Testosterone suppresses phospholipase D, causing sex differences in leukotriene biosynthesis in human monocytes

Abstract

Sex disparities in inflammation have been reported, but the cellular and molecular basis for these discrepancies is unknown. Monocytes are central effector cells in immunity and possess high capacities to produce proinflammatory leukotrienes (LTs). Here, we investigated sex differences in the activation of 5-lipoxygenase (5-LO), the key enzyme in LT biosynthesis, in human peripheral monocytes. In cells from females, 5-LO product formation was 1.8-fold higher than in cells from males, as evaluated by HPLC. When female monocytes were resuspended in plasma from males, 5-LO products were significantly lower than in female plasma. Interestingly, 5α-dihydrotestosterone (5α-DHT, 10 nM) repressed LT synthesis in female cells down to the levels observed in males, while estradiol (100 nM) was without effect, and progesterone (100 nM) caused only a slight inhibition. 5α-DHT (10 nM) caused ERK phosphorylation and inhibition of phospholipase D (PLD), as evaluated by Western blot and measurement of PLD activity via radioenzymatic diacylglyceride (DAG) and nonradioactive choline assays. Accordingly, PLD activity and DAG formation were 1.4- to 1.8-fold lower in male vs. female monocytes connected to increased ERK phosphorylation. Our data indicate that ERK activation by androgens in monocytes represses PLD activity, resulting in impaired 5-LO product formation due to lack of activating DAGs.

Oxylipid Profile of Low‐Dose Aspirin Exposure: A Pharmacometabolomics Study

“…We also observed higher levels of 5‐LOX products (namely, the eicosapentaenoic acid–derived 5S‐HEPE, the LA‐derived 9‐HODE, the AA‐derived 5‐HETE, and the α‐linolenic acid–derived 9‐HOTrE) in women. This is in accordance with previous findings that androgens downregulate 5‐LOX product formation 9, 10, 37, 38. Most of these constitutive differences in oxylipid levels persisted after aspirin treatment and no sex‐specific effect of aspirin on oxylipid was observed, consistent with our previous findings in this population 4, 9, 11, 12, 39…”

Selective upregulation of TNFα expression in classically-activated human monocyte-derived macrophages (M1) through pharmacological interference with V-ATPase

“…Peripheral blood mononuclear cells (PBMC) were isolated by dextran sedimentation and density centrifugation on lymphocyte separation medium (LSM 1077, PAA Laboratories, Pasching, Austria). Monocytes from the PBMC fraction were isolated by adherence for 1 hr at 37 °C and 5% CO2 to culture flasks (2  10 7 cells/mL RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin) [24].…”

Targeting V-ATPase in primary human monocytes by archazolid potently represses the classical secretion of cytokines due to accumulation at the endoplasmic reticulum

“…Platelets and peripheral blood mononuclear cells (PBMC) were isolated by dextran sedimentation and centrifugation on LSM 1077 lymphocyte separation medium (PAA Laboratories, Pasching, Germany), as described [19,20]. To obtain monocytes, PBMC were collected, washed two times with cold PBS, and monocytes were separated by adherence for 1.5 h at 37 °C to culture flasks (210 7 cells/mL RPMI 1640 medium containing 2 mM L-glutamine and 100 U/mL penicillin plus 100 µg/mL streptomycin).…”

“…Cells were preincubated with compounds or DMSO (30 min, 37 °C), 2% human serum was added and cells were stimulated with LPS (100 ng/mL) for 5 min (p38 MAPK, ERK-1/2, Akt, SAPK/JNK) or 15 min (IκB). For analysis of BiP expression, monocytes were plated in monocyte medium, pre-incubated with compounds or DMSO (30 min, 37 °C) and stimulated with LPS (1 µg/ml) for 24 h. Cells were placed on ice, washed twice with PBS and lysed by NP-40 (1%) as described [19]. Lysates were centrifuged (10,000g, 4 °C, 5 min), and protein concentrations in the supernatants were determined using a Protein Assay (Bio-Rad Laboratories, CA).…”