Peripheral blood mononuclear cells preferentially activate 11-oxygenated androgens.

Lina Schiffer,Ildem Akerman,Punith Kempegowda,Wiebke Arlt,Angela E Taylor,Alicia Bossey,Dagmar Scheel-Toellner,Karl-Heinz Storbeck

doi:10.1530/eje-20-1077

Lina Schiffer, Ildem Akerman + Show 6 more

Open Access

https://doi.org/10.1530/eje-20-1077

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Abstract

ObjectiveAndrogens are important modulators of immune cell function. The local generation of active androgens from circulating precursors is an important mediator of androgen action in peripheral target cells or tissues. We aimed to characterize the activation of classic and 11-oxygenated androgens in human peripheral blood mononuclear cells (PBMCs).MethodsPBMCs were isolated from healthy male donors and incubated ex vivo with precursors and active androgens of the classic and 11-oxygenated androgen pathways. Steroids were quantified by liquid chromatography-tandem mass spectrometry. The expression of genes encoding steroid-metabolizing enzymes was assessed by quantitative PCR.ResultsPBMCs generated eight-fold higher amounts of the active 11-oxygenated androgen 11-ketotestosterone than the classic androgen testosterone from their respective precursors. We identified the enzyme AKR1C3 as the major reductive 17β-hydroxysteroid dehydrogenase in PBMCs responsible for both conversions and found that within the PBMC compartment natural killer cells are the major site of AKRC13 expression and activity. Steroid 5α-reductase type 1 catalyzed the 5α-reduction of classic but not 11-oxygenated androgens in PBMCs. Lag time prior to the separation of cellular components from whole blood increased serum 11-ketotestosterone concentrations in a time-dependent fashion, with significant increases detected from two hours after blood collection.Conclusions11-Oxygenated androgens are the preferred substrates for androgen activation by AKR1C3 in PBMCs, primarily conveyed by natural killer cell AKR1C3 activity, yielding 11-ketotestosterone the major active androgen in PBMCs. Androgen metabolism by PBMCs can affect the results of serum 11-ketotestosterone measurements, if samples are not separated in a timely fashion.Significance statementWe show that human peripheral blood mononuclear cells (PBMCs) preferentially activate 11-ketotestosterone rather than testosterone when incubated with precursors of both the classic and the adrenal-derived 11-oxygenated androgen biosynthesis pathways. We demonstrate that this activity is catalyzed by the enzyme AKR1C3, which we found to primarily reside in natural killer cells, major contributors to the anti-viral immune defense. This potentially links intracrine 11-oxygenated androgen generation to the previously observed decreased NK cell cytotoxicity and increased infection risk in primary adrenal insufficiency. In addition, we show that PBMCs continue to generate 11-ketotestosterone if the cellular component of whole blood samples is not removed in a timely fashion, which could affect measurements of this active androgen in routine clinical biochemistry.

Highlights

A critical step in androgen activation is the conversion of the androgen precursor androstenedione (A4) to the active androgen testosterone (T) [1, 2, 3], which occurs in the testes catalyzed by 17β-hydroxysteroid dehydrogenase type 3 (HSD17B3)
While we could not detect the expression of steroid 5α-reductase type 2 (SRD5A2) and 17β-hydroxysteroid dehydrogenase type 2 (HSD17B2) in any of the samples from 14 donors using our Quantitative PCR (qPCR) assay, we detected consistently high expression of steroid 5α-reductase type 1 (SRD5A1) in samples from all donors (n = 14) and detected 17β-hydroxysteroid dehydrogenase type 4 (HSD17B4) expression in samples from 6 of 14 donors
This indicates that SRD5A1 is responsible for the 5α-reduction we observe in our peripheral blood mononuclear cells (PBMCs) incubations with androgen substrates while HSD17B4 catalyzes the oxidative, inactivating 17β-hydroxysteroid dehydrogenase activity we observed (Fig. 1A), consistent with previously published findings [6]. 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) expression was detectable in samples from 13/14 donors, while HSD11B2 mRNA was not detected

Summary

Introduction

A critical step in androgen activation is the conversion of the androgen precursor androstenedione (A4) to the active androgen testosterone (T) [1, 2, 3], which occurs in the testes catalyzed by 17β-hydroxysteroid dehydrogenase type 3 (HSD17B3). A number of studies have shown gradual increases in T measured in serum, if the blood sample is left unseparated from cellular components for an increased period of time, using either immunoassays [19, 20, 21] or ultra-high performance liquid chromatographytandem mass spectrometry (LC-MS/MS) for steroid quantification [22, 23] One of those studies investigated the effect of time before separation on 11KT concentrations measured in serum and observed a much more pronounced increase in 11KT than T over time [23], which could have a differential impact on the measurement of circulating 11-oxygenated androgen concentrations

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