 2.5 mg/kg OxPAPC i.p. and/or 100 μM of Ht-31 immediately before infection with 104 CFU E. coli. At 10 hours after infection, PLF was harvested and bacterial CFUs enumerated. Data are mean ± SEM of 2 independent experiments from n = 7–9 mice/group; **P < 0.01 versus carrier. (D) Mice received 2.5 mg/kg DMPC or OxPAPC i.p. followed by i.p. injection of vehicle or Ht-31 (OxPAPC–Ht-31), after which they were infected with 104 CFU E. coli. Survival was monitored every 2 hours; n = 12 mice/group. P values indicate differences between OxPAPC versus DMPC or OxPAPC versus OxPAPC-Ht-31, respectively.

Figure 4

Figure 4. The AKAP WAVE1 mediates OxPL inhibition of phagocytosis in peritoneal macrophages.

AKAP-Lbc (150 bp), Wave1 (116 bp), and Gravin (136 bp) mRNA expression in (A) RAW 264.7 or (B) primary peritoneal macrophages; GAPDH (372 bp). (C) Western blot verifying silencing of Wave1 (82 kD) in RAW 264.7 cells; β-actin control (37 kD). (D) Scrambled control and shRNA (targeting Wave1) cells incubated with carrier, DMPC, or OxPAPC (10 μg/ml) and stained with phalloidin (green) and PI for nuclei (red). Original magnification, ×800. (E) Phagocytosis of E. coli (60 minutes) assayed in scrambled control and shRNA-Wave1 cells preincubated with carrier, DMPC, or OxPAPC (5 μg/ml). Data depicted are mean ± SEM of triplicates, *P < 0.05 versus corresponding carrier/DMPC. (F) mRNA expression and (G) Western blot for Wave1 in WT and Wave1–/– primary peritoneal macrophages (WAVE1 82 kD; β-actin 39 kD). (H) Primary peritoneal macrophages of WT and Wave1–/– mice incubated with carrier, DMPC, or OxPAPC (10 μg/ml) and stained with phalloidin (green) and PI (red). (I) Phagocytosis of FITC-labeled E. coli (60 minutes) by WT and Wave1–/– peritoneal macrophages analyzed after prior incubation with DMPC or OxPAPC (5 μg/ml). Data are mean ± SEM of triplicates of 2 independent experiments; **P < 0.01 versus corresponding DMPC.

Figure 5

Figure 5. Chimeric _Wave1_-KO mice are rendered unresponsive to OxPL.

(A–D) WT mice reconstituted with WT or Wave1–/– bone marrow were treated with DMPC or OxPAPC (2.5 mg/kg) i.p. and infected with 104–5 CFU E. coli i.p. (A) Peritoneal, (B) liver, and (C) blood CFU counts were enumerated 10 hours after infection, and (D) survival was monitored every 2 hours. Data are from n = 9–12 mice/group and are presented as mean ± SEM; *P < 0.05 versus corresponding DMPC control.

Figure 6

Figure 6. OxPL in human PDF inhibit phagocytosis in the presence of WAVE1.

(A) Levels of oxidized phosphocholine were measured using the EO6 mAb or an isotype control in PDF from 2 patients undergoing peritoneal dialysis. (B) Murine primary peritoneal macrophages were incubated for 15 minutes with PDF of 1 representative patient (patient A), and phagocytosis of E. coli after 60 minutes was assessed by FACS. Ig depletion was done with protein G beads. (C) PLF from Ldlr–/–Rag–/– mice on normal diet (ND) or high-fat diet (HFD) was placed on primary peritoneal macrophages in the presence or absence of Ht-31 (100 μM) and phagocytosis of E. coli after 60 minutes. (D) RAW 264.7 macrophages were either preincubated with EO6 antibody or isotype control (1 μg/ml) for 1 hour, then with OxPL or DMPC (5 μg/ml) for 15 minutes. Phagocytosis of E. coli was assessed after 60 minutes by FACS. Control assays were done in RPMI. (E) PDF of patient A was subjected to chloroform extraction, and the resultant water or lipid fraction was added to RAW 264.7 cells 15 minutes prior to addition of E. coli. Phagocytosis was assayed after 60 minutes by FACS. Control assays were done in RPMI. (F) RAW 264.7 macrophages were incubated for 15 minutes with IgG-depleted PDF that had been pretreated with either EO6 or isotype control antibody (10 μg/ml) for 1 hour, and phagocytosis of E. coli after 60 minutes was assessed by FACS. (G) WT and Wave1–/– primary peritoneal macrophages were incubated with Ig-depleted PDF from patient A, and phagocytosis of E. coli was determined was analyzed. Data are mean ± SEM of at least duplicate experiments; *P < 0.05; **P < 0.01; ***P < 0.001 versus corresponding control.

References

    1. Hampton MB, Kettle AJ, Winterbourn CC. Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood. 1998;92(9):3007–3017. - PubMed
    1. Miller YI, et al. Oxidation-specific epitopes are danger-associated molecular patterns recognized by pattern recognition receptors of innate immunity. Circ Res. 2011;108(2):235–248. doi: 10.1161/CIRCRESAHA.110.223875. - DOI - PMC - PubMed
    1. Binder CJ, et al. Innate and acquired immunity in atherogenesis. Nat Med. 2002;8(11):1218–1226. doi: 10.1038/nm1102-1218. - DOI - PubMed
    1. Hansson GK, Libby P. The immune response in atherosclerosis: a double-edged sword. Nat Rev Immunol. 2006;6(7):508–519. doi: 10.1038/nri1882. - DOI - PubMed
    1. Yoshimi N, et al. Oxidized phosphatidylcholine in alveolar macrophages in idiopathic interstitial pneumonias. Lung. 2005;183(2):109–121. doi: 10.1007/s00408-004-2525-0. - DOI - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources