RELAZIONE ATTIVITA’ SCIENTIFICA ALL’ESTERO
Dott.ssa Simona Pace
(Università Federico II di Napoli)
Relazione finale - Simona Pace, Borse di ricerca per giovani Soci SIF. Istituzione di provenienza: Department of Pharmacy, University of Naples Federico II. Istituzione ospitante. Department of Pharmaceutical/Medicinal Chemistry (Friederich Schiller Universität Jena, Germany).
Since October, 2013 until October, 2014 I attended the laboratories of Prof. Dr. Oliver Werz at the Department of Pharmaceutical/Medicinal Chemistry (Friederich Schiller Universität Jena, Germany) to carry out my research project.
TITLE:SEX MODULATION OF LEUKOTRIENE BIOSYNTHESIS
INTRODUCTION
Leukotrienes (LTs) are bioactive lipid mediators which are produced during the inflammation process by different immune cells such as macrophages (MO), monocytes and neutrophils. They derive from arachidonic acid (AA) through the action of 5-lipoxygenase (5-LO), which converts AA to LTA4, representing the first step in the synthesis of all LTs [1]. In fact, LTA4 can be hydrolyzed to the chemotactic agent LTB4 by LTA4 hydrolase or conjugated with reduced glutathione by LTC4 synthase to form LTC4. This eicosanoidis then metabolized by sequential proteolytic hydrolysis to LTD4 and LTE4, which are together known as cysteinyl-LTs (cys-LTs) with vascular actions.
LT biosynthesis is regulated by different mechanisms and among them post-translational modifications of 5-LO, including phosphorylation at serine residues, Ca2+ binding, interactions with certain phospholipids and glycerides and interactions with 5-LO-activating protein and coactosin-like protein [1]. Also, LT synthesis is strongly influenced by the intracellular localization of 5-LO and the amount of trafficable enzyme. Thus, 5-LO is a soluble protein in the cytosol or nucleoplasm of resting cells, and translocates to perinuclear membranes when intracellular Ca2+ levels increase or/and after its phosphorylation by mitogen-activated protein kinase (MAPK) [2-5].
LTs play a pivotal role in various immune diseases (asthma, multiple sclerosis [6]) in which there is a well known sex bias. Recently, it has been shown the sex modulation of LT biosynthesis in human whole blood, human neutrophils and monocytes. In particular, after in vitro stimulation, the cells from female donors showed an higher production of LTs [7-8]. This effect was related to a down regulation of 5-LO products by testosterone. In particular male sex hormones repressed LT biosynthesis in human neutrophils and monocytes by affecting 5-LO localization via differential activation of extracellular signal-regulated kinase (ERK) [7] and repressing phospholipase D, respectively [8].
Then, we demonstrated also in vivo in an acute inflammatory model in which LT plays a pivotal role, such as mouse zymosan-induced peritonitis, that the inflammatory reaction (cell recruitment and vascular permeability), as well as LT biosynthesis were higher in female then male mice. This sex difference in LT production was due to a different subcellular localization of 5-LO in peritoneal macrophages (PM) of mice. In particular, differences in basal 5-LO localization (i.e., higher levels of nuclear 5-LO in unstimulated PM from males) resulted in a reduced trafficable 5-LO protein in stimulated male PM, correlating to lower 5-LO product synthesis versus female cells [9].
AIM
The aim of this project was to elucidate the role of testosterone in the observed sex biased LT biosynthesis. In particular, based on our previous findings, we focused on the effect of testosterone on mouse resident PM (cells responsible for the in vivo production of LTs in zymosan-induced peritonitis) and on human monocyte-derived macrophages.
METHODS
Zymosan-induced peritonitis in mice
Female adult CD1 mice (8 weeks) were treated intraperitoneally (i.p.) with a solution of 5α dihydrotestosterone (5α-DHT 0.5 mg/kg, Sigma Aldrich) or vehicle (DMSO 2%) 30 minutes before peritonitis induction. In particular, peritonitis was induced by i.p. injection (0.5 mL) of zymosan (2 mg/mL in saline, boiled and washed). At selected time points after zymosan injection (0, 15, 30, 240 min), mice were euthanized and peritoneal exudates were collected using 2 mL of PBS. Supernatants were collected and frozen at -80°C for eicosanoid analysis. Levels of LTC4 and 5-HETE in the peritoneal exudates were measured by Ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) after solid phase extraction.
Solid phase extraction (SPE)
The solid phase extraction was performed with RP18-columns containing a polymeric reversed phase. The sample was added after conditioning the column with 1 mL 100% Methanol and 1 mL H2O. After washing the columns two times with 0.5 mL H2O, eicosanoids were eluted with 300 μL 100% Methanol. The supernatant obtained after two centrifugation steps (15000 x g, 5 min, 4°C) was used for the UPLC-MS/MS run.
Determination of eicosanoid production by UPLC-MS/MS
UPLC-MS/MS analyses were carried out on an Acquity UPLC BEH C18 column (1.7 μm, 2.1×50 mm, Waters, Milford, MA, USA) using an AcquityTM UPLC system (Waters) and a QTRAP 5500 Mass Spectrometer (AB Sciex, Darmstadt, Germany) equipped with a Turbo V™ Source and electrospray ionization (ESI) probe. Cell products (4 μL injection) were separated at a flow rate of 0.8 mL/min and a column temperature of 45 °C. The solvents for the mobile phase were water/acetonitrile (90/10; solvent A) and acetonitrile (solvent B) both acidified with 0.07% (v/v) formic acid. Isocratic elution at A/B=30% was performed for 2 min, and followed by a linear gradient to 70% B within 5 min. HPLC solvents were from VWR (Darmstadt, Germany). Lipid mediators were detected by multiple reaction monitoring in the negative ion mode using a dwell time of 10 ms. The ion spray voltage was set to 4500 V, the heater temperature to 500 °C, the declustering potential to 50–120 eV, the entrance potential to 10 eV and the collision cell exit potential to 11–22 eV, the spray gas pressure to 50 psi, the Turbo V gas pressure to 80 psi and the curtain gas pressure to 20 psi.
UPLC-MS/MS analysis
Automatic peak integration was performed with Analyst 1.6 software (AB Sciex, Darmstadt, Germany) using IntelliQuan default settings. Data were normalized on the internal standard PGB1.
Peritoneal macrophages
Resident PM were obtained by lavage of the peritoneal cavity of male and female CD1 mice (8 weeks) with 7 mL of cold Dulbecco’s modified Eagles medium (DMEM) with heparin (5 U m/L). PM were then centrifuged at 500 ´ g 4°C for 5 min, resuspended at 1´107cells/mL and 0.5 mL of cell suspension were added to a 15 mL conical polypropylene tube for each experimental condition. Cells from female mice were then incubated at 37°C with 5α-DHT (10 nM), or vehicle (DMSO 0.5%) for 15 minutes, while cells from male mice where incubated at 37°C with β-estrogen (100nM) or vehicle (DMSO 0.5%) for 15 minutes. Then, PM were stimulated with ionophore A23187 (4.5 mL, final concentration 2.5 mM, 15 min), EDTA at a final concentration of 4 mM was added at the end of incubation time to A23187-stimulated cells. All samples were then centrifuged at 500 ´ g at 4°C for 5 min. Supernatants were collected and frozen at -80°C for measurement of LTC4 by EIA (Cayman Chemical Company; Aurogene, Rome, Italy).
Isolation of monocytes and differentiation in macrophages
For the isolation of human monocytes, peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats (Institute of Transfusion Medicine at University Hospital Jena, Germany) from male and female donors as described previously [8]. Then, monocytes were isolated by adherence for 1 h at 37°C to culture flask (20x106/mL of RPMI). Finally male monocytes were seeded in culture flasks at the concentration of 106/mL of RPMI containing 5% of stripped fetal calf serum (FCS) in presence or absence of 5α-DHT (30nM), while, female monocytes were seeded with 5% of stripped FCS in presence or absence of β-estrogen (30nM). Macrophage colony stimulating factor (M-CSF) at the concentration of 25 ng/ml was added in the culture medium for six days to differentiate monocytes in macrophages (MO).
Macrophage polarization
After six days, MO were scraped and plated for two hours in 24/well plate at the concentration of 4 x 105 to adhere for two hours in culture medium in presence or absence of sexual hormones. Then, female and male MO were polarized to the M1 (principal effectors of the inflammatory process [10]) phenotype by treatment for 24 hours with LPS (100 ng/mL) and IFN-y (100 ng/mL). To induce the production of LTs, M1 were stimulated with A23187 (2.5 µM, 10 min 37°C) in presence or absence of AA (10µM, 10 min 37°C) in PBS containing 0.1 % glucose and 1mM CaCl2. Supernatants, were then analyzed by UPLC-MS/MS after SPE.
RESULTS
Effect of 5α-DHTon 5-LO products in zymosan-induced peritonitis in mice
To assess if the sex difference in LT biosynthesis observed in vivo was due to testosterone, we pre-treated female mice with 5α-DHT (0.5 mg/kg) or vehicle (DMSO 2%) before zymosan injection. According to our previous findings [9], LTC4 was the main 5-LO metabolite produced during the peritonitis model with a slight increase within the first 30 min and a sharp reduction after 60 min. At 30 min, mice pre-treated with 5α-DHT produced significantly (p<0.001) less LTC4 compared to mice pre-treated with vehicle. Moreover, 5α-DHT was able to reduce also the production of another 5-LO metabolite, 5-HETE at the same time points.
Effect of 5α-DHTon 5-LO products in resident peritoneal macrophages
During the first 60 min after zymosan injection, PM are the cells responsible for LT production in the peritoneal cavity [11]. Thus, to verify if the sex difference in LT production observed in vivo was due to an effect of sexual hormones on these cells, we collected PM from male and female mice and stimulated them in vitro with A23187 after a pre-treatment with sexual hormones. 5α-DHT (10nM) well as β-estrogen (100nM)did not make any effect on LTC4 production in PM.
LTB4 biosynthesis in human monocyte-derived M1 macrophages
Monocyte-derived M1 macrophages were selected as in vitro model to study if sexual hormones act directly on macrophages or during the differentiation process. First, we evaluated if there is a sex biased LTB4 (the main 5-LO product in this experimental condition)formation by stimulation of monocyte-derived M1 macrophages grown in standard condition (without addition of sexual hormones in the medium). The stimulation of M1 with A23187 alone or in combination with AA produced a significant increase of LTB4 production (p<0.001) in both sexes. No sex difference was evident after the stimulation.
Sex differences in LTB4 biosynthesis in human monocyte-derived M1 macrophages cultured with sexual hormones
Next, to keep the monocytes under the stimulus of the sexual hormones, during the differentiation and polarization process, in the culture medium of male monocytes was added 5α-DHT 30 nM, while, in the culture medium of female monocytes was added 30 nM β-estrogen. After stimulation of monocyte derived M1 with A23187, female showed higher levels of LTB4 compared to male.
CONCLUSIONS
Our data show that sexual hormones play a pivotal role on the sex-related difference in LT biosynthesis during the in vivo differentiation of monocytes in macrophages. In particular, the absence of the effect of 5α-DHT on PM led us to think that macrophages are not susceptible themselves to testosterone activity, while this hormone acts on these cells when they are monocytes. In fact, when human monocytes were differentiated in macrophages for six days in absence of hormones, the sex difference in LTB4 production was abolished. On the contrary, when the sexual hormones were added to the culture medium during the polarization and differentiation process, after stimulation with A23187 there was a sex difference in LTB4 production. Future experiments will focus on the elucidation of the mechanism of action of testosterone during differentiation.
REFERENCES
[1] Rådmark O, Werz O, Steinhilber D, Samuelsson B. 5-Lipoxygenase: regulation of expression and enzyme activity. Trends BiochemSci 2007;32:332-4.
[2] Funk CD. Prostaglandins and leukotrienes: advances in eicosanoid biology. Science 2001;294:1871-5.
[3] Peters-GoldenM, Brock TG.Intracellular compartmentalization of leukotriene biosynthesis. Am J RespirCrit Care Med 2000;161:S36-40.
[4] WerzO, Klemm J, Samuelsson B, Rådmark O. Phorbol ester up-regulates capacities for nuclear translocation and phosphorylation of 5-lipoxygenase in Mono Mac 6 cells and human polymorphonuclear leukocytes. Blood 2001;97:2487-95.
[5] Werz O, Bürkert E, Fischer L, Szellas D, Dishart D, Samuelsson B, Rådmark O, Steinhilber D. Extracellular signal-regulated kinases phosphorylate 5-lipoxygenase and stimulate 5-lipoxygenase product formation in leukocytes. FASEB J 2002;16:1441-3.
[6] Whitacre C C. Sex differences in autoimmune disease. Nat Med 2001;2:777-80.
[7] Pergola C, Dodt G, Rossi A, Neunhoeffer E, Lawrenz B, Northoff H, Samuelsson B, Rådmark O, Sautebin L, Werz O. ERK-mediated regulation of leukotriene biosynthesis by androgens: a molecular basis for gender differences in inflammation and asthma.ProcNatlAcadSci U S A 2008;105:19881-6.
[8] Pergola C, Rogge A, Dodt G, Northoff H, Weinigel C, Barz D, Rådmark O, Sautebin L, Werz O. Testosterone suppresses phospholipase D, causing sex differences in leukotriene biosynthesis in human monocytes. FASEB J 2011;25:337787.
[9] Rossi A, Pergola C, Pace S, Rådmark O, Werz O, Sautebin L.In vivo sex differences in leukotriene biosynthesis in zymosan-induced peritonitis.Pharmacol Res. 2014 Sep;87:1-7.
[10] Bystrom J, Evans I, Newson J, Stables M, Toor I, van Rooijen N, Crawford M, Colville-Nash P, Farrow S, Gilroy DW.Resolution-phase macrophages possess a unique inflammatory phenotype that is controlled by cAMP. Blood, 2008; 112(10):4117-27.
[11] Kolaczkowska E, Shahzidi S, Seljelid R, van Rooijen N, Plytycz B. Early vascular permeability in murine experimental peritonitis is co-mediated by resident peritoneal macrophages and mast cells: crucial involvement of macrophage-derived cysteinyl-leukotrienes.Inflammation 200