Depletion of NSPs abrogated estrogen-mediated promotion of protease activity in splenocytes.

<p>(A) Western blot analysis of NE, PR3, MPO, and CC/DPPI proteins in the splenocytes of placebo and estrogen-treated wild type (WT) and NSP^-/- knock out B6 mice. β-actin was probed as protein loading control. (B) Analysis of protease and elastase activity in splenocytes from placebo- and estrogen-treated WT and NSP^-/- mice with EnZCheck elastase assay kit. The graph shows the mean ± SEM (n≥4). Unpaired student <i>t</i> tests (placebo <i>vs</i> estrogen) and paired student <i>t</i> test (WT/estrogen; without inhibitor <i>vs</i> with inhibitor) were preformed; ***, <i>p</i> < 0.001; and ns, not significant.</p

The protease and elastase activities are increased in splenocytes from estrogen-treated mice when compared to placebo controls.

<p>(A) The total protease activity in freshly isolated splenocytes from placebo- and estrogen-treated B6 mice was determined by EnzChek Protease Assay kit. The graph shows the mean ± SEM (n≥7). (B) The chymotrypsin-like serine protease activity in whole living splenocytes was detected with FLISP^™ serine protease detection kit. Freshly isolated splenocytes were stained with FSFCK (upper left panel) or FSLCK (upper right panel) FLISP reagent, which detected chymotrypsin-like serine proteases favoring phenylalanine and leucine, respectively. The FAM signal intensity in living splenocytes (gated on propidium iodide (PI) negative cells) was analyzed by flow cytometry. The representative flow scatter plots are shown. The table in the lower panel shows the FAM value (mean ± SEM) summarized from the Flow data (n≥6). (C) The elastase activity in freshly isolated splenocytes from placebo- and estrogen-treated B6 mice was determined by EnZCheck elastase assay kit with or without the addition of inhibitor in the reaction. Mean activity ± SEM (n≥4) is shown. (D) The graph demonstrates that the activity of purified porcine pancreatic elastase was completely blocked by a selective inhibitor, N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone at 100 μM concentration. Either unpaired student <i>t</i> test (placebo <i>vs</i> estrogen) or paired student <i>t</i> test (without inhibitor <i>vs</i> with inhibitor) were performed; *, <i>p</i><0.05, ** <i>p</i><0.01, and ***, <i>p</i> <0.001.</p

NSP expression levels are upregulated in the splenocytes from estrogen-treated B6 mice when compared to placebo controls.

<p>(A) Real-time RT-PCR analysis of the relative mRNA expression levels of NE, PR3, and CG in the splenocytes from placebo- and estrogen-treated B6 mice. The graph represents the means ± SEMs (n ≥ 4). Student <i>t</i> tests (placebo <i>vs</i> estrogen) were preformed; *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; and ***, <i>p</i> < 0.001. (B and C) Western blot analysis of NE and PR3 (B), CC/DPPI (C) protein expression levels in the whole splenocyte extracts from placebo- and estrogen-treated mice. β-actin was probed as a protein loading control.</p

Neutrophil percentages are increased in the splenocytes of three different strains of spontaneous lupus-prone mice.

<p>(A-C) The flow cytometric analysis of neutrophils (CD11b⁺GR1⁺) in the splenocytes from MRL-<i>lpr (A)</i>, B6-<i>lpr(B)</i>, NZB/W_F1 (C) lupus mice and their respective control MRL, B6, and NZW mice. Representative FACS plots are shown. The graphs show the summary of flow analysis of neutrophil percentage in splenocytes of different strains of lupus mice (means ± SEMs, n ≥4). (D-F) Real-time RT-PCR analysis of the relative expression levels of NE, PR3, CG, and MPO in the splenocytes from MRL-<i>lpr (D)</i>, B6-<i>lpr (E)</i>, NZB/W_F1 (F) lupus mice and their respective control MRL, B6, and NZW mice. The graphs show means ± SEM (n ≥4). Unpaired student <i>t</i> test (MRL <i>vs</i> MRL-<i>lpr</i>; and B6 <i>vs</i> B6-<i>lpr</i>, NZW <i>vs</i> NZB/W_F1); *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; and ***, <i>p</i> < 0.001. (G) Western blot analysis of NE, PR3, and MPO protein expression in splenocytes from MRL-<i>lpr</i>, B6-<i>lpr</i>, NZBW_F1, and their respective control MRL, B6, and NZW. β-actin was probed as protein loading control.</p

Depletion of neutrophils <i>in vitro</i> from splenocytes has limited effect on LPS-induced inflammatory responses in splenocytes.

<p>The splenocytes from placebo-and estrogen-treated mice were treated with anti-mouse Ly6G, or anti-mouse Gr-1 antibody and magnetic beads to deplete neutrophils. The No Ab control received no any specific antibody treatment. (A-C) The graphs summarize the flow cytometric analysis data of the percentage of CD11b⁺Ly6G⁺, CD4⁺ and CD19⁺ cells in the splenocytes after neutrophil depletion. (D) Real-time RT-PCR analysis of the relative mRNA expression levels of NE, PR3, and CG in the splenocytes from estrogen-treated B6 mice after neutrophil depletion. The graph represents the means ± SEMs (n = 2). (E-G) The neutrophil-depleted splenocytes from placebo- and estrogen-treated B6 mice were stimulated with LPS for 24h, and then supernatant were collected to analyze the production of IFNγ, IL-6, and MCP-1 by ELISA. The graphs (A-C and E-G) show means ± SEM (n = 2 for placebo with anti-Gr-1 treatment; n≥4 for the other treatment groups). Paired student <i>t</i> test (No Ab control <i>vs</i> anti-Ly6G or anti-Gr-1); *, <i>p</i> < 0.05; **, <i>p</i> <0.01; and ***, <i>p</i> < 0.001.</p

Depletion of NSPs has no obvious effect on estrogen-mediated promotion of inflammatory molecules and neutrophils.

<p>The splenocytes from placebo- and estrogen-treated wild type (WT) and NSP^-/- knock out mice were stimulated with LPS for the indicated times. The culture supernatant was collected to measure cytokines IFNγ (A), IL-1β (B), IL-6 (C), IL-10 (D), TNFα (E), chemokine MCP-1 (F), and inflammatory molecule NO (G). The expression level of iNOS protein in LPS activated splenocytes (24hr) was measured by Western blotting (H). The graphs show means ± SEMs (n = 3 for wild type B6 mice and n = 5 for NSP^-/- mice). Unpaired student <i>t</i> test (placebo <i>vs</i> estrogen); *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; and ***, <i>p</i> < 0.001.</p

Estrogen treatment increases neutrophil percentages in wild type B6 mice.

<p>(A-C) Flow cytometry analysis. Red blood cell-depleted whole splenocytes (A), peripheral blood (B), and bone marrow (C) cells from placebo- and estrogen-treated mice were stained with neutrophil surface markers FITC conjugated anti-Gr1 and APC conjugated anti-CD11b antibodies. Shown are the representative FACS plots from at least three independent experiments accompanied by the summary graphs showing the percentages (means ± SEMs) of neutrophils in the spleen, blood, and bone marrow cells from placebo- and estrogen-treated mice (n≥4). (D) The total splenocyte count in placebo- and estrogen-treated mice. (E) The total CD11b⁺GR1⁺ splenic neutrophil count in placebo- and estrogen-treated mice. (F) Real-time RT-PCR analysis of the expression of MPO in splenocytes from placebo- and estrogen-treated mice. The graphs show means ± SEMs (n≥4). Unpaired student <i>t</i> tests (placebo <i>vs</i> estrogen) were performed. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; and ***, <i>p</i> < 0.001. (G) Western blot analysis of MPO protein expression levels in the splenocytes from placebo- and estrogen-treated mice. β-actin was probed as protein loading control.</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

Fungal burden 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) Quantitative fungal cultures in lung homogenates, B) serum galactomannan, C) BALF galactomannan. n = 10 mice per genotype subjected to infection (A. fum), and n = 1 mouse per genotype subjected to sham-infection. No significant differences occurred in quantitative lung fungal cultures, serum galactomannan, and BALF galactomannan between <i>Aspergillus</i>-infected WT mice and NE^−/−×CG^−/− mice.</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