In vitro bactericidal assay Mouse bone marrow was isolated from the femur and the tibia. Cells were suspended in phosphate buffered saline containing.5% BSA and 2 mm EDTA and filtered through a cell strainer. Red blood cells were removed and neutrophils were enriched by negative selection using an EasySep system (StemCell Technologies, Vancouver, BC, Canada). We routinely obtain 4-5 million cells per mouse, and >9% of them are morphologically mature neutrophils (Fig. S8). Purified neutrophils were treated with different concentrations of SF167 or DMSO (vehicle control) for 3 min at 37 C then washed twice with HBSS. Live E. coli particles (strain 19138; American Type Culture Collection, 12.5 1 5 cfu) were opsonized with mouse serum (final concentration 12.5%) at 37 C for 3 min and then incubated with pretreated neutrophils (2.5 1 5 cfu) at 37 C for 1 hour. Reactions were stopped and neutrophils were lysed with sterile water (1:1). Samples were then serially diluted and spread on Luria broth (LB) agar plates. Colony numbers were determined after overnight incubation at 37 C. Gentamicin protection assay Neutrophil intracellular bactericidal activity was measured using a gentamicin protection assay. Briefly, mouse bone marrow derived neutrophils were treated with SF167 or DMSO (vehicle control) for 3 min at 37 C then washed twice with HBSS. Cells were incubated with serum-opsonized live E. coli particles (as described above) at a ratio of 1:1 at 37 C for 1 hour. Gentamicin was then added at the final concentration of 1 g/ml to kill extracellular bacteria. After additional 1 hour incubation, cells were washed twice with HBSS and lysed with 1 ml sterile water at room temperature. Viable intracellular bacteria were quantified by subsequent plating the lysed samples on LB agar plates. The results were normalized by phagocytosis indexes. Phagocytosis assay Fluorescein-conjugated Zymosan bioparticles (Molecular Probes) were reconstituted in HBSS and opsonized with 12.5% mouse serum at 37 C for 3 min. Mouse bone marrow derived neutrophils were incubated with serum-opsonized bioparticles at a ratio of 1:1 (neutrophils : bioparticles) at 37 C for 1 hour. Negative controls were incubated on ice instead. The fluorescence of extracellular particles was 1
quenched by addition of 1 μl of.25 mg/ml trypan blue. The number of internalized particles was counted under a fluorescence microscope (Olympus lx17, 1X objective). Phagocytosis efficiency was expressed as the number of the internalized particles per 1 neutrophils (Phagocytosis index). Binding efficiency was expressed as the number of the binding particles per 1 neutrophils (Binding index). More than 1 cells were counted from random fields per coverslip for each group. Bacterial burden and BALF total protein levels Bronco alveolar lavage (BAL) samples were collected from mice at 24 hr after bacteria challenge. For bacterial burden assay, 1 ml of cold PBS/15 mm EDTA was injected to the lung and withdrew while gently massaging lungs. This was repeated with 1 ml PBS/15 mm EDTA buffer. BALF were then pooled and serially diluted with ice-cold sterile PBS. Aliquots of 1 l were spread on LB agar plates (E.coli), chocolate II agar (S. pneumonia), or Columbia agar with 5% sheep blood (S. aureus). Colonies were counted after overnight incubation. For BALF total protein assay, BAL was done with 1 ml of ice cold PBS/15 mm EDTA. Protein concentration was measured using the Bio-Rad protein assay reagent. The standard curve was constructed using BSA. Histopathology Lungs were collected 24 hr after infection. Whole lungs were inflated and fixed with Bouin s solution. The specimens were embedded in paraffin and 6 m thick sections were stained with hematoxylin and eosin and examined by light microscopy. Nonquantitative histological analysis was performed by a pathologist blinded for groups. IPLab software (BD Biosciences) was used to manually trace edema containing regions in the tissue section. The pixel area of each edema containing region was calculated using the same IPLab software ( area measurement modular). The edema formation was calculated as the percentage of pixel area of all the edema containing regions relative over the pixel area of the whole image. 2
3 min 16 hr DMSO DMSO SF: 125 nm 7AAD SF: 25 nm 7AAD SF: 25 nm SF: 5 nm Annexin V Annexin V Figure S1. Treatment with SF167 does not significantly affect neutrophil spontaneous death. Human neutrophils (1 x 1 6 ) were treated with indicated concentrations of SF167 at 37 C for 3 min and washed twice with PBS. After being cultured in RPMI164 plus 1% FBS for indicated time, the amounts of healthy and apoptotic cells were determined by AnnexinV-APC and 7-Amino-actinomycin D (7-AAD) staining as previously described (Zhu et al., 26).
A fmlp 1µM fmlp 1µM ionomycin B [Ca2 2+] ( F34/38) ) [Ca2 2+] ( F34/38) ) F34/38 Figure S2. Treatment with SF167 does not alter fmlp-induced calcium signaling in neutrophils. Mouse bone marrow-derived neutrophils were pretreated with or without SF167 at 37 C for 3 min, and then loaded with 1 µm of Fluo-4-AM and 2 µm of Fura-Red AM in 1 ml PBS at RT for 45 min. Cells were washed hdtwice with ihpbs and resuspended ddi in (A) HBSS (no calcium, no magnesium) + 1% FBS + 2 nm HEPES + 1 mm EDTA or (B) HBSS (with calcium and magnesium) + 1% FBS + 2 nm HEPES. Cells were allowed to settle at least 15 min at RT before being stimulated with 1 M of fmlp. The changes in intracellular calcium ([Ca 2 ]i) were monitored for 5 min after stimulation. Black line, DMSO vehicle control; blue line, 5 nm of SF167; brown line, 25 nm of SF167; green line,125 nm of SF167. Arrows, addition of fmlp or ionomycin. The signal detected in (A) in the absence of extracellular calcium represents calcium release from intracellular calcium stores.
DMSO control DMSO control DMSO 3nM FITC-fMLP 6nM FITC-fMLP 3nM FITC-fMLP + 1 M fmlp 6nM FITC-fMLP + 1M fmlp control 12nM FITC-fMLP 12nM FITC-fMLP + 1M fmlp Events SF:5nM control 3nM FITC-fMLP 3nM FITC-fMLP + 1 M fmlp SF:5nM control 6nM FITC-fMLP 6nM FITC-fMLP + 1 M fmlp SF:5nM control 12nM FITC-fMLP 12nM FITC-fMLP + 1 M fmlp FITC Figure S3. Treatment with SF167 does not change surface expression of fmlp receptors in neutrophils. Human neutrophils (.5 x 1 6 ) treated with or without 5 nm of SF167 (37 C for 3 min) were pre-blocked on ice for 45 min in HBSS supplemented with 1% BSA/.2% sodium azide. Cells were then incubated with 3 nm, 6 nm, or 12 nm of FITC-fMLP (Invitrogen, Carlsbad, CA) in the same medium (for 1 hour on ice), in the presence or absence of 1 M of unlabeled fmlp, washed once with HBSS, and immediately analyzed by flow cytometry. Neutrophils were gated by forward/side scatter. Specific binding of FITC-fMLP was determined by subtracting geometric mean fluorescence intensity in the presence of 1 M unlabeled fmlp from the geometric mean fluorescence intensity in the absence of 1 M unlabeled fmlp.
DMSO SF:1nM SF:1nM SF:1nM Figure S4. PTEN inhibitor SF167 does not have bactericidal activity. Live E. coli particles (strain 19138; American Type Culture Collection, 12.5 1 5 cfu) were incubated with indicated concentrations of SF167 in HBSS at 37 C for 1 hour (total volume 1 µl). Samples were then diluted and spread on LB agar plates. Colonies in each sample were counted after overnight incubation at 37 C.
A Neutropenic mice Recipient i mice Induction of neutropenia with Cy Intraperitoneal injection of ftg(3%) (3%), 2.5 hours Mixed (1:1) population was iv injected into the inflamed mice Donor mice CMTMR labeling -1.5 hours after neutrophil transfusion, collect cells from peritoneal cavity. CMTM MR CMTMR+ Input control Bone marrow derived d neutrophils 1.5h after iv injection CMFDA labeling B C D CMTMR+ Relativ ve amount of neutr rophils (CMDMR/CMFDA A) 1.5 1.5 Input control Peritoneal lavage - Determine the ratio of (CMTMR + cells)/(cmfda )( + cells) by FACS analysis. red blood cells Neutrophils lymphocytes CMFDA Figure S5. Recruitment of transfused neutrophils to the inflamed peritoneal cavity. (A) Schematic representation of the experimental system for neutrophil transfusion. Neutrophils isolated from wild-type mice were treated with or without SF167 for 3 min at 37 C, washed twice with PBS, and then labeled with CMTMR or CMFDA. Labeled cells were mixed (1:1) as indicated and then transfused intravenously (via tail vein) to neutropenic mice that had been challenged with 3% thioglycollate (TG) for 2.5 hr. Peritoneal lavage was harvested 1.5 hr after the neutrophil transfusion. (B) Labeling with fluorescent dye only does not affect neutrophil recruitment. Bone marrow derived neutrophils were labeled with CMFDA or CMTMR. The amount of adoptively transferred neutrophils recruited to the peritoneal cavity were analyzed using a FACSCanto II flow cytometer and FACSDiva software. (C) Relative recruitment of neutrophils was calculated as the ratio of indicated populations in the peritoneal lavage. Data shown are mean ± SD of three mice. (D) Forward and side scatter shows that most of the non-neutrophil populations in the inflamed peritoneal cavity are red blood cells with small amount of lymphocytes.
Cy: 15 mg/kg ip 1mg/kg ip Bacteria challenge & Neutrophil transfusion Days 1 2 3 4 5 6 7 Peripheral blood neutrophils (x1million n/ml) 1.5 1.2.9.6.3 d1 d2 d3 d4 d5 d6 d7 WT.87.18.1.13.15 Figure S6. Schematic of mouse neutropenia model. Mouse neutropenia was induced by two intraperitoneal injections of cyclophosphamide (Cy) at a total dose of 25 mg/kg (15 mg/kg on day 1 and 1 mg/kg on day 4). On day 5, Cy-treated mice contained approximately 9% fewer circulating neutrophils than untreated or saline-treated group. The profound neutropenia persisted through days 6 and 7.
DMSO control SF167 Arbitrary Lig ght Units 5 4 3 2 1 (-) fmlp (+) fmlp t Units Arbirary Ligh 5 4 3 2 1 (-) fmlp (+) fmlp 5 1 15 2 25 5 1 15 2 25 Time (sec) Time (sec) TNFa priming TNFa priming + SF167 trary Light Units Arbit 5 (-) fmlp 4 (+) fmlp 3 2 1 5 1 15 2 25 Time (sec) Arbitra ary Light Units 5 (-) fmlp 4 (+) fmlp 3 2 1 5 1 15 2 25 Time (sec) Figure S7. PTEN inhibitor SF167 can further enhance ROS production in mouse neutrophils primed with TNFa. Bone marrow derived mouse neutrophils were treated with TNFa (5ng/mL), SF167 (5nM), TNFa+SF167, or DMSO alone at 37ºC for 3 min in HBSS (.2% BSA) buffer, washed twice with HBSS, and then stimulated with HBSS or 2 µm of fmlp. ROS production was monitored in the presence of 5 M of isoluminol,.8u of HRP in a luminometer at 37 C. Chemiluminescence (arbitrary light units) was recorded at indicated time points.
Figure S8. The purity of isolated mouse neutrophils. Mouse bone marrow was isolated from the femur and the tibia. Cells were suspended in phosphate buffered saline containing.5% BSA and 2 mm EDTA and filtered through a cell strainer. Red blood cells were removed and neutrophils were enriched by negative selection using an EasySep system (StemCell Technologies, Vancouver, BC, Canada). We routinely obtain 4-5 million cells per mouse, and >9% of them are mature neutrophils.