DEPARTMENT OF BIOLOGICAL AND 
ENVIRONMENTAL SCIENCES
ROLE OF ANDROGEN RECEPTOR SIGNALING IN THE 
ACTIVATION AND DIFFERENTIATION OF 
PLASMACYTOID DENDRITIC CELLS
FAIZA TAHSIN RASHID
Degree project for Master of Science (120 hec) with a major in Biology
BIO727, Degree project in Physiology and Cell Biology and 60 hec
Second cycle 
Semester/year: Autumn 2022, Spring 2023
Supervisor: Mattias Svensson, Department of Rheumatology and Inflammation Research
Examiner: Malin Celander, Department of Biological and Environmental Sciences
https://doi.org/10.3389/fimmu.2022.986840
Table of Contents
Abstract…………………………………………………………………….….…….2
1. Introduction…………………………………………………………….….……...3
2. Aim…………………………………………………………………….…….……7
3. Materials and Methods………………………………………….……….….…….8
4. Results………………………………………………………………….…………13
5. Discussion…………………………………………………………….…….…….19
6. Conclusion………………………………………………………….………….…20
References………………………………………………………………………...…21
Acknowledgments………………………………………………………….…….…23
Popular Science Summary………………………………………………….…….…24
1
Abstract
Androgens are steroid hormones essential for the development and maintenance of male 
reproductive organs and secondary sexual characteristics. Plasmacytoid dendritic cells (pDCs) are 
immune cells that act as the first line of defense against viral infections. These cells have 
specialized receptors such as the toll-like receptors (TLR7) that recognize viral nucleic acids and 
initiate the production of antiviral cytokines such as Type I interferons (IFN-α1 and IFN-α2). 
Androgen receptor signaling might affect pDC functions throughout their activation and maturation 
in response to viral infections via the androgen receptor (AR), a nuclear hormone receptor that 
binds to hormones such as testosterone, an androgen hormone. Androgens have also been shown 
to promote the replication and intensity of some viruses, most notably SARS-CoV-2, an RNA virus. 
Understanding the mechanism of androgen signaling is critical for developing effective antiviral 
therapies. Considering this, we stimulated human peripheral mononuclear cells (PBMCs) with a 
TLR7 agonist such as Gardiquimod, an AR agonist R1881, and the synthetic androgen named 
Testosterone propionate. An antagonist namely G15 for the G-protein coupled estrogen receptor 
was also used. These compounds are used to activate the AR in order to assess its role in interferon 
production. Furthermore, qPCR was used to examine the gene expression of IFN⍺1, IFN⍺2, IRF7, 
and MX1 using GAPDH as the endogenous control. Flow cytometry was also performed after the 
cells were stained with BDCA2, CD3, CD19, CD123, and IFN⍺2 to evaluate the pDC population 
and interferon production. The findings showed an increase in IFN response when the PBMCs were 
stimulated with Gardiquimod and the agonists, with individual differences. To date, relatively few 
studies have been conducted on the role of androgens in immune system regulation. Further 
research can generate effective treatment targets and lessen the impact that certain infections have 
on human health.
2
1. Introduction
1.1 Sex Hormones and the Immune System
The immune system is comprised of two types: the innate immune system which is made up of a 
variety of physical, chemical, and cellular mechanisms that act as the first line of defense against 
pathogens, and the adaptive immune system which responds to specific antigens (Chaplin 2010). 
Sex plays a significant role in the innate immune system, and gender disparities in immunity have 
been thoroughly documented in years of scientific research. In general, women have stronger 
immunological responses than men. This is assumed to be owing to the effects of sex hormones on 
the immune system, such as estrogen and testosterone. Estrogen has been demonstrated to stimulate 
the immune system, whilst testosterone can suppress it. Moreover, the ability to respond to 
infection and vaccination is sex-biased (Ruggieri et al. 2016). Furthermore, many immune-related 
genes such as TLR7, TLR8, IRAK1, etc. are expressed on the X chromosome. Because females have 
two X chromosomes, they have a greater number of immune-related genes than men who have 
only one X chromosome. However, this increased immunological response in women may 
contribute to a higher occurrence of autoimmune diseases, which occur when the immune system 
mistakenly attacks the body's own tissues (Rojas-Villarraga et al. 2010). For example, Systemic 
lupus erythematosus (SLE), Rheumatoid Arthritis (RA), and Multiple Sclerosis prevail more in 
women than men. On the contrary, men are more susceptible to infectious diseases. An analysis of 
death among 17 million adults during COVID-19 showed that being female was a robust protective 
factor (Williamson et al. 2020). Despite both genders exhibiting similar vulnerability, men faced a 
greater likelihood of being infected and experiencing a higher mortality risk (Ramírez-Soto, 
Ortega-Cáceres, and Arroyo-Hernández 2021). The involvement of sex differences in viral 
infections is evident although the mechanisms have quite not been explored extensively. An 
example of how testosterone affects a variety of cell types in response to infections and 
vaccinations is shown in Fig 1 (Trigunaite, Dimo, and Jørgensen 2015).
Fig 1: Testosterone regulation in males is responsible for immune responses.
3
1.2 Androgen Receptor Signaling
Androgens are a group of steroid hormones, and they are primarily produced in the testes of males 
and in lower amounts in the adrenal glands of both sexes. There are five types of androgens where 
testosterone is produced in Leydig cells and adrenal glands, and a more potent form of testosterone 
named dihydrotestosterone (DHT) is the one responsible for male sexual characteristics and male 
reproductive organs. The effects of androgens are mediated through androgen receptor (AR). The 
AR is a ligand-dependent nuclear receptor, meaning it is activated by binding to steroid hormones 
such as androgens (Sever and Glass 2013). The classical pathway by which AR acts is illustrated 
in Fig 2 below.
Fig 2: (1) The enzyme 5-reductase in the basal epithelial cells of the prostate converts testosterone to DHT. 
(2) DHT enters epithelial cells' cytoplasm and binds to AR. (3) Ligand interaction causes a conformational 
change in AR, causing heat shock proteins (HSPs) to dissociate to enable nuclear translocation. (4) In the 
nucleus, AR dimerizes (6) Various co-activators attach to AR within the nucleus, and the AR-DBD enhances 
nucleic acid binding at androgen response elements (ARE). This stimulates the results in chromatin 
remodeling. This permits TATA-binding protein (TBP), general transcription factors (GTF), and RNApolII 
to bind and activate transcription. (7) AR that is not bound to ligands is recycled back to the cytoplasm in 
preparation for future ligand binding (“Molecular Cell Biology of Androgen Receptor Signalling” 2010).
4
1.3 Complete Androgen Insensitivity Syndrome
The modulation of AR function is dependent on mutations that arise in various loci on the AR gene 
located on the X chromosome. These mutations might result in a reduction or complete loss of gene 
expression. Due to the presence of only one X chromosome in males, these mutations are expressed 
in all cells, whereas in females, the second copy of the X chromosome counteracts their effects 
leading them to be silent carriers (Quigley et al. 1995). Androgen Insensitivity Syndrome (AIS) is 
a functional consequence of the AR mutation where individuals are insensitive to androgens 
including the variation in the absence of phenotypic characteristics (Hughes et al. 2012). The 
severity of this condition varies in different degrees from partial, mild to complete.
Complete Androgen Insensitivity Syndrome (CAIS), an X-linked disease is a severe form of AIS 
where individuals are completely resistant to androgens. This is an extremely rare genetic disorder 
that occurs in 1 in 20,000 to 64,000 male births (Ahmed et al. 2000). Individuals with CAIS carry 
the 46, XY karyotype and testes. However, they exhibit the female phenotype with normal female 
external genitalia and breast development (Quigley et al. 1995). It is common for these individuals 
to go undiagnosed until they reach puberty and when they are examined to have an inguinal hernia. 
Individuals with CAIS are considered for this project because they are unable to respond to 
androgens completely and to see how they respond to similar experimental conditions besides 
healthy male individuals. Hence, they can be assigned as the control group.
5
1.4 Plasmacytoid Dendritic Cells
Fig 3: There are three signaling pathways of pDC activation and differentiation. Following viral or 
nucleic acid activation, TLR7, and TLR9 move from the ER to the endosomes to interact with their 
RNA or DNA agonists. TLR conformational alterations activate MyD88 (myeloid differentiation 
primary-response gene 88), which subsequently connects with TRAF6 (tumor necrosis factor 
(TNF) receptor-associated factor 6), BTK (Bruton's tyrosine kinase), and IRAK4 (interleukin-1-
receptor-associated kinase 4). To spread downstream signals, the “MyD88-TRAF6-IRAK4” 
complex activates IRF7 (interferon-regulatory factor 7), TAK1 (transforming-growth-factor-
activated kinase 1), nuclear factor-kB (NF-kB), and IRF5. TRAF3, IRAK1, IKK (NF-B kinase 
inhibitor), osteopontin (OPN), and phosphoinositide 3-kinase (PI3K) are the primary activators of 
IRF7. Following ubiquitylation and phosphorylation, IRF7 translocate to the nucleus and initiates 
type I interferon transcription (Lund et al. 2003; Gilliet, Cao, and Liu 2008).
6
Plasmacytoid dendritic cells (pDCs), also known as "interferon-producing cells," are a type of 
specialized subset of dendritic cells that are antigen-presenting cells found in lymphoid organs and 
consist of 0.4% of peripheral blood mononuclear cells (Blom et al. 2000). These cells can express 
low levels of MHC II molecules which can be upregulated when activated and present to CD4+ T 
cells. pDCs recognize single-stranded viral RNA through pattern recognition receptors such as the 
toll-like receptor 7 (TLR7) as shown in the case of HIV and Influenza Virus (Beignon et al. 2005; 
Cella et al. 2000). Moreover, they can also recognize double-stranded DNA viruses through Toll-
like receptor 9 such as Herpes Simplex Virus-2 (HPV) (Lund et al. 2003). When encountering viral 
nucleic acids, this specialized subgroup of dendritic cells is the initial responder in the innate 
immune system against invaders, secreting higher levels of Type 1 Interferons, primarily IFN alpha 
as an anti-viral response (Coccia et al. 2004) as shown in Fig (c). Studies have suggested that 
females have higher expression of TLR7 as well as higher production of IFN alpha (Pisitkun et al. 
2006). Moreover, these cells can also secrete cytokines that can activate Natural Killer (NK) cells 
to mediate the lysis of target cells (McKenna, Beignon, and Bhardwaj 2005; Swiecki et al. 2010). 
An impairment of pDC function can lead to chronic conditions of certain diseases such as HIV 
(McKenna, Beignon, and Bhardwaj 2005). In humans, male mononuclear cells generate lower type 
I IFNs in response to TLR7 ligands and more IL-10 in response to TLR9 ligands than females 
(Meier et al. 2009). Recent studies have shown that pDC impairment is a critical factor in causing 
severe medical conditions in patients infected by the Sars-Cov2 virus (Zhou et al. 2020). Looking 
at how pDCs have contributed to the innate immune system, it is important to study these cells.
2. Aim
To assess the role of androgen receptor signaling on Type I Interferon by pDCs. Evaluation of 
Interferon production by pDCs will be done after PBMC stimulation of healthy male individuals 
and CAIS patients and then compared to see if the gene and protein expression levels varied among 
the two groups.
7
3. Materials and methods
Table: Materials required
Reagents Company Catalog number
Gibco ™ RPMI 1640 media Thermo Fisher Scientific 61870-010
TheraPEAK ™ XVIVO ™ - 15 Lonza BE02-060F
media
Gardiquimod Invivogen tlr-gdq-5
R1881 Sigma Aldrich 965-93-5
Testosterone Propionate Sigma Aldrich 86541-5G
G15 TOCRIS 2B/277935
2-Mercaptaethanol gibco 21985-023
Benzonase Endonuclease Sigma Aldrich 1.01695.0001
IC Fixation Buffer Invitrogen 00-8222-49
Permeabilization Buffer Invitrogen 00-8333-56
CD3 BioLegend 300430
CD19 BioLegend 302230
BDCA2 BD Biosciences 566427
CD123 BioLegend 306010
IFN-⍺2 BD Pharmingen ™ 560097
Fc block BD Biosciences 564220
Ficoll cytiva 17144003
DMSO SIGMA D2438
8
3.1 Isolation of PBMCs from the blood by Ficoll
Fresh blood was taken from healthy volunteers at Sahlsgrenska University Hospital in Heparin 
tubes, which were inverted three times and kept at room temperature for 30 minutes before being 
put into a single 50ml falcon tube rinsed with PBS. Blood was diluted in PBS RT at a 1:1 ratio and 
divided into two 50 ml falcon tubes. 15 ml Ficoll was introduced to a fresh 50 ml falcon tube. We 
added the diluted blood at a 45-degree angle into the Ficoll at a maximum of 20 mL of blood into 
the tube and centrifuged at 400g for 40 minutes at room temperature, acceleration of 5, and brake 
of 0. The lymphocyte-containing top layer was collected and placed in 10 ml PBS. After that, 50 
ml PBS was added and centrifuged for 20 minutes at 4℃, acceleration: 7, and deceleration: 7. After 
centrifugation, the supernatant was discarded by pouring and 50 ml cold PBS was added and 
centrifuged again. The supernatant was poured out again and 1ml cold PBS was added, pipetted up 
and down to mix, and set for counting.
3.2 Freezing of PBMCs
First, we centrifuge and resuspend cells of 500 µl in FBS and put them on ice. 500 µl with 20% 
Dimethyl sulfoxide (DMSO) was prepared where 100 µl is DMSO with 400 µl of FBS. 500 µl of 
cell suspension was added to each cryovial. Then we added the 20% DMSO+FBS solution to the 
vials so that there is 10% DMSO in each vial. The vials were then put immediately in Mr. Frosty 
Freezing Container under the -80℃ freezer for two days and then transferred to the -150℃ freezer.
3.3 Thawing of PBMCs
A total of four individuals with buffy coats (Male 1: B488; Male 2: B044, Male 3: B479; Male 4: 
11.22) with the highest cell count were included in this project. 20 ml of RPMI+10% charcoal Fetal 
Bovine Serum (FBS)+1%PenStrip+50 µl 2-Mercaptaethanol (BME) was taken and 0.5 µl of 
Benzonase Endonuclease (BE) was added to it and 4 ml was set aside in another falcon tube. The 
reason for using charcoal FBS is to deplete the media of hormones. 4 vials each containing the 
same buffy coat were thawed in a hot water bath for 30 seconds and then with a pipette, 1 ml of it 
was added slowly to the 16 ml RPMI media that we made earlier and rested for 10 minutes. 
Centrifugation was done at 250g, 9 acceleration (acc), 7 deceleration (dec), for 10 minutes at 22℃. 
After discarding the supernatant, 3 ml of the mix was added to it and centrifuged again at 7 acc, 5 
dec, and for 10 minutes. The supernatant was discarded and 1ml of RPMI media was added to it. 
Then we calculated the amount of media for each well (500 µl) and cell suspension and add it to a 
48-well plate. For example, 500 µl multiplied by 12 wells, we need 6ml in total. Therefore, 5 ml 
media was added to the 1 ml of cell suspension. 
9
3.4 Stimulation of PBMCs
The initial concentration of R1881 was 35 mM; to make it 350 µM, 1 µl was added to 99 µl of 
complete media, the step was repeated, and 1.7 µl was put into the wells, with each well having 
1.19 µM. The second R1881 concentration would be 3.5 µM, thus we added 1 µl of the 350 µM 
concentration to 99 µl of complete media. The wells were then filled with 1.7 µl of 11.9 nM 
solution. For two different time points such as 0 and 3 hours, we calculated different concentrations 
of Gardiquimod, a TLR7 agonist, R1881, an AR agonist, Testosterone Propionate, and an 
antagonist namely G15. The starting concentration of testosterone propionate was 20 millimolar. 1 
µl of it was added to 1 ml of media, resulting in a concentration of 20 µM, the step was repeated to 
obtain a concentration of 20 nM, and the concentration was 100 pM after we added 2.5 µl to the 
wells. For the second concentration, 10 pM, we took 1 µl of 20 nM and mixed it with 99 µl media 
before adding 2.5 µl to the well. In the instance of G15, an antagonist for GPER, we use a 
concentration of 0.5 µg, so we add 1 µl to 1 ml of media and 5 µl to the wells. All of this in their 
final concentrations were added to the wells and incubated at 37 ℃ for two hours. For the 
Gardiquimod, we add 9 µl of it to 21 µl media, then add 15 µl of the mix to 15 µl media, and finally 
add 5 µl to the wells. The final concentration of 1.5 µg per ml was added after two hours of 
incubation and incubated for 3 hours. RL buffer was prepared with 5 ml of media and 50 µl of 
BME. 500 µl from the wells were taken into Eppendorf tubes and centrifuged at 200 g, 22 ℃, for 
10 minutes, and 350 µl of RL buffer was added to the wells. The supernatant was discarded while 
we pipetted the RL buffer up and down the wells and collect it into the tubes with the pellet. The 
tubes were then vortexed for 10 seconds at -80 ℃.
3.5 Purification of RNA
Total RNA purification Plus Micro Kit with product code no. 48400 from Norgen Biotek Corp was 
used for RNA extraction and purification according to the manufacturer's protocol.
3.6 cDNA synthesis
The iScript cDNA Synthesis Kit (BioRad) Cat#1708891 was used to generate cDNA according to 
the manufacturer's instructions. Nanodrop was used to determine the concentration of RNA. 
3.7 qPCR
To perform quantitative PCR, The SsoAdvanced Universal SYBR Green Supermix from BioRad 
#L001894 B was used following the recommended protocols provided by the manufacturer. Pre-
designed qPCR primers were obtained from Sigma Aldrich. The qPCR was done in duplicates.
10
3.8 Statistical data
Microsoft Excel was used to analyze the data from the qPCR which typically contained cycle 
threshold (Ct) values that were organized with each column representing a sample and each row 
representing the target gene and its specific Ct value. We calculated the ΔCt (delta Ct) value for 
each sample by subtracting the Ct value of the reference gene from the Ct value of the target gene. 
Next, we calculated the ΔΔCt (delta-delta Ct) value for each sample by subtracting the average ΔCt 
value of the control group from the ΔCt value of the experimental group. Then we calculated the 
fold change in gene expression known as the Relative Quantification (RQ) value for each sample 
by using the formula 2^-ΔΔCt. The data was further analyzed on the software GraphPad Prism 9.
3.9 Flow cytometry
X-Vivo ™ 15 media which is a serum-free media designed for hematopoietic cells was used beside 
the RPMI media following the same protocol of cell stimulation. Brefeldin A (1000x) which is a 
Golgi apparatus blocker was added taking 5 µl of it for all the wells and resting for 3 hours. 
Afterward, the Fc block solution was prepared by calculating first. 1 µl of Fc block was needed for 
100 µl of FACS buffer and 50 µl of Fc block for each sample. The next step was surface staining 
for which 2 µl of CD3, 2 µl of CD19, 2 µl of BDCA2, and 0.5 µl of CD123 were taken. For 25 
samples, 2 x 25=50 µl for the first three antibodies, and for the last one, 12.5 µl was taken. The 
cocktail in a falcon tube contained 2500-165=2335 µl of FACS buffer and the antibodies that we 
just calculated but we took 165 µl with 3 µl of viability dye. The cells were collected by pipetting 
up and down and then 300 µl of PBS+EDTA was added to the cells, pipetted up and down, and 
collected. Centrifugation of the tubes was done at 300 g, for 5 minutes. The supernatant was sucked 
out and then 50 µl of Fc block solution was added to it, pipetted up and down, and added to the 
flow plate. The cocktail mix of 100 µl was added to the plate and rested for 30 minutes. A buffer 
mix with IC fixation buffer which is used to fix cells before permeabilization and 100 µl FACS 
buffer were prepared. We needed 200 µl x 25 = 5 ml so we added 2500 µl of the IC fixation buffer 
and 2500 µl of FACS solution. After 30 minutes, we added 200 µl of FACS to wash and centrifuge 
the well plate at 1500 rpm, 3 mins, 4℃. We repeated the steps two more times. We added the 
fixation buffer to the wells, sealed the plate, and put it in the fridge overnight. The next day, we 
need to permeabilize the cells for intracellular staining i.e., making holes in the membrane. So, we 
added a permeabilization buffer 100 µl to the wells to dilute and centrifuge at 1500 rpm, for 3 
minutes. Meanwhile, we prepared 12.5 µl Fc block and 1250 µl of Permeabilization buffer to 
permeabilize the cells for intracellular staining. The solution was discarded by inverting and 
centrifugation twice. The IFN-α2 was added for intracellular staining. 5 µl of antibody in 95 µl of 
permeabilization buffer was taken. For 25 samples, 125 µl of antibody and 2375 µl of 
permeabilization buffer was calculated. 50 µl of Fc block and 95 µl of IFN-α2 were added to the 
plate, sealed, and put in room temperature for one hour. FACS buffer was added taking 100 µl, 
washed, centrifuged twice at 1500 rpm for 3 minutes. 200 µl of FACS buffer was added to the 
wells, pipetted up and down, and with the plate sealed, it was run in FACS. 
11
3.10 Gating strategy for flow cytometry
Fig 4: The gating strategy for flow analysis is to target leukocytes, followed by single cells, the 
viability of cells, the exclusion of T and B cells, and lastly, the pDCs. BDCA2 (Flurochrome: 
BV421) and CD123 (Fluorochrome: PE) pDC markers distinguish the pDC population (Dzionek 
et al. 2000). Finally, we can see the amount of IFN alpha 2. 
The data is further analyzed in the software FlowJo and GraphPad Prism 9.
3.11 Ethical declaration
Any human blood cells used in my research were collected in accordance with ethical and 
regulatory standards. Necessary precautions were taken to ensure the safe handling, storage, and 
disposal of these cells, including adhering to biosafety regulations and guidelines.
Ethical permit number: 2019-00205/2022-02545-2.
12
4. Results
At first, we decided to test different concentrations of Gardiquimod which is a TLR7 agonist 
meaning that it activates the TLR7 receptor. In order to observe its effects on the expression of the 
targeted genes, we tried three different concentrations such as 0.75 µg/ml, 1.5 µg/ml, and 3 µg/ml.
4.1 Gene expression by qPCR after stimulation with Gardiquimod
Figure 5: Different concentrations (0.75 µg/ml, 1.5 µg/ml, and 3 µg/ml) of Gardiquimod, a TLR7 
agonist show different effects of gene regulation on one male individual at different time points. 
From Figure 5, we saw an upregulated gene expression of the targeted genes such as IFN-α1, IFN-
α2, IRF7, and MX1 at the concentration of 1.5 µg/ml at 3 hours. IFN-α1 and IFN-α2 are the 
common type of IFN alpha that are produced as an antiviral response and IRF7 and MX1 are IFN 
responsive genes with IRF7 responsible as a transcription factor for IFN production. At 6 hours, 
there is a peak with a downregulation reaching the lowest levels at 24 hour-mark. At 0.75 µg/ml, 
the gene expression levels were not significant. Moreover, at concentration of 3 µg/ml, there was 
not significant gene expression levels either. 
13
4.2 Gene expression by qPCR after stimulation with Gardiquimod and R1881
After testing the Gardiquimod, we proceeded further with the R1881, an AR agonist which 
activates the AR receptor. We added the R1881 in two different concentrations with the 
Gardiquimod. We are looking to see what kind of effect could result when the TLR7 receptor and 
the AR are activated. 
Figure 6: The IFN-α2 gene expression varied when PBMCs from three male individuals were 
thawed in RPMI and stimulated with Gardiquimod, a TLR7 agonist, and two concentrations of 
R1881 which is an AR receptor agonist (350 µM, 3.5 µM). The symbols (+) and (-) indicates the 
presence and absence of the stimulants. 
Figure 6 indicates the differences in the gene expression levels of IFN-α2 among three male 
individuals. Male 1 had the highest upregulation when stimulated with Gardiquimod and the higher 
concentration of R1881. However, with the lower concentration of R1881 and Gardiquimod, there 
was a significant downregulation in the gene expression levels. In the case of Male 
4.3 Gene expression by qPCR after stimulation with Gardiquimod and Testosterone 
Propionate
We stimulated PBMCs with Testosterone Propionate which is a synthetic androgen in two different 
concentrations and Gardiquimod. We expected the synthetic androgen to suppress the gene 
expression of IFN-⍺2 given the theory that androgens are responsible for having 
immunosuppressive effects against viral infections.
14
Figure 7: After thawing PBMCs from male people in RPMI media and stimulating them with 
Gardiquimod, a TLR7 agonist, and two doses (100 pM, 10 pM) of Testosterone Propionate, a 
synthetic androgen, IFN-⍺2 gene expression levels differed. 
As we see from Figure 7 that when stimulated with Gardiquimod and Testosterone Propionate, 
Male 1 had the highest upregulation of the gene at both concentrations, whereas Male 3 had a minor 
upregulation with the higher dose. On the contrary, Male 2 did not show any effects.
4.4 Gene expression by qPCR after stimulation with Gardiquimod and G15
Figure 8: IFN-⍺2 productions varied after stimulating PBMCs from male subjects in RPMI 
medium with Gardiquimod, a TLR7 agonist, and G15, an antagonist for the G-protein coupled 
estrogen receptor. The (+) and (-) symbols indicate whether stimulants are present.
It can be seen in the above figure that all three male individuals had a higher gene expression of 
IFN-⍺2 when stimulated with the Gardiquimod and G15, an antagonist in contrast to the previous 
stimulants. The expectation was that G15 would block the expression of IFN alpha, but it did have 
any effect after stimulation, and we saw a higher expression.
15
4.5 Effect of R1881 on IFN-⍺2 protein expression by flow cytometry
After obtaining data from the qPCR, we evaluated the protein expression of IFN-⍺2 by flow 
cytometry.
Figure 9: IFN-⍺2 protein expression in RPMI media analyzed by flow cytometry showed a slight 
if not significant upregulation in all three male individuals in the higher concentration of the AR 
agonist, R1881, and Gardiquimod.
Figure 10: When stimulated in the X-VIVO ™ 15 media, there seems to be a downregulation of 
the IFN-⍺2 protein expression in two male individuals following stimulation with R1881 and 
Gardiquimod.
Here, we observe different effects of the stimulants in different media such as the RPMI and 
XVIVO ™ 15. We decided to go for serum-free media to confirm the effects of the stimulants since 
serum can contain hormone-binding proteins. In the XVIVO ™ 15 media, we see consistency in 
the downregulation of the protein expression in two male individuals that showed a bit of 
upregulation in the RPMI media which was interesting. 
16
4.6 Effect of Testosterone Propionate on IFN-⍺2 protein expression by flow cytometry
 
Figure 11: Flow cytometry analysis of IFN-⍺2 protein expression in RPMI medium revealed a 
slight, albeit not significant, decrease in one male individual while little upregulation can be seen 
in the other two males when stimulation was performed with the synthetic androgen, Testosterone 
Propionate, and Gardiquimod.
Figure 12: In two male individuals, stimulation of PBMCs with Testosterone Propionate and 
Gardiquimod showed downregulation in Male 1 with the lower concentration and Male 2 in both 
concentrations of the synthetic androgen compound.
Figures 11 and 12 show the effects of the Testosterone Propionate on both RPMI and XVIVO ™ 
15 media. There was a slight upregulation of protein expression when it came to Male 3 in RPMI 
media while in XVIVO, it had a rather downregulation which from theory shows that androgens 
have suppressive effects. The difference could be due to the presence of some hormone-binding 
proteins in serum such as estrogen-binding proteins that could trigger the upregulation of the 
stimulant used in this scenario.
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4.7 Effect of G15 on IFN-⍺2 protein expression by flow cytometry
Figure 13: PBMCs of three male individuals when stimulated with G15, an antagonist for GPER 
along with Gardiquimod, a TLR7 agonist in RPMI media show a slight upregulation in two male 
individuals.
Figure 14: PBMCs after 3 hours of stimulation with Gardiquimod and G15 showed 
downregulation in protein expression of IFN-⍺2 in two male individuals. 
To ensure the effect of testosterone, we tested G-15 as estrogen is known to promote IFN 
production. Here in Figure 13 and 14, similar results are observed with enhanced interferon levels 
after stimulation was done with Gardiquimod and G15 in RPMI media. In the X VIVO ™ 15 media, 
we could see downregulation which means that the G15 is able to block the protein expression even 
though it is not significant. These results are in line with previous studies done on estrogen 
signaling.
18
5. Discussion
The purpose of this project was to evaluate the production of IFN-⍺2 secreted by plasmacytoid 
dendritic cells as a means of fighting off infectious agents such as viruses. In the event of COVID-
19, failure in pDC response was a critical factor for inducing the severity of the patient’s conditions 
(Venet et al. 2023). The buffy coats that were selected for this study were based on the donors 
having a higher number of cell counts ranging from 10 to 20 million. The findings of this project 
suggest that the IFN-⍺2 production which is the most common cytokine out of the 13 subtypes of 
human IFN alpha secreted after encountering viruses varied between individuals (Yang et al. 2020; 
Zhang et al. 2020). Among the four individuals, Male 1 had the highest level of the targeted gene 
and protein expression which could be due to the presence of an early infection before donating 
blood since their pDCs will have already been primed. The upregulation of IFN-⍺2 when stimulated 
with R1881, an AR agonist which activates the AR, in RPMI media, contrasted with the theory that 
androgens possess immunosuppressive effects which was rather surprising. The effect of 
testosterone propionate which is a synthetic androgen diluted to the lowest concentrations for their 
potency showed similar results with upregulation in the targeted gene expression in Males 3 and 4. 
The effect of the antagonist G15 was shown to have upregulation when used with the RPMI media. 
This led us to seek an alternative for RPMI media since it could be a possibility that the FBS present 
in the media following charcoal depletion which was done in order to remove sex hormones from 
the serum that could contain hormone-binding proteins such as estrogen-binding proteins might be 
responsible. FBS is not only variable, but it also differs from the fluid to which cells are naturally 
exposed (Baker 2016). Given that the stimulants are potent in nature, only a little amount of 
hormones in FBS can induce a response. Over the last few decades, there has been research to find 
alternatives to FBS with no potential outcome. One of the top suggestions for using alternatives 
was to use depletion or serum-free media. Animal-free media could be a suggestion, but in that 
case, it would still need to be chemically defined in a way that promoted certain cell types. The X-
VIVO ™ 15 media which is a serum-free media and completely devoid of hormones or any other 
factors of undetermined composition was used to further evaluate the effects of the stimulants and 
how they influence the production of IFN-⍺2. The results gave different expression levels of IFN-
⍺2 protein that was quite the opposite of what we obtained from RPMI media. The protein 
expression levels were downregulated in all the individuals. Although the differences in the 
downregulation were not immensely significant, it, however, points towards androgens exerting 
inhibitory effects. This indicates that FBS in the RPMI media could have had a role in the 
upregulation. 
All these results were surprising based on previous observations regarding the role of estrogens 
and testosterone on pDCs. It is to be noted that the presence of sex hormones is not the sole factor 
for modulating innate or adaptive immune responses against infectious diseases. Other 
epidemiological factors include increased age, being male, genetic factors, and underlying medical 
conditions (Pijls et al. 2021). This brings us to the sample size which was a limitation in this study 
due to time constraints. Another limitation was a delay in collecting samples from CAIS patients 
which would have otherwise been taken as a control group thus the data could not be compared. 
The studies are still ongoing with the next step would be to perform the same experiments on the 
CAIS patients repeatedly so the data could be compared. So, there could be more clear insights into 
the androgen receptor and its role in the innate immune system. 
19
6. Conclusion
The data obtained from this project concludes that androgens can play a major role in the innate 
immune system. The theory that this hormone has immunosuppressive effects on the innate 
immune system through impaired pDC activation and differentiation leading to a reduced amount 
of IFN-⍺2 generations and that being male is a demographic factor against viral diseases can be 
realized from the final sets of experiments that were done in the X-VIVO ™ media. Since 
autoimmune diseases prevail in women, emphasis has been given to studying the role of estrogens. 
With the research still ongoing and after the limitations are overcome, we can expect that this could 
lead to an in-depth understanding of how the androgen receptor signaling works and contributes to 
disease severity. While there are androgen deprivation therapies that are being used to treat prostate 
cancers, further research in this area can lead to the development of potential therapeutic strategies 
such as anti-viral therapies to treat male patients to fight off infectious diseases and provide optimal 
disease management for both genders.
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Acknowledgments
First and foremost, I would like to express my heartfelt gratitude to Mattias Svensson, my thesis 
supervisor, for giving me the opportunity to work at his laboratory and for his insightful feedback, 
and constructive criticism during the whole research process. I am truly grateful for it.
I want to extend my deepest appreciation to Negar Ayoubzadeh, my lab supervisor for her 
invaluable guidance, expertise, and consistent motivation which have played an important role in 
defining the direction and quality of my work.
I would like to address the contributions of my friends and classmates, who provided me with 
valuable insights, worthwhile discussions, and moral support during moments of doubt. Their 
friendship and camaraderie have added to the fulfillment and enjoyment of this academic quest.
Lastly, I would like to offer my heartfelt gratitude and appreciation to everyone else who has helped 
me along the way to completing my master’s thesis. This accomplishment would not have been 
achieved without their direction, encouragement, and constant support.
Thank you all for being a part of this important milestone in my life.
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Popular Science Summary
Do androgens put men at higher risk of infection against viral diseases?
Our bodies are complex machines consisting of countless interconnected processes, and the 
immune system is our body's natural defense mechanism that protects us from foreign invaders 
such as bacteria, viruses, and fungi. Sex hormones are one of the key soldiers to building that 
defense. These hormones do so through secreting different pro-inflammatory cytokines that relay 
signals that help our body's immune system to fight off invaders. Because of their unique sex 
hormones such as androgens and estrogens, men and women may fall ill in slightly different ways. 
Men may be more likely to get sick from viruses, whereas women may be more susceptible to 
autoimmune illnesses. It's as though each gender has its very own kryptonite. For a long time, 
scientists have been researching how the female sex hormone, estrogen impacts our bodies, but 
they have not done studies on the male sex hormone, androgen as thoroughly. Based on this fact, I 
have done my project to discover more about how androgens aid our immune systems to combat 
viral diseases. 
At first, we collected blood samples from male donors and isolated peripheral blood mononuclear 
cells (PBMCs) which are made up of different types of cells such as monocytes, lymphocytes, 
natural killer cells, and most importantly, dendritic cells which are the first responders to viral 
pathogens. We begin stimulating the PBMCs with different concentrations of TLR7 agonists which 
are substances that bind to and activate certain receptors in the body which in this case is the TLR7 
receptor and begin the production of interferons such as IFN-⍺1 and IFN-⍺2. They can disrupt viral 
protein synthesis and viral replication, limit viral assembly, and promote the destruction of infected 
cells. Interferons also increase the activity of natural killer cells and cytotoxic T cells, both of which 
are required for eliminating virus-infected cells. We then analyzed the data by molecular techniques 
such as qPCR and flow cytometry. Now, what we expected to see is that there would not be a 
tremendous amount of IFN production since different studies supported androgens having 
suppressive effects on the immune system. However, in some cases, there seemed to be the opposite 
of our expectations. We believe that it had something to do with the media that we used earlier that 
could have possibly contained some sort of hormonal residues to have triggered a higher production 
of the desired cytokine.
To ensure effective research methods, it is essential for all researchers to adopt innovative 
approaches. Individuals with complete androgen insensitivity syndrome (CAIS) are significant in 
this perspective. CAIS patients have this disorder because of mutations in the androgen receptor 
on the X chromosome otherwise known as X-linked recessive disorder, which results in a complete 
lack of androgens. Given that testosterone has no effects on these patients, they provide an 
appropriate basis for comparison with healthy individuals in order to investigate the differences in 
interferon production. After comparison, it would be possible to conclude the importance of 
androgen signaling in fighting off viral infections. Understanding androgen receptor signaling is 
necessary in order to develop therapies in the future.
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