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Blood, Vol. 112, Issue 13, 5202-5211, December 15, 2008
CD18-dependent activation of the neutrophil NADPH oxidase during phagocytosis of Escherichia coli or Staphylococcus aureus is regulated by class III but not class I or II PI3Ks
Blood Anderson et al.
112: 5202
Supplemental materials for: Anderson et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 96.8 KB)
- Figure S1. Effect of DPI and absence of gp91phox on S. aureus and E. coli-induced ROS generation (JPG, 47.1 KB)
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Human peripheral and mouse bone marrow derived neutrophils (from WT or gp91phox−∕− mice as indicated) were prepared, primed with TNFα and GM-CSF and, as indicated, preincubated with 3 µM DPI, in the presence of luminol as described in “Materials and Methods”. Cells were added to serum-opsonised S. aureus (black) or E. coli (grey). (i) Chemiluminesence was recorded as described in Fig. 1. All experiments were performed in duplicate and data (mean ± SEM) are accumulated light emission for a combination of at least two experiments, expressed as a percentage of the response in DMSO vehicle controls. (ii) Neutrophils and bacteria were incubated in suspension for 7min at 37°C. Non-phagocytosed bacteria were lysed with lysostaphin incubation as described in Document 1. Suspensions were cytospun onto glass coverslips, fixed in 4% paraformaldehyde and phagocytosed bacteria counted using DIC images on a Zeiss LSM 510 META point-scanning microscope. Data are the number of bacteria phagocytosed per neutrophil (mean ± SEM) collected for at least 50 cells, expressed as a percentage of phagocytosed bacteria in DMSO controls.
- Figure S2. Effect of PI3K inhibition/knockdown on phagocytosis of S. aureus and E. coli by neutrophils and GFP-PX RAW cells (JPG, 51.4 KB)
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(i) Primed human (black) or mouse (grey) neutrophils were pre-treated with 0-100nM wortmannin as indicated for 10min, prior to addition of RITC-labeled, serum-opsonised S. aureus (squares) or E. coli (triangles) at a ratio of 1:20. Neutrophils and bacteria were incubated in suspension for a further 7min at 37°C. Non-phagocytosed bacteria were quenched by trypan blue or lysostaphin incubation as described in Document 1. Suspensions were cytospun onto glass coverslips, fixed in 4% paraformaldehyde and phagocytosed bacteria were counted using DIC images on a Zeiss LSM 510 META point-scanning microscope. Data are the number of bacteria phagocytosed per neutrophil (mean ± SEM) collected for at least 50 cells, expressed as a percentage of phagocytosed bacteria in the absence of wortmannin (11.48, 9.27 for S. aureus in human and mouse neutrophils respectively, and 13.82, 11.43 for E. coli.). (ii) GFP-PX-RAW cells were adhered to glass coverslips and pre-treated with 0-100nM wortmannin for 10min prior to addition of RITC-labeled, serum-opsonised S. aureus (squares) or E. coli (triangles). Phagocytosis was quantified as described above. Data are the number of bacteria phagocytosed per cell (mean ± SEM) collected for at least 50 cells, expressed as a percentage of phagocytosed bacteria in the absence of wortmannin (6.87,10.67 for S. auerus and E. coli respectively). (iii) GFP-PX-RAW cell populations expressing control (CON), or two independent Vps34 shRNAi constructs (Vps34.1, Vps34.2) were prepared as described in “Materials and Methods”. Cells were adhered to glass coverslips and incubated with RITC-labeled serum-opsonised S. aureus. Phagocytosis was quantified as described above. Data are the number of bacteria phagocytosed per cell (mean ± SEM) collected for at least 50 cells, expressed as a percentage of phagocytosed bacteria in CON cells (100% = 5.56).
- Figure S3. Effect of general and Class I PI3K isoform selective inhibitors on S. aureus and E. coli induced ROS formation in unprimed neutrophils (JPG, 38.3 KB)
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Unprimed bone marrow neutrophils, derived from wild type mice (WT) or mice lacking p110γ (p110γ−∕−) (i), or unprimed human neutrophils (ii) were pre-incubated with either 100nM wortmannin, (or DMSO vehicle control) or a combination of 0.1 µM TGX221 (TGX), 3 µM YM-024 (YM), or 3 µM IC87114 (IC) as indicated, for 10min prior to addition of serum-opsonised S. aureus (black) or E. coli (grey). ROS responses were measured over 40 min, as described in Fig. 1. Data (mean ± SEM, n ≥ 3) are accumulated light emission, expressed as a percentage of response in the absence of inhibitor (DMSO control).
- Figure S4. Effect of RNAi-mediated knock-down of Class III PI3K expression on ROS responses to S. aureus and E. coli in PLB-985 cells (JPG, 53.5 KB)
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PLB-985 cells were transduced with lentivirus directing expression of non-silencing control shRNAi (CON), or two independent Class III PI3K shRNAi constructs (Vps34.1, Vps34.2), GFP-sorted, and differentiated as described in Document 1. (A) shRNAi treated PLB985 cells were harvested, sonicated into SDS-sample buffer, subjected to SDS-PAGE, and immunoblotted for Class III PI3K, or actin as a loading control. Shown is one representative blot of three. Quantitation of Vps34 expression in Vps34.1 and Vps34.2 PLB985 cell lines yielded an average of 87.31 ± 4.81% and 70.1 ± 4.15% of control Vps34 expression respectively. (B) Control (black diamonds) and two Vps34 (open squares and triangles) differentiated shRNAi PLB985 cell populations were harvested, incubated with luminol, and ROS generation measured over time in response to serum-opsonised S. aureus (i) or E. coli (ii) as described in Fig. 1. Data shown are mean ± range for duplicate measurements in one representative experiment of two.
- Figure S5. Measurement of superoxide generation in S. aureus phagosomes using reduction of NBT (JPG, 41.2 KB)
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Primed p40phox+/+ or p40phoxR58A/R58A mouse neutrophils were incubated with NBT, in the presence or absence of 100nM wortmannin, prior to incubation with serum-opsonised S. aureus, as described in Document 1. Non-phagocytosed S. aureus were lysed by lysostaphin, and samples were cytospun onto glass coverslips, fixed and washed as described in Document 1. Dark formazan deposition was detected by DIC imaging on a Zeiss confocal microscope, and quantified using LSM 510 Image browser software. (i) Representative DIC images. (ii) Quantitation of the dark, phagosomal formazan deposits (mean ± SEM, n ≥ 60), expressed as a percentage of the WT value.
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