Pseudomonas aeruginosa ExoU augments neutrophil transepithelial migration

Excessive neutrophil infiltration of the lungs is a common contributor to immune-related pathology in many pulmonary disease states. In response to pathogenic infection, airway epithelial cells produce hepoxilin A3 (HXA3), initiating neutrophil transepithelial migration. Migrated neutrophils amplify this recruitment by producing a secondary gradient of leukotriene B4 (LTB4). We sought to determine whether this two-step eicosanoid chemoattractant mechanism could be exploited by the pathogen Pseudomonas aeruginosa. ExoU, a P. aeruginosa cytotoxin, exhibits phospholipase A2 (PLA2) activity in eukaryotic hosts, an enzyme critical for generation of certain eicosanoids. Using in vitro and in vivo models of neutrophil transepithelial migration, we evaluated the impact of ExoU expression on eicosanoid generation and function. We conclude that ExoU, by virtue of its PLA2 activity, augments and compensates for endogenous host neutrophil cPLA2α function, leading to enhanced transepithelial migration. This suggests that ExoU expression in P. aeruginosa can circumvent immune regulation at key signaling checkpoints in the neutrophil, resulting in exacerbated neutrophil recruitment

<i>Pseudomonas aeruginosa</i> ExoU augments neutrophil transepithelial migration

<div><p>Excessive neutrophil infiltration of the lungs is a common contributor to immune-related pathology in many pulmonary disease states. In response to pathogenic infection, airway epithelial cells produce hepoxilin A₃ (HXA₃), initiating neutrophil transepithelial migration. Migrated neutrophils amplify this recruitment by producing a secondary gradient of leukotriene B₄ (LTB₄). We sought to determine whether this two-step eicosanoid chemoattractant mechanism could be exploited by the pathogen <i>Pseudomonas aeruginosa</i>. ExoU, a <i>P</i>. <i>aeruginosa</i> cytotoxin, exhibits phospholipase A2 (PLA2) activity in eukaryotic hosts, an enzyme critical for generation of certain eicosanoids. Using in vitro and in vivo models of neutrophil transepithelial migration, we evaluated the impact of ExoU expression on eicosanoid generation and function. We conclude that ExoU, by virtue of its PLA2 activity, augments and compensates for endogenous host neutrophil cPLA2α function, leading to enhanced transepithelial migration. This suggests that ExoU expression in <i>P</i>. <i>aeruginosa</i> can circumvent immune regulation at key signaling checkpoints in the neutrophil, resulting in exacerbated neutrophil recruitment.</p></div

Bacterial ExoU is associated with increased neutrophil transepithelial migration and LTB₄ generation.

<p>Inverted H292 monolayers were infected with various doses of matched strains of either (A, C) PAO1 or (B, D) PA14 that express or lack ExoU. Neutrophils were provided to the basolateral surface and allowed to migrate. Neutrophil migration was reported as a percent of Control. The magnitude of the neutrophil migration response to parental strains PAO1 Vector or PA14 at infection doses of 1.59E+07 and 1.34E+09 respectively were considered the Control and set at 100%. We assayed (A, B) total neutrophil migration, as well as (C, D) total LTB₄ in the apical space following 2h migration. Data are shown as mean +/- SD, and are representative of multiple independent experiments. *p ≤ 0.05, **p < 0.01, ***p < 0.001 indicate comparison between ExoU lacking or expressing genetically matched strains at the same concentration.</p

ExoU expression is associated with increased LTB₄ and PMN infiltration independently of any detectible ExoU-associated cytotoxicity at early stages in an in vivo model of acute pneumonia.

<p>Adult 6–8 week old female C57BL/6J mice were challenged by intranasal inoculation of either PA14 or PA14ΔexoU. Bronchial alveolar lavage (BAL) samples were collected at 6hpi and total cell counts were performed. (A) Total cells were stained for Ly6G and analyzed by flow cytometry. Protein extracts were prepared from BAL samples and analyzed for (B) neutrophil elastase/ELA2 and (C) myeloperoxidase/MPO. (D) BAL samples were spun down and supernatant liquids were tested for levels of LTB₄ via ELISA. (E) LDH levels were assessed in the cell-free supernatant to assess cytotoxicity. (F) In independent experiments, 6–8 week old C57BL/6J female mice were infected and lung tissues were harvested 6hpi post infection to assess bacterial burden. Data are representative of five animals per condition and are shown as mean +/- SEM, and are representative of multiple independent experiments. *p ≤ 0.05, **p < 0.01, indicate comparison between PA14 and PA14ΔexoU. n.s. indicates a non-statistically significant difference.</p

ExoU-associated PLA2 activity enhances production of epithelial arachidonic acid and PGE₂.

<p>Lung epithelial monolayers were infected with matched strains of PAO1 or PA14 that either express or lack ExoU. (A, B) Arachidonic acid release was assessed following infection of confluent 24w plate-grown A549 cells with ExoU-expressing or -lacking strains of <i>P</i>. <i>aeruginosa</i>. Control cells stably express irrelevant control RNAi, while cPLA2α iRNA cells express inhibitory RNA targeting <i>cpla2α</i> mRNA. (C, D) Total PGE₂ production was measured in the supernatant of infected A549 cells by immunoassay following 1h infection with the indicated strain, and a subsequent 2h incubation following epithelial wash. Total PGE₂ (E, F) levels were measured in lipid-extracted supernatants by LC/MS/MS following infection of non-transfected H292 lung epithelial cells. Data are represented as means +/- SD and are representative of multiple independent experiments. **p < 0.01, ***p < 0.001, indicate comparison between ExoU lacking or expressing genetically matched strains. n.s. indicates a non-statistically significant difference. †† p <0.01, ††† p <0.001 indicate comparison to HBSS control.</p

ExoU compensates for absence of cPLA2α, but not 5-LOX in a mouse in vitro model of neutrophil transepithelial migration.

<p>Mouse lung epithelial MLE12 monolayers were grown on inverted transwells and infected with paired PAO1 strains that express or lack ExoU. Bone marrow from (A, C) cPLA2α-/- mice or their littermate controls, or from (B, D) ALOX5-/- or WT mice were provided in the basolateral chamber to assess migration of bone marrow neutrophils. (A, B) Apical supernatant was assayed for total LTB₄ content following 2h migration period. (C, D) Migration of neutrophils was assessed following epithelial infection with PAO1 Vector or PAO1 +ExoU. Neutrophil migration was reported as percent of control. The neutrophil migration response of WT neutrophils to the parental strain PAO1 Vector was set to 100%. Data are shown as mean +/- SD, and are representative of multiple independent experiments. *p ≤ 0.05, **p < 0.01, ***p < 0.001 between the indicated conditions. n.s. indicates a non-statistically significant difference. ††p <0.01, †††p <0.001 indicates comparison to HBSS control.</p

ExoU circumvents suppression of cPLA2α activity.

<p>Neutrophils were treated with pharmacological inhibitors of the LTB₄ biosynthetic pathway U0126 (20μM), and MK886 (2μM) for 1h prior to migration, and allowed to migrate across untreated H292 epithelial transwell monolayers in response to infection or exogenous gradients of IL-8. (A) Apical supernatant was sampled following migration and assayed for total LTB₄ by immunoassay. Migration of neutrophils treated with (B) U0126, (C) MK886, or their respective vehicle controls was assessed by myeloperoxidase assay. Neutrophil migration was measured as % of Control, in which the migration response of vehicle treated neutrophils to either parental strain (PAO1 Vector or PA14) were set to 100%. Data are shown as mean +/- SD, and are representative of multiple independent experiments. **p < 0.01, ***p < 0.001 between the indicated conditions. n.s. indicates a non-statistically significant difference.</p