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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
Files in this Data Supplement:
- Figure S6. Disruption of PTEN enhances pulmonary neutrophil accumulation and reduces bacterial burden during bacterail pneumonia in irradiation (IR)-induced neutropenic mice (JPG, 96.5 KB)
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(A) PTEN depletion does not affect the amount of circulating neutrophils. The complete blood count (CBC) and white blood cell count of each sample were analyzed using a Hemavet 850 hematology system. (B) Histologic analysis of lungs reveals bacterial colonies and polymerized fibrin in the pulmonary parenchyma. Lungs were fixed with Bouin’s solution, and 6-µm sections were stained with H&E. (C) Total numbers of neutrophils in BALF were quantified as described in Fig. 1A. (D) Bacterial killing in inflamed lungs. Live bacteria in lung homogenates were assessed using the colony assay as described in Fig. 3B-C. (E) 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. All data are presented as mean ± SD, n ≥ 4 mice in each group. *P < 0.05, **P < 0.01, ***P < 0.001 versus wild type.
- Figure S7. Recruitment of adoptively transferred neutrophils to the inflamed lungs is independent of the method for neutrophil staining (JPG, 53.2 KB)
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(A) Wild-type neutrophils were labeled with CFSE or SNARF-1. Labeled neutrophils were mixed (about 1:1) and then injected intravenously (via tail vein) into wild-type neutropenic recipient mice that have been challenged with 105 cfu of E. coli for 2.5 hr (see Fig. 5). BAL fluids were harvested 2.5 hr after the injection of cell mixture. The amount of adoptively transferred neutrophils recruited to the lungs was analyzed using a BD FACSCanto II flow cytometer and BD FACSDiva software. (B) Relative recruitment of neutrophil was calculated as the ratio of CFSE+ to Snarf-1+ cells in the BALF. Data shown are mean ± SD of n = 3 mice. No significant difference was observed between CFSE and SNARF-1 labeled neutrophils in their ability to accumulate in the inflamed lungs (the ratio remained to be “1” in the BALF).
- Figure S8. Alveolar macrophages (JPG, 97.1 KB)
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Wild-type and PTEN-knockout mice were anesthetized and intratracheally instilled with 1 × 106 cfu of E. coli. Bronchoalveolar lavage (BAL) was performed with PBS/15mM EDTA (10 × 1mL) in each group. BALF total cells were first 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. Differentiation of BALF leukocytes by FACS analysis was based on the different expression of surface markers. Exudate macrophages were recognized as CD11b+F4/80+ cells; resident macrophages were recognized as CD11b-F4/80+ cells; and neutrophils were recognized as CD11b+F4/80− cells.
- Figure S9. Pneumonia associated bronchial hyperreactivity to methacholine is reduced in myeloid-specific PTEN knockout mice (JPG, 48.2 KB)
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Methacholine, a synthetic choline ester that acts as a non-selective muscarinic receptor agonist, can provoke narrowing of the airways (bronchoconstriction) and thus decrease pulmonary compliance and increase the resistance. Changes in airway resistance and dynamic compliance were measured in response to increasing doses of methacholine as described in Fig. 7. Results are expressed as mean ±SD (n ≥ 4 mice in each group). *P < 0.05 versus baseline values of airway resistance and dynamic compliance. Treatment with cyclophosphamide alone (without lung infection) did not induce airway hypersensitivity in response to increasing doses of methacholine in wild type or PTEN-KO mice. Although bronchial hyperreactivity to methacholine is not a typical symptom of human bacterial pneumonia, mice with severe lung inflammation have elevated sensitivity and react to lower doses of inhaled methacholine. In PTENnull mice, such hyperreactivity is drastically reduced, which is consistent with the alleviated lung inflammation and damage in these mice.
- Figure S10. Disruption of PTEN enhances pulmonary neutrophil accumulation and acute injury in LPS-induced pneumonia in neutropenic mice (JPG, 89.5 KB)
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Age- and sex-matched wt and PTEN-knockout mice were pretreated with Cy (250mg/kg) as described in the “Materials and Methods” and then challenged with LPS (5mg/kg) for indicated time. (A) PTEN depletion increased neutrophil recruitment. Bronchoalveolar lavage was performed with PBS/15 mM EDTA (10 × 1mL) 24h after LPS challenge. Total numbers of neutrophils in BALF were quantified as described in Fig. 1A. (B) Histologic analysis of lungs reveals polymerized fibrin in the pulmonary parenchyma. Lungs were fixed with Bouin’s solution, and 6-µm sections were stained with H&E. (C) 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. All data are presented as mean ± SD, n ≥ 4 mice in each group. *P < 0.05 versus wild type. (D) The survival rate of neutropenic mice. The difference in survival was not statistically significant (P >0.05 by log-rank test).
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