Robert Frei

Adipose triglyceride lipase acts on neutrophil lipid droplets to regulate substrate availability for lipid mediator synthesis

In humans, mutations in ATGL lead to TG accumulation in LDs of most tissues and cells, including peripheral blood leukocytes. This pathologic condition is called Jordans’ anomaly, in which functional consequences have not been investigated. In the present study, we tested the hypothesis that ATGL plays a role in leukocyte LD metabolism and immune cell function. Similar to humans with loss‐of‐function mutations in ATGL, we found that global and myeloid‐specific Atgl−/− mice exhibit Jordans’ anomaly with increased abundance of intracellular TG‐rich LDs in neutrophil granulocytes. In a model of inflammatory peritonitis, lipid accumulation was also observed in monocytes and macrophages but not in eosinophils or lymphocytes. Neutrophils from Atgl−/− mice showed enhanced immune responses in vitro, which were more prominent in cells from global compared with myeloid‐specific Atgl−/− mice. Mechanistically, ATGL−/− as well as pharmacological inhibition of ATGL led to an impaired release of lipid mediators from neutrophils. These findings demonstrate that the release of lipid mediators is dependent on the liberation of precursor molecules from the TG‐rich pool of LDs by ATGL. Our data provide mechanistic insights into Jordans’ anomaly in neutrophils and suggest that ATGL is a potent regulator of immune cell function and inflammatory diseases.

Activation of EP4 receptors prevents endotoxin‐induced neutrophil infiltration into the airways and enhances microvascular barrier function

Pulmonary vascular dysfunction is a key event in acute lung injury. We recently demonstrated that PGE2, via activation of E‐prostanoid (EP)4 receptors, strongly enhances microvascular barrier function in vitro. The aim of this study was to investigate the beneficial effects of concomitant EP4 receptor activation in murine models of acute pulmonary inflammation.

Cannabinoid receptor 2 augments eosinophil responsiveness and aggravates allergen-induced pulmonary inflammation in mice.

Accumulation of activated eosinophils in tissue is a hallmark of allergic inflammation. The endocannabinoid 2‐arachidonoylglycerol (2‐AG) has been proposed to elicit eosinophil migration in a CB2 receptor/Gi/o‐dependent manner. However, it has been claimed recently that this process may also involve other mechanisms such as cytokine priming and the metabolism of 2‐AG into eicosanoids. Here, we explored the direct contribution of specific CB2 receptor activation to human and mouse eosinophil effector function in vitro and in vivo.

EP4 receptor prevents endotoxin‐induced neutrophil infiltration into the airways and enhances microvascular barrier function

Pulmonary vascular dysfunction is a key event in acute lung injury. We recently demonstrated that PGE2, via activation of E‐prostanoid (EP)4 receptors, strongly enhances microvascular barrier function in vitro. The aim of this study was to investigate the beneficial effects of concomitant EP4 receptor activation in murine models of acute pulmonary inflammation.

Neutrophil effector responses are suppressed by secretory phospholipase A2 modified HDL

Secretory phospholipase A2 (sPLA2) generates bioactive lysophospholipids implicated in acute and chronic inflammation, but the pathophysiologic role of sPLA2 is poorly understood. Given that high-density lipoprotein (HDL) is the major substrate for sPLA2 in plasma, we investigated the effects of sPLA2-mediated modification of HDL (sPLA2-HDL) on neutrophil function, an essential arm of the innate immune response and atherosclerosis. Treatment of neutrophils with sPLA2-HDL rapidly prevented agonist-induced neutrophil activation, including shape change, neutrophil extracellular trap formation, CD11b activation, adhesion under flow and migration of neutrophils. The cholesterol-mobilizing activity of sPLA2-HDL was markedly increased when compared to native HDL, promoting a significant reduction of cholesterol-rich signaling microdomains integral to cellular signaling pathways. Moreover, sPLA2-HDL effectively suppressed agonist-induced rise in intracellular Ca²⁺ levels. Native HDL showed no significant effects and removing lysophospholipids from sPLA2-HDL abolished all anti-inflammatory activities. Overall, our studies suggest that the increased cholesterol-mobilizing activity of sPLA2-HDL and suppression of rise in intracellular Ca²⁺ levels are likely mechanism that counteracts agonist-induced activation of neutrophils. These counterintuitive findings imply that neutrophil trafficking and effector responses are altered by sPLA2-HDL during inflammatory conditions.

Phosphoinositide-dependent protein kinase 1 (PDK1) mediates potent inhibitory effects on eosinophils.

Prostaglandin E2 (PGE2) protects against allergic responses via binding to prostanoid receptor EP4, which inhibits eosinophil migration in a PI3K/PKC‐dependent fashion. The phosphoinositide‐dependent protein kinase 1 (PDK1) is known to act as a downstream effector in PI3K signaling and has been implicated in the regulation of neutrophil migration. Thus, here we elucidate whether PDK1 mediates inhibitory effects of E‐type prostanoid receptor 4 (EP4) receptors on eosinophil function. Therefore, eosinophils were isolated from human peripheral blood or differentiated from mouse BM. PDK1 signaling was investigated in shape change, chemotaxis, CD11b, respiratory burst, and Ca2+ mobilization assays. The specific PDK1 inhibitors BX‐912 and GSK2334470 prevented the inhibition by prostaglandin E2 and the EP4 agonist ONO‐AE1‐329. Depending on the cellular function, PDK1 seemed to act through PI3K‐dependent or PI3K‐independent mechanisms. Stimulation of EP4 receptors caused PDK1 phosphorylation at Ser396 and induced PI3K‐dependent nuclear translocation of PDK1. EP4‐induced inhibition of shape change and chemotaxis was effectively reversed by the Akt inhibitor triciribine. In support of this finding, ONO‐AE1‐329 induced a PI3K/PDK1‐dependent increase in Akt phosphorylation. In conclusion, our data illustrate a critical role for PDK1 in transducing inhibitory signals on eosinophil effector function. Thus, our results suggest that PDK1 might serve as a novel therapeutic target in diseases involving eosinophilic inflammation.

Altered Inhibitory Function of the E-Type Prostanoid Receptor 4 in Eosinophils and Monocytes from Aspirin-Intolerant Patients

Prostaglandin (PG) E2 has been implicated in the pathogenesis of aspirin-exacerbated respiratory disease (AERD). E-type prostanoid (EP) receptor 4 is known to confer inhibitory signals to eosinophils and monocytes, amongst others. In this study, we investigated whether the responsiveness of eosinophils and monocytes to PGE2 and EP4 receptor activation is altered in AERD patients. While the expression of the EP4 receptor in eosinophils was unaltered in AERD patients, inhibition of eosinophil chemotaxis by PGE2 or the EP4 agonist CAY10598 was less pronounced in AERD patients as compared to healthy control subjects. In monocytes, we found no changes in basal or lipopolysaccharide (LPS)-stimulated PGE2 synthesis, but the response to EP4 receptor activation with respect to inhibition of LPS-induced tumor necrosis factor-α release was reduced in AERD patients, especially in the presence of aspirin (acetylsalicylic acid). Our data point towards a decreased sensitivity of inhibitory EP4 receptor that may play a role in AERD.

Contents Vol. 94, 2014

C. Antoniou, Athens J. Ahrens, Hannover L.Z. Benet, San Francisco, Calif. M.L. Billingsley, Hershey, Pa. A. Breckenridge, London K.K. Burkhart, Silver Spring, Md. G. Coruzzi, Parma S. Dhein, Leipzig T.L. Goodfriend, Madison, Wisc. L.S. Harris, Richmond, Va. M. Hirafuji, Ishikari-Tobetsu M.D. Hollenberg, Calgary, Alta. M. Inui, Yamaguchi K. Ishii, Yamagata C. Joukhadar, Boston, Mass. Y. Kamisaki, Osaka N. Kaplowitz, Los Angeles, Calif. A. Kawabata, Osaka A. Levitzki, Jerusalem B.R. Lucchesi, Ann Arbor, Mich. W.E. Müller, Frankfurt am Main N.H. Neff , Columbus, Ohio J.P. O’Callaghan, Morgantown, W.Va. K. Pennypacker, Tampa, Fla. S. Saha, Kansas City, Mo. C. Scarpignato, Nantes C.D. Smith, Charlestown, S.C. E. Taira, Iwate C. Zeng, Chongqing City International Journal of Experimental and Clinical Pharmacology

The role of CB2 receptor ligands in human eosinophil function

Background Eosinophils play a key role in allergic diseases such as bronchial asthma and atopic dermatitis. A prominent feature of these diseases is the accumulation of eosinophils in inflamed tissue induced by several chemoattractants like prostaglandin (PG) D2 or eotaxins. After the discovery of the endocannabinoid system and investigation of several endogenous and synthetic ligands, evidence has accumulated that cannabinoids, especially CB2 receptor ligands, may play a major role in mediating inflammatory responses. Elevated levels of 2-arachidonoylglycerol (2AG; a CB1/CB2 agonist) were found in tissues of mouse models of allergic inflammation, suggesting a possible involvement in leukocyte recruitment.