Brain microglia express steroid-converting enzymes in the mouse

Abstract

In the CNS, steroid hormones play a major role in the maintenance of brain homeostasis and it's response to injury. Since activated microglia are the pivotal immune cell involved in neurodegeneration, we investigated the possibility that microglia provide a discrete source for the metabolism of active steroid hormones. Using RT-PCR, our results showed that mouse microglia expressed mRNA for 17beta-hydroxysteroid dehydrogenase type 1 and steroid 5alpha-reductase type 1, which are involved in the metabolism of androgens and estrogens. Microglia also expressed the peripheral benzodiazepine receptor and steroid acute regulatory protein; however, the enzymes required for de novo formation of progesterone and DHEA from cholesterol were not expressed. To test the function of these enzymes, primary microglia cultures were incubated with steroid precursors, DHEA and AD. Microglia preferentially produced delta-5 androgens (Adiol) from DHEA and 5alpha-reduced androgens from AD. Adiol behaved as an effective estrogen receptor agonist in neuronal cells. Activation of microglia with pro-inflammatory factors, LPS and INFgamma did not affect the enzymatic properties of these proteins. However, PBR ligands reduced TNFalpha production signifying an immunomodulatory role for PBR. Collectively, our results suggest that microglia utilize steroid-converting enzymes and related proteins to influence inflammation and neurodegeneration within microenvironments of the brain.

Fig. 1. Purity of in vitro cultures of primary microglia (1°MG) from cfms-EGFP mice

Purity of 1°MG cultures obtained from cfms-EGFP neonatal (day 2) mice was verified by fluorescence microscopy (A–B) for expression of EGFP, as well as by FACS analysis using EGFP expression and staining with the macrophage marker CD11b (C). Isotope control staining is shown in (D). Scale bar = 20µM.

Fig. 2. Expression summary of steroid converting proteins in microglia

Diagram of the steroidogenic pathway showing the main steroidogenic enzymes and their steroid substrates. The boxed enzymes are those expressed in resting microglia, the shadowed ones were not expressed.

Fig. 3. In vivo LPS stimulation reduces steroidogenic enzyme expression in ex vivo microglia

RT-PCR analysis of steroidogenic enzymes in FACS-sorted microglia from adult mice treated for 24hr with an IP injection of saline (hatched bars) or 1mg/kg LPS (open bars). Bars represent the % expression of each enzyme with respect to control samples and normalized to the housekeeping gene L27A. Values are the mean ± the SEM of three animals. *, P < 0.05 vs. control.

Fig. 4. LPS+INFγ increases the expression of steroidogenic enzymes in cultured 1°MG

RT-PCR analysis of steroidogenic enzymes in cultured 1°MG. A–D) 1°MG cells were stimulated for 24hr with 100ng/ml LPS+ 10ng/ml INFγ (LPS), 10ng/ml INFγ (INFγ), LPS-conditioned media (LCM), 1mM db-cAMP (db-cAMP), or vehicle (Veh) (hatched bars). Bars represent the % expression of each enzyme with respect to vehicle and normalized to the housekeeping gene L27A. Values are the mean ± the SEM of 2–3 independent experiments done in triplicate. *, **, P < 0.05, P < 0.001 vs. Veh, respectively.

Fig. 5. Metabolism of H³-DHEA by 1°MG

TLC analysis of H³-DHEA metabolism in 1°MG. Cells were incubated for 24hr with H³-DHEA and metabolites extracted from culture supernatants were resolved by TLC and counted on a scintillation counter. A) Resting microglia (MG, open bars) showed a significant conversion of DHEA compared to the no-cell controls (Control, grey bars), with the only product being Adiol. B) LPS (100ng/ml LPS+ 10ng/ml INFγ) stimulation of the cells (hatched bars) didn’t alter the metabolic activity or profile of steroids produced from DHEA in microglia. Bars represent the % of total H³ radioactivity collected from the TLC assay. Values are the mean ± the SEM of 2–3 independent experiments done in triplicate. ***, P < 0.0001 vs. MG.

Fig. 6. 1°MG convert H³-AD into downstream steroids

TLC analysis of H³-AD metabolism in 1°MG. Cells were incubated for 24hr with H³-AD and metabolites extracted from culture supernatants were resolved by TLC and counted on a scintillation counter. A) Resting microglia (MG, open bars) showed a significant conversion of AD compared to the no-cell controls (Control, grey bars). The main products of AD were testosterone (T), 5αAndrostanedione (5αAD), and dihydrotestosterone (DHT). B) LPS (100ng/ml LPS+ 10ng/ml INFγ) stimulation (hatched bars) slightly decreased the conversion of AD compared to vehicle treated cells (open bars), but not the production of downstream steroids. Bars represent the % of total H³ radioactivity collected from the TLC assay. Values are the mean ± the SEM of 2–3 independent experiments done in triplicate. **,***, P < 0.001, P < 0.0001 vs. MG, respectively.

Fig. 7. PBR ligands selectively modulate TNFα production in 1°MG

Cells were incubated for 24hr with LPS (100ng/ml LPS+ 10ng/ml INFγ) with a 10-minute pre-treatment of the PBR ligands Ro (10pM) and PK (10pM), or 1µM corticosterone as positive control. Culture supernatants were assayed for TNFα (A), IL-6 (B), and nitric oxide (NO) by ELISA and Greiss assay, respectively. Bars represent the % of total cytokines detected in the LPS controls. Mean cytokine levels measured for the LPS+INFγ controls were: TNFα (32ng/ml), IL-6 (70ng/ml) and NO (35mM). Un-stimulated cells had no detectable cytokine production. Bars represent the mean ± S.E.M. of 2–3 independent experiments done in triplicate. *, P < 0.05 vs. LPS+INFγ.

Fig. 8. Adiol is an effective estrogen receptor agonist

Estrogen receptor luciferase reporter assay reflected by luciferase expression from an ERE-Luciferase containing plasmid transfected into the neuronal cell line EtC.1. Bars represent luciferase activity determined by luminometry in cells treated with increasing doses of estrogen (E2) (open bars) or Adiol (grey bars). The third group of columns is from cells pre-treated 30 min with 100nM ICI 182,780 and then treated with the high doses of E2 and Adiol (ICI). Values are given in fold over basal ± the SEM, from one representative experiment done in triplicate.