PAO1ΔcheYZBAW do not respond to CFBE41o- wounds.

<p><b>A.</b> Images from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150109#pone.0150109.s008" target="_blank">S8 Movie</a> showing CFBE41o- cells incubated in Ringer containing Syto 11-loaded PAO1ΔcheYZBAW (2 MOI) and PI before (t = -1 min) and at 2, 5 and 30 mins after wounding. <b>B.</b> Quantitation of PAO1ΔcheYZBAW (green trace), live epithelial cells (blue) and dead epithelial cells (red) near wound (yellow circle in A) under control conditions and then for 30 mins following wounding (arrow). <b>C.</b> Quantitation of PAO1-GFP (green trace), live epithelial cells (blue) and dead epithelial cells (red) in the adjacent non-wound region (white circle in A) under control conditions and then following wounding (arrow). <b>D.</b> Average number of PAO1ΔcheYZBAW (normalized to bacteria in field before wounding) at t = 0 mins (before the wound) and at 2–5 mins and 15 mins after wounding. Data are avg +/- SD, n = 4 experiments.</p

Flagella are required for swimming toward, and pili are required for binding of <i>P</i>. <i>aeruginosa</i> to wounded CFBE41o- cells.

<p>CFBE41o- monolayers were incubated in Ringer and wounded in the presence of <i>P</i>. <i>aeruginosa</i> strains PAK-GFP or Syto 11-labeled PAKΔfliC or Syto 11-labeled PAKΔpilA. After 30 mins, cells were rinsed and placed in the incubator for 2 hrs, then rinsed and imaged (DIC and green fluorescence). Yellow arrowheads show path of wound in healing CFBE41o- cells. <b>A.</b> PAK-GFP, <b>B.</b> PAKΔfliC, <b>C.</b> PAKΔpilA, <b>D.</b> Average (+/- SD, n = 3) number of bacteria bound along wounds of CFBE41o- cell monolayers.</p

Similar responses of PAO1-GFP to wounds in CFBE41o- cells incubated in pH 8 vs pH 6 solutions.

<p>CFBE41o- cells (labeled with BCECF to for visualization) incubated in Ringer buffered to pH 6.0 or pH 8.0 and containing PAO1-GFP (2 MOI) were wounded and imaged. <b>A.</b> Images from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150109#pone.0150109.s006" target="_blank">S6 Movie</a> showing PAO1-GFP and epithelial cells bathed in pH 6 Ringer in control conditions (t = -1 min) and after wounding (edge of wound marked by yellow triangles) epithelial cells (t = 2, 5 and 15 min). <b>B.</b> Images from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150109#pone.0150109.s007" target="_blank">S7 Movie</a> showing PAO1-GFP and unlabeled epithelial cells bathed in pH 8 in control conditions (t = -1 min) and after wounding (edge of wound marked by yellow triangles) epithelial cells (t = 2, 5 and 15 min). <b>C.</b> Quantitation of PAO1-GFP near the wounded CFBE41o^- cells (green circle in A) bathed in pH 6 Ringer beginning when wound removed epithelial cells (arrow). <b>D.</b> Quantitation of PAO1-GFP near the wounded CFBE41o^- cells (green circle in B) bathed in pH 8 Ringer beginning when wound removed epithelial cells (arrow). <b>E.</b> Average number of PAO1-GFP (normalized to bacteria in field before wounding) in the epithelial wounds (i.e., in regions shown by green circles in A and B) during control conditions (t = 0 mins), at peak of bacterial migration to the wound (2–5 mins) and after 15 mins for the pH 6 and pH 8 experiments. Data are avg +/- SD (n = 4–5 experiments).</p

Time course of changes in CFBE41o- epithelia and PAO1-GFP following wounding.

<p><b>A.</b> Images from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150109#pone.0150109.s001" target="_blank">S1 Movie</a> showing overlays of DIC, fura-2 (blue), PI (red nuclei) and PAO1-GFP in control conditions (t = -1 min) and after wounding CFBE41o- cells (t = 2, 5 and 30 min). <b>B.</b> Quantitation of PAO1-GFP (green trace), live epithelial cells (blue) and dead epithelial cells (red) in the region of the wound shown by the yellow circle in A under control conditions and then following wounding (arrow). Fluorescence intensities were measured on background-subtracted images and expressed in arbitrary fluorescence units. <b>C.</b> Quantitation of PAO1-GFP (green trace), live epithelial cells (blue) and dead epithelial cells (red) in the adjacent non-wound region (white circle in A) under control conditions and then following nearby wounding (arrow). <b>D.</b> Average number of PAO1-GFP (normalized to bacteria in field before wounding) in the epithelial wounds (i.e., in regions shown by yellow circles in A) during control conditions (t = 0 min), at peak of bacterial migration to the wound (2–5 mins) and when bacteria returned to random swimming but some remained immobilized on both dead and live cells near the wound (t = 15 mins). Data are avg +/- SD (n = 5 experiments).</p

Chemotaxis and Binding of <i>Pseudomonas aeruginosa</i> to Scratch-Wounded Human Cystic Fibrosis Airway Epithelial Cells

<div><p>Confocal imaging was used to characterize interactions of <i>Pseudomonas aeruginosa</i> (PA, expressing GFP or labeled with Syto 11) with CF airway epithelial cells (CFBE41o-, grown as confluent monolayers with unknown polarity on coverglasses) in control conditions and following scratch wounding. Epithelia and PAO1-GFP or PAK-GFP (2 MOI) were incubated with Ringer containing typical extracellular salts, pH and glucose and propidium iodide (PI, to identify dead cells). PAO1 and PAK swam randomly over and did not bind to nonwounded CFBE41o- cells. PA migrated rapidly (began within 20 sec, maximum by 5 mins) and massively (10–80 fold increase, termed “swarming”), but transiently (random swimming after 15 mins), to wounds, particularly near cells that took up PI. Some PA remained immobilized on cells near the wound. PA swam randomly over intact CFBE41o- monolayers and wounded monolayers that had been incubated with medium for 1 hr. Expression of CFTR and altered pH of the media did not affect PA interactions with CFBE41o- wounds. In contrast, PAO1 swarming and immobilization along wounds was abolished in PAO1 (PAO1ΔcheYZABW, no expression of chemotaxis regulatory components <i>cheY</i>, <i>cheZ</i>, <i>cheA</i>, <i>cheB</i> and <i>cheW</i>) and greatly reduced in PAO1 that did not express amino acid receptors <i>pctA</i>, <i>B</i> and <i>C</i> (PAO1ΔpctABC) and in PAO1 incubated in Ringer containing a high concentration of mixed amino acids. Non-piliated PAKΔpilA swarmed normally towards wounded areas but bound infrequently to CFBE41o- cells. In contrast, both swarming and binding of PA to CFBE41o- cells near wounds were prevented in non-flagellated PAKΔfliC. Data are consistent with the idea that (i) PA use amino acid sensor-driven chemotaxis and flagella-driven swimming to swarm to CF airway epithelial cells near wounds and (ii) PA use pili to bind to epithelial cells near wounds.</p></div

Role of leaked amino acids in swarming of PAO1 to wounded CFBE41o- cells.

<p>CFBE41o- cells (labeled with BCECF for visualization) were incubated in Ringer containing PAO1-GFP, with Syto 11-labelled PAO1ΔpctA,B,C or PAO1-GFP+tryptone (1% w/v) (2 MOI). <b>A.</b> Images from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150109#pone.0150109.s009" target="_blank">S9 Movie</a> showing PAO1-GFP and epithelial cells in control conditions (t = -1 min) and after wounding (yellow triangles; t = 2, 5 and 15 min). <b>B.</b> Images from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150109#pone.0150109.s010" target="_blank">S10 Movie</a> showing PAO1-ΔpctABC and epithelial cells in control conditions (t = -1 min) and after wounding (yellow triangles; t = 2, 5 and 15 min). <b>C.</b> Images from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150109#pone.0150109.s011" target="_blank">S11 Movie</a> showing PAO1-GFP and epithelial cells with 1% tryptone in control conditions (t = -1 min) and after wounding (yellow triangles; t = 2, 5 and 15 min). <b>D.</b> Quantitation of PAO1-GFP near the wounded CFBE41o^- cells (green circle in A). <b>E.</b> Quantitation of PAO1-ΔpctABC near the wounded CFBE41o^- cells (green circle in B). <b>F.</b> Quantitation of PAO1-GFP near the wounded CFBE41o^- cells (green circle in C) in the presence of tryptone. <b>G.</b> Average numbers of bacteria (PAO1-GFP, PAO1ΔpctABC, and PAO1-GFP + tryptone) near the wound (in green circles, normalized to number before the wound) were measured before (t = 0) and after wounding (t = 2–5 and 15 mins). Avg +/- SD, n = 4–9 for each strain.</p

Similar responses of PAO1-GFP to epithelial wounds in CF vs. CFTR-corrected CFBE41o- cells.

<p>CFBE41o- and CFTR-corrected CFBE41o- cells were incubated in Ringer containing PAO1-GFP (2 MOI) and then wounded and imaged for 20 mins. <b>A.</b> Images from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150109#pone.0150109.s004" target="_blank">S4 Movie</a> showing PAO1-GFP and unlabeled CFBE41o^- epithelial cells in control conditions (t = -1 min) and after wounding (yellow triangles) CFBE41o- cells (t = 2, 5 and 15 min). <b>B.</b> Images from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150109#pone.0150109.s004" target="_blank">S4 Movie</a> showing PAO1-GFP and unlabeled CFTR-corrected CFBE41o^- epithelial cells in control conditions (t = -1 min) and after wounding (wound edge shown by yellow triangles) CFTR-corrected CFBE41o- cells (t = 2, 5 and 15 min). <b>C.</b> Quantitation of PAO1-GFP near the wounded CFBE41o^- cells (green circle in A) and non-wounded cells (red circle in A) under control conditions and then following wounding (arrow). <b>D.</b> Quantitation of PAO1-GFP near the wounded CFBE41o^- cells (green circle in B) and non-wounded cells (red circle in B) under control conditions and then following nearby wounding (arrow). <b>E.</b> Average number of PAO1-GFP (normalized to bacteria in field before wounding) in the CF and CFTR-corrected epithelial wounds (i.e., in regions shown by green circles in A and B) during control conditions (t = 0 mins), at peak of bacterial migration to the wound (2–5 mins) and after 15 mins. Data are avg +/- SD (n = 3 experiments).</p

Altered chemotaxis of PAKΔfliC but not PAKΔpilA to wounds of CFBE41o- cell monolayers.

<p>CFBE41o- monolayers incubated in Ringer containing <i>P</i>. <i>aeruginosa</i> strains PAK-GFP, PAKΔflic and PAKΔpilA (2 MOI) were imaged under control conditions and following wounding. <b>A.</b> Images from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150109#pone.0150109.s012" target="_blank">S12 Movie</a> showing PAK-GFP and epithelial cells in control conditions (t = -1 min) and after wounding (wound edge shown by yellow triangles; t = 2, 5 and 15 min). <b>B.</b> Images from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150109#pone.0150109.s013" target="_blank">S13 Movie</a> showing PAK-ΔfliC and epithelial cells in control conditions (t = -1 min) and after wounding (wound edge shown by yellow triangles; t = 2, 5 and 15 min). <b>C.</b> Images from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150109#pone.0150109.s014" target="_blank">S14 Movie</a> showing PAK-ΔpilA and epithelial cells in control conditions (t = -1 min) and after wounding (wound edge shown by yellow triangles; t = 2, 5 and 15 min). <b>D.</b> Quantitation of PAK-GFP near wounded CFBE41o^- cells (green circle in A) and in a control region (red circle in A). <b>E.</b> Quantitation of PAO1-ΔfliC near wounded CFBE41o^- cells (green circle in B) and in a control region (red circle in B). <b>F.</b> Quantitation of PAK-ΔpilA near wounded CFBE41o^- cells (green circle in C) and in a control region (red circle in C) in the presence of tryptone. <b>G.</b> Average numbers of bacteria (PAK-GFP, PAK-ΔfliC, and PAK-ΔpilA, normalized to number present before the wound) accumulating near scratch-wounded epithelia at times t = 0, 2–5 and 15 mins. Avg +/- SD, n = 3 for each strain.</p

Cellulose Nanofibril Hydrogel Tubes as Sacrificial Templates for Freestanding Tubular Cell Constructs

The merging of defined nanoscale building blocks with advanced additive manufacturing techniques is of eminent importance for the preparation of multiscale and highly functional materials with <i>de novo</i> designed hierarchical architectures. Here, we demonstrate that hydrogels of cellulose nanofibrils (CNF) can be processed into complex shapes, and used as a sacrificial template to prepare freestanding cell constructs. We showcase our approach for the fabrication of hollow fibers using a controlled extrusion through a circular die into a coagulation bath. The dimensions of the hollow fibers are tunable, and the final tubes combine the nanofibrillar porosity of the CNF hydrogel with a submillimeter wall thickness and centimeter-scale length provided by the additive manufacturing technique. We demonstrate that covalent and supramolecular cross-linking of the CNFs can be used to tailor the mechanical properties of the hydrogel tubes within 1 order of magnitude and in an attractive range for the mechanosensation of cells. The resulting tubes are highly biocompatible and allow for the growth of mouse fibroblasts into confluent cell layers in their inner lumen. A detailed screening of several cellulases enables degradation of the scaffolding, temporary CNF hydrogel tube in a quick and highly cell-friendly way, and allows the isolation of coherent cell tubes. We foresee that the growing capabilities of hydrogel printing techniques in combination with the attractive features of CNFssustainable, globally abundant, biocompatible and enzymatically degradablewill allow making plant-based biomaterials with hierarchical structures and on-demand degradation useful, for instance, to engineer complex tissue structures to replace animal models, and for implants

In ferret trachea, PGE₂ stimulated <i>I</i>_<i>sc</i> is mediated by CFTR and Ca²⁺-activated Cl^- channels.

<p><b>A.</b> Representative <i>I</i>_<i>sc</i> trace with vertical deflections indicating the change in <i>I</i>_<i>sc</i> after a 1 mV pulse was applied (every 1 minute). Ferret trachea was exposed to serosal to mucosal Cl^- gradient with equivalent bilateral HCO₃^-. PGE₂ (1 μM, serosal) was added to ferret trachea after a baseline period of ≥ 10 minutes, with CFTR_inh-172 (20 μM, mucosal) added after 30 minutes. <b>B.</b> Representative <i>I</i>_<i>sc</i> trace of ferret trachea incubated in CFTR_inh-172 (20 μM, mucosal) for at least 30 minutes prior to PGE₂ (1 μM, serosal) stimulation. After 30 minutes, niflumic acid (100 μM, mucosal) was added. <b>C.</b> Change in PGE₂-stimulated <i>I</i>_<i>sc</i> (mean ± SEM, n ≥ 5) in ferret trachea, with comparisons between no inhibition, CFTR inhibition, or CFTR and Ca²⁺-activated Cl^- inhibition. Asterisks denote significance by Student’s t-test (*, P < 0.05, **, P < 0.01). Mean percent inhibition compared to PGE₂ stimulation alone noted.</p