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Blood, Vol. 113, Issue 20, 4930-4941, May 14, 2009
Targeted deletion of tumor suppressor PTEN augments neutrophil function and enhances host defense in neutropenia-associated pneumonia
Blood Li et al.
113: 4930
Supplemental materials for: Li et al
Mice
The conditional PTEN-knockout mice (PTEN loxP/loxP) and the myeloid-specific Cre mice were purchased from Jackson Laboratories (Bar Harbor, ME). The experimental myeloid-specific PTENknockout mice were generated as previously described.1,2 Six-to-12-week-old mice were used in all experiments. Mouse bone marrow neutrophils were prepared as described by Zhu et al.1 All procedures involving mice were approved and monitored by the Children’s Hospital Animal Care and Use Committee. Histopathology
Lungs were collected 8h or 24h after infection and fixed by intratracheal instillation of Bouin’s solution at 23 cm H2O pressure. Tissues were embedded in paraffin, and 6-µm thick sections were stained with hematoxylin and eosin and examined by light microscopy. IPLab software (BD Biosciences) was used to manually trace edema containing regions of 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 to the pixel area of the whole image. Neutrophil recruitment
Wild-type and PTEN-knockout mice were anesthetized and instilled with bacteria as described above. After 8h or 24h mice were euthanized by CO2. The chest cavity was opened, and a catheter was tied to the trachea. Bronchoalveolar lavage (BAL) was performed (1 mL of PBS/15mM EDTA ×10) in each group. The BAL fluid (BALF) was centrifuged at 450 g for 10 min, and the total and differential cell counts were determined from the pelleted cell fraction. The number of neutrophils in alveolar air spaces was quantified by morphometric analyses of histological lung sections as previously described.3 For morphometric examinations, investigators were blinded to the identities of the mice. BALF macrophage
BALF samples were collected as described above and resuspended in PBS-2% BSA. Cells were blocked with anti-CD16/32 antibody (1µg/million cells) for 5 min on ice, and then stained with FITC conjugated anti-F4/80 antibody (1:100) and PE conjugated anti-CD11b antibody (1:100) for 30 min on ice. After washed twice with PBS, cells were resuspended in PBS. The amount of exudate macrophages, resident macrophages, and neutrophils were analyzed using a Becton-Dickinson FACSCanto II flow cytometer and FACSDiva software (Fig. S8). BALF cytokine and chemokine levels and total protein levels
BALF samples were obtained from mice at 24h after E.coli challenge. BAL was done with 1 ml of cold PBS/15mM EDTA flushed back and forth three times. The levels of TNF-α, IL-1β, IL-6, MIP-2, and KC in the BALF were measured with ELISA kits following a protocol provided by the manufacturer (R&D Systems). Protein concentration was measured in the BALF using the Bio-Rad protein assay reagent. The standard curve was constructed using BSA.4 In vitro cytokine and chemokine release by alveolar macrophages
BALF samples were collected from unchallenged WT and PTEN-knockout mice. Cells were cultured in RPMI 1640-10% FBS medium for 2 hours at density of 105 cells/mL. Unattached cells (Less than 5%) were removed by washing twice before addition of LPS (Escherichia coli 055:B5; Sigma-Aldrich) at the final concentration of 1µg/ml. Microscopy examination confirmed that more that 95% attached cells were morphologically mature macrophages. Supernatants were collected at indicated times and secreted cytokines and chemokines were measured by ELISA kits (R&D Systems) as described above. IL-1 levels were too low to be detected under our experimental condition. In situ detection of apoptosis
Lung sections were stained using a TACS™ TDT Kit by following the protocol provided by the manufacturer (R&D Systems). Neutrophils were recognized by their characteristic donut-shaped nucleus and the relatively small size. Cell death was determined as the ratio of dead (TUNEL-positive) cells to total cell number. At least 200 cells were counted for each data point. Neutrophil apoptosis in inflamed peritoneal cavity
Peritonitis was induced by intraperitoneal injection of 3% thioglycollate (TG) solution as previously described.5 Peritoneal lavage fluids were collected from wild-type and PTEN-knockout mice 5 hr after TG injection. Peritoneal neutrophils were separated by centrifugation over a two-layer Percoll gradient (80%/57%). Purified neutrophils were identified by Wright-Giemsa staining (Donut-shape, segmented nucleus). We routinely obtain 5–10 million cells per mouse, and >95% of them are morphologically mature neutrophils. Purified neutrophils were labeled with intracellular fluorescent dye 5- (and -6)-carboxyfluorescein diacetate succinimidyl esters (CFSE, final concentration 5 µM) or 5- (and -6)-chloromethyl SNARF-1 acetate (SNARF-1, final concentration 5 µM). Cultured neutrophils labeled with CFSE or SNARF-1 did not have altered lifespans (data not shown). Labeled cells were mixed (1:1) and then injected intraperitoneally (106 cells in 200 µl PBS) into wild-type mice that had been challenged with 1 ml 3% TG for 2.5 hr. The concentration of transplanted neutrophils left in the peritoneal cavity was analyzed 15 hr after the injection. Relative apoptosis of wild-type and PTEN-null neutrophils was calculated as the ratio of indicated populations in the peritoneal cavity (Fig. 2C). Bacterial burden
Lungs were homogenized with 10 ml PBS. Lung homogenates were then serially diluted in ice-cold sterile PBS, and aliquots were spread on Luria broth (LB) agar plates. After overnight incubation at 37°C, colonies were counted, and bacterial viability was expressed as colony-forming units (cfu) per lung.6 In vitro bactericidal assay
Mouse bone marrow was isolated from the femur and the tibia. Bone marrow neutrophils are separated by centrifugation over a three-layer Percoll gradient (78%/69%/52%) as previously described.2 We routinely obtain 4–5 million cells per mouse, and >90% of them are morphologically mature neutrophils. Live E. coli particles (strain 19138; American Type Culture Collection, 12.5 ×105 cfu) were opsonized with mouse serum (final concentration 12.5%) at 37°C for 30 min and then incubated with neutrophils (2.5 × 105 cfu) in HBSS (without mouse serum) at 37°C for 1 hour. Samples were then serially diluted and spread on LB agar plates. The number of live E. coli cells in each sample was 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 neutrophils were primed with 100ng/mL PMA (sigma) at 37°C for 15 min before added to serum-opsonized live E. coli particles (as described above) at a ratio of 1:10, and incubated at 37°C for 1 hour. Gentamicin was then added at the final concentration of 100µg/ml to kill extracellular bacteria. After additional 1 hour incubation, cells were washed twice with HBSS and lysed with 1ml sterile water at room temperature. Samples were serially diluted and spread on LB agar plates. Colonies were counted after overnight incubation at 37°C. Phagocytosis assay
Fluorescein-conjugated E. coli and Zymosan bioparticles (Molecular Probes) were reconstituted in HBSS and opsonized with 12.5% mouse serum at 37°C for 30min. Mouse bone marrow neutrophils were primed with 100ng/mL PMA(sigma) at 37°C for 15 min, then added to serum-opsonized bioparticles at a ratio of 1:10 (neutrophils : bioparticles), and incubated at 37°C for 1 hour. Negative controls were incubated on ice for 1 hour. The assays were terminated by cooling the cells on ice. The fluorescence of extracellular particles was quenched by addition of 100 µL of 0.25 mg/mL trypan blue. After 1 minute incubation at room temperature, cells were washed and resuspended in ice-cold HBSS. The number of internalized particles was counted under a fluorescence microscope (Olympus lX17, 100× objective). Phagocytosis efficiency was expressed as the number of the internalized particles per 100 neutrophils (Phagocytosis index). Binding efficiency was expressed as the number of the binding particles per 100 neutrophils (Binding index). More than 100 cells were counted from random fields per coverslip for each group. Latex beads (1.3 um, Bangs Laboratories Inc.) were used to study the fusion of lysosome with phagosome as described by Huynh et al.7 Briefly, bone marrow derived neutrophils were incubated with mouse serum opsonized beads (neutrophil:bead = 1:10) at 37°C for 1 hour in serum-free DMEM. Cells were washed twice with PBS, labeled with LysoTracker Green DND- 26 (Molecular Probes) at 37°C for 15 min, and then examined under fluorescence microscope (Olympus lX17, 100X objective). Fusion of phagosome with lysosome was expressed as percentage of LysoTracker-positive phagosomes relative to the total amount of phagosomes (the number of engulfed Latex beads). Superoxide production during phagocytosis
Superoxide production was measured essentially as described by Subramanian et al.1 Briefly, Serumopsonized E. coli or Zymosan bioparticles (Molecular Probes) were mixed with luminal (final concentration: 50µM), SOD (80U/ml), and catalase (2000U/ml) on ice. Right before starting the recording on the luminometer, bone marrow derived wt and PTEN null neutrophils were added to each well and luminiscence was recorded at fixed time intervals for 1h. Myeloperoxidase activity assay
Myeloperoxidase (MPO) activity, an indicator of tissue neutrophil accumulation, was determined using an EnzChek® Myeloperoxidase (MPO) Activity Assay Kit (Invitrogen). Briefly, lungs were cut out, washed with sterile saline, blotted to dry, frozen in liquid nitrogen, and stored at -80°C. The frozen tissue was ground in liquid nitrogen and homogenized in 1 ml of ice-cold 50-mM potassium phosphate buffer, pH 6.0, containing 0.5% hexadecyltrimethylammonium bromide. Homogenates were centrifuged at 12,000 × g for 15 min at 4°C. The supernatants were harvested and assayed for MPO activity according to the manufacture’s instructions. Total protein concentration was measured using Bio-Rad protein assay reagent with a standard curve constructed using BSA. MPO activity was standardized to protein concentration. Pulmonary mechanics
Mice were anesthetized with pentobarbitol (50 mg/kg), and instruments were placed for measuring pulmonary mechanics according to a protocol provided by the manufacturer (BUXCO Electronics, Troy, NY). Mice were tracheostomized, intubated, and mechanically ventilated at a tidal volume of 0.2 ml and a frequency of 150 breaths/min as previously described (48). Baseline lung resistance (RL) and responses to aerosolized saline (0.9% NaCl) were first measured, followed by responses to increasing doses of aerosolized acetyl-b-methylcholine chloride (methacholine; MCh) (Sigma-Aldrich, St. Louis, MO) (0.32 to 40 mg/ml). The pulmonary resistance and compliance were calculated using BioSystem XA software (BUXCO Electronics, Troy, NY). DISCUSSION Neutropenia and related lung infection are the most important dose-limiting toxicities in anti-cancer chemotherapy and radiotherapy. One obvious strategy for treating such infections is to give broadspectrum antibiotics to neutropenic patients. However, not all patients respond to antibiotic treatment. In addition, this method increases the risk of inducing antibiotic resistance. In fact, current guidelines for preventing neutropenia-related pneumonia advise against the use of prophylactic antibiotics because they do not decrease the mortality rate and they increase the potential of developing antibiotic resistance.8,9 An alternative approach is G-CSF treatment.10 G-CSF is a hematopoietic cytokine produced by monocytes, fibroblasts, and endothelial cells. It has several functions in normal, steadystate hematopoiesis, such as regulating neutrophil production and release from the bone marrow, neutrophil progenitor proliferation and differentiation, and the functional activation of neutrophils. It is now used clinically to restore the neutrophil number in patients with neutropenia-related pneumonia by stimulating the bone marrow to produce more neutrophils. G-CSF therapy does not have the disadvantages associated with antibiotic resistance and has been shown to reduce hospitalization and improve quality of life. However, this therapy often does not work before the bone marrow is recovered, and it has side-effects such as bone pain, headache, fatigue, and nausea.10–13 Thus, there is a great need for developing new treatments for neutropenia-related pneumonia. PtdIns(3,4,5)P3 is an essential cellular signaling molecule, and its level is regulated by phosphatidylinositol 3′-kinases (PI3 kinase or PI3K),14,15 the tumor suppressor PTEN,16,17 and SHIP (SH2-containing inositol 5′-phosphatase, a phosphatidylinositol 5′-phosphatase, which converts PtdIns3,4,5P3 to PtdIns3,4P2)17,18. In this study, we elevated PtdIns(3,4,5)P3 signaling by disrupting PTEN. However, other ways of elevating PtdIns(3,4,5)P3 signaling, such as disrupting SHIP or over-expressing constitutively active PI3 kinase, might also alleviate neutropenia-related pneumonia. In addition, the strength of PtdIns(3,4,5)P3 signaling can be regulated by mechanisms independent of its level in the plasma membrane. PtdIns(3,4,5)P3 exerts its function by mediating protein translocation by binding to its PH domains. This membrane translocation is crucial for those proteins to fulfill their functions in PtdIns(3,4,5)P3-mediated cellular processes, such as cell survival, proliferation, growth, differentiation, polarization, chemotaxis, cytoskeletal rearrangement, and membrane trafficking.19–21 PH-domain membrane translocation was previously thought to depend solely on concentrations of PtdIns(3,4,5)P3 in the membrane.16,22 We recently demonstrated that two inositol phosphates, InsP7 and Ins(1,3,4,5)P4, compete for PH domain binding with PtdIns(3,4,5)P3 both in vitro and in vivo, providing another level of regulation for PH domain membrane translocation.23 In neutrophils, Ins(1,3,4,5)P4 is the major negative regulator, and its level was mainly regulated by InsP3 kinase B (InsP3KB).5 PtdIns(3,4,5)P3 signaling is much enhanced in InsP3KB-null neutrophils. These neutrophils exhibit enhanced superoxide production, chemoattractant-induced polarization, and chemotaxis. Their recruitment to inflamed peritoneal cavity is also elevated.5 It will be intriguing to see whether elevating PtdIns(3,4,5)P3 signaling by disrupting InsP3KB also enhances neutrophil function and thus reduces the severity of lung inflammation in neutropenia-associated pneumonia. One caveat of using PtdIns(3,4,5)P3 signaling activators as immune enhancers in neutropeniaassociated pneumonia is that hyperactivation of PtdIns(3,4,5)P3 signaling might also induce cancer. Nevertheless, induction of cancer after elevated PtdIns(3,4,5)P3 signaling is a progressive process and usually takes several months or even years. For example, in myeloid-specific PTEN-knockout mice, we did not find any tumors until 3 months after birth (unpublished data). It also took several months to develop leukemia in PTEN fl/fl Mx-Cre mice.24,25 When used as an immune enhancer, PtdIns(3,4,5)P3 pathway activation will only be given for several days; thus it is unlikely that this type 10 of treatment will lead to tumorigenesis. More studies are needed to determine the minimal and maximal dosages for effective treatment. REFERENCES 1. Zhu D, Hattori H, Jo H, et al. Deactivation of phosphatidylinositol 3,4,5-trisphosphate/Akt signaling mediates neutrophil spontaneous death. Proc Natl Acad Sci U S A. 2006;103:14836–14841.
2. Subramanian KK, Jia Y, Zhu D, et al. Tumor suppressor PTEN is a physiologic suppressor of chemoattractant-mediated neutrophil functions. Blood. 2007;109:4028–4037.
3. Mizgerd JP, Peschon JJ, Doerschuk CM. Roles of Tumor Necrosis Factor Receptor Signaling during Murine Escherichia coli Pneumonia. Am J Respir Cell Mol Biol. 2000;22:85–91.
4. Su X, Johansen M, Looney MR, Brown EJ, Matthay MA. CD47 deficiency protects mice from lipopolysaccharide-induced acute lung injury and Escherichia coli pneumonia. J Immunol. 2008;180:6947–6953.
5. Jia Y, Subrahmanyam KK, Erneux C, et al. Inositol 1,3,4,5-tetrakisphosphate negatively regulates PtdIns(3,4,5)P3 signaling in neutrophils. Immunity. 2007;27:453–467.
6. Aujla SJ, Chan YR, Zheng M, et al. IL-22 mediates mucosal host defense against Gramnegative bacterial pneumonia. Nat Med. 2008;14:275–281.
7. Huynh KK, Eskelinen EL, Scott CC, Malevanets A, Saftig P, Grinstein S. LAMP proteins are required for fusion of lysosomes with phagosomes. Embo J. 2007;26:313–324.
8. Lo N, Cullen M. Antibiotic prophylaxis in chemotherapy-induced neutropenia: time to reconsider. Hematol Oncol. 2006;24:120–125.
9. Leibovici L, Paul M, Cullen M, et al. Antibiotic prophylaxis in neutropenic patients: new evidence, practical decisions. Cancer. 2006;107:1743–1751.
10. Anderlini P, Przepiorka D, Champlin R, Korbling M. Biologic and clinical effects of granulocyte colony-stimulating factor in normal individuals. Blood. 1996;88:2819–2825.
11. Joos L, Tamm M. Breakdown of pulmonary host defense in the immunocompromised host: cancer chemotherapy. Proc Am Thorac Soc. 2005;2:445–448.
12. Viscoli C, Varnier O, Machetti M. Infections in patients with febrile neutropenia: epidemiology, microbiology, and risk stratification. Clin Infect Dis. 2005;40 Suppl 4:S240–245.
13. Leung AN, Gosselin MV, Napper CH, et al. Pulmonary infections after bone marrow transplantation: clinical and radiographic findings. Radiology. 1999;210:699–710.
14. Cantrell DA. Phosphoinositide 3-kinase signalling pathways. J Cell Sci. 2001;114:1439–1445.
15. Engelman JA, Luo J, Cantley LC. The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat Rev Genet. 2006;7:606–619.
16. Maehama T, Dixon JE. PTEN: a tumour suppressor that functions as a phospholipid phosphatase. Trends Cell Biol. 1999;9:125–128.
17. Di Cristofano A, Pandolfi PP. The multiple roles of PTEN in tumor suppression. Cell. 2000;100:387–390.
18. Rauh MJ, Kalesnikoff J, Hughes M, Sly L, Lam V, Krystal G. Role of Src homology 2- containing-inositol 5′-phosphatase (SHIP) in mast cells and macrophages. Biochem Soc Trans. 2003;31:286–291.
19. Rickert P, Weiner OD, Wang F, Bourne HR, Servant G. Leukocytes navigate by compass: roles of PI3Kgamma and its lipid products. Trends Cell Biol. 2000;10:466–473.
20. Hemmings BA. PtdIns(3,4,5)P3 gets its message across. Science. 1997;277:534.
21. Downward J. Mechanisms and consequences of activation of protein kinase B/Akt. Curr Opin Cell Biol. 1998;10:262–267.
22. Cantley LC. The phosphoinositide 3-kinase pathway. Science. 2002;296:1655–1657.
23. Luo HR, Huang YE, Chen JC, et al. Inositol pyrophosphates mediate chemotaxis in Dictyostelium via pleckstrin homology domain-PtdIns(3,4,5)P3 interactions. Cell. 2003;114:559–572.
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25. Yilmaz OH, Valdez R, Theisen BK, et al. Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature. 2006;441:475–482.
Files in this Data Supplement:
- Figure S1. Neutrophil recruitment during E. coli pneumonia (JPG, 102 KB)
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Mice were challenged with intratracheally instilled with E. coli for indicated time and their lungs were fixed with Bouin’s solution at 23 cm H2O pressure. Tissues were embedded in paraffin, and 6 µm thick sections were stained with hematoxylin and eosin (H&E). (A) Staining of histological lung sections shows emigrated neutrophils and polymerized fibrin in the pulmonary parenchyma. (B) Neutrophil recruitment was quantified as volume fraction of the alveolar air space (see Fig. 1C). Data are presented as mean ± SD, n ≥ 4 mice in each group. *P < 0.05, **P < 0.01 versus wild type.
- Figure S2. No significant differences were observed in the amount of circulating neutrophils between wild type and PTEN KO mice (JPG, 22.5 KB)
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Mice were infected by intratracheal instillation of 106 cfu E. coli and euthanized at indicated time points. Orbital peripheral blood (250 µl) was collected into K2-EDTA coated tubes. Complete blood count (CBC) and white blood cell count of each sample were analyzed using a Hemavet 850 hematology system. Data shown are means ± SD (n=3).
- Figure S3. Neutrophil recruitment and lung injury during LPS-induced pneumonia (JPG, 19.2 KB)
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Mice were challenged with intratracheally instilled LPS (5mg/kg body weight) for indicated time. (A) Neutrophils in bronchoalveolar lavage fluid (BALF). BAL was performed with PBS/15mM EDTA (10 × 1mL) and the total number of cells in the lungs was counted by hemocytometer. Differential cell counts were conducted on cytospin preparations stained with a modified Wright-Giemsa stain (Volu-Sol, Inc. Salt Lake City, UT). Neutrophils were recognized by their lobular or segmented nuclei. The percentage of pulmonary neutrophils in the whole population (%PMN) was determined accordingly. Total number of pulmonary neutrophils (#PMN) recruited was calculated as follows: #PMN = cell density × volume × %PMN. (B) BALF total protein. BAL was performed with 1mL of PBS/15mM EDTA flushed back and forth three times. Protein accumulated in the inflamed lung was measured using a Bio-Rad protein assay kit. The standard curve was constructed using BSA. Data are presented as mean ± SD, n ≥ 4 mice in each group. **P < 0.01 versus wild type.
- Figure S4. Apoptotic neutrophils were hardly detected in bronchoalveolar lavage fluids (BALF) (JPG, 59.2 KB)
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Neutrophil recruitment to the lungs was induced by intratracheal instillation of 106 cfu of E. coli. BALFs were collected from wild type and PTEN knockout mice 24 hours after the instillation. Apoptotic cells were detected by Annexin V-FITC and 7-AAD staining. Ten thousand cells were analyzed using a BD FACSCanto II flow cytometer and BD FACSDiva software. As shown in right panels, most BALF neutrophils are viable (double negative). Noticeably, at 24 hours after E. coli instillation, most cells in BALF are neutrophils.
- Figure S5. Mouse neutropenia was induced by intraperitoneal injection of cyclophosphamide (Cy) (JPG, 69.6 KB)
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As shown in the left panel, 200mg/kg/day Cy injection for 4 days was able to induce severe neutropenia. However, a comparable neutropenia can also be induced using a relatively low dose - two intraperitoneal injections at a total dose of 250 mg/kg (150 mg/kg on day 1 and 100 mg/kg on day 4) (right panel). On day 5, Cy-treated mice contained approximately 90% fewer circulating neutrophils than untreated or saline-treated group. The profound neutropenia persisted through days 6 and 7. These schemes can be used to induce neutropenia in both wild type and PTEN knockout mice. Body weight was also measured once a day for 6 days after Cy injection. Since induction with lower dose of Cy caused less severe weight loss, we used this scheme in all the experiments described in this study.
Additional supplemental figures can be found here.
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