Patient-Derived Airway Secretion Dissociation Technique To Isolate and Concentrate Immune Cells Using Closed-Loop Inertial Microfluidics

Assessment of airway secretion cells, both for research and clinical purposes, is a highly desired goal in patients with acute and chronic pulmonary diseases. However, lack of proper cell isolation and enrichment techniques hinder downstream evaluation and characterization of cells found in airway secretions. Here, we demonstrate a novel enrichment method to capture immune-related cells from clinical airway secretions using closed-loop separation of spiral inertial microfluidics (C-sep). By recirculating the output focusing stream back to the input reservoir and running continuously with a high flow processing rate, one can achieve optimal concentration, recovery and purity of airway immune cells from a large volume of diluent, which was not readily possible in the single-pass operation. Our method reproducibly recovers 94.0% of polymorphonuclear leukocytes (PMNs), with up to 10⁵ PMNs in clear diluted buffer from 50 μL of airway secretions obtained from mechanically ventilated patients. We show that C-sep isolated PMNs show higher neutrophil elastase (NE) release following activation by phorbol 12-myristate 13-acetate (PMA) than cells isolated by conventional mucolytic method. By capturing cells without chemically disrupting their potential function, our method is expected to expand the possibility of clinical <i>in vitro</i> cell based biological assays for various pulmonary diseases such as acute respiratory distress syndrome, pneumonia, cystic fibrosis, and bronchiectasis

Duffy mediated chemokine endocytosis in DIH cells is caveolin-1, clathrin, flotillin-1 independent.

<p>DIH cells were transfected with SiRNA targeted against caveolin-1 (A&B), flotillin-1(C&D), clathrin (E, F &G), and control scramble sequence RNA (ScRNA). At 72 hour post-transfection, endocytosis experiments were performed and cells lysates were analysed by SDS-PAGES and immunoblotted with caveolin-1, clathrin, flotillin-1 and β-actin antisera. (E) DIH cells transfected with SiCLAC or control SiRNA and were incubated with ¹²⁵I-transferrin for various time points then were washed with binding buffer or acid wash buffer, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029624#s4" target="_blank">Materials and Methods</a>. The mean ± SEM from three independent experiments is shown.</p

CXCL1 endocytosis is CtBP1 and PAK1 independent.

<p>DIH cells were transfected with SiRNA targeted against CtBP1 (A) or PAK1 (B) and control scramble sequence RNA (ScRNA). At 72 hour post-transfection, endocytosis experiment was performed and cells lysates were analysed by SDS-PAGES and immunoblotted with CtBP1, PAK1 and β-actin antisera. The mean ± SEM from three independent experiments is shown.</p

Silencing of Dynamin II expression partially inhibited chemokine endocytosis.

<p>(A) DIH cells were pretreated with dynasore prior to incubation with ¹²⁵I-CXCL1/GRO-α for the specified time points, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029624#s4" target="_blank">Materials and Methods</a>. Dynasore inhibited CXCL1 internalization in DIH cells. * <i>p</i><0.05 dynasore treatment <i>vs</i> control, paired t test. (B) DIH cells were transfected with SiRNA targeted against dynamin II and control SiRNA (ScRNA). At 72 hour post-transfection, endocytosis experiments were performed and cells lysates were analysed by SDS-PAGES and immunoblotted with dynamin II and β-actin antisera. The mean ± SEM from three independent experiments is shown.</p

Cholesterol is required for CXCL1 endocytosis in DIH cells.

<p>(A) DIH cells were pre-treated with 2-hydroxypropyl-β-Cyclodextrin prior to incubation with ¹²⁵I -CXCL1/GRO-α for various time points, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029624#s4" target="_blank">Materials and Methods</a>. 2-hydroxypropyl-β-cyclodextrin showed inhibition of chemokine internalization that could be reversed with cholesterol repletion. * <i>p</i><0.01, 10 mM 2-hydroxypropyl-β-Cyclodextrin treatment <i>vs</i> control, two way ANOVA. ** <i>p</i><0.05 10 mM 2-hydroxypropyl-β-Cyclodextrin treatment <i>vs</i> repletion with cholesterol, two way ANOVA. (B) DIH cells were pre-treated with 2-hydroxypropyl-β-Cyclodextrin, prior to the addition of ¹²⁵I -CXCL1/GRO-α with or without unlabeled excess CXCL1 for 120 minutes, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029624#s4" target="_blank">Materials and Methods</a>. Data presented are the combined results from at least three independent experiments.</p

Addition of amiloride, inhibition of actin polymerization, or PKC inhibition decreased CXCL1 internalization in DIH cells.

<p>DIH cells were pre-treated with various inhibitors prior to incubation with ¹²⁵I-CXCL1/GRO-α for the specified time points, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029624#s4" target="_blank">Materials and Methods</a>. (A) Amiloride significantly inhibited DARC-mediated CXCL1 internalization in DIH cells. * <i>p</i><0.05 amiloride treatment <i>vs</i> control, paired t test. (B) Actin polymerization inhibitor cytochalasin D decreased CXCL1 internalization in DIH cells. * <i>p</i><0.05 cytochalasin D treatment <i>vs</i> control, paired t test. (C) PKC inhibitor Gö6976 also showed inhibition of CXCL1 internalization. * <i>p</i><0.05 Gö6976 treatment <i>vs</i> control, paired t test. Shown is the mean ± SEM from three experiments.</p

PDGF enhances chemokine internalization in DIH cells and co-localization with early endosomal antigen.

<p>(A) DIH cells were stimulated with or without PDGF for 10 min, then DARC (red) and EEA (green) or LAMP (green) co-localization were detected using confocal imaging. Blue, actin. (B) DIH cells were pretreated with PDGF 50 ng/ml for 10 min prior to endocytosis experiments. By 120 minutes, PDGF increased DARC-mediated CXCL1 internalization. * <i>p</i><0.05 PDGF treatment <i>vs</i> control, paired t test. Cells incubated in the absence of PDGF served as a control for internalization. Data presented are the combined results of at least three independent experiments. (C) DIH cells were pretreated with or without PDGF 50 ng/ml for 10 min prior to surface staining for DARC as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029624#s4" target="_blank">Materials and Methods</a>.</p

Duffy antigen mediates CXCL1 binding and endocytosis in DIH cells.

<p>MIH cells and DIH cells were incubated with ¹²⁵I-CXCL1/GRO-α at the designated time points then were washed with binding buffer or acid wash buffer, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029624#s4" target="_blank">Materials and Methods</a>. (A) Internalized ¹²⁵I-CXCL1/GRO-α counts in MIH and DIH cells. * <i>p</i><0.01 MIH cells <i>vs</i> DIH cells, paired t test. (B) Percentage of ¹²⁵I-CXCL1/GRO-α internalization in MIH and DIH cells. * <i>p</i><0.001 MIH cells <i>vs</i> DIH cells, paired t test. Experiments were conducted in triplicates and the mean ± SEM from two independent experiments is shown. (C) DIH cells were labeled with anti-Duffy 2C3 Fy6 antibody or isotype mouse IgG1 antibody and surface expression of Duffy examined by flow cytometry. (D) DIH cells do not show detectable surface CXCR2 expression following immune-labeling with anti-human CXCR2 or isotype mouse IgG1 antibody. (E) Peripheral leukocytes were isolated from a healthy volunteer and immune-labeled with anti-human CXCR2 or isotype IgG1 control antibody. Histogram shows gated population of granulocytes expressing CXCR2. Flow cytometric examination of surface DARC and CXCR2 expression on DIH cells was performed 2 independent times. CXCR2 expression on human peripheral granulocytes was performed once.</p

Majority of Duffy antigen is present on the surface of endothelial cells within vesicles and plasma membrane extensions.

<p>Duffy antigen was detected on membrane surfaces by pre-embedding TEM in DIH cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029624#s4" target="_blank">Materials and Methods</a>. All panels are imaged from different cells. Duffy antigen (5 nm gold, arrows) was seen located associated with plasma membrane extensions and invaginations. These structures are large (>100 nm) and have no consistent morphology, suggesting they are macropinosomes.</p

Duffy antigen co-localizes with caveolin-1 but not with clathrin.

<p>The DIH cells were stimulated with or without CCL2 100 ng/ml for 120 minutes. Figure A shows a typical single plane confocal optical section from the lamellipodial region of a cell, with Panel B focusing at a specific area on the cell outlined by the rectangle in A. Panel B is then separated into specific color signals to accentuate the localization status of each protein. Cells are stained for Duffy (Panel C, blue), caveolin (Panel D, green) and clathrin (Panel E, red). Some Duffy positive foci colocalize with the caveolin signal (arrows) while no Duffy signal is seen colocalized with the red clathrin signal(arrowheads). Figure (F-H), by immuno-EM, caveolin-1 was labeled with 10 nm gold particle- (▾), Duffy antigen was labeled with 5 nm gold particle- (<b>↓</b>), colocalization between caveolin and Duffy is observed in flask-like membrane structures- (Δ).</p