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

CD73-Generated Extracellular Adenosine Promotes Resolution of Neutrophil-Mediated Tissue Injury and Restrains Metaplasia in Pancreatitis

Pancreatitis is currently the leading cause of gastrointestinal hospitalizations in the US. This condition occurs in response to abdominal injury, gallstones, chronic alcohol consumption or, less frequently, the cause remains idiopathic. CD73 is a cell surface ecto-5\u27-nucleotidase that generates extracellular adenosine, which can contribute to resolution of inflammation by binding adenosine receptors on infiltrating immune cells. We hypothesized genetic deletion of CD73 would result in more severe pancreatitis due to decreased generation of extracellular adenosine. CD73 knockout (CD7

Targeting LIF-mediated paracrine interaction for pancreatic cancer therapy and monitoring.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis largely owing to inefficient diagnosis and tenacious drug resistance. Activation of pancreatic stellate cells (PSCs) and consequent development of dense stroma are prominent features accounting for this aggressive biology1,2. The reciprocal interplay between PSCs and pancreatic cancer cells (PCCs) not only enhances tumour progression and metastasis but also sustains their own activation, facilitating a vicious cycle to exacerbate tumorigenesis and drug resistance3-7. Furthermore, PSC activation occurs very early during PDAC tumorigenesis8-10, and activated PSCs comprise a substantial fraction of the tumour mass, providing a rich source of readily detectable factors. Therefore, we hypothesized that the communication between PSCs and PCCs could be an exploitable target to develop effective strategies for PDAC therapy and diagnosis. Here, starting with a systematic proteomic investigation of secreted disease mediators and underlying molecular mechanisms, we reveal that leukaemia inhibitory factor (LIF) is a key paracrine factor from activated PSCs acting on cancer cells. Both pharmacologic LIF blockade and genetic Lifr deletion markedly slow tumour progression and augment the efficacy of chemotherapy to prolong survival of PDAC mouse models, mainly by modulating cancer cell differentiation and epithelial-mesenchymal transition status. Moreover, in both mouse models and human PDAC, aberrant production of LIF in the pancreas is restricted to pathological conditions and correlates with PDAC pathogenesis, and changes in the levels of circulating LIF correlate well with tumour response to therapy. Collectively, these findings reveal a function of LIF in PDAC tumorigenesis, and suggest its translational potential as an attractive therapeutic target and circulating marker. Our studies underscore how a better understanding of cell-cell communication within the tumour microenvironment can suggest novel strategies for cancer therapy

Mutant KRas-Induced Mitochondrial Oxidative Stress in Acinar Cells Upregulates EGFR Signaling to Drive Formation of Pancreatic Precancerous Lesions

The development of pancreatic cancer requires the acquisition of oncogenic KRas mutations and upregulation of growth factor signaling, but the relationship between these is not well established. Here, we show that mutant KRas alters mitochondrial metabolism in pancreatic acinar cells, resulting in increased generation of mitochondrial reactive oxygen species (mROS). Mitochondrial ROS then drives the dedifferentiation of acinar cells to a duct-like progenitor phenotype and progression to PanIN. This is mediated via the ROS-receptive kinase protein kinase D1 and the transcription factors NF-κB1 and NF-κB2, which upregulate expression of the epidermal growth factor, its ligands, and their sheddase ADAM17. In vivo, interception of KRas-mediated generation of mROS reduced the formation of pre-neoplastic lesions. Hence, our data provide insight into how oncogenic KRas interacts with growth factor signaling to induce the formation of pancreatic cancer

Persistent salmonellosis causes pancreatitis in a murine model of infection.

Pancreatitis, a known risk factor for the development of pancreatic ductal adenocarcinoma, is a serious, widespread medical condition usually caused by alcohol abuse or gallstone-mediated ductal obstruction. However, many cases of pancreatitis are of an unknown etiology. Pancreatitis has been linked to bacterial infection, but causality has yet to be established. Here, we found that persistent infection of mice with the bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) was sufficient to induce pancreatitis reminiscent of the human disease. Specifically, we found that pancreatitis induced by persistent S. Typhimurium infection was characterized by a loss of pancreatic acinar cells, acinar-to-ductal metaplasia, fibrosis and accumulation of inflammatory cells, including CD11b+ F4/80+, CD11b+ Ly6Cint Ly6G+ and CD11b+ Ly6Chi Ly6G- cells. Furthermore, we found that S. Typhimurium colonized and persisted in the pancreas, associated with pancreatic acinar cells in vivo, and could invade cultured pancreatic acinar cells in vitro. Thus, persistent infection of mice with S. Typhimurium may serve as a useful model for the study of pancreatitis as it relates to bacterial infection. Increased knowledge of how pathogenic bacteria can cause pancreatitis will provide a more integrated picture of the etiology of the disease and could lead to the development of new therapeutic approaches for treatment and prevention of pancreatitis and pancreatic ductal adenocarcinoma

<i>S</i>. Typhimurium colonize and persist in the pancreas, associate with pancreatic acinar cells <i>in vivo</i>, and can invade pancreatic acinar cells <i>in vitro</i>.

<p>(<i>A–C</i>) Bacterial loads per gram of pancreas (A), liver (B) and spleen (C) tissue harvested from C57BL/6J <i>Nramp1^G169</i> mice (n = 3 per group) at indicated times after infection with <i>S</i>. Typhimurium. (<i>D</i>) Representative confocal images of pancreatic tissue sections harvested from C57BL/6J <i>Nramp1^G169</i> mice (n = 3 per group) infected with <i>S</i>. Typhimurium expressing GFP. Tissue sections were stained with Alexa Fluor 594 phalloidin (red) and DAPI (blue). Arrows point to GFP-expressing <i>S</i>. Typhimurium. (<i>E</i>) Invasion of cultured pancreatic acinar cells (line 266-6) by wild-type or <i>invA</i>-deficient <i>S</i>. Typhimurium as measured by gentamicin protection assay. (<i>F and G</i>) Detection of GFP associated with cultured pancreatic acinar cells (line 266-6) infected with wild-type or <i>invA</i>-deficient <i>S</i>. Typhimurium expressing GFP. Data shown in (A–D) show mean with spread from (A–C), or are representative of (D), two independent experiments. Data shown in (E–G) show mean with SEM from (E and G), or are representative of (F), four independent experiments. Data in (E and G) were analyzed using a two-tailed, paired Student’s t-test; p values<0.05 were considered to be statistically significant. Asterisks indicate statistically significant differences (***p<0.001, **p<0.01).</p

<i>S</i>. Typhimurium infection induces pancreatitis.

<p>(<i>A and B</i>) Histological analysis of pancreatic tissue sections from C57BL/6J <i>Nramp1^G169</i> mice (n = 3–4 per group) left uninfected or infected for 10 days with <i>S</i>. Typhimurium (STm). Tissue sections were stained using H&E, cytokeratin 19, or Picrosirius Red Stain Kit. In addition, tissue sections were subjected to IHC using antibodies specific for collagen I (A) or F4/80 or Ly6B.2 (B). Scale bars for H&E = 200 μm and for IHC = 100 μm. (<i>C</i>) Quantitation of IHC data shown in (B). (<i>D and E</i>) Expression of surface F4/80 and CD11b by cells harvested from pancreata of C57BL/6J <i>Nramp1^G169</i> mice (n = 3–4 per group) left uninfected or infected for 10 days with STm as measured using flow cytometry. Numbers in (D) refer to CD11b⁺ F4/80⁺ cells as percentages of the total numbers of cells. (<i>F and G</i>) Expression of surface Ly6C and Ly6G by CD11b⁺ cells present in pancreata of C57BL/6J <i>Nramp1^G169</i> mice (n = 3–4 per group) left uninfected or infected for 10 days with STm as measured using flow cytometry. Numbers in (F) refer to CD11b⁺ Ly6C^hi Ly6G⁻ and CD11b⁺ Ly6C^int Ly6G⁺ cells as percentages of the total numbers of CD11b⁺ cells. Data are representative of (A, B, D and F), or show mean with SEM from (C, E and G), two independent experiments. Data were analyzed using a two-tailed, paired Student’s t-test; p values<0.05 were considered to be statistically significant. Asterisks indicate statistically significant differences (***p<0.001, *p<0.05).</p

<i>S</i>. Typhimurium LPS induces pancreatic inflammation.

<p>(<i>A</i>) Histological analysis of pancreatic tissue sections from mock-treated or LPS-treated C57BL/6J mice (n = 4 per group). Tissue sections were stained using H&E or subjected to IHC using antibodies specific for F4/80. Scale bars for H&E = 200 μm and for IHC = 100 μm. (<i>B</i>) Quantitation of IHC data shown in (A). (<i>C and D</i>) Expression of surface F4/80 and CD11b by cells harvested from pancreata of mock-treated or LPS-treated C57BL/6J mice (n = 4 per group) as measured using flow cytometry. Numbers in (C) refer to CD11b⁺ F4/80⁺ cells as percentages of the total numbers of cells. (<i>E and F</i>) Expression of surface Ly6C and Ly6G by CD11b⁺ cells present in pancreata of mock-treated or LPS-treated C57BL/6J mice (n = 4 per group) as measured using flow cytometry. Numbers in (E) refer to CD11b⁺ Ly6C^hi Ly6G⁻ and CD11b⁺ Ly6C^int Ly6G⁺ cells as percentages of the total numbers of CD11b⁺ cells. Data are representative of (A, C, and E), or show mean with SEM from (B, D and F), two independent experiments. Data were analyzed using a two-tailed, paired Student’s t-test; p values<0.05 were considered to be statistically significant. Asterisks indicate statistically significant differences (***p<0.001, *p<0.05).</p

Pancreatitis progresses with persistent <i>S</i>. Typhimurium infection.

<p>(<i>A and C</i>) Histological analysis of pancreatic tissue sections from C57BL/6J <i>Nramp1^G169</i> mice (n = 3–4 per group) left uninfected or infected for 60 days with <i>S</i>. Typhimurium (STm). Tissue sections were stained using H&E, cytokeratin 19, or Picrosirius Red Stain Kit. In addition, tissue sections were subjected to IHC using antibodies specific for collagen I (A) or F4/80 or Ly6B.2 (C). Scale bars for H&E = 200 μm and for IHC = 100 μm. (<i>B and D</i>) Quantitation of IHC data shown in (A and C). (<i>E and F</i>) Expression of surface F4/80 and CD11b by cells harvested from pancreata of C57BL/6J <i>Nramp1^G169</i> mice (n = 3–4 per group) left uninfected or infected for 60 days with STm as measured using flow cytometry. Numbers in (E) refer to CD11b⁺ F4/80⁺ cells as percentages of the total numbers of cells. (<i>G and H</i>) Expression of surface Ly6C and Ly6G by CD11b⁺ cells present in pancreata of C57BL/6J <i>Nramp1^G169</i> mice (n = 3–4 per group) left uninfected or infected for 60 days with STm as measured using flow cytometry. Numbers in (G) refer to CD11b⁺ Ly6C^hi Ly6G⁻ and CD11b⁺ Ly6C^int Ly6G⁺ cells as percentages of the total numbers of CD11b⁺ cells. Data are representative of (A, C, E and G), or show mean with SEM from (B, D, F and H), two independent experiments. Data were analyzed using a two-tailed, paired Student’s t-test (B and D) or a one-way ANOVA (F and H); p values<0.05 were considered to be statistically significant. Asterisks indicate statistically significant differences (***p<0.001).</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