Deficiencies of the Lipid-Signaling Enzymes Phospholipase D1 and D2 Alter Cytoskeletal Organization, Macrophage Phagocytosis, and Cytokine-Stimulated Neutrophil Recruitment

Cell migration and phagocytosis ensue from extracellular-initiated signaling cascades that orchestrate dynamic reorganization of the actin cytoskeleton. The reorganization is mediated by effector proteins recruited to the site of activity by locally-generated lipid second messengers. Phosphatidic acid (PA), a membrane phospholipid generated by multiple enzyme families including Phospholipase D (PLD), has been proposed to function in this role. Here, we show that macrophages prepared from mice lacking either of the classical PLD isoforms PLD1 or PLD2, or wild-type macrophages whose PLD activity has been pharmacologically inhibited, display isoform-specific actin cytoskeleton abnormalities that likely underlie decreases observed in phagocytic capacity. Unexpectedly, PA continued to be detected on the phagosome in the absence of either isoform and even when all PLD activity was eliminated. However, a disorganized phagocytic cup was observed as visualized by imaging PA, F-actin, Rac1, an organizer of the F-actin network, and DOCK2, a Rac1 activator, suggesting that PLD-mediated PA production during phagocytosis is specifically critical for the integrity of the process. The abnormal F-actin reorganization additionally impacted neutrophil migration and extravasation from the vasculature into interstitial tissues. Although both PLD1 and PLD2 were important in these processes, we also observed isoform-specific functions. PLD1-driven processes in particular were observed to be critical in transmigration of macrophages exiting the vasculature during immune responses such as those seen in acute pancreatitis or irritant-induced skin vascularization

Distinct Roles for Hematopoietic and Extra-Hematopoietic Sphingosine Kinase-1 in Inflammatory Bowel Disease

<div><p>Sphingosine kinase 1 (SK1), one of two SK enzymes, is highly regulated and has been shown to act as a focal point for the action of many growth factors and cytokines. SK1 leads to generation of sphingosine-1-phosphate (S1P) and potentially the activation of S1P receptors to mediate biologic effects. Our previous studies implicated SK1/S1P in the regulation of inflammatory processes, specifically in inflammatory bowel disease (IBD). These studies were conducted using a total body knockout mouse for SK1 and were unable to determine the source of SK1/S1P (hematopoietic or extra-hematopoietic) involved in the inflammatory responses. Therefore, bone marrow transplants were performed with wild-type (WT) and SK1-/- mice and colitis induced with dextran sulfate sodium (DSS). Irrespective of the source of SK1/S1P, bone marrow or tissue, DSS induced colitis in all mice; however, mice lacking SK1 in both hematopoietic and extra-hematopoietic compartments exhibited decreased crypt damage. Systemic inflammation was assessed, and mice with WT bone marrow demonstrated significant neutrophilia in response to DSS. In the local inflammatory response, mice lacking SK1/S1P in either bone marrow or tissue exhibited decreased induction of cytokines and less activation of STAT3 (signal transducer and activator of transcription 3). Interestingly, we determined that extra-hematopoietic SK1 is necessary for the induction of cyclooxygenase 2 (COX2) in colon epithelium in response to DSS-induced colitis. Taken together our data suggest that hematopoietic-derived SK1/S1P regulates specific aspects of the systemic inflammatory response, while extra-hematopoietic SK1 in the colon epithelium is necessary for the autocrine induction of COX2 in DSS-induced colitis.</p></div

Mice with WT bone marrow exhibit significant neutrophilia following DSS-induced colitis.

<p><b>A</b>) Whole blood was collected and analyzed for neutrophil counts. <b>B</b>) Neutrophil counts are normalized to total lymphocytes and expressed as neutrophil-lymphocyte ratio. Data represent mean ±SD, n≥6 for each treatment group; *p<0.05, ***p<0.001 and ****p<0.001 vs strain untreated, #p<0.05 as compared to WT^WTBM DSS treated, %p<0.05 as compared to SK1^SK1BM DSS. X-axis: regular text refers to the host genotype and the superscript to the bone marrow genotype.</p

STAT3 phosphorylation requires dual sources of SK1/S1P in acute DSS-induced colitis.

<p>Phospho-STAT3 (Ser727) was examined by IHC. <b>A–D</b>) untreated mice; <b>E–H</b>) DSS treated mice. Scale bars  = 20 µm. Regular text refers to the host genotype and the superscript to the bone marrow genotype.</p

Dual sources of SK1/S1P regulate IL-1β and IL-6 expression in DSS-treated mice.

<p>Expression levels of <b>A</b>) IL-1β, <b>B</b>) IL-6, and <b>C</b>) TNFα in colon tissues were determined using real time-RTPCR and normalized to β-actin. Data represent mean ±SEM, n≥3 for each treatment group; *p<0.05, **p<0.01, and ****p<0.001 vs strain untreated, #p<0.05 as compared to WT^WTBM DSS. X-axis: regular text refers to the host genotype and the superscript to the bone marrow genotype.</p

DOCK2 mislocalizes in the absence of PLD1 or PLD2.

<p>A) Confocal microscopy of WT, <i>Pld1</i>^−/−, and <i>Pld2</i>^−/− BMDM stimulated with human IgG-coated beads for 5 min followed by immunostaining for DOCK2 (green) and rhodamine phalloidin staining of F-actin (red); *, region magnified in inset. B) Graph of distinct and indistinct DOCK2 cups as well as actin cups, quantitated as in Fig. 2. C) Western blot of DOCK2 in WT, <i>Pld1</i>^−/−, and <i>Pld2</i>^−/− BMDM, best representative images from at least 3 blots. Bar, 7.5 µm. ***  =  p<0.0001.</p

PLD deletion results in abnormal phagosomal cup formation.

<p>Confocal microscopy of primary BMDM transfected via nucleofection with an EGFP-fused Spo20-PA sensor to image PA (green) in unstimulated WT BMDM (A) or in WT (B), <i>Pld1</i>^−/− (C), <i>Pld2</i>^−/− (D), or FIPI-treated WT (E) BMDM that were cultured with opsonized beads for 5 min. F-actin was visualized using rhodamine phalloidin (red). Scale bar, 5 µm. Best representative images from at least 3 sets of independently-isolated BMDMs. F) Circles shown indicating thickness of cup in WT BMDM superimposed on <i>Pld1</i>^−/− and <i>Pld2</i>^−/− cups, with quantitation of the thickness of the PA-visualized cups. At least 10 cups were measured for each experimental setting, and the experiments were performed 3 or more times. ***  =  p<0.0001.</p

<i>Pld1</i>^−/− and <i>Pld2</i>^−/− neutrophils exhibit impaired migration but only <i>Pld1</i>^−/− neutrophils have impaired tissue extravasation.

<p>A) Quantitation of chemoattractant-stimulated <i>in vitro</i> migration through 4 µm pore transwell filters of neutrophils isolated from the blood of WT, <i>Pld1</i>^−/− and <i>Pld2</i>^−/− mice. WT macrophages stimulated with LPS (1 µg/mL for 2 hrs) were used to generate conditioned media that was used as the chemoattractant. Blood neutrophils were allowed to migrate towards the conditioned media for 30 min. The experiment was performed at least 3 times using independently prepared BMDM. B) Quantitation of circulating neutrophil recruitment to the peritoneum. LPS (1 mg/kg) was injected into the peritoneum 4 hrs prior to sacrifice. The peritoneum was lavaged using HBSS to collect the recruited neutrophils; n = 3 for each type. C) IHC of pancreata stained with Ly6B.2 to detect neutrophils (circled) following induction of pancreatitis. Arrows indicate blood vessels and fibrotic border. D) Quantitation of neutrophil recruitment to the pancreas following acute pancreatitis. WT, n = 9; <i>Pld1</i>^−/−, n = 5; <i>Pld2</i>^−/−, n = 7. E) Quantitation of Evan's blue dye leakage into the ear following irritation with mineral oil. Results are the average of 3 independent experiments. *  =  p<0.05, **  =  p<0.005.</p

F-actin organization in resting and activated macrophages.

<p>A) Confocal images of BMDM transfected via nucleofection with the Spo20-GFP PA sensor (green), serum starved for 2 hrs, and then plated on fibronectin-coated coverslips for 30 min, fixed, and stained for F-actin using rhodamine phalloidin (red). Images are representative best of at least 3 separate experiments from which at least 3 macrophages were visualized. Arrow, podosome; arrowhead, cortical actin; *, region magnified in inset. B) Western blot analysis of BMDM macrophages treated as above. The experiment was repeated at least 3 times using BMDM prepared from different mice. C) Confocal images of intraperitoneal (IP) macrophages from WT, <i>Pld1</i>^−/−, and <i>Pld2</i>^−/− mice serum-starved for 2 hrs and plated on fibronectin-coated coverslips. Macrophages were stained with rhodamine phalloidin to visualize F-actin. Podosomes are circled and arrows indicate cortical actin. Images are representatives of at least 3 experiments. D) Graph summarizing average cell diameter of IP macrophages on fibronectin. Data collected from at least 3 fields of vision from 3 independent mice. E) Graph representing the diameters of podosomes from IP macrophages plated on fibronectin. Data collected from at least 3 fields of vision from 3 separate mice. Bar, 7.5 µm. *  =  p<0.05, **  =  p<0.005, ***  =  p<0.0001.</p

Rac1 recruitment and localization is abnormal in the absence of PLD1 or PLD2.

<p>A) Confocal microscopy of WT, <i>Pld1</i>^−/−, and <i>Pld2</i>^−/− BMDM stimulated with human IgG-coated beads for 5 min followed by immunostaining for Rac1 (green) and staining for F-actin (rhodamine phalloidin, red). *, shown magnified in inset. B) Graph of distinct (example: top panel, left) and indistinct (examples: top panel, right) Rac1 cups. Type of cup (distinct versus indistinct) was normalized to the number of beads per field. Quantitation was performed using Zeiss intensity maps as well as MATLAB parameters as described in Materials and Methods. At least 3 fields and 20 cups were scored for each condition from 5 independent experiments. C) Confocal microscopy using an activation state-sensitive anti-Rac1 monoclonal antibody to visualize activated Rac1 in WT, <i>Pld1</i>^−/−, and <i>Pld2</i>^−/− BMDM stimulated with human IgG-coated beads for 5 min. D) Western blot of PBD-pull down assay to assess GTP-bound Rac1 in WT, <i>Pld1</i>^−/−, and <i>Pld2</i>^−/− BMDM stimulated with human IgG-coated beads for 5 min; total Rac1 in the lysates is shown as a loading control. The western was repeated at least 3 times with similar results. Bar, 7.5 µm. *  =  p<0.05, **  =  p<0.005, ***  =  p<0.0001.</p