R E S E A R CH AR T I C L E

Suppressed oligodendrocyte steroidogenesis inmultiple sclerosis: Implications for regulation ofneuroinflammation

Roobina Boghozian1,2 | Brienne A. McKenzie1 | Leina B. Saito1 | Ninad Mehta1 |

William G. Branton3 | JianQiang Lu4 | Glen B. Baker5 | Farshid Noorbakhsh2 |

Christopher Power1,3,5

1Department of Medical Microbiology &

Immunology, University of Alberta

Edmonton, Alberta, Canada

2Department of Medical Microbiology &

Immunology, School of Medicine, Tehran

University of Medical Sciences, Tehran, Iran

3Department of Medicine, University of

Alberta Edmonton, Alberta, Canada

4Department of Laboratory Medicine &

Pathology, University of Alberta Edmonton,

Alberta, Canada

5Depatment of Psychiatry, University of

Alberta Edmonton, Alberta, Canada

Correspondence

Christopher Power, Division of Neurology,

HMRC 6-11, University of Alberta,

Edmonton, Alberta.

Email: chris.power@ualberta.ca

or

Farshid Noorbakhsh, Department of

Immunology, Tehran University of Medical

Sciences, Tehran Iran.

Email: f-noorbakhsh@tums.ac.ir

AbstractMultiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system

(CNS). Neurosteroids are reported to exert anti-inflammatory effects in several neurological disor-

ders. We investigated the expression and actions of the neurosteroid, dehydroepiandrosterone

(DHEA), and its more stable 3b-sulphated ester, DHEA-S, in MS and associated experimental

models. CNS tissues from patients with MS and animals with experimental autoimmune encepha-

lomyelitis (EAE) displayed reduced DHEA concentrations, accompanied by diminished expression

of the DHEA-synthesizing enzyme CYP17A1 in oligodendrocytes (ODCs), in association with

increased expression of inflammatory genes including interferon (IFN)-g and interleukin (IL)-1b.

CYP17A1 was expressed variably in different human neural cell types but IFN-g exposure selec-

tively reduced CYP17A1 detection in ODCs. DHEA-S treatment reduced IL-1b and 26 release

from activated human myeloid cells with minimal effect on lymphocyte viability. Animals with EAE

receiving DHEA-S treatment showed reduced Il1b and Ifng transcript levels in spinal cord com-

pared to vehicle-treated animals with EAE. DHEA-S treatment also preserved myelin basic protein

immunoreactivity and reduced axonal loss in animals with EAE, relative to vehicle-treated EAE

animals. Neurobehavioral deficits were reduced in DHEA-S-treated EAE animals compared with

vehicle-treated animals with EAE. Thus, CYP17A1 expression in ODCs and its product DHEA

were downregulated in the CNS during inflammatory demyelination while DHEA-S provision

suppressed neuroinflammation, demyelination, and axonal injury that was evident as improved

neurobehavioral performance. These findings indicate that DHEA production is an immunoregula-

tory pathway within the CNS and its restoration represents a novel treatment approach for

neuroinflammatory diseases.

K E YWORD S

CYP17A1, DHEA, EAE, multiple sclerosis, neuroinflammation, neurosteroid

1 | INTRODUCTION

Multiple sclerosis (MS) is a debilitating disease of the central nervous

system (CNS) which affects motor, sensory, visual, bowel, bladder, and

mental functions in people during the prime of life. The cause of MS is

unknown although it is widely regarded as a progressive inflammatory

demyelinating disease leading to neurodegeneration over time (Ellwardt

& Zipp, 2014; Herz, Zipp, & Siffrin, 2010; Noorbakhsh, Overall, & Power,

2009). Axonal damage and neuronal cell body injury and loss accompany

leukocyte infiltration and demyelination. Imaging studies suggest that

axonal injury associated with cerebral atrophy constitutes a major mech-

anism by which physical disability evolves during MS progression (Lee,

Biemond, & Petratos, 2015). The contribution of immunopathogenesis

as a determinant of MS is supported by large-scale genetic studies

1590 | VC 2017Wiley Periodicals, Inc. wileyonlinelibrary.com/journal/glia Glia. 2017;65:1590–1606.

Received: 24 August 2016 | Revised: 26 May 2017 | Accepted: 26 May 2017

DOI: 10.1002/glia.23179

showing linkages to multiple immune genes, especially to the major his-

tocompatability complex (MHC) locus on chromosome 6 (Caillier et al.,

2008; Mangalam, Rajagopalan, Taneja, & David, 2008). Several environ-

mental risk factors implicated in MS include reduced sunlight exposure

and vitamin D3 serum levels as well as cigarette smoking and specific

infections (Matias-Guiu, Oreja-Guevara, Matias-Guiu, & Gomez-Pinedo,

2016; Morandi, Tarlinton, & Gran, 2015; Watad et al., 2016). Both

innate and adaptive immunity participate in the pathogenesis of MS and

its established model, experimental autoimmune encephalomyelitis

(EAE), as indicated by the presence of activated lymphocytes and

increased cytokine and chemokine production by macrophages, micro-

glia and astrocytes (Gomez Perdiguero, Schulz, & Geissmann, 2013;

Grebing et al., 2016; Hendriks, Teunissen, de Vries, & Dijkstra, 2005;

Mayo, Quintana, & Weiner, 2012). Mechanisms of demyelination and

ensuing neurodegeneration (axonal and neuronal damage) remain uncer-

tain although inflammatory molecules including cytokines, chemokines,

prostaglandins, reactive oxygen species (Caruso et al., 2014; Lassmann,

2014) and proteases have been implicated in demyelination and axonal/

neuronal injury (Huber & Irani, 2015; Lam et al., 2016; Radbruch et al.,

2016; Takahashi, Giuliani, Power, Imai, & Yong, 2003). Several

mechanisms by which inflammatory mediators contribute to cytotoxicity

have been identified, including exacerbation of glutamate excitotoxicity

by proinflammatory cytokines, damage to DNA, lipids, and proteins by

ROS, and induction of apoptosis by death-receptors ligands (Kharel,

McDonough, & Basu, 2016; Ohl, Tenbrock, & Kipp, 2016; Sulkowski,

Dabrowska-Bouta, Kwiatkowska-Patzer, & Struzynska, 2009).

Steroid hormones are produced in the healthy CNS, exerting their

effects by binding to intracellular receptors that translocate to the

nucleus and bind to response elements in regulatory promoter regions

of specific genes. Multiple studies demonstrate that steroid molecules

also bind to specific neurotransmitter receptors and alter neuronal

excitability (Campbell & Herbison, 2014; Carver & Reddy, 2013;

Ishihara et al., 2016). Steroid molecules that act as neuromodulators in

this manner are termed neurosteroids and include pregnanolone,

progesterone, pregnenolone, pregnenolone sulphate (pregnenolone-S),

dehydroepiandrosterone (DHEA), and dehydroepiandrosterone sul-

phate (DHEA-S) and a number of 3a-reduced neurosteroids including

allopregnanolone (Figure 1A) (Shulman & Tibbo, 2005). Neurosteroids

originate as circulating steroid hormones or are produced locally within

the brain from cholesterol (Baulieu, Robel, & Schumacher, 2001).

Neurosteroids are synthesized within multiple cell types including

neurons, oligodendrocytes and astrocytes (Mellon & Deschepper,

1993; Plassart-Schiess & Baulieu, 2001). Herein, we use the terms

neurosteroid and neuroactive steroid interchangeably although we

recognize that not all neurosteroids are neuroactive.

Neurosteroids act as allosteric modulators of neurotransmitter

receptors, including g-aminobutyric acid (GABA), glutamate, serotonin

(5-HT), and r1 opioid receptors (Puthenkalam et al., 2016). Growing

evidence suggests involvement of neurosteroids in the pathophysiol-

ogy and pharmacotherapy of a number of neurological disorders (Dury,

Ke, & Labrie, 2016). Indeed, several neurosteroids and synthetic deriva-

tives have been tested in clinical trials with benefits in diseases such as

epilepsy, and were well tolerated by patients (Reddy & Estes, 2016).

Awareness is growing of the importance of steroid biology in the brain

and links to pathways implicated in inflammation and cell survival

(Arbo, Benetti, & Ribeiro, 2016; Chatzopoulou et al., 2016; El-Etr et al.,

2015; Gorska, Kuban-Jankowska, Milczarek, & Wozniak, 2016;

Hirahara et al., 2013; Patel & Katyare, 2006; Weng & Chung, 2016). In

previous studies, the neurosteroids, allopregnanolone and its synthetic

derivative ganaxolone, displayed anti-inflammatory and cytoprotective

actions in models of MS (Noorbakhsh et al. 2011, Paul et al., 2014).

Multiple reports describe robust anti-inflammatory and neuroprotective

effects for DHEA and DHEA-S using in vitro and in vivo models of

neuroinflammation (Aggelakopoulou et al., 2016; Du, Khalil, & Sriram,

2001; Traish, Kang, Saad, & Guay, 2011).

In this study, we investigated the involvement of the DHEA-

associated neurosteroid pathway (Figure 1A) in MS and associated

experimental models. CNS tissues from patients with MS and animals

with EAE displayed reduced DHEA levels and diminished expression of

the DHEA-synthesizing enzyme, CYP17A1. In vitro CYP17A1 expres-

sion was detected in several human neural cell types but exposure

to IFN-g, a proinflammatory T cell-derived cytokine known to be

pathogenic in MS/EAE, significantly suppressed CYP17A1 expression

in oligodendrocytes (ODCs). Treatment with the 3b-sulphate ester of

DHEA (DHEA-S) reduced in vitro inflammatory gene expression,

enhanced in vivo axonal and myelin preservation, and improved

neurobehavioral outcomes in EAE. These findings indicated that the

DHEA synthesis pathway is an important regulatory component of MS

pathogenesis and might be directed to restore neurological functions in

patients with MS.

2 | METHODS AND MATERIALS

2.1 | Ethics statement

The use of autopsied brain tissues was approved (Pro0002291) by the

University of Alberta Human Research Ethics Board (Biomedical), and

written informed consent documents were signed for all autopsied brain

tissues including frontal cortex and white matter collected from age- and

sex-matched subjects, nonMS patients (n514) and MS patients (n510)

with disease phenotypes (Supporting Information Table 1), samples

stored at 2808C. Human fetal tissues were obtained from 15 to 20

week aborted fetuses which collected with the written informed consent

of the donor (Pro00027660), as approved by the University of Alberta

Human Research Ethics Board (Biomedical). All animal experiments were

approved by the University of Alberta Health Sciences Animal Care and

Use Committee (Pro 00000317). All animals were housed and monitored

on a regular schedule, and experiments were performed according to

the Canadian Council on Animal Care (http://www.ccac.ca/en) and local

animal care and use committee guidelines.

2.2 | Neurosteroid analyses

Gas chromatography-mass spectrometry (GC-MS) analysis was used to

measure levels of DHEA and pregnenolone in CNS tissues, using a

BOGHOZIAN ET AL. | 1591

modification of a procedure we described previously (Noorbakhsh et al.,

2011). Protein from tissues and cell culture supernatants were precipi-

tated by the addition of methanol followed by centrifugation. The super-

natant was retained, and the steroids were isolated using solid-phase

extraction with Oasis 30-mg HLB plates (Waters, Mississauga, ON,

Canada). The samples were then eluted with dichloromethane: methanol

(90:10 v/v), taken to dryness, and derivatized with heptafluorobutyryli-

midazole (Fisher, Mississauga, ON, Canada) and the resultant derivatives

FIGURE 1 Cyp17A1 and DHEA levels are reduced in MS white matter. (a) Neuroactive steroid synthesis pathway. (b) Pregnenolone levels weresimilar in MS and nonMS patients’ cortex and white matter but (c) DHEA levels were suppressed in white matter from MS patients compared to

nonMS patients as measured by GC-MS. (d) CYP17A1 transcript levels were reduced in MS white matter, assessed by RT-PCR while CD3E, (e),IFNG (f) and IL1B (g) transcript levels were induced in white matter from MS (n56) compared to nonMS (n56) patients. (Student t-test, *p<0.05)

1592 | BOGHOZIAN ET AL.

were analyzed by GC combined with negative ion chemical ionization

MS (an Agilent 6890 GC coupled to a 5973N mass selective detector;

Agilent Technologies, Santa Clara, CA). Standard curves were run in par-

allel with each assay.

2.3 | Cell cultures

Human fetal astrocytes, neurons and microglia were isolated based on

differential culture conditions, as previously described (Afkhami-Goli

et al., 2007; Walsh et al., 2014). Briefly, fetal brain tissues were

dissected, meninges were removed, and a single cell suspension was

prepared through enzymatic digestion for 60 min with trypsin (2.5%)

and DNase I (0.2 mg/ml), followed by passage through a 70-lm cell

strainer. Cells were washed twice with fresh medium and plated in T-

75 flasks (flasks were coated with poly-L-ornithine for neurons culture)

at 6 to 8 3 107 cells/flask. Cultures were maintained in MEM supple-

mented with Earle Salts, FBS (10%), L-glutamine (2 mM), sodium pyru-

vate (1 mM), nonessential amino acids (1%), dextrose (0.1%), penicillin

(100 U/ml), streptomycin (100 lg/ml), amphotericin B (0.5 lg/ml), and

gentamicin (20 lg/ml). For microglial cells, mixed cultures were main-

tained for 1–2 weeks at which point astrocytes and neurons formed an

adherent cell layer with microglia loosely attached or free floating in

the medium. Cultures were gently rocked for 20 min to suspend the

weakly adhering microglia in medium, which were then collected,

washed and plated. Astrocyte cultures were passaged using trypsin

(0.25%) EDTA (1 mM) once cultures were confluent for 4–6 times until

the neurons were eliminated. Purity of cultures was verified as previ-

ously reported by our group (Afkhami-Goli et al., 2007). Primary human

fetal neurons were prepared using the same method but were grown

in the presence of cytosine arabinoside (25 lM) (Sigma-Aldrich) and

L-ornithine coated flasks. Human MO3.13 cells (ODC cell line)

(McLaurin, Trudel, Shaw, Antel, & Cashman, 1995; Buntinx et al., 2003)

were cultured in DMEM, supplemented with glucose, L-glutamine,

sodium bicarbonate (Sigma-Aldrich) with FBS (10%), penicillin (100

U/ml) and streptomycin (100 lg/ml). Cells were differentiated for 4

days using DMEM supplemented with phorbol 12-myristate 13-

acetate (PMA) (100 nM), penicillin (100 U/ml) and streptomycin

(100 lg/ml) (Buntinx et al., 2003). Human peripheral blood lymphocyte

(PBL) samples were prepared from healthy volunteers as previously

reported (Power et al., 1998). Then, cells were isolated by Ficoll-

Hypaque (Sigma-Aldrich) density gradients (1.077 g/ml). PBLs were

prepared by adhering monocytes during incubation of cells for 6 h in

cell culture plastic flasks. Afterwards, cells were plated in 96-well plates

5 3 104 cells each well and exposed to vehicle (Dimethyl sulfoxide

(DMSO)) or DHEA-S (16, 80, 400, 2,000,10,000 nM) (Sigma-Aldrich).

Human myeloid (U937) cells were maintained in RPMI1640 with FBS

(10%), penicillin (100 U/ml), streptomycin (100 lg/ml). For differentia-

tion cells were grown for 3 days in media with PMA (50 nM) in 12-well

plates at a density of 1 3 106 cells per well. Cells were then exposed

to lipopolysaccharide (LPS) (50 ng/ml) (Sigma-Aldrich) and vehicle

(DMSO) (1.0%) (Sigma-Aldrich) or DHEA-S (50, 100, 200, 400 nM)

(Steraloids). Culture supernatants were harvested at the indicated time

points and stored at 2808C until tested for IL-1b and IL-6 by enzyme-

linked immunosorbent assay (ELISA) (Bio-Rad DY201 and DY206).

2.4 | Western blot analysis

Western blots were performed on cultured cells and also on autopsied

and necropsied tissue samples. All cell types were cultured as men-

tioned in the previous section. Media was aspirated and the cells were

washed twice with PBS. For tissue samples, the tissue was homoge-

nized using FastPrep®-24 (MP Bio Medical). The homogenized tissue

was used for protein extraction. Protein was isolated using Qiagen

AllPrep DNA/RNA/Protein Mini Kit (Qiagen). The protein extraction

was performed according to manufacturer’s instruction. The protein

samples were boiled in 958C for 20 min and stored at 2208C. Western

blot followed by reduced protein samples electrophoretic separation

on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE) (BioRad) at 120 V for 1 h. Proteins were transferred at

48C onto nitrocellulose membranes for 1 h (Hybond N; GE Healthcare).

Membranes were blocked for 1 h in room temperature with Odyssey

blocking buffer (Li-Cor Biosciences, Lincoln, NE, USA). Western blot anal-

ysis was performed using the following primary antibodies: anti-

CYP17A1 antibody (H-48) (sc-66849, Santa Cruz Biotechnology, Inc.),

anti-CYP17A1 antibody (M-80) (sc-66850, Santa Cruz Biotechnology,

Inc.), anti-Cytochrome P450 17A1 antibody [EPR6293] (ab125022,

Abcam, Cambridge, MA), anti-MBP antibodies (SMI94R, Covance) and

anti-b-Actin antibody (A2228, Sigma-Aldrich). The secondary antibodies

used were goat anti-rabbit Alexa Fluor 680 antibody (Thermo Scientific)

and donkey anti-mouse IRDye800 antibody (Rockland). All antibodies

were diluted in Odyssey® Blocking Buffer1Tween®-20 (0.2%) (OBBT).

Proteins were visualized using an Odyssey Infrared Imaging System (LI-

COR). Immunoreactivity was quantified by using the Odyssey Infrared

Imaging System (LI-COR) and represented graphically (Wang et al., 2007).

2.5 | Polymerase chain reaction (PCR)

For gene expression analyses, total RNA was extracted from cultured

cells, mouse spinal cord and human brain (frontal lobe) using TRIzol (Invi-

trogen) followed by first strand complementary DNA synthesis total RNA

(1.0 mg) of prepared from cultured cells, spinal cord and human brain sam-

ples, together with Superscript II reverse transcriptase (Invitrogen, Carls-

bad CA) and random primers (Roche). Specific genes were quantified by

real-time RT-PCR using i-Cycler IQ5 system (Bio-Rad, Mississauga, ON,

Canada). The individual primers used in the real-time PCR are provided

(Supporting Information Table 2). Semi-quantitative PCR analysis was

performed by monitoring, in real time, the increase of fluorescence of

SYBR Green dye on the Bio-Rad detection system (iQTM SYBR Green

Supermix Bio-Rad), as previously reported (Maingat et al., 2013), and was

expressed as relative fold change (RFC) compared to untreated cells and

healthy controls, respectively, as previously reported by our group

(Acharjee et al., 2010; Johnston et al., 2001; Paul et al., 2014; Ramasw-

amy et al., 2013; van Marle, Antony, Silva, Sullivan, & Power, 2005).

Briefly, real-time fluorescence measurements were performed and a

threshold cycle (CT) value for each gene of interest was calculated by

BOGHOZIAN ET AL. | 1593

determining the point at which the fluorescence exceeded a threshold

limit (12-fold increase above the standard deviation of the initial baseline).

To confirm single-band production, we performed melt-curve analysis

and subsequently confirmed it by electrophoresis and ethidium bromide

staining. All data were normalized against GAPDH and/or ACTB mRNA

level for human samples and against hprt and/or gapdh for mice and

expressed relative to sham-injected controls.

2.6 | Experimental autoimmune encephalomyelitis

(EAE) induction and assessment

C57BL/6 female mice (10–12 weeks old) were immunized with

MOG35–55 peptide emulsified with complete Freund’s adjuvant (CFA)

(Hooke laboratories, EK-0115, Lawrence, MA, USA) according to the

manufacturer’s instructions. Mice were also injected intraperitoneally

with pertussis toxin (300 ng) (Sigma-Aldrich) in PBS (100 ml) on the day

of immunization and 2 days later. For DHEA-S treatment, animals

received daily intraperitoneal injections of DHEA-S (5.0 or 10.0 mg/kg)

after the onset of clinical signs until the end of the experiment (day

30). Control animals received daily injections of normal saline/DMSO

(1.0%) (vehicle). Animals were assessed daily and scored for disease

severity up to 30 days following EAE induction using an established

0–15 point scale (Ellestad et al., 2009). Briefly, for better differentiation

of functional outcomes we used a 0–15 scoring scale and the final score

was the sum of the state of the tail and all of the four limbs scores. For

tail function, a score of 0 referred to no symptoms, 1 was partial paraly-

sis, while 2 was full paralysis. For the hind or forelimbs, each assessed

separately, a grade of 0 referred to no symptoms, 1 was a weak and

shaking walk with that limb, 2 was a limb that was dragged but which

still had minor movements, while the score of 3 was full paralysis of that

limb (Giuliani et al., 2005). Spinal cords and hindbrains were collected at

the peak of the disease (Day 20 post immunization) and day 30, after

animals were anesthetized and transcardially perfused with PBS.

2.7 | Immuno-fluorescence and -histological analyses

Human brain samples (frontal cortex and adjacent white matter) were

fixed in 10% formalin. Paraffin-embedded sections (10lm) were pre-

pared for histological and double and triple antibody immunolabeling

studies (Day, McCarty, Ockerse, Head, & Rohn, 2016). Mice underwent

trans-cardiac perfusion with PBS through the left cardiac ventricle at

day 30 post-EAE induction. Spinal cords were removed, post-fixed in

paraformaldehyde (PFA) (4%) for 24 h, and processed for paraffin

embedding and subsequent sectioning (10 lm). Immunohistochemical

and histochemical labeling of human and mouse CNS tissues was

performed as previously reported (Tsutsui et al., 2004). Briefly, tissue

sections were deparaffinized and hydrated using decreasing concentra-

tions of ethanol. Human tissue sections were histologically stained

with Luxol fast blue (LFB) and hematoxylin. Mouse sections were

stained by Bielschowsky’s silver impregnation and the axonal number

was quantified. Spinal cords were scanned to provide digital images

and quantitative analysis of axonal numbers per mm2 was performed

by Image J Fiji (Schindelin et al., 2012). Antigen retrieval was performed

by boiling the slides in trisodium citrate buffer (pH 6.0) (0.01 M) for 10

min. Endogenous peroxidases were inactivated by incubating sections

in hydrogen peroxide (0.3%) (Caledon) for 20 min. To prevent nonspe-

cific binding, sections were pre-incubated with normal goat serum

(10%) for 1 h at room temperature. For immunohistochemical detec-

tion of macrophage/microglial, astrocyte, T lymphocytes and myelin

immunoreactivity, as previously described (Noorbakhsh et al., 2006),

antibodies against ionized calcium binding adaptor molecule 1 (Iba-1)

(1:500; Wako), glial fibrillary acidic protein (GFAP) (1:200; DAKO,

Copenhagen, Denmark), CD3 (1:50; Santa Cruz Biotechnology Inc.),

myelin basic protein (1:1,000; Sternberger Monoclonal), CYP17A

(1:100, Santa Cruz Biotechnology, Inc.), followed by application of

appropriate biotinylated secondary antibodies. For immunofluorescent

studies, tissue sections were de-paraffinized by incubation for 1 h at

608C followed by one 10 min and two 5 min incubations in toluene

baths through decreasing concentrations of ethanol to distilled water.

Antigen retrieval was performed by boiling in 10 mM sodium citrate

(pH 6.0) for 1 h. Slides were blocked with HHFH buffer (1.0 mM

HEPES buffer, 2% (v/v) horse serum, FBS (5% (v/v)), sodium azide

(0.1% (w/v)) in Hank’s balanced salt solution (HBSS)) for 4 h at room

temperature. Human sections were incubated with a mixture of mouse

anti-GFAP (1:200) (BD Pharmingen), rat anti-MBP (1:200) (Abcam),

monoclonal rabbit anti-CYP17A1 (1:125) (Abcam) mouse slides were

incubated with a cocktail of mouse anti-CNPase (1:50) and monoclonal

rabbit anti-CYP17A1 (1:125) (Abcam) overnight at 48C. Primary anti-

bodies was removed by PBS washes (5 min x3) and slides were incu-

bated for 3 min in 0.22 lm filtered Sudan black dissolved (1% (w/v)) in

ethanol (70%) and washed an additional 3 times in PBS. A mixture of

FITC-conjugated goat anti-rat IgG (1:500), Alexa 568 goat anti mouse

IgG, Alexa 647 goat anti-rabbit IgG (Abcam) was incubated for 2 h and

washed 33 in PBS and mounted with prolong diamond mounting

media containing DAPI. Slides were imaged with Wave FX Spinning

Disc confocal microscope (Zeiss).

2.8 | Statistical analyses

Statistical analysis for neurobehavioral studies was performed using

the Mann–Whitney U test. ANOVA followed by a Tukey–Kramer Mul-

tiple Comparisons test was used for statistical comparisons between

more than two groups. Regression analyses were performed using

Pearson’s correlation. Comparisons between two groups were per-

formed by unpaired Student’s t-test or by ANOVA with Tukey-Kramer

or Bonferroni as post hoc tests, using GraphPad Instat 3.0 (GraphPad

Software, San Diego, CA). As indicated in figure legends, p<0.05 was

considered significant.

3 | RESULTS

3.1 | Reduced CYP17A1 and DHEA in white

matter from MS patients

In the present studies, the neurosteroid synthesis pathway underlying

DHEA synthesis was examined (Figure 1A) because of its implicated

1594 | BOGHOZIAN ET AL.

effects on neuroinflammation and cellular survival. In human brains,

levels of pregnenolone, the precursor to DHEA, were similar in MS and

nonMS patients’ cortex and in white matter (Figure 1B). In contrast,

DHEA levels were suppressed in white matter from MS patients com-

pared to nonMS patients while there was no difference in cortical

DHEA levels between patient groups (Figure 1C). CYP17A1 transcript

levels, the enzyme responsible for DHEA synthesis, was reduced

significantly in MS white matter compared to nonMS white matter (Fig-

ure 1D) although CYP11A levels, the enzyme responsible for pregneno-

lone synthesis, did not differ in white matter between groups

(Supporting Information Figure 1A). To investigate the extent of

immune activation in the same human white matter samples, transcript

expression of CD3E (Figure 1E), IFNG (Figure 1F), IL1B (Figure 1G), and

CD68 (Supporting Information Figure 1B) were all found to be signifi-

cantly increased in MS white matter compared to nonMS patients’

white matter.

While CYP17A1 transcript expression was reduced in MS patients’

white matter, it was imperative to delineate which cells expressed

CYP17A1 in the context of inflammatory demyelination. Morphological

analyses of white matter from nonMS and MS patients revealed that

Luxol Fast Blue (LFB) staining was preserved in nonMS patients’

white matter (Figure 2Ai), while reduced LFB staining, indicative of

demyelination (D), was evident in MS patient tissue sections (Figure

2Aii). Similarly, in nonMS patients there was minimal evidence of T cell

infiltration (Figure 2Bi) while there was abundant evidence of CD3E1

cells in the perivascular space and parenchyma in MS patients’ white

matter (Figure 2Bii). CD68 expression was minimal in nonMS patients

(Supporting Information Figure 1C), while there was notable infiltration

of CD681 macrophages in MS patient brains. CYP17A1 immunoreac-

tivity was diffusely detected in glial cells in nonMS patients (Figure

2Ci), but it was diminished within regions of demyelination in MS

patient white matter (Figure 2Cii) with increased expression along the

border of the lesion; phagocytic CYP17A11 macrophages were evi-

dent near the lesion margin of demyelinating lesions (Figure 2Cii, inset).

Immunofluorescent labelling disclosed co-localization of CYP17A1 (red)

with GFAP (yellow, astrocytes) and MBP (green, ODCs) in DAPI1

(blue) cells in white matter (Figure 2Di) in nonMS sections, although

CYP17A1 immunoreactivity was similarly detected in MS patients’

section but with areas of punctate immunoreactivity and reduced

abundance (Figure 2Dii). These findings were extended by immunolab-

eling oligodendrocytes with anti-CNPase antibodies that was

FIGURE 2 CYP17A1 immunoreactivity is diminished in MS-associated demyelination. (a) Luxol fast blue/H&E staining of cere-bral white matter showed diminished staining in MS-associateddemyelination (d) with associated cellular infiltrates (aii) comparedto nonMS patients’ white matter (ai). (b) CD3E immunoreactivitydetected by immunocytochemistry was increased in MS brains (bii)compared to nonMS brain (bi). (c) CYP17A1 immunoreactivity wasevident in nonMS patients’ white matter but reduced in MS-associated demyelination d) (cii) compared to nonMS patients (ci);

CYP17A1 was also concentrated in cells resembling phagocyticmacrophages (cii, inset). (di) CYP17A1 immunoreactivity (red), asindicated by fluorescence, was co-localized with GFAP1 astrocytes(yellow) and MBP1 ODCs (green) as well as other nucleatedDAPI1 (blue) cells in nonMS white matter but exhibited a particu-late appearance in MS white matter (dii). (ei) CYP17A1 immunore-activity (red), as indicated by fluorescence, was co-localized withCNPase1 ODCs (yellow) and nucleated DAPI1 (blue) cells innonMS white matter but exhibited a particulate appearance in MSwhite matter (eii)

BOGHOZIAN ET AL. | 1595

co-localized with CYP17A1 immunoreactivity in nonMS patient sec-

tions (Figure 2Ei) although there was a paucity of both CNPase and

CYP17A1 immunoreactivity in white matter of MS patients (Figure

2Eii), emphasizing the loss of oligodendrocytes and associated

CYP17A1 expression in MS lesions.

To verify the above observations, western blot analyses of white

matter from MS and nonMS patients revealed that MBP immunoreac-

tivity (33 kDa) was apparent in the white matter (Figure 3A) but was

reduced in MS brains (Figure 3B). CYP17A1 immunoreactivity was evi-

dent at 47 kDa and 42 kDa (Figure 3A) and its expression was also

diminished in MS patients’ white matter (Figure 3B). These data sug-

gested that with increased neuroinflammation in cerebral white matter

of MS patients, there was a corresponding decline in CYP17A1 expres-

sion levels in conjunction with reduced MBP expression that was

accompanied by diminished DHEA levels (Figure 1). There was also

increased CYP17A1 immunoreactivity in myeloid cells, reflecting phag-

ocytosis or perhaps induction of CYP17A1 expression in these cells.

3.2 | Glial CYP17A1 expression and responses

As CYP17A1 immunoreactivity was apparent in multiple cell types in

vivo and both protein and mRNA encoded by this gene had been previ-

ously reported in human neural cell lines (Rachel, Brown, & Papadopou-

los, 2000), it was essential to examine its regulation using cultured

human brain cells. Western blotting of human astrocytes, microglia,

neurons, and an ODC cell line disclosed that CYP17A1 immunoreactiv-

ity was detected in all cell types, although it appeared to be compara-

tively increased in microglia and in the ODC cell line, in which it again

showed an immuno-labeled doublet at 47 and 42 kDa (Figure 4A).

CYP17A1 transcript levels in all cell types showed limited correlation

with protein detection (Supporting Information Figure 3A). Western

blot analysis showed that stimulation of the ODC cell line with IFN-g

suppressed CYP17A1 immunoreactivity (Figure 4B). Conversely, IFN-g

exposure to human astrocytes resulted in induction of CYP17A1

immunoreactivity (Figure 4C) that was concentration-dependent (Sup-

porting Information Figure 3B). Similarly, IFN-g exposure to human

microglia showed that CYP17A1 expression was also induced (Figure

4D). Of note, TNF-a did not affect CYP17A1 expression in the same

glial cells (data not shown). To assess neurosteroid release from each

neural cell in a resting state, supernatant levels were measured reveal-

ing that neurons produced all of the neurosteroids although superna-

tants the ODC cell line showed the highest DHEA levels when

normalized based on cell numbers (Supporting Information Figure 2).

These data indicated that all neural cell types expressed CYP17A1

although its expression levels were influenced differentially depending

on the cell type, with suppression in ODC cell lines and induction in

myeloid and astrocytic cells following exposure to IFN-g. However,

based upon the loss of CYP17A in MS brains (Figure 3C), the induction

of CYP17A1 in some cell types did not restore the loss of CYP17A1 in

ODCs.

3.3 | DHEA-S regulates innate immune responses

As CYP17A1 is responsible for the synthesis of DHEA and its sulfated

ester, DHEA-S, which is more stable than DHEA, the effects of DHEA-

S on immune activation were examined in leukocytes. DHEA-S treat-

ment of human PBLs, stimulated with IL-2 and PHA, revealed that

DHEA-S exerted no effects on lymphocyte viability at a wide range of

FIGURE 3 MBP and CYP17A1 expression are reduced in MS white matter. (a) Immunoblotting disclosed MBP (33kDa) and CYP17A1immunoreactivity (47 and 42 kDa) in MS and nonMS patients’ white matter but (b) graphic representation showed both MBP and (c)CYP17A1 immunoreactivities were diminished in MS white matter (Student t-test, *p<0.05)

1596 | BOGHOZIAN ET AL.

concentrations (16–2,000 nM) after exposure for 24 h (Figure 5A).

However, at a high concentration, DHEA-S (10,000 nM) resulted in a

small decrease in overall viability of PBLs (Figure 5A). These analyses

were explored further by examining the effects of DHEA-S on human

myeloid cell activation; in U937 cells activated with LPS, DHEA exerted

a concentration-dependent inhibition of IL-1b and IL-6 production (Fig-

ure 5B). These results were complemented by studies in human micro-

glia exposed to LPS which again showed reduced IL-1b and IL-6

expressed at 24 h following activation with LPS with DHEA-S treat-

ment (Figure 5C). These studies implied that while DHEA-S did not

have substantial effects on T cell viability or proliferation, it did exert

anti-inflammatory effects in human myeloid cells.

3.4 | CYP17A1 expression in EAE

Since DHEA expression levels as well as CYP17A1 levels were dimin-

ished in cerebral white matter from patients with MS, the present anal-

yses were extended in vivo using the EAE animal models. Induction of

EAE with MOG/CFA/PTX resulted in disease onset at day 10 and was

carried forward to day 30. Analysis of hindbrains from EAE and control

animals showed that DHEA levels were diminished in EAE animals’

tissues (Figure 6A) but conversely pregnenolone levels were increased

in the EAE CNS samples (Supporting Information Figure 4A). Western

blotting of CNS tissues showed that CYP17A1 expression was detecta-

ble at 47 and 42 kDa, as observed in Figures 3 in both groups (Figure

6B), but its expression was diminished in EAE animals at day 30 post-

induction (Figure 6C). Comparison of neurobehavioral scores at days

20 and 30 post-EAE induction with molecular changes in the spinal

cord showed that f4/80 transcript levels were correlated positively

with severity of disease (r250.514, p<0.05) while Cyp17a1 were neg-

atively correlated with disease severity (r250.713, p<0.01) but Cd3e

levels were not correlated with disease severity scores. These studies

verified the findings of reduced CYP17A1 expression white matter

from patients with MS by showing that CYP17A1 and DHEA levels

were reduced in this animal model of MS and associated with disease

severity.

3.5 | DHEA-S treatment ameliorates EAE severity

To pursue these latter findings further, animals with EAE or CFA expo-

sure were treated with DHEA-S (10 mg/kg) at the onset of neurobeha-

vioral deficits. Examination of MhcII transcript expression revealed that

FIGURE 4 CYP17A1 expression in different neural cell types. (a) CYP17A1 was detectable in human astrocytes, microglia, neurons and anODC cell line, measured by western blotting; doublet immunoreactivity was seen at 47 and 42 kDa in ODC cell lines. (b) CYP17A1immunoreactivity is reduced in ODC cell lines following IFN-g exposure. (c) CYP17A1 immunoreactivity was increased in astrocytes and (d)microglia after IFN-g exposure. (n54 for each cell type; ANOVA with Bonferroni post hoc comparisons, *p<0.05)

BOGHOZIAN ET AL. | 1597

it was increased in the EAE animals’ spinal cords but DHEA-S treat-

ment had minimal effect on its expression at peak disease (Figure 7A).

Ifng transcript expression was increased in EAE animals and showed

significant reduction in expression at peak disease with DHEA-S treat-

ment (Figure 7B). Il1b was also increased, by 20-fold, in EAE animals’

spinal cords at day 20 post-induction, but this gene’s transcripts were

significantly diminished with DHEA-S treatment (Figure 7C). Il6 tran-

script levels were induced in spinal cords from EAE animals but DHEA-

S treatment resulted a trend toward reduced expression (Figure 7D).

Cyp17a1 expression was largely unchanged in healthy controls and

CFA-exposed animals, but its transcript expression was reduced in spi-

nal cords of EAE animals at peak disease, which was rescued by treat-

ment with DHEA-S (Figure 7E). In contrast, steroid sulfatase (Tremlett,

Zhao, Devonshire, & Neurologists) expression, the enzyme responsible

for DHEA-S synthesis was unchanged in control and EAE animals,

regardless of the presence or absence of DHEA-S treatment

(Figure 7F). These data showed that DHEA-S suppressed expression of

inflammatory genes in the CNS during EAE.

FIGURE 5 DHEA-S influences leukocyte immune activation. (a)Exposure of human PBLs to DHEA-S did not affect cell viability, asmeasured by trypan blue staining except at 10,000 nM at whichthere was a small reduction in cell viability (24 h). (b) In humandifferentiated myeloid cells (U937), LPS induced IL-1b and IL-6production, assessed by ELISA, which was suppressed by DHEA-Streatment at different concentrations (72 h). (c) LPS induced IL-6and IL-1b in human microglial cells, which was suppressed byDHEA-S treatment at 24 h (all experiments were performed intriplicate and repeated at least twice; ANOVA with Bonferroni posthoc analyses *p<0.05)

FIGURE 6 DHEA and CYP17A1 are suppressed in the CNS ofmice with EAE. (a) DHEA levels were reduced in the hindbrains ofanimals with EAE at day 30 post-induction, based on GC-MS. (b)CYP17A1 immunoreactivity on western blotting was apparent inthe hindbrains of animals with EAE and healthy control animals atday 30 post-induction at 47 and 42 kDa. (c) Graphic representationshowed significant reduction in CYP17A1 immunoreactivity inhindbrains of animals with EAE at day 30 post-induction. (Studentt-test *p<0.05)

1598 | BOGHOZIAN ET AL.

FIGURE 7 DHEA-S treatment of EAE modulates CNS immune activation. (a) Mhc II transcript levels, assessed by RT-PCR were increasedin EAE animals’ spinal cords in the presence of vehicle or DHEA-S treatment. (b) Ifng was increased in EAE animals but suppressed byDHEA-S treatment. Similarly, Il1b (c) and IL6 (d) levels were increased in EAE spinal cords but suppressed by DHEA-S treatment. (e)Cyp17a1 transcript levels were diminished significantly in EAE animals at day 20 post-induction, while (f) Sts levels in spinal cord wereinduced in EAE animals, but unaffected by DHEA-S treatment (n56 for all groups; ANOVA with Bonferroni post hoc analyses *p<0.05)

BOGHOZIAN ET AL. | 1599

3.6 | DHEA-S treatment of EAE improvedneuropathological outcomes

The above molecular findings were extended in morphological analyses

of spinal cords from control (CFA-exposed) and EAE animals showing

that CD3e1 T cells were increased in the EAE animals’ spinal cord white

matter and this effect was diminished by DHEA-S treatment (Figure 8A).

Analysis of myeloid cell activation in EAE and control spinal cords

showed that Iba-1 immunoreactivity in microglia and macrophages was

markedly enhanced in the spinal cords of EAE animals and this immuno-

reactivity was diminished by DHEA-S treatment (Figure 8B). Similarly,

analysis of myelin basic protein (MBP) immunoreactivity was markedly

diminished in EAE animals, with preservation in EAE animals receiving

DHEA-S treatment compared to controls (Figure 8C). CYP17A1 expres-

sion was detectable in glial cells in control animals’ spinal cords but was

markedly reduced during EAE although DHEA-S treatment preserved its

expression (Figure 8D). Immunofluorescent labelling showed that

in CFA-exposed animals, CNPase immunoreactivity in ODCs was

co-localized with CYP17A1 and DAPI but reduced in EAE animals’ spinal

cords, albeit with punctate immunoreactivity, although DHEA-S treat-

ment increased CYP17A1 expression (Fig. 8E). Finally, analysis of axonal

integrity showed intact silver stained axons in spinal cords from CFA-

exposed controls, but the density of axons was diminished in EAE ani-

mals’ spinal cords compared to EAE animals receiving DHEA-S (Figure

8F). Graphic representation showed that axonal counts were reduced

significantly in EAE animals compared to the CFA animals, as well as the

EAE animals with DHEA-S treatment (Figure 8G). In summary, these

data showed that DHEA-S exerted both anti-inflammatory and neuro-

protective effects in EAE.

3.7 | Neurobehavioral deficits in EAE are

reduced by DHEA-S treatment

Neurobehavioral analyses were performed daily in EAE animals with

and without DHEA-S treatment, as well as controls. These data

showed that the severity of disease was significantly diminished over

time in the DHEA-S-treated EAE animals compared to vehicle-treated

EAE animals (Figure 9A), which was also apparent in maximal disease

scores (Figure 9B). Treatment of animals with a lower (5 mg/kg) con-

centration of DHEA-S at the time of disease onset also improved neu-

rological outcomes in EAE (Supporting Information Figure 5). In

summary, these data indicated that CYP17A1 was expressed in the

CNS, with reduced RNA and protein levels in brains and spinal cords of

FIGURE 8 DHEA-S improves neuropathological outcomes in EAE.(a) CD3E immunoreactivity detected by immunocytochemistry wasincreased in EAE animal spinal cords (aii) but suppressed by DHEA-S treatment in EAE (aiii). (b) Iba-1 immunoreactivity was markedlyincreased in the spinal cords of EAE animals (bii), which was dimin-ished by DHEA-S treatment (biii). (c) MBP immunoreactivity wasreduced in EAE animals at day 20 post-induction (cii), but pre-served with DHEA-S treatment (ciii). (d) CYP17A1 immunoreactiv-ity was detected sparsely in spinal cords of EAE animals (dii), but

was restored by DHEA-S treatment (diii). (e) Immunofluorescentlabelling showed CNPase (green) and CYP17A1 (red) co-localizationwith DAPI (blue) in CFA exposed animals (ei) that was reduced inEAE animals (eii) but was preserved in DHEA-S treated animals(eiii). Bielchowsky silver staining showed diminished numbers ofaxons in lumbar spinal cords of EAE animals (fii), which was pre-served by DHEA-S treatment (fiii). (g) Quantitative analysis of axo-nal counts/mm2 showed that DHEA-S preserved axons during thecourse of EAE compared to vehicle treated animals (Kruskal-Walliswith Dunn’s post hoc analyses, *p<0.05)

1600 | BOGHOZIAN ET AL.

humans with MS and animals with EAE, but DHEA-S treatment exerted

anti-inflammatory effect that reduced myelin and axonal injury.

4 | DISCUSSION

The present results, to the best of our knowledge, represent the first in

vivo delineation of protein expression and function for an important

neurosteroid synthesis enzyme, CYP17A1, in individual CNS cell types

in humans. Moreover, CYP17A1 expression and its chief product,

DHEA, (Figure 1A) were suppressed in white matter from MS patients

and animals with EAE. These studies also show that CYP17A1 expres-

sion in ODCs was diminished in MS white matter and was also selec-

tively downregulated in an ODC cell line by IFN-g exposure, while

myeloid cells show an up-regulation of CYP17A1 with neuroinflamma-

tion, possibly due to phagocytosis of dying/dead oligodendrocytes or

as host protective response. Provision of DHEA-S as a treatment

improved neurological outcomes in EAE, possibly through its anti-

inflammatory effects. These observations suggest that although neuro-

steroid synthesis pathways are vulnerable during inflammatory demye-

lination, exogenous neuroactive steroids as therapies might serve as

modulators by which inflammation can be controlled within the CNS.

Neurosteroid synthesis within the CNS is susceptible to the effects

of inflammation and demyelination, as reported for allopregnanolone

synthesis in MS patients by our group and others (Caruso et al., 2014;

Leitner, 2010; Luchetti, Huitinga, & Swaab, 2011; Melcangi, Panzica, &

Garcia-Segura, 2011; Noorbakhsh et al., 2011). This wide ranging effect

on neurosteroid pathways might reflect ubiquitous neurosteroid

enzyme expression in multiple CNS cell types, as implied by our find-

ings of CYP17A1 detection in all neural cells examined, although there

is differential vulnerability depending on the individual neurosteroid

pathway, the cell type and anatomic site (involving largely white

FIGURE 9 DHEA-S treatment improved neurobehavioral outcomes in EAE. (a) Intraperitoneal treatment with DHEA-S of EAE animalsbeginning at disease onset showed a reduction of severity of disease that was sustained over time compared to EAE animals treatedwith vehicle only. (b) The maximum neurobehavioral deficit score in each DHEA-S-treated animal was significantly less compared tovehicle-treated EAE animals. (Kruskal-Wallis with Dunn’s post hoc analyses, *p<0.05)

BOGHOZIAN ET AL. | 1601

matter). Moreover, in an ODC cell line and astrocytes, CYP17A1 immu-

noreactivity was apparent as a doublet, with the major signal at the

predicted 47 kDa and a second signal at �42 kDa, perhaps indicative

of a splice variant or a post-translational modification such as proteo-

lytic cleavage (Figure 3). Nonetheless, analyses of different human neu-

ral cell types showed that DHEA levels were highest in supernatants

from resting ODCs compared to astrocytes, microglia and neurons

(Supporting Information Figure 2), which is altered in a disease circum-

stance such as MS or EAE. The reduction of CYP17A1 and DHEA in

both MS and EAE likely reflects the selective vulnerability of ODCs to

inflammatory stimuli, for example, IFN-g, an established Th1 cytokine

contributor to inflammatory demyelination (Bsibsi et al., 2014; Horiu-

chi, Itoh, Pleasure, & Itoh, 2006; Wang et al., 2014). By contrast, sup-

pression of allopregnanolone synthesis in our previous report was

mediated by microRNAs (Noorbakhsh et al., 2011); the same microRNA

database was interrogated for microRNAs targeting CYP17A1 and

associated enzymes but induction of CYP17A1-specific microRNAs in

MS white matter was not found (data not shown). The loss of

CYP17A1 expression and accompanying reduction in DHEA levels

within the CNS in the current study differs from a recent report in

which neurosteroids were increased in cerebrospinal fluid (CSF) from

relapsing-remitting MS patients compared to controls. However, blood

DHEA levels were suppressed in patients with MS, especially in those

with fatigue (Foroughipour, Norbakhsh, Najafabadi, & Meamar, 2012;

Tellez et al., 2006). These dichotomous findings likely reflect different

cellular sources of DHEA in CSF versus brain, perhaps from circulating

activated leukocytes in CSF, as suggested by our findings in which IFN-

g activated myeloid cells showed induction of CYP17A1 (Figure 4D).

IFN-g is known to act directly on ODCs, causing induction of endoplas-

mic reticulum (ER) stress with protective consequences (Lin et al.,

2007); it is intriguing that CYP17A1 is expressed in the ER and in some

MS models induction of ER stress is associated with worsened out-

comes (Deslauriers et al., 2011; McMahon, McQuaid, Reynolds, & Fitz-

Gerald, 2012; Mhaille et al., 2008).

The actions of DHEA and DHEA-S are diverse, with effects on

neurotransmitter receptor function, especially glutamatergic and

GABAergic transmission, but also repressive effects on gene expression

including inflammatory genes (Acaz-Fonseca, Avila-Rodriguez, Garcia-

Segura, & Barreto, 2016; Kipper-Galperin, Galilly, Danenberg, & Bren-

ner, 1999; Koziol-White et al., 2012). Earlier studies of DHEA treat-

ment in EAE showed that it exerted anti-inflammatory effects,

particularly in circulating leukocytes, resulting in diminished severity of

EAE (Du et al., 2001). In another study, treatment of EAE with a syn-

thetic derivative (HE3286) of a natural steroid, b-AET, improved

aspects of optic neuritis (Khan, Dine, Luna, Ahlem, & Shindler, 2014). In

the present study, treatment with DHEA-S was implemented because

it is a downstream product of CYP17A1, is more stable than DHEA,

and is detectable in human blood. Of note, DHEA-S treatment (10mg/

kg) initiated at the time of EAE induction delayed the disease onset

(Supporting Information Figure 6A) and also reduced the overall sever-

ity of disease (Supporting Information Figure 6B), pointing to its sus-

tained benefits with longer term treatment. In a previous study from

our group, DHEA-S was a highly effective modulator of inflammation,

especially in myeloid cells (Maingat et al., 2013) and in other studies

(Ben-Nathan et al., 1992; Romanutti, Bruttomesso, Castilla, Galagovsky,

& Wachsman, 2010) but was not neuroprotective per se. DHEA-S is

highly induced in blood in several diseases in humans, although the

impact of blood-derived DHEA-S on brain function remains uncertain

(Klouche et al., 2007; Oberbeck, 2004). Of interest, DHEA-S readily

crossed the blood-brain barrier in an experimental feline model of HIV/

AIDS, resulting in reduced neuroinflammation and neuronal preserva-

tion (Maingat et al., 2013). The loss of CYP17A1 expression in ODCs

and accompanying reduced CNS DHEA levels likely reflects disruption

of an important mechanism by which inflammation within the CNS is

constitutively regulated; provision of DHEA-S appeared to reverse

this disturbance and curtail neuroinflammation leading to improved

outcomes in EAE.

Several challenges were encountered in the current studies. Most of

the patients with MS included in this study had progressive MS (Table 1)

and the pathogenic circumstances might be different in relapsing-

remitting MS; patients with relapsing-remitting MS rarely come to

autopsy making this issue a difficult point to resolve. Importantly, all of

the female patients in the present studies were post-menopausal obviat-

ing concerns of altered DHEA or DHEA-S levels due to sex although the

multiple polymorphisms documented in CYP17A1 might also impact on

the current results (Nogueira et al., 2011). While CYP17A1 expression

was reduced in the CNS of both patients with MS and mice with EAE,

selective knockdown of CYP17A1 in ODCs remains to be performed in

vivo. A Cyp17a1 knockout mouse was generated in the past, which

resulted in embryonic lethality (Bair & Mellon, 2004) while in a chimeric

Cyp17A1 mouse with reduced DHEA levels, a neurological phenotype

was not apparent (Liu, Pocivavsek, & Papadopoulos, 2009); these obser-

vations suggest that an intervening stimulus, such as inflammation, might

be required for neuropathogenic outcomes and the suppression of

CYP17A1 might be a downstream effect resulting from the collective

pathogenic mechanisms implicated in neuroinflammation. Future studies

will need to address the exact in vivo consequences of downregulated

CYP17A1 expression in ODCs in the context of neuroinflammation.

DHEA is available as an over-the-counter nutritional supplement

with a range of putative beneficial effects (on aging, neurocognitive

impairments, cardiovascular disease, immune, and allergic disorders) albeit

none are specific indications for its use at present (Castanho et al., 2014;

de Menezes et al., 2016; Dillon, 2005). A previous study suggested that

through DHEA’s action on NFjB signaling, it could enhance a Th2 pheno-

type in activation lymphocytes (Du, Khalil & Sriram, 2001; Sun, Yang,

Zang, & Wu, 2010). Other studies indicate a restorative function in lym-

phocytes for DHEA, including in HIV/AIDS (Jacobson, Fusaro, Galmarini,

& Lang, 1991; Oberbeck et al., 2002; Oberbeck et al., 2001). However, its

mechanisms of action likely vary depending on the targeted cell type and

organ. Given its plethora of therapeutic benefits with its limited side-

effect profile, consideration should be given exploring the effects of

DHEA or DHEA-S as treatments for MS and related conditions in the

future, perhaps in concert with glucocorticoids.

These studies were supported by the Alberta Innovates – Health

Solutions (AI-HS) CRIO Project, Canada Foundation for Innovation

1602 | BOGHOZIAN ET AL.

and the University Hospital Foundation (CP, GBB). RB was sup-

ported by a research award from the Department of Immunology at

Tehran University of Medical Sciences. BAM was supported by Stu-

dentships from the Canadian Institutes of Health Research and

AIHS. CP is supported by a Canada Research Chair in Neurologic

Infection and Immunity. The authors thank Dr. Vassilios Papadopou-

los for invaluable discussion of these studies and Gail Rauw for

expert technical assistance.

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