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\begin{document}

\title{Revisiting bupropion anti-inflammatory action: involvement of the
TLR2/TLR4 and JAK2/STAT3}



\author[1]{Alireza Karimollah}%
\author[1]{Anahid Hemmatpur}%
\author[1]{Taha Vahid}%
\affil[1]{Shahid Sadoughi  University of Medical Sciences and Health Services}%


\vspace{-1em}



  
  \date{May 28, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
There are accumulating reports regarding poor response to the common
antidepressant therapy. Antidepressant resistance has often been
associated with activation of the inflammatory system. Accordingly,
major depressive disorder (MDD) patients displaying inflammation prior
to the treatment are less responsive to antidepressants. We hypothesized
that the inefficacy of antidepressant therapy in some patients could be
due to the drugs' inflammatory mode of action that remained overshadowed
by their substantial therapeutic value. Bupropion is a common-used
antidepressant that is prescribed for seasonal affective disorders and
smoking cessation as well. Nevertheless, there are some reports
regarding inflammation induction and depressive behavior exacerbation in
response to bupropion. Here, we put a spot on bupropion and investigate
the alterations of innate and adaptive immunity cytokines and the
influence on immune signaling pathways. Therefore, we treated
LPS-stimulated human peripheral mononuclear cells (PBMCs) with different
doses of bupropion. Pro-/ anti-inflammatory cytokines (TNF-a, \selectlanguage{english}IL-1\selectlanguage{ngerman}ß,
IL-17, and IL-10) on both transcriptional and translational levels are
assessed as well as the involvement of the JAK2 /STAT3, TLR2, and TLR4
signaling in this process. Bupropion decreased IL-17A, TNF-\selectlanguage{english}a, and IL-1\selectlanguage{ngerman}ß
protein levels in the cultures. Nonetheless, the results regarding the
target genes expression were controversial. Surprisingly, IL-1ß, TNF-\selectlanguage{english}a,
and IL-17A genes expression increased following bupropion treatment.
TLR2, TLR4, JAK2, and STAT3 gene expression also rose in response to
bupropion. Our findings suggest that bupropion possesses
pro-inflammatory properties especially at concentrations of 50 and 100
and would rather be co-administrated with anti-inflammatory agents at
least in patients with inflammatory conditions.%
\end{abstract}%



\sloppy


\textbf{Revisiting bupropion anti-inflammatory action: involvement of
the TLR2/TLR4 and JAK2/STAT3}

\textbf{Short title: Bupropion \& inflammatory response}

\textbf{Alireza Karimollah \textsuperscript{1}*, Anahid
Hemmatpur\textsuperscript{2}, Taha Vahid \textsuperscript{1}}

\textsuperscript{1}Department of Pharmacology, School of Pharmacy,
Shahid Sadoughi University of Medical Sciences and Health Services,
Shohaday-e-Gomnam Blvd., Alem Sq., Yazd, Iran

\textsuperscript{2}Department of biochemistry, School of medicine,
Shahid Sadoughi University of medical sciences and Health Services,
Shohaday-e-Gomnam Blvd., Alem Sq., Yazd, Iran

\textbf{* Corresponding author}

\textbf{Alireza Karimollah, PhD}

\textbf{E-mail address: karim2560@yahoo.com}

\textbf{Tel.: +98 (35) 3820 3410}

\textbf{Fax: +98 (35) 3725 4750}

\textbf{Principal investigator statement}

The authors confirm that the PI for this paper is Alireza Karimollah and
that he had direct clinical responsibility for patient.

Word count: 2827

Table count: 1

Figure count: 4

What is already known about this subject?

\begin{itemize}
\tightlist
\item
  A considerable proportion of depressed patients do not respond to the
  common antidepressant therapy.
\item
  Inflammation is linked to the depression pathophysiology and
  inflammatory status prior to the treatment determines the success of
  medication in some MDD patients.
\item
  Bupropion is a common-used antidepressant with the confirmed
  immunomodulatory properties.
\end{itemize}

What this study adds:

\begin{itemize}
\tightlist
\item
  This study revealed that despite the attributed anti-inflammatory
  properties to bupropion, it enhances gene expression of
  pro-inflammatory cytokines including IL-1\selectlanguage{ngerman}ß, TNF-\selectlanguage{english}a, and IL-17A.
\item
  Bupropion upregulates gene expression of TLR2, TLR4 and JAK2/STAT3
  signaling molecules.
\item
  Bupropion would better get administrated with anti-inflammatory
  agents.
\end{itemize}

\textbf{ABSTRACT}

There are accumulating reports regarding poor response to the common
antidepressant therapy. Antidepressant resistance has often been
associated with activation of the inflammatory system. Accordingly,
major depressive disorder (MDD) patients displaying inflammation prior
to the treatment are less responsive to antidepressants. We hypothesized
that the inefficacy of antidepressant therapy in some patients could be
due to the drugs' inflammatory mode of action that remained overshadowed
by their substantial therapeutic value. Bupropion is a common-used
antidepressant that is prescribed for seasonal affective disorders and
smoking cessation as well. Nevertheless, there are some reports
regarding inflammation induction and depressive behavior exacerbation in
response to bupropion. Here, we put a spot on bupropion and investigate
the alterations of innate and adaptive immunity cytokines and the
influence on immune signaling pathways. Therefore, we treated
LPS-stimulated human peripheral mononuclear cells (PBMCs) with different
doses of bupropion. Pro-/ anti-inflammatory cytokines (TNF-a, \selectlanguage{english}IL-1\selectlanguage{ngerman}ß,
IL-17, and IL-10) on both transcriptional and translational levels are
assessed as well as the involvement of the JAK2 /STAT3, TLR2, and TLR4
signaling in this process. Bupropion decreased IL-17A, TNF-\selectlanguage{english}a, and IL-1\selectlanguage{ngerman}ß
protein levels in the cultures. Nonetheless, the results regarding the
target genes expression were controversial. Surprisingly, IL-1ß, TNF-\selectlanguage{english}a,
and IL-17A genes expression increased following bupropion treatment.
TLR2, TLR4, JAK2, and STAT3 gene expression also rose in response to
bupropion. Our findings suggest that bupropion possesses
pro-inflammatory properties especially at concentrations of 50 and 100
and would rather be co-administrated with anti-inflammatory agents at
least in patients with inflammatory conditions.

\textbf{Key words:} Bupropion, response, TLR2, TLR4, JAK2/ STAT3



\textbf{INTRODUCTION}

The interactions between the immune and nervous systems are the major
concern of psychoneuroimmunology. Current literature repeatedly
highlighted the role of inflammation in depression pathogenesis and
relapse \textsuperscript{1,2}. Regarding, cytokine production pattern is
indicated to be dysregulated in MDD patients with inflammatory etiology.
Several meta-analyses reported elevated levels of TNF-a, \selectlanguage{english}IL6, and IL-1\selectlanguage{ngerman}ß
in the blood and brain of MDD patients \textsuperscript{3,4}. IL-17 role
in the pathophysiology of depression is also
established\textsuperscript{5}. Furthermore, mild stimulation of the
immune system with lipopolysaccharides (LPS) could induce the symptoms
of depression\textsuperscript{6,7}. Therefore, the contribution of
inflammatory signaling pathways to the pathophysiology of the disorder
also took into consideration in recent documentations. In this regard,
studies reported an imbalanced toll-like receptor (TLR)-mediated
inflammatory response in MDD. TLR4 mRNA and protein levels were found to
be increased in both the periphery and CNS of MDD patients
\textsuperscript{4,8}. The Janus kinase/signal transducer and activator
of transcription (JAK/STAT) inflammatory signaling pathway is also
proposed to be pathologically linked to depression \textsuperscript{9}.
Regarding, evidence indicated that JAK2/STAT3 is crucial to neuroimmune
system maintenance.

Antidepressants are believed to shift the immune balance toward
anti-inflammatory responses. However, provocation of inflammatory
responses (e.g. increased TNF-\selectlanguage{english}a and IL-6 levels) under treatment with
serotonin and norepinephrine reuptake inhibitors (SNRIs) have been
reported previously in both human and mouse models \textsuperscript{4}.
Despite the development of various classes of antidepressants, over 30\%
of patients don't achieve remission and 60\% of remitted patients
develop a new depressive episode \textsuperscript{2,3}. Recent studies
suggest that inflammatory status prior to the treatment determines the
success of medication in MDD patients at least in the inflammatory form
of the disease \textsuperscript{1}. Moreover, MDD patients with multiple
failed antidepressant treatments showed higher plasma levels of TNF and
IL-6 \textsuperscript{10}. Despite the existence of relatively large
data discussing cytokine protein levels as immune system mediators in
response to antidepressants, expression of correlated genes following
antidepressant administration has remained disregarded.

During systemic inflammation, pro-inflammatory cytokines can pass
through leaky parts of blood-brain barrier and induce neuroinflammation.
Penetrating the brain, cytokines modulate the synthesis, release, and
reuptake of neurotransmitters relevant to the mood including serotonin,
norepinephrine, and dopamine \textsuperscript{11}. Evidence implied that
bupropion decreases IL-1\selectlanguage{ngerman}ß, TNF-\selectlanguage{english}a, and IFN-\selectlanguage{greek}γ \selectlanguage{english}levels while increases IL-10
levels \textsuperscript{12}. Given that bupropion is a
norepinephrine--dopamine reuptake inhibitor (NDRI) and a serotonin
(5-HT) type 3A receptor inhibitor possessing anti-inflammatory
properties \textsuperscript{13}, it is expected to have a double point
advantage as an antidepressant. Meanwhile, in an animal study conducted
by Helaly et al, bupropion exerted an inflammatory effect at therapeutic
doses evidenced by positive NF-\selectlanguage{greek}κ\selectlanguage{english}B/ p65 stained microglia and neurocytes,
obvious inflammation in hippocampus along with a moderate rise in the
mean of serum IL- 6 levels \textsuperscript{14}. Moreover, the
association between bupropion administration and a couple of
inflammatory conditions is stated in a considerable number of case
reports \textsuperscript{15-18}. Some investigations have also stated
the possibility of bupropion-induced psychotic
symptoms\textsuperscript{19}.

Based on the aforementioned observations, we hypothesize that the
absence of the desired response to antidepressants, in our case
bupropion, in some patients could be due to the not previously-noticed
pro-inflammatory properties of the drug. To test the hypothesis, we
evaluated the modulation of protein and gene expression of TNF-a, \selectlanguage{english}IL-1\selectlanguage{ngerman}ß,
IL-17, and IL-10 by bupropion. Gene expression of immunomodulatory
signaling molecules including TLR2, TLR4, JAK2, and STAT3 was also
assessed.

\textbf{MATRIAL AND METHODS}

\textbf{Ethical statement}

It is important to note that the volunteers gave consent to take part in
the study. Tests with the use of human samples have been approved by
University ethical committee with ethical code:
ir.ssu.medicine.rec.1395.337.

\textbf{PBMC isolation, culture, and treatment}

Mutual interaction of microglia and immune cells is indicated to be
present in depression. Therefore concerning microglia's central role in
the development of depressive behavior, studies represented MDD as a
microgliopathy \textsuperscript{20}. Microglia are the present immune
cells of the central nervous system and despite some differences, they
share a similar progeny with peripheral immune cells. Given their
similar progeny, microglia-like cells have been induced from the human
peripheral blood monocyte cells in culture \textsuperscript{21,22}.
Therefore, PBMCs response could reflect the drug impact on both
peripheral and microglia in patients as previously
indicated\textsuperscript{23}. Furthermore, according to the literature
inflammatory status of the monocytes in MDD patients determines the
response rates to some antidepressants \textsuperscript{1}. Therefore,
we chose the PBMCs model to explore bupropion immunomodulatory effects.
PBMCs were isolated from the whole blood collected from 5 healthy
volunteers via Ficoll (Baharafshan, Iran) gradient centrifugation and
washed three times subsequently with RPMI-1640 medium (BioIdea, Iran).
Cells were suspended in RPMI 1640 medium thereafter (supplemented with
100\selectlanguage{english} U of penicillin/ml, 100\selectlanguage{english} \selectlanguage{greek}μ\selectlanguage{english}g/ml streptomycin, 2 mM L-glutamine, and
10\% Fetal Bovine Serum FBS) (FBS, Biochrom, Berlin, Germany). The cell
viability was evaluated using trypan blue (Innoclon, Iran), and cells
with a viability of more than 95\% were selected for culture. Cells were
transferred in duplicate into the wells of a 96-well microplate at the
densities of 10\textsuperscript{4} cells/well. The microculture plate
was incubated at 37\selectlanguage{ngerman}ºC under 5\% CO2 with 90\% humidity. To induce cell
proliferation, phytohemagglutinin (10 \selectlanguage{greek}μ\selectlanguage{english}g/ml) (PHA, Sigma-Aldrich, USA)
was added to the wells in the presence or absence of bupropion. LPS (100
ng/ml) was also added to the test culture and incubated at 37\selectlanguage{ngerman}°C, 5\%
CO2, for 24 hours. Finally, bupropion was administered to PBMCs at
different concentrations (25, 50, 100, 200 \selectlanguage{greek}μ\selectlanguage{english}M). Following 48-hour
incubation, plates were centrifuged at 1700 RPM for 10 minutes, and
Supernatants get collected.

\textbf{Cytokine Array and Enzyme-Linked Immunosorbent Assay (ELISA)}

After 72 h co-treatment of cells with PHA+ LPS and bupropion, the plate
was centrifuged for 10 min, the supernatant removed, and get aliquoted.
Cytokines concentration of IL-1\selectlanguage{greek}β, \selectlanguage{english}IL-17, TNF\selectlanguage{greek}α, \selectlanguage{english}and IL-10 were determined
using ELISA kits (R\&D Systems, US) according to the manufacturer's
protocol. Optical density measurements took at 450 nm.

\textbf{RNA isolation and RT-PCR}

Total RNA isolated from 200 \selectlanguage{greek}µ\selectlanguage{english}L bupropion treated and untreated PBMCs
using Total RNA Mini Kit (Yektatajhiz, Iran) and cDNA synthesized
afterward using a cDNA synthesis kit (Yektatajhiz, Iran) according to
the manufacturer's instruction. Relative mRNA expression of target genes
including tumor necrosis factor-a (TNF-a), Interleukine1(IL-1\selectlanguage{greek}β\selectlanguage{english}), Janus
Kinase 2 (JAK2), and Signal transducer and activator of transcription 3
(STAT3) quantification conducted on Step One Plus real-time polymerase
chain reaction (PCR) system (Applied Biosystems, ABI, Foster City, CA,
USA) using SYBR Green qPCR Master Mix (Applied Biosystems, ABI, Foster
City, CA, USA). \selectlanguage{greek}β-\selectlanguage{english}actin mRNA levels used for results' normalization.
Primer sequences are mentioned in Table 1.

\textbf{Statistical Analysis}

Statistical analysis performed using GraphPad Prism 6.07 Software. Data
analyzed performing one way analysis of variance (ANOVA) followed by
Sidak's multiple comparisons test. Data presented as mean \selectlanguage{ngerman}± Standard
Deviation (SD). A P value of less than 0.05 was considered statistically
significant.



\textbf{RESULTS}

\textbf{Bupropion effect on concentration of target cytokines in PBMCs
culture}

To discover the alterations in pro-inflammatory cytokines profile in
response to bupropion, LPS-PHA treated peripheral blood mononuclear
cells get treated with different concentrations of bupropion. As
depicted in graphs (Fig. 1A-C), bupropion administration significantly
decreased the secretion of pro-inflammatory cytokines including TNF-\selectlanguage{english}a,
IL-1\selectlanguage{ngerman}ß, and IL-17 levels in media assessed by ELISA. We further evaluated
the levels of anti-inflammatory cytokine IL-10; bupropion treatment
resulted in a significant increase in IL-10 concentration in the test
cell culture (Fig.1D).

\textbf{Bupropion effect on gene expression of target cytokines in PBMCs
culture}

In search of the inflammatory response to bupropion treatment at the
gene expression level, we assessed the relative expression of
pro-inflammatory interleukins performing real-time PCR. Interestingly,
mRNA levels of pro-inflammatory cytokines including TNF-\selectlanguage{english}a, IL-1\selectlanguage{ngerman}ß, and
IL-17 were upregulated in cultures following bupropion incubation (Fig.
2A-C). Bupropion treatment also increased mRNA levels of IL-10 compared
to the untreated culture (Fig. 2D).

\textbf{Bupropion effect on the gene expression of TLR2 and TLR4}

TLRs are key components of innate immunity and orchestrate immune
responses by inducing the production of many cytokines. Concerning the
recognized role of TLR2, TLR4 in depression, and as the representative
of innate and adaptive immunity we focused on TLR2, TLR4 signaling
pathways. Bupropion addition to LPS-treated PBMCs significantly
increased gene expression of both receptors (Fig. 3).



\textbf{Tropisetron effect on the gene expression of JAK2 and STAT3}

Several cytokines signal through the Janus kinase/signal transducer and
activator of transcription (JAK/STAT) pathway which is involved in the
development and modulation of immune responses. Among the human JAK four
family members, JAK2 is the most common kinase activated by two-thirds
of ligands. Following activation, STAT3 can promote the production of
both inflammatory cytokines like IL-17 and IL-6 and anti-inflammatory
cytokines (like TGF-\selectlanguage{greek}β \selectlanguage{english}and IL-10) \textsuperscript{24}. We focused on
this kinase and related transcription factor to elucidate the molecular
signaling pathway that might account for the bupropion immunomodulatory
actions. Bupropion treatment led to a considerable rise in gene
expression of JAK2 at 50 and 100 concentrations (Fig. 4A). We also found
out that LPS-treated PBMCs significantly enhanced STAT3 gene expression
in the presence of bupropion (Fig. 4B).

\textbf{Dose-related effects of bupropion}

Based on our findings, bupropion immunomodulatory effect varied
depending on the concentration. Results demonstrated that bupropion
exerts the most inflammatory action in 50 and 100 \selectlanguage{greek}μ\selectlanguage{english}M evidenced by a
sharp increase in mRNA levels of target pro-inflammatory cytokines
(TNF-a, \selectlanguage{english}IL-1\selectlanguage{ngerman}ß, and IL-17) and considerable upregulation of TLR2, TLR4,
JAK2, and STAT3 genes.

\textbf{DISCUSSION}

Bupropion is a common antidepressant that has been prescribed for over
20 years as a sole antidepressant or with other classes of
antidepressants. Meanwhile, literature has reported the low response
rates to bupropion in the patients with treatment-refractory
depression\textsuperscript{25}. Low-grade inflammation is believed to be
involved in the poor responsiveness to regular
antidepressants\textsuperscript{1}. Moreover, despite the repeatedly
mentioned anti-inflammatory effects, some controversies are reported
regarding bupropion action including the development of some
inflammatory abnormalities such as type 1 hypersensitivity reactions
(erythema multiformeis) and serum sickness-like reaction
\textsuperscript{26,27}. Moreover, Eller et al observed elevated levels
of IL-8 in response to bupropion \textsuperscript{28}. Bupropion
administration also failed to lower interleukins secretion in
monocyte-derived macrophages collected from HIV positive women
\textsuperscript{29}. Therefore, we sought to determine bupropion
inflammatory effects and underlying pathways in LPS-stimulated PBMCs.
Present results revealed that bupropion might induce inflammatory
responses via upregulation of TLR2, TLR4, JAK2, and STAT3 and
consequently the pro-inflammatory cytokines gene expression.

In consistence with previous reports \textsuperscript{30,31}, bupropion
inhibited secretion of TNF-\selectlanguage{english}a, IL-1-\selectlanguage{ngerman}ß by LPS-stimulated PBMCs. Data
addressing transcriptional changes in cytokine genes following bupropion
administration is almost scarce. Surprisingly, bupropion increased mRNA
levels of TNF-\selectlanguage{english}a, IL-1-\selectlanguage{ngerman}ß, in the present study. However, in an animal
study conducted by Helaly et al TNF-\selectlanguage{english}a gene expression reported being
down-regulated by bupropion \textsuperscript{14}. Studies concerning
changes in IL-17 levels in response to bupropion are limited. In 2013
Kim et al failed to find a significant change in IL-17 levels under
bupropion treatment \textsuperscript{32}. However, an interesting study
indicated that a higher baseline IL-17 level is associated with a
greater reduction in depression severity in a bupropion-SSRI treatment
arm \textsuperscript{5}. Assessing IL-17A expression at both
translational and transcriptional levels, we figured out that bupropion
significantly increased the cytokine gene expression whereas it somehow
inhibited the protein levels. Th17 cells and its major cytokine product,
IL-17A, promote activation of microglia and astrocytes that may
contribute to the neuroinflammation, neuronal damage, and consequently
lead to depression. In addition to the Th17 cells, CD8+ cells, invariant
natural killer T cells, monocytes, neutrophils, and \selectlanguage{greek}γδ \selectlanguage{english}T cells also
secrete IL-17A. IL-17A facilitates monocyte penetration through the
blood-brain barrier, enhances the blood-brain barrier permeability, and
extends the damage by attracting cytokine-producing
cells\textsuperscript{33}. Reaching the brain, IL-17A can activate
microglia via present IL-17 receptors and indirectly induce the release
of cytokines and chemokines that exacerbates the inflammation.

The abnormally high level of pro-inflammatory cytokines in MDD reflects
the overactive TLR-mediated signaling. Evidence indicated that both
protein and mRNA expression of TLRs including TLR2 and TLR4 are higher
in the central nervous system and peripheral blood of depressed
patients. Bupropion capability to attenuate the deregulated expression
of TLR4 and TLR2 has been suggested by few
studies\textsuperscript{34,35}. However, our obtained results concerning
TLR4 expression profile differed depending on the drug concentration.
Bupropion inhibited TLR4 expression at 25 and 200 \selectlanguage{greek}μ\selectlanguage{english}M, though it enhanced
TLR4 expression at 100 \selectlanguage{greek}μ\selectlanguage{english}M. Bupropion also increased TLR2 gene expression
in stimulated PBMCs. Microglia, the immune cells of the brain, also
express TLR2 and TLR4 and provide pro-/anti-inflammatory cytokines as
mediators for the neuroimmune system. Moreover, microglia showed an
enhanced response to cytokines in MDD patients. Inflammation-induced by
the activation of TLRs in microglia plays a substantial part in the
pathology of depression \textsuperscript{36,37}. Furthermore, TLR4 mRNA
and protein reported being higher in both the periphery and CNS of MDD
patients \textsuperscript{4}. J. Hines et al demonstrated that
intraperitoneal injection of Tat-TLR4 interfering peptides improves
LPS-induced sickness behavior, an important contributor to the
occurrence of depressive-like behavior, via rescuing the microglia
morphology changes and cytokine production that are normally induced by
LPS \textsuperscript{38}. More importantly, negative regulators of the
TLR signaling pathway have been suggested as potential predictive
biomarkers of response to antidepressant treatment.

In search of signaling pathways underlying bupropion immunomodulatory
action, Tsai et al reported that bupropion did not affect LPS-induced
phospho-p65 and phospho-JNK, and phospho-ERK and phospho-p38 expression
in THP-1 cells \textsuperscript{7}. Besides the well-known role in the
peripheral immune responses, a growing body of evidence proposed that
the JAK2-3 signaling pathway is associated with neuroinflammation.
JAK2-3 signaling and consequent secretion of TNF-\selectlanguage{greek}α \selectlanguage{english}and IL-1\selectlanguage{greek}β \selectlanguage{english}by
microglia promote neuronal death and can consequently result in anxiety
and depression \textsuperscript{11,39}. JAK2 and 3 genes stated being
over-expressed in both MDD patients and mice model with inflammation-
induced depressive behavior \textsuperscript{40,41}. Blocking JAK2-3
signaling inhibited pro-inflammatory cytokines release and attenuated
neuroinflammation caused by activated microglia \textsuperscript{42}.
Moreover, intrathecal injection of a JAK2-3 cascade inhibitor
considerably normalized the spinal inflammatory profile caused by
peripheral inflammation \textsuperscript{43,44}. JAK2-STAT3inhibitors
have been proposed as a beneficial strategy in the treatment of
depression \textsuperscript{9,45,46}. Therefore, we investigated the
gene expression of JAK2 and 3 in LPS-treated PBMCs after bupropion
treatment. JAK2 expression was upregulated at 50 and 100 \selectlanguage{greek}μ\selectlanguage{english}M
concentrations of the drug. 3 expression was also increased considerably
at all concentrations of bupropion.

Taken together bupropion might contribute to peripheral and CNS
inflammation via modulating TLR2/TLR4 and JAK2/STAT3 signaling pathways,
particularly at moderate concentrations. We concluded that lack of
expected response to bupropion in MDD patients with inflammatory
etiology could be due to this unnoticed mode of action. Therefore,
bupropion would better get administrated with anti-inflammatory agents.
In confirmation of our results, studies demonstrated that bupropion
combination with anti-inflammatory agents yielded a better
outcome\textsuperscript{47,48}. Of note, bupropion is an antidepressant
with complex mechanisms of action including the recently discovered
effect on the serotonin receptors. Dopamine, norepinephrine,
acetylcholine, and serotonin each exert a specific immunomodulatory
effect. Therefore, more comprehensive studies should get conducted to
understand the net immunomodulatory effect of bupropion under both
inflammatory and immunologically-balanced status in patients with
depressive-like behavior. Our findings also notify the importance of a
personalized medication approach in MDD patients regarding bupropion
prescription.

\textbf{ACKNOWHEDMENTS}

We thank all people in Shahid Sadoughi University of medical sciences
who kindly help us to perform this investigation.

\textbf{CONFLICT OF INTEREST}

The authors declare that there are no conflicts of interest.

\textbf{FUNDING INFORMATION}

This research did not receive any specific grant from funding agencies
in the public, commercial, or not-for-profit sectors.

\textbf{DATA AVALABILITY STATEMENT}

The data that support the findings of this study are available from the
corresponding author upon reasonable request.

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\textbf{Table 1. qPCR primer sequences}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\textbf{Primer} & \textbf{Forward} & \textbf{Reverse}\tabularnewline
\midrule
\endhead
-actin \selectlanguage{greek}β & \textbf{5'-} \textbf{CTGGAACGGTGAAGGTGACA -3'} & \textbf{5'-}
\textbf{AAGGGACTTCCTGTAACAATGCA -3'}\tabularnewline
TLR2 & \textbf{5'- GGGTTGAAGCACTGGACAAT -3'} & \textbf{5'-
TTCTTCCTTGGAGAGGCTGA -3'}\tabularnewline
TLR4 & \textbf{5'-} \textbf{TGGAAGTTGAACGAATGGAATGTG -3'} & \textbf{5'-
ACCAGAACTGCTACAACAGATACT -3'}\tabularnewline
JAK2 & \textbf{5'- CCGATCTGTGTAGCCGGTTT -3'} & \textbf{5'-
GTAAGGCAGGCCATTCCCAT -3'}\tabularnewline
STAT3 & \textbf{5'- TCCTGAAGCTGACCCAGGTA -3} & \textbf{5'-
TCCTCACATGGGGGAGGTAG -3}\tabularnewline
IL-1-\selectlanguage{ngerman}ß & \textbf{5'- CAGAAGTACCTGAGCTCGCC -3} & \textbf{5'-
AGATTCGTAGCTGGATGCCG -3}\tabularnewline
TNF-a & \textbf{5'- CCGATGGGTTGTACCTTGTC -3} & \textbf{5'-
GTGGGTGAGGAGCACGTAGT -3}\tabularnewline
IL-17 & \textbf{5'- TGGAGGCCATAGTGAAGG -3} & \textbf{5'-
TAGTGCTGAGGAGATGTTGC -3}\tabularnewline
IL-10 & \textbf{5'- ACCTCGACTCGCCTACAAAG -3} & \textbf{5'-
GGCCACATGGTGGACAAT -3}\tabularnewline
\bottomrule
\end{longtable}

FIGURE LEGENDS

Figure 1. Bupropion effect on the secretion of the
pro-/anti-inflammatory cytokines. Bupropion treatment significantly
suppressed (A) IL-1\selectlanguage{ngerman}ß, (B) TNF-\selectlanguage{english}a, and (C) IL-17 concentration while
increased the (D) IL-10 levels in PBMCs media. The results are presented
as mean \selectlanguage{ngerman}± S.D, \selectlanguage{english}* p \textless{} 0.05, \selectlanguage{english}*\selectlanguage{english}*\selectlanguage{english}* p \textless{} 0.001, and \selectlanguage{english}*\selectlanguage{english}*\selectlanguage{english}*\selectlanguage{english}* P
\textless{} 0.0001. Statistical analysis was performed by one-way
analysis of variance (ANOVA) followed by the Sidak's multiple
comparisons test.

Figure 2. Bupropion effect on gene expression of target cytokines gene.
Bupropion significantly enhanced the gene transcription of (A) IL-1ss,
(B) TNF-\selectlanguage{english}a, (C) IL-17, and (D) IL-10. Fold changes are calculated with
the 2\textsuperscript{-[?][?]Ct} method. The results are presented as mean \selectlanguage{ngerman}±
S.D, \selectlanguage{english}* p \textless{} 0.05, \selectlanguage{english}*\selectlanguage{english}*\selectlanguage{english}*p \textless{} 0.001, and \selectlanguage{english}*\selectlanguage{english}*\selectlanguage{english}*\selectlanguage{english}* P
\textless{} 0.0001. Statistical analysis was performed by one-way
analysis of variance (ANOVA) followed by the Sidak's multiple
comparisons test.

Figure 3. Bupropion effect on gene expression of TLRs. Treatment of
LPS+PHA-stimulated cells with bupropion promoted the mRNA expression of
(A) TLR2 and (B) TLR4. Fold changes are calculated with the
2\selectlanguage{english}\textsuperscript{-[?][?]Ct} method. The results are presented as mean +-
S.D, \selectlanguage{english}* p \textless{} 0.05, \selectlanguage{english}*\selectlanguage{english}*\selectlanguage{english}*p \textless{} 0.001, and \selectlanguage{english}*\selectlanguage{english}*\selectlanguage{english}*\selectlanguage{english}* P
\textless{} 0.0001. Statistical analysis was performed by one-way
analysis of variance (ANOVA) followed by the Sidak's multiple
comparisons test.

Figure 4. Bupropion effect on gene expression of JAK2 /STAT3. Treatment
of LPS+PHA-stimulated cells with bupropion promoted the mRNA levels of
(A) JAK2 and (B) STAT3. Fold changes are calculated with the
2\selectlanguage{english}\textsuperscript{-[?][?]Ct} method. The results are presented as mean +-
S.D, \selectlanguage{english}* p \textless{} 0.05, \selectlanguage{english}*\selectlanguage{english}*\selectlanguage{english}*p \textless{} 0.001, and \selectlanguage{english}*\selectlanguage{english}*\selectlanguage{english}*\selectlanguage{english}* P
\textless{} 0.0001. Statistical analysis was performed by one-way
analysis of variance (ANOVA) followed by the Sidak's multiple
comparisons test.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/fig-1/fig-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/fig-2/fig-2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/fig-3/fig-3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/fig-4/fig-4}

\end{center}
\end{figure}

\selectlanguage{english}
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