Phospholipase D isoforms differentially regulate leukocyte responses to acute lung injury

Abstract

Phospholipase D (PLD) plays important roles in cellular responses to tissue injury that are critical to acute inflammatory diseases, such as the acute respiratory distress syndrome (ARDS). We investigated the expression of PLD isoforms and related phospholipid phosphatases in patients with ARDS, and their roles in a murine model of self-limited acute lung injury (ALI). Gene expression microarray analysis on whole blood obtained from patients that met clinical criteria for ARDS and clinically matched controls (non-ARDS) demonstrated that PLD1 gene expression was increased in patients with ARDS relative to non-ARDS and correlated with survival. In contrast, PLD2 expression was associated with mortality. In a murine model of self-resolving ALI, lung Pld1 expression increased and Pld2 expression decreased 24 h after intrabronchial acid. Total lung PLD activity was increased 24 h after injury. Pld1^-/- mice demonstrated impaired alveolar barrier function and increased tissue injury relative to WT and Pld2^-/- , whereas Pld2^-/- mice demonstrated increased recruitment of neutrophils and macrophages, and decreased tissue injury. Isoform-specific PLD inhibitors mirrored the results with isoform-specific Pld-KO mice. PLD1 gene expression knockdown in human leukocytes was associated with decreased phagocytosis by neutrophils, whereas reactive oxygen species production and phagocytosis decreased in M2-macrophages. PLD2 gene expression knockdown increased neutrophil and M2-macrophage transmigration, and increased M2-macrophage phagocytosis. These results uncovered selective regulation of PLD isoforms after ALI, and opposing effects of selective isoform knockdown on host responses and tissue injury. These findings support therapeutic strategies targeting specific PLD isoforms for the treatment of ARDS.

Keywords: Phospholipase D; acute lung injury; acute respiratory distress syndrome; microarray.

FIGURE 1. PLD genes are differentially regulated in peripheral blood during ARDS.

PLD1 and PLD2 gene expression fold change in ARDS/Non-ARDS (A) at presentation to the ICU, and (B) at day 7. (C) PLD1 and PLD2 gene expression fold change in ARDS/Non-ARDS patients from pooled microarray datasets obtained in external cohorts, *P < 0.05. (D) PLD1 and PLD2 gene expression fold change at presentation in ARDS survivors/nonsurvivors. Gene expression is expressed in log2

FIGURE 2. PLD isoforms and phospholipid phosphatase genes are differentially regulated in lungs after acute lung injury.

(A) Model of selective intratracheal HCl instillation to the left lung, and tissue harvesting at 24 h. Gene expression of (B) Pld1, (C) Pld2, (D) Plpp1, (E) Plpp2, and (F) Plpp6 in control and lungs 24 h after intratracheal HCl. Gene expression is expressed in fold change relative to housekeeping gene (GAPDH, see Materials and Methods). Results are expressed as mean ± SEM, N > 10, *P < 0.05

FIGURE 3. PLD deficiency impacts alveolar barrier disruption.

(A) Total lung PLD activity in wild-type (WT) mice, and mice deficient in Pld1 and Pld2 gene, at t = 0 and t = 24 h after intrabronchial HCl. Results are expressed as mean ± SEM, N ≥ 10, *P < 0.05. (B) Albumin levels in BAL fluid from WT, Pld1^−/−, and Pld2^−/− animals 24 h after intrabronchial HCl. (C) Evans blue dye levels in left lung homogenate 24 h after intrabronchial HCl and treatment with a selective inhibitor of PLD1 (PLD1in, black) or PLD2 (PLD2in, gray), or vehicle (Veh, hatched). Results are expressed as mean ± SEM, N ≥ 10, *P < 0.05. (D) Representative lung sections from WT (injured left lung and uninjured right lung from same animal), Pld1^−/−, and Pld2^−/− animals

FIGURE 4. PLD1 and PLD2 regulate leukocyte recruitment after ALI.

(A) Representative flow cytometry dot plots and histograms from BAL gated on total cell population. Neutrophils, CD45+ CD11b+ Ly6G+; Macs, total macrophages, CD45+ F4/80+; iMacs; inflammatory macrophages, CD45+ F4/80+ CD11c− CD11b+ SiglecF− Ly6C+; exMacs; exudative macrophages, CD45+ F4/80+ CD11c+ CD11b+ SiglecF+ Ly6C+; rAM, resident alveolar macrophages, CD45+ F4/80+ CD11c+ CD11b− SiglecF+ Ly6C−. (B–G) Enumeration of (B) total cell count and differential cell count including (C) neutrophils, (D) total macrophages, (E) rAM, (F) ExMacs, and (G) iMacs, by flow cytometry analysis of BAL fluid after intrabronchial acid. Results are expressed as mean ± SEM. n = > 10 per group. *P < 0.05

FIGURE 5. PLD genetic deficiency is associated with increased macrophage–neutrophil interactions.

(A) Representative dot plots of macrophage–neutrophil interactions as determined by Ly6G staining gated on macrophage subsets in BAL obtained 24 h after intrabronchial acid. (B) Absolute count and (C) percentage of iMacs that interact with neutrophils. (D) Absolute count and (E) percentage of exMacs that interact with neutrophils. Results are expressed as mean ± SEM. n ≥ 6 per group. *P < 0.05

FIGURE 6. PLD isoform knockdown is associated with selective regulation of leukocyte function.

(A and B) Differentiated human M2 macrophages and neutrophils were transfected with either scrambled siRNA (controls), or a pool of PLD1 siRNAs, or a pool of PLD2 siRNAs (see Materials and Methods). (A) Chemotaxis across transwell membrane toward MCP-1(10 nM) was determined for the indicated lengths of time; cells migrated at the bottom of the wells were stained and counted. Averages from 7 fields were plotted. (B) Phagocytosis of green-fluorescent latex beads (0.1 mg/mL) was determined and expressed as the average of beads inside macrophages by fluorescence. (C and D) Differentiated human neutrophils were transfected with either scrambled siRNA (controls), or a pool of PLD1 siRNAs, or a pool of PLD2 siRNAs (see Materials and Methods). (C) Neutrophil transmigration across transwell membrane toward IL-8(10nM) was determined for the indicated lengths of time; cells migrated at the bottom of the wells were stained and counted. Averages from 7 fields were plotted. *P < 0.05 versus control. (D) Neutrophil superoxide anion release in response to vehicle (veh), f-MLP (150 nM), and PMA (50 ng/mL). Data presented are averages + SEM from 4 separate biologic experiments each in duplicate. *P < 0.05 versus veh, #P < 0.05 versus siControl