MMP-9 Cleaves SP-D in a Dose- and Time-dependent Manner.

<p>(A) Dose and (B) time course digestions of SP-D by MMP-9. For the dose course (A), the reaction loaded onto lane 2 contained 62 ng/mL MMP-9 and the concentration was raised 3-fold serially to 5 µg/mL MMP-9 at lane 6. Reactions were incubated for 4 hours. For the time course (B), the length of digestion in minutes is listed above the lane. SP-D concentration was 20 µg/mL for both experiments, and MMP-9 concentration was 5 µg/mL for the time course. For the Native PAGE (C), lane 1 contains intact SP-D and lane 2 contains cleaved SP-D. An arrow indicates the band corresponding to MMP-9.</p

Cleaved SP-D Retains its Ability to Bind <i>E. coli</i> LPS.

<p>Examination of the ability of SP-D to bind to LPS-coated plates. In (A), the ELISA was performed using 2-fold serially diluted SP-D samples. In (B), 1 µg/mL intact or cleaved SP-D was analyzed in triplicate with MMP-9 as a control and NE-cleaved SP-D as a negative control. The average of PBST alone ran in triplicate was subtracted from all values. While intact and MMP-9-cleaved SP-D in PBST are significantly different from PBST alone (p≤0.001 and p<0.05, respectively) and NE-cleaved SP-D (p≤0.001 and p<0.05, respectively), MMP-9 along with intact and cleaved SP-D in PBST with maltose were not significantly different from PBST or PBST with maltose. For (B), error bars represent standard deviation.</p

Cleaved SP-D Fails to Increase Phagocytosis by MH-S Cells.

<p>Examination and quantitation of phagocytosis of <i>E. coli</i> by MH-S cells was performed using (A) a gentamicin protection assay and (B and C) flow cytometry. For (A), all conditions are significantly different (p<0.05) from 1 µg/mL intact SP-D. An asterisk (*) denotes p≤0.001 when compared to 1 µg/mL intact SP-D. Columns marked with # are significantly different when compared to 0.5 µg/mL intact SP-D (p<0.05). For (B), intact SP-D causes MH-S cells to have significantly higher mean fluorescence intensity (MFI) than all other conditions (p≤0.001). Cleaved SP-D is significantly different when compared to both MMP-9 and PBS controls (p≤0.001). For (C), flow data was gated on forward and side scatter to select for MH-S cells. For both (A) and (B), error bars represent standard deviation.</p

Determining if a bacterial mimetic (LPS) can cause the release of EVs in the lung – Nanosight imaging.

<p>Mice were challenged with the aerosolised vehicle of endotoxin-free saline or LPS (1 mg/ml) in Perspex chambers for 30 minutes. Animals were sacrificed and BALF obtained 6 hours after challenge. The samples were then centrifuged (900 g) to remove the white blood cells and debris. The presence of EVs was imaged using Nanosight technology.</p

Determining if a bacterial mimetic (LPS) can cause the release of EVs in the lung – Cytokine release.

<p>Mice were challenged with the aerosolised vehicle of endotoxin-free saline or LPS (1 mg/ml) in Perspex chambers for 30 minutes. Animals were sacrificed and BALF obtained 6 hours after challenge. The samples were centrifuged (900 g) to remove the white blood cells and debris, and then treated with vehicle (PBS) or ATP<i>γS</i> (10⁻³ M), and incubated for a further 4 hours and subsequent cytokine release was analysed by ELISA. Data shown as mean +/− S.E.M. (A: IL-1β, B: IL-18, C: IL-1α).</p

Determining whether the ATP/P2X₇ axis is central to the exacerbation response <i>in vivo.</i>

<p>Mice (n = 8 per group) were challenged with the aerosolised vehicle of endotoxin-free saline or LPS (1 mg/ml) in Perspex chambers for 30 minutes. Four hours later the mice were intranasally dosed with saline (2 ml/kg) or ATPγs (0.001 mg/kg) whilst under light anaesthesia (4% isoflurane in oxygen). The mice received oral vehicle or P2X₇ inhibitor, A438079, 30 minutes prior to the ATP challenge, 4 hours after the challenge and 1 hour prior to cull. Twenty four hours after the LPS exposure the mice were culled and lavaged. IL-1β (A) and neutrophil (B) numbers were measured in the BALF. Data shown as mean +/− S.E.M. An unpaired T-test was used for the statistical analysis. *  = P = 0.0378 (Panel A); *  = P = 0.0162 (Panel B).</p

Demonstration that LPS-induced release of EVs can enhance IL-1β and neutrophil levels and change disease phenotype in model known to have increased levels of ATP.

<p>Mice (n = 8 per treatment group) were exposed to either room air (control) or CS (3R4F cigarettes) using a negative pressure system. Mice were subjected to 2 periods of CS exposure (500 ml/minute) per day (4 hours apart) for 3 consecutive days. On the morning of the third challenge day, the mice were exposed to aerosolised vehicle of endotoxin free saline or LPS (1 mg/ml) in Perspex chambers for 30 minutes. Animals were culled and BALF and lung tissue samples were collected 24 hours after LPS treatment. IL-1β levels were measured in the BALF and neutrophil numbers were determined in the BALF and lung tissue. In separate BALF samples collected from parallel smoke or LPS driven challenges ATP levels were measured (Panel A). Data shown as mean +/− S.E.M. (A: ATP #  = P = 0.0023, Mann-Whitney; B: IL-1β #  = P = 0.0009, Mann-Whitney; C: BALF neutrophil number, #  = P = 0.0431, Students T test; D: lung tissue neutrophil number; #  = P = 0.0006, Mann-Whitney).</p

Determining if a bacterial mimetic (LPS) can cause the release of EVs in the lung – Signalling.

<p>Mice (n = 6 per group) were challenged with the aerosolised vehicle of endotoxin-free saline or LPS (1 mg/ml) in Perspex chambers for 30 minutes. Animals were sacrificed and BALF obtained 6 hours after challenge. The samples were then centrifuged (900 g) to remove the white blood cells and then pre-treated with inhibitors (P2X₇ antagonist A 438079 (10⁻⁶ M) or caspase-1 inhibitor VX 765 (10⁻⁷ M)) and incubated for 1 hour. Samples were then treated with vehicle (PBS) or ATP<i>γS</i> (10⁻³ M), and incubated for a further 4 hours and subsequent cytokine release was analysed by ELISA. Data shown as mean +/− S.E.M. (A: IL-1β, B: TNFα). *  = P = 0.0138 (One way ANOVA followed by a Bonferroni's Multiple Comparison test).</p

Demonstration of the concept that a bacterial mimetic insult can cause EV release.

<p>THP-1 cells were treated with RPMI (vehicle) or LPS (0.1 µM) and incubated overnight and samples were collected and centrifuged to remove the cells. The _supernatants were collected and split into two equal fractions: non-ultracentrifuged (EV-rich – left side) and ultracentrifuged (EV-_{deficient – right side}). The samples were pre-treated with vehicle (DMSO, 0.1%, V/V) or P2X₇ antagonist (AZ 11645373; 10⁻⁷ M). Samples were incubated for one hour and then treated with vehicle (PBS) or exogenous ATP<i>γS</i> (10⁻³ M). The samples were then incubated for a further 4 hours prior to ELISA assessment for cytokines (A: IL-1β, B: IL-18, C: TNFα, D: MMP-9). The data is shown as mean +/− S.E.M.</p

Human translation data: exogenous ATP increases IL-1β/IL-18 level in samples collected from LPS challenged healthy subjects.

<p>Healthy subjects were challenged with inhaled LPS and BALF was collected 6(10⁻³ M) and incubated for 4 hours; cytokine release was analysed by ELISA. Panel A shows the paired IL-1β data. Panel B, C and D represents the levels of IL-1β, IL-18 and TNFα, respectively. Data shown as mean +/− S.E.M. Statistical analysis using a paired T-test.</p