NET formation in young and old trauma patients and volunteers.

<p>NET formation in PMN isolated from young healthy volunteers (n = 10, <b>A and E</b>), young trauma patients (n = 7, <b>B and F</b>), old healthy volunteers (n = 6, <b>C and G</b>), and old trauma patients (n = 5, <b>D and H</b>) was evaluated. <b>A-D</b>: PMN were immediately fixed after attachment. <b>E-H</b>: PMN were stimulated with 20 nM PMA for 3–4 h.</p

MtDNA-induced NET formation is independent of NADPH oxidase.

<p>PMN were pre-treated with 10 μM DPI for 1 h before the stimulation (B and D). Then either 20 nM PMA (A and B) or 50 μg/mL mtDNA (C and D) was applied for 3–4 h. NETs were detected as described in Materials and Methods. Experiments were repeated more than three times. Magnification x20.</p

Plasma mtDNA levels are higher in the elderly.

<p><b>A</b>. MtDNA concentration was assessed in plasma obtained from subjects from four groups: 1. Young healthy (n = 11, average age 23 years); 2. Elderly healthy (n = 7, average age 72 years); 3. Young trauma (n = 9, average age 37 years); and 4. Elderly trauma (n = 6, average age 83 years). All pairs showed significant difference except between old healthy vs. young trauma; p<0.001, One Way ANOVA, followed by Tukey’s Test. <b>B</b>. ISS is shown for young and older trauma patients listed in <b>A</b>. p = 0.164 (Student’s t-test).</p

MtDNA induces NET formation.

<p>Freshly isolated PMN from young volunteers were attached to coverslips for 1 h and treated with either medium (untreated) or with purified mtDNA (50 μg/mL) for 4 h. NET formation was detected as described in Materials and Methods. Experiments were repeated more than three times. Magnification x40.</p

WT mice and NE^−/−×CG^−/− mice were resistant to <i>Burkholderia cepacia</i> infection, whereas p47<i>^phox−/−</i> mice were highly susceptible.

<p>A) Kaplan-Meier survival curves in WT, p47<i>^phox</i>^−/− and NE^−/−×CG^−/− mice administered intraperitoneal <i>B. cepacia</i> (4×10⁷ CFUs/mouse). Log-rank analysis, p<0.0002 comparing WT with p47<i>^phox</i>^−/− mice and p<0.0002 comparing NE^−/−×CG^−/− mice with p47<i>^phox</i>^−/− mice. n = 10 mice per genotype. B) In separate experiments, mice (n = 5 per genotype) were administered the same inoculum of <i>B. cepacia</i>, and quantitative cultures were performed at 24 h. WT and NE^−/−×CG^−/− mice cleared infection, whereas bacterial infection persisted in the peritoneum and spleens of p47<i>^phox</i>^−/− mice. Circles, no growth. *, p<0.03; **, p<0.01.</p

Lung histology and airway inflammation in WT and NE^−/−×CG^−/− mice after <i>A. fumigatus</i> administration.

<p>Mice were administered <i>A. fumigatus</i> (1.25×10⁷ conidia per mouse) by oropharyngeal aspiration and sacrificed on day 3. A) BALF leukocyte recovery and B) percent lung inflammation were similar in WT and NE^−/−×CG^−/− mice. Representative lung histology from WT (C and D) and NE^−/−×CG^−/− mice (E and F). Predominantly peribronchovascular neutrophilic and lymphohistiocytic inflammation occurred in both genotypes (C and E; H&E, 40×). GMS staining (400×) of lung sections from WT (D) and NE^−/−×CG^−/− (F) mice showed what appeared to be degenerated hyphal fragments, but no evidence of intact invasive hyphae. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028149#s2" target="_blank">Results</a> are representative of 15 WT and 10 NE^−/−×CG^−/− mice. By comparison, p47<i>^phox−/−</i> mice administered <i>A. fumigatus</i> at 0.1% of this inoculum (1.25×10⁴ conidia per mouse) and sacrificed on day 3 had evidence of fungal pneumonia characterized by G) multiple foci of neutrophilic consolidation (H&E, 40×), and H) hyphal parenchymal invasion (arrow) (GMS, 400×).</p

Peritoneal and splenic granulocytic MDSCs from tumor-bearing mice suppress T cell proliferation independently of NADPH oxidase.

<p>Ly6G-enriched PECs and splenocytes from MOSEC-bearing WT and p47<i>^phox−/−</i> mice (day 90) were co-cultured with splenocytes from non-tumor-bearing WT mice (E∶T ratio: 1∶1). A) Ly6G-enriched PECs completely suppressed anti-CD3/B7.1-stimulated CD4⁺ and CD8⁺ T cell proliferation. PECs from 3 mice per genotype were evaluated. B) In Ly6G-enriched splenocytes, the majority of cells had a granulocytic morphology (arrows), and 86% of CD11b⁺ cells expressed granulocytic MDSC markers (Ly6G⁺Ly6C^low). C) Ly6G-enriched splenocytes from WT and p47<i>^phox−/−</i> mice modestly suppressed anti-CD3/B7.1-stimulated CD4⁺ and CD8⁺ T cell proliferation. N = 3 mice per genotype were used in this experiment, and results are representative of 3 experiments. Comparison between genotypes: p = NS.</p

Effect of NADPH oxidase in local and systemic accumulation of MDSCs in tumor-bearing mice.

<p>A) Representative quantification of MDSCs. Splenocytes from WT and p47<i>^phox−/−</i> mice at day 42 and 90 after MOSEC administration were analyzed for MDSC accumulation. Gating on myeloid (CD11b⁺) cells, the proportion of monocytic MDSCs (R1; Ly6C⁺Ly6G⁻) and granulocytic MDSCs (R2; Ly6G⁺Ly6C^Low) significantly increased at day 90 versus day 42. All gates were set based on isotypes. This approach was used to quantify MDSCs in PECs, lymph nodes, and spleens. B) Proportion of MDSCs in myeloid PECs on day 42 and 90. The proportion with granulocytic and monocytic MDSC markers was greater in advanced (day 90) versus early (day 42) stage tumor burden in both genotypes. C) In draining lymph nodes, there was a trend toward increased monocytic MDSC accumulation in p47<i>^phox−/−</i> versus WT mice at day 42 but not at day 90. There was no effect of NADPH oxidase on granulocytic MDSC accumulation at either time point. D) In spleens, there was an increased accumulation of MDSCs, particularly granulocytic MDSCs, in mice with advanced versus early disease, but no effect of mouse genotype. Data (± SEM) are from at least 3 mice per genotype per time point, and are representative of 3 separate experiments. Comparison between genotypes: p = NS.</p

to tumor progression requiring euthanasia is not altered by NADPH oxidase.

<p>Kaplan-Meier plots of WT and NADPH oxidase-deficient (p47<i>^phox−/−</i>) mice (10 mice/group) showed similar survival after i.p. MOSEC challenge (log-rank, p = 0.25).</p

Lung histology in WT and DPPI^−/− mice on day 3 after oropharyngeal <i>A. fumigatus</i> (1.25×10⁷ conidia per mouse) administration.

<p>In both WT (A) and DPPI^−/− (B) mice, mild predominantly peribronchovascular inflammation occurred (H&E, 100×). No evidence of invasive hyphae was present with GMS staining (400×) in either WT (C) or DPPI^−/− (D) mice. n = 5 mice per genotype.</p