Journal of Psychiatric Research 105 (2018) 95–102

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Journal of Psychiatric Research

journal homepage: www.elsevier.com/locate/jpsychires

Second generation atypical antipsychotics olanzapine and aripiprazole T

reduce expression and secretion of inflammatory cytokines in human
immune cells
Britta Stapela,b, Irina Sieveb, Christine S. Falkc, Stefan Bleicha, Denise Hilfiker-Kleinerb,
Kai G. Kahla,∗
a
Department of Psychiatry, Social Psychiatry and Psychotherapy, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
b
Department of Cardiology and Angiology, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
c
Institute of Transplant Immunology, Integrated Research and Treatment Center Transplantation, IFB-Tx, Hannover Medical School, Carl-Neuberg Str. 1, 30625,
Hannover, Germany

A R T I C LE I N FO A B S T R A C T

Keywords: Schizophrenia and major depression are associated with alterations in peripheral inflammatory markers, and
Schizophrenia anti-inflammatory therapy has been proposed as a promising add-on approach in the pharmacologic treatment of
Major depression both disorders. Second-generation atypical antipsychotics are currently first-line drugs in the treatment of
Cytokines schizophrenia and are also used as augmentation strategies in treatment-resistant major depression.
Inflammation
Furthermore, these drugs have been reported to exhibit distinct metabolic side effects and to influence in-
Olanzapine
flammatory processes.
Aripiprazole
In this study, we used ex vivo stimulation of primary human peripheral blood mononuclear cells (PBMC) from
healthy blood donors with atypical antipsychotics olanzapine or aripiprazole to examine effects on cytokine
production independent from metabolic side effects and disease status.
Both olanzapine and aripiprazole stimulation decreased mRNA levels of IL-1β, IL-6, and TNF-α and resulted
in diminished protein concentrations of IL-6 and TNF-α in conditioned medium of stimulated PBMC. A multiplex
approach revealed additional downregulation of IL-2; MIP-1β and IP-10 secretion. Similarly, olanzapine and
aripiprazole stimulation of the human monocytic cell line THP-1 resulted in a significant decrease in expression
and secretion of IL-1β and TNF-α. Our results suggest that atypical antipsychotics directly influence immune cell
function and thereby highlight the importance to factor in potential side effects of drugs routinely used in
treatment of schizophrenia and major depression on inflammatory processes when considering anti-in-
flammatory drug therapy as an additional treatment option.

1. Introduction
Schizophrenia is a severe, chronic, neurodevelopmental mental
health disorder clinically characterized according to DSM-V by delu-
sions, hallucinations, disorganized speech, disorganized behavior or
negative symptoms, of which 2 must be present over a 1month period
(being delusions, hallucinations or disorganized speech one of them).
Genetic and environmental factors both contribute to the etiology of
schizophrenia. Recent studies suggest that immunological factors,
especially those associated with inflammation, impact disease devel-
opment and progression, which could in turn explain the high incidence
of autoimmune diseases observed in schizophrenic patients (Benros
et al., 2011; Fernandes et al., 2016). Cytokines, which are produced by
various cell types and in turn present potent pleotropic modulators of
inflammatory reactions, constitute a potential readout for chronic in-
flammatory processes.
Serum levels of distinct cytokines were found to be elevated in pa-
tients with first-episode, drug-naïve schizophrenia, which indicates
regulation independent of antipsychotic drug treatment, while addi-
tional cytokines were reported to be regulated depending on clinical
status (Miller et al., 2011). In this regard, a meta analysis found ele-
vated levels of soluble interleukin-2 receptor (sIL-2R), interferon (IFN)-
γ, and tumor necrosis factor (TNF)-α in first-episode, drug-naïve pa-
tients that remained high in chronically ill patients and were in-
dependent of disease state (Miller et al., 2011). Contrarily, serum levels
of IL-1β, IL-6, and transforming growth factor (TGF)-β were found to be

∗
Corresponding author.
E-mail address: kahl.kai@mh-hannover.de (K.G. Kahl).

https://doi.org/10.1016/j.jpsychires.2018.08.017
Received 27 April 2018; Received in revised form 10 August 2018; Accepted 10 August 2018
0022-3956/ © 2018 Elsevier Ltd. All rights reserved.
B. Stapel et al. Journal of Psychiatric Research 105 (2018) 95–102

consistently increased only in schizophrenic patients during an ex- of cytokines previously associated with schizophrenia and major de-
acerbation of symptoms while no significant difference to healthy vo- pression in response to treatment with SGAs by use of an ex vivo setting
lunteers was found during periods of clinical stability (Miller et al., utilizing PBMC from healthy blood donors, thereby obtaining results
2011). independent from metabolic side-effects or disease state. We choose
Furthermore, peripheral inflammation is linked to an increased risk two SGAs, olanzapine and aripiprazole, which are characterized by
for neurodegenerative diseases including Alzheimer's disease and de- distinct metabolic side effects and displayed an opposing influence on
mentia, while major depression is associated with alterations in per- cellular glucose metabolism in an ex vivo setting (Lett et al., 2012;
ipheral cytokine levels (Dowlati et al., 2010; Howren et al., 2009; Stapel et al., 2017). To the best of our knowledge, this is the first study
Jenkinson et al., 1989; Sardi et al., 2011). In this regard, it has been examining effects of olanzapine and aripiprazole on cytokine and che-
reported that alterations in immune response and cytokine levels might mokine expression in human primary cells ex vivo.
directly impact on neurotransmission in the brain by influencing the
dopaminergic, serotonergic, noradrenergic, and glutamatergic neuro-
2. Material and methods
transmission (Miller et al., 2013).
Based on these observations, peripheral inflammation has been
2.1. Isolation, cultivation and treatment of human peripheral mononuclear
considered a potential target for pharmacological treatment of affective
cells and of the human monocytic cell line THP-1
disorders and schizophrenia. In a randomized-controlled trial, the cy-
clooxygenase (COX)-2 inhibitor celecoxib was used as add-on treatment
Human PBMC were isolated from leucocyte reduction filters ob-
to reboxetine in major depression. After 6 weeks of treatment, the re-
tained from healthy individuals at the transfusion unit of Hannover
boxetine plus celecoxib group showed significantly greater improve-
Medical School by use of a density centrifugation utilizing biocoll re-
ment in Hamilton Depression Scale compared to the reboxetine plus
agent (Biochrom) as described previously (Stapel et al., 2017). Physical
placebo group (Muller, 2017; Muller et al., 2006). In the context of
and mental health was confirmed by standardized health questionnaires
schizophrenia, a randomized, double-blind study found that celecoxib
and clinical impression. Further, current drug treatment was an ex-
as add-on therapy improved outcome in patients with acute psychotic
clusion criterium for blood donation.
exacerbation when compared to treatment with risperidone alone.
Cells were cultured at a density of 2*106 cells per mL in RPMI
Further, cognitive function was significantly improved in the risper-
medium (Gibco) containing 10% heat-inactivated FCS (Biochrom), pe-
idone plus celecoxib group (Muller et al., 2002, 2005). However,
nicillin/streptomycin (100 units/100 μg per mL, Gibco), and 2 mM L-
therapeutic benefit of the anti-inflammatory actions of COX-2 inhibi-
glutamine (Gibco) and cultured overnight at 37 °C with 5% CO2 in a
tion appears to be limited to early disease stages and failed to improve
dedicated cell culture incubator. The following day cells were treated
outcome in a chronic setting (Rapaport et al., 2005). Additional anti-
with olanzapine (10−4 M, Selleckchem) or aripiprazole (10−5 M,
inflammatory therapies as treatment option in schizophrenia are cur-
Selleckchem) for 72 h. Control cells were treated with an equal volume
rently discussed and could utilize specific monoclonal antibodies tar-
of the solvent DMSO (0.1%, Sigma). At the end of each experiment, cell
geting distinct cytokines, for instance IL-6 or the soluble IL-6 receptor.
number was determined.
Therefore, a detailed understanding of potential pro- or anti-in-
The human monocytic cell line THP-1 that originally derived from
flammatory actions of drugs already established in the pharma-
an acute monocytic leukemia patient was previously shown to maintain
cotherapy of schizophrenia are required (Miller and Buckley, 2016).
monocytic characteristics for up to 20 month (Tsuchiya et al., 1980).
Some studies suggest that second generation atypical antipsychotics
THP-1 cells were cultured in RPMI medium supplemented as described
(SGA), which according to current guidelines are first line drugs in
above. Cells were passaged twice a week and seeded at a density of
treatment of schizophrenia and also find use in therapy of treatment-
0.2*106. Cells were seeded at a density of 0.5*106 for analysis of mRNA
resistant depression (Nelson and Papakostas, 2009), distinctly affect
expression and at a density of 1.0*106 for generation of conditioned cell
cytokine levels in vivo (Baandrup et al., 2016; Handley et al., 2016;
culture supernatants. Stimulation with olanzapine (10−4 M) and ar-
Lehman et al., 2004). However, different SGAs exhibit distinct meta-
ipiprazole (10−5 M) was carried out for 72 h. Cell number was de-
bolic side effects including weight gain, hyperlipidemia, and diabetes
termined at the end of each experiment.
mellitus and increase the risk for metabolic syndrome and cardiovas-
Conditioned medium from PBMC or THP-1 cells was collected after
cular events (Raedler, 2010). Importantly, obesity and diabetes mellitus
stimulation with olanzapine or aripiprazole as described above. Cell
are considered parameters of metabolic syndrome, which in turn is
culture supernatant was centrifuged at 300 x g for PBMC and 100 x g for
associated with low-grade inflammation and alterations in serum levels
THP-1 cells to eliminate cells and cell debris and medium was stored at
of pro-inflammatory cytokines IL-6 and TNF-α (Expert Panel on
−80 °C. Medium was concentrated 5-fold by use of centrifugal filter
Detection and Adults, 2001; Monteiro and Azevedo, 2010; Srikanthan
units (Merck Millipore) with a cut-off of 3 kDa in accordance to the
et al., 2016). In this regard, it was shown that olanzapine induced
manufacturer's protocol.
weight gain in a rat model, which was associated with elevated serum
levels of IL-1β and IL-8 and increased levels of pro-inflammatory IL-1β,
IL-6 and TNF-α in white and brown adipose tissue and in the hy- 2.2. RNA isolation, cDNA synthesis, semi-quantitative real-time PCR
pothalamus (Davey et al., 2012; Zhang et al., 2014).
Therefore, measurement of serum cytokines does not allow for Total RNA was isolated by use of TRIzol® Reagent (Life
distinction between direct effects of SGAs on immune cells and indirect Technologies) as indicated in the manufacturer's protocol and reverse
actions brought forward by complex metabolic changes; neither does it transcription of one μg RNA was performed with Superscript III
allow for a distinction of contributing cell types. (Invitrogen) and random hexamer primer in accordance to the manu-
A previous study used immune-challenged peripheral blood mono- facturer's instructions. For semi-quantitative real time PCR SYBR Green
nuclear cells (PBMC) to compare effects of the typical antipsychotic qPCR Master Mix (Thermo Fisher) in the AriaMx Real-Time PCR System
haloperidol and three atypical antipsychotics, clozapine, quetiapine, (Agilent Technologies) was used. Primer pairs were designed to span at
and risperidone, with regard to secretion of a limited cytokine panel least one exon junction and respective primer sequences, accession
and found distinct effects on secretion of IL-4, IL-10 and IFN-γ in cells numbers of target-mRNAs, and product sizes are summarized in table
from patients with first-episode schizophrenia, thereby indicating that S1. Data analysis was performed by use of the mean CT-value from each
some antipsychotics directly influence cytokine production by immune PCR reaction that was performed in triplicate. Fold-change values were
cells in an ex vivo setting (Al-Amin et al., 2013). calculated by use of the 2-ΔΔCT formula (Pfaffl, 2001) with 18S rRNA
In the present study, we assessed changes in the expression pattern values used for normalization.

96
B. Stapel et al.
2.3. Detection of cytokine concentrations in cell culture supernatants by
ELISA
Levels of cytokines in conditioned medium of PBMC or THP-1 cells
were assessed by use of commercially available ELISA systems (R&D
Systems) in accordance to the manufacturer's protocol. The following
assay systems were used: human IL-1β (DLB50), human IL-6 (D6050),
human IL-10 (D1000B), and human TNF-α (DTA00C).
2.4. Analysis of cytokine concentrations in cell culture supernatants by
multiplex assays
A chemokine and cytokine profile of PBMC-derived conditioned
medium was compiled using the Bio-Plex Human Cytokine Group 1 27-
Plex Assay (M500KCAF0Y, Bio-Rad, Hercules, USA) in accordance to
the manufacturer's instruction. The assay was performed with 25 μL cell
culture supernatant diluted with 25 μL sample diluent. Chemokines and
cytokines that were above the detection threshold value in all assessed
samples were analyzed and are depicted.
2.5. Statistical analysis
GraphPad Prism 5.0 software was used to perform all statistical
analysis. All data were considered as normally distributed. P values
were calculated by use of two-tailed one sample t-test for data sets
depicted in Fig. 1, or by one-way ANOVA followed by Dunnet's Multiple
Comparison Test for data sets depicted in Figs. 2–5. Differences were
considered to be statistically significant for P < 0.05. As this is a pilot
exploratory study we did not adjust for multiple comparisons as re-
commended in the dedicated literature (Bender and Lange, 2001;
Sainani, 2009). However, we recognize that the type one error might be
inflated without the additional adjustment of P values and confidence
intervals. In all figures, data are depicted as mean ± SEM. Respective
statistical tests as well as n-numbers (representing numbers of experi-
ments performed in independent cell isolations for PBMC or on cells
from different passage number for THP-1 cells) are also detailed in the
respective figure legends; n-numbers ranged from 3 to 4 for the mul-
tiplex and ELISA measurements as well as mRNA expression in THP-
1 cells and from 4 to 6 for analysis of mRNA expression in PBMC.
3. Results
3.1. Ex vivo treatment with atypical antipsychotics decreases mRNA
expression of pro-inflammatory cytokines in PBMC
We previously reported that treatment with indicated concentra-
tions of olanzapine or aripiprazole did not significantly impact on
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Journal of Psychiatric Research 105 (2018) 95–102
survival of PBMC ex vivo excluding unspecific cell toxic effects to in-
fluence obtained results (Stapel et al., 2017). Furthermore, previous
results showed that both treatments did not significantly impact on the
composition of PBMC subpopulations albeit ex vivo culture in itself
altered distribution of subpopulations when compared to values de-
scribed for freshly isolated PBMC (Kleiveland, 2015; Stapel et al.,
2017). Blood levels of several pro-inflammatory cytokines were de-
scribed to be upregulated in the context of schizophrenia and major
depressive disorder (Dowlati et al., 2010; Howren et al., 2009; Miller
et al., 2011). To test whether SGAs olanzapine and aripiprazole directly
affect cytokine expression in PBMC without influence from interfering
clinical values or other cell types, we assessed mRNA levels of relevant
pro- and anti-inflammatory cytokines after ex vivo treatment with
olanzapine and aripiprazole. Treatment with either SGA decreased
mRNA expression of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α
significantly albeit the effect of aripiprazole was more pronounced than
that observed with olanzapine treatment (Fig. 1A and B). In contrast,
both antipsychotics increased mRNA expression of anti-inflammatory
IL-10, olanzapine by trend, aripiprazole significantly, while no effect on
TGF-β1 expression was detected (Fig. 1C and D).
3.2. Olanzapine and aripiprazole treatment diminishes secretion of IL-6 and
TNF-α protein in PBMC ex vivo
In line with decreased mRNA expression of pro-inflammatory cy-
tokines, analysis of protein levels by ELISA showed diminished levels of
IL-6 and TNF-α in conditioned medium from PBMC after treatment with
olanzapine or aripiprazole when compared to medium from respective
DMSO-treated control cells (Fig. 2A and B). Levels of IL-1β were below
the detection level of the assay. In contrast to the observed increase in
IL-10 mRNA, levels of secreted IL10 were significantly reduced in re-
sponse to either olanzapine or aripiprazole (Fig. 2C and D).
3.3. Treatment with olanzapine and aripiprazole decreases mRNA
expression of pro-inflammatory cytokines in monocytic THP-1 cells
As indicated above we could previously show that neither addition
of olanzapine nor with aripiprazole resulted in significant alterations in
the composition of cellular subpopulations of PBMC ex vivo (Stapel
et al., 2017). However, we found that after 72 h of ex vivo culture CD3+
T cells represented more than 80% of the total cell count, while CD14+
monocytes contributed less than 2% in all experimental groups (Stapel
et al., 2017). Therefore, to test the effect of olanzapine and aripiprazole
on cytokine expression specifically in monocytes, the monocytic cell
line THP-1 was treated with these SGAs for 72 h. Similarly to results
obtained in PBMC, both olanzapine and aripiprazole treatment resulted
in a significant decrease in mRNA expression of pro-inflammatory
Fig. 1. Olanzapine or aripiprazole stimulation of
PBMC reduces mRNA expression of pro-in-
flammatory cytokines ex vivo. Bar graphs sum-
marizing mRNA expression of pro-inflammatory cy-
tokines IL-1β, IL-6 and TNF-α in PBMC after 72 h of
olanzapine (Olan, 10−4 M; A) or aripiprazole (Arip;
10−5 M; B) stimulation and graphs showing mRNA
levels of anti-inflammatory IL-10 and TGF-β1 after
stimulation with Olan (C) or Arip (D) compared to
corresponding control (Ctrl) cells. Values for controls
were set as 100% in each cell preparation. Numbers
of independent cell preparations for were as follows:
n = 4 for IL-6; n = 5 for IL-10; n = 6 for IL-1β, TNF-
α and TGF-β1. Results are depicted as mean ± SEM.
P-values were assessed by two-tailed one sample t-
test; ****P < 0.0001, ***P < 0.001, **P < 0.01,
*P < 0.05 versus respective control group.
B. Stapel et al.
cytokines IL-1β and TNF-α (Fig. 3A and B). As observed in PBMC, the
effect of aripiprazole was more pronounced than that of olanzapine. In
contrast to PBMC, IL-6 and IL-10 mRNA level were below the detection
limit in THP-1 cells.
3.4. Olanzapine and aripiprazole decrease secretion of IL-1β and TNF-α in
monocytic THP-1 cells
In line with reduced mRNA levels, treatment with olanzapine and
aripiprazole resulted in reduced levels of secreted pro-inflammatory
cytokines IL-1β and TNF-α in THP-1-derived conditioned medium
(Fig. 4A and B). While levels of IL-10 in supernatants of THP-1 cells
were low when compared to those detected in conditioned medium
from PBMC, protein levels were detectable in all samples. Treatment
with olanzapine and aripiprazole resulted in a significant decrease in IL-
10 content (Fig. 4C and D).
3.5. Treatment with olanzapine and aripiprazole reduces secretion of
several cytokines and chemokines in PBMC ex vivo cultures
To assess more global changes in secretion of cytokines and che-
mokines by PBMC in response to treatment with olanzapine and ar-
ipiprazole, levels of 27 cytokine and chemokine were assessed in con-
centrated supernatants using the Luminex-based multiplex technique.
Treatment with both SGAs resulted in similar changes in levels of se-
creted cytokines and chemokines. Treatment with both SGAs resulted in
a significant decrease in the levels of secreted IL-2, IL-6, IFN-γ inducible
protein (IP)-10 (CXCL10), and macrophage inflammatory protein
(MIP)-1β (CCL4, Fig. 5A and B). Furthermore, IL-9 was reduced by
trend after addition of olanzapine and significantly downregulated after
aripiprazole treatment, while conversely anti-inflammatory IL-1
computed by use of one-way ANOVA followed by Dunnet's Multiple Comparison Test. ***P < 0.001, **P < 0.01 versus respective control group.
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Journal of Psychiatric Research 105 (2018) 95–102
Fig. 2. Protein content of pro- and anti-in-
flammatory cytokines in cell culture super-
natants of PBMC after stimulation with olanza-
pine or aripiprazole. Bar graphs showing relative
protein content of pro-inflammatory cytokines IL-6
and TNF-α (measured by ELISA) in supernatants of
PBMC stimulated with olanzapine (Olan; 10−4 M; A)
or aripiprazole (Arip; 10−5 M; B) and quantification
of anti-inflammatory IL-10 in cell culture super-
natants of PBMC after stimulation with Olan (C) or
Arip (D). Values of controls were set as 100% in each
independent cell isolation. Results are depicted as
mean ± SEM and data were obtained from n = 3
(TNF-α) or n = 4 (IL-6 and IL-10) cell isolations from
individual blood donors. P-values were determined
by use of one-way ANOVA followed by Dunnet's
Multiple Comparison Test. ***P < 0.001 versus re-
spective controls.
receptor antagonist (IL-1RA) was decreased by trend in the aripiprazole
group and significantly downregulated after treatment with olanzapine.
Both antipsychotics resulted in a reduction of IL-9, IL-10, MIP-1α
(CCL3) and TNF-α secretion by trend when compared to corresponding
controls, while no regulation of IL-12, IL-15, fibroblast growth factor
(FGF)-β, granulocyte-macrophage stimulating-growth factor (GM-CSF),
monocyte chemoattractant protein (MCP)-1 (CCL2), platelet-derived
growth factor (PDGF)-bb or vascular endothelial growth factor (VEGF)
was observed (Fig. 5A and B). While none of the analyzed proteins was
significantly upregulated, olanzapine treatment resulted in an increase
of RANTES (CCL5) by trend while aripiprazole resulted in heightened
IL-8 (CXCL8) levels that however were not statistically significant
(Fig. 5A and B).
4. Discussion
In the present study, we assessed effects of two SGAs previously
described to exhibit an unequal risk for metabolic side effects and to
distinctly affect cellular glucose metabolism, on cytokine production by
ex vivo treatment of PBMC from healthy individuals (Stapel et al.,
2017).
We report a similar influence of olanzapine and aripiprazole on
cytokine expression and secretion in the identical experimental setting.
Both SGAs significantly decreased mRNA expression of three pro-in-
flammatory cytokines, IL-1β, IL-6, and TNF-α, which previously were
reported to be increased in the context of schizophrenia and depression,
in PBMC-derived mRNA as well as in serum samples (Himmerich et al.,
2008; Miller et al., 2011; Song et al., 2009). In line with mRNA results
both, IL-6 and TNF-α protein, was significantly decreased in condi-
tioned medium from PBMC after SGA treatment.
Furthermore, both SGAs resulted in upregulation of IL-10 on the
Fig. 3. Olanzapine or aripiprazole stimulation
reduces expression of pro-inflammatory cyto-
kines in monocytic THP-1 cells. Bar graphs de-
picting mRNA expression of pro-inflammatory cyto-
kines IL-1β and TNF-α in THP-1 cells after 72 h of
olanzapine (Olan, 10−4 M, A) or aripiprazole (Arip,
10−5 M, B) stimulation. Values for controls were set
as 100% in each individual experiment and data de-
rived from n = 3 independent cell culture passages.
Results are depicted as mean ± SEM. P-values were
B. Stapel et al.
mRNA level but significantly decreased protein concentrations in su-
pernatants from treated PBMC. Notably, the observed decrease in IL-10
concentration in supernatant of SGA-treated cells was less pronounced
than that observed for IL-6 and TNF-α. The discrepancy in mRNA and
protein might reflect different kinetics in regulation of mRNA expres-
sion and protein secretion. IL-10 is a cytokine that excites anti-in-
flammatory properties by inhibition of pro-inflammatory cytokine
production and was found to be involved in the pathophysiology of
schizophrenia (Fiorentino et al., 1991). On the one hand, IL-10 levels
were reported to be decreased in acute relapsed inpatients (Miller et al.,
2011), and a meta analysis suggests an SNP and two haplotypes of IL-10
to be associated with schizophrenia (Gao et al., 2014).
A multiplex approach confirmed downregulation of IL-6, TNF-α,
and IL-10, albeit the latter two did not reach statistical significance, and
furthermore revealed that both SGAs caused a significant decrease in IL-
2 and interferon-γ-inducible protein (IP)-10 secretion. Previous studies
did not find significant changes in IL-2 levels in serum from schizo-
phrenic patients independent of the disease status (Miller et al., 2011).
However, in the context of major depression a study found increased IL-
2 levels in the responder group when compared to non-responders
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Journal of Psychiatric Research 105 (2018) 95–102
Fig. 4. Protein content of pro- and anti-in-
flammatory cytokines in conditioned medium of
olanzapine or aripiprazole stimulated monocytic
THP-1 cells. Bar graphs depicting protein content of
pro-inflammatory cytokines IL-1β and TNF-α after
olanzapine (Olan, 10−4 M; A) or aripiprazole (Arip,
10−5 M; B) stimulation for 72 h in conditioned
medium of THP-1 cells measured by ELISA. Protein
content of anti-inflammatory cytokine IL-10 in THP-1
culture medium is depicted after stimulation with
Olan (C) or Arip (D). Values for controls (Ctrl) were
set as 100% in each individual experiment and data
derived from n = 3 independent cell culture pas-
sages. Results are depicted as mean ± SEM. P-values
were derived by use of one-way ANOVA followed by
Dunnet's Multiple Comparison Test. ***P < 0.001;
**P < 0.01; *P < 0.05 versus respective control
group.
before the start of antidepressant therapy even after control for BDI-II
levels and BMI (Schmidt et al., 2016) and an increase in serum con-
centrations of its soluble receptor, a marker of T cell activation (Rubin
et al., 1985), was found in the context of first episode psychosis and in
stable medicated outpatients (Miller et al., 2011). As sIL-2R binds IL-2
with a similar affinity as membrane bound IL-2R, the observed increase
in serum levels could be interpreted as immunosuppressive and the
downregulation of IL-2 by olanzapine and aripiprazole might amplify
this effect (Rubin et al., 1986).
Interestingly, we found a slight albeit not significant decrease in IL-
17 levels after olanzapine/aripiprazole treatment. Other groups found
low IL-17 in first episode psychosis (Borovcanin et al., 2012), and in-
creased IL-17 levels in the presence of various neuroleptic agents except
aripiprazole and olanzapine (Himmerich et al., 2011). The observed
differences may be explained by different models used. In the study by
Himmerich et al., whole blood was used and cytokine expression was
stimulated with recombinant TSST-1 (a staphylococcal secreted exo-
toxin), while we used unstimulated PBMC. However, the significance of
IL-17 in schizophrenia is still a matter of debate, since two recent meta-
analyses failed to show altered IL-17 levels in first-episode psychosis, or
Fig. 5. Protein content of cytokines in cell culture
supernatants of PBMC after stimulation with
olanzapine or aripiprazole. Bar graphs depicting
results of multiplex analysis performed on condi-
tioned medium from PBMC after stimulation with
olanzapine (Olan, 10−4 M, A) or aripiprazole (Arip,
10−5 M, B) for 72 h. Respective CC and CXC no-
menclature is provided on the right. Results are de-
picted as mean ± SEM with control cells of each
independent cell preparation set as 100%. Data de-
rived from n = 3 independent cell isolations. P-va-
lues were computed by use of one-way ANOVA fol-
lowed by Dunnet's Multiple Comparison Test. No
correction for multiple testing was performed;
***P < 0.001, **P < 0.01, *P < 0.05 versus re-
spective controls.
B. Stapel et al.
an association between antipsychotic treatment and IL-17 level altera-
tion (Capuzzi et al., 2017; Fang et al., 2018).
IP-10, a chemokine secreted in response to type I and II IFNs, acts as
a chemoattractant for activated T cells (Farber, 1997). While IP-10 le-
vels in the context of schizophrenia were not previously studied, ele-
vated IP-10 serum levels that normalized after antidepressant drug
therapy were described in patients with major depressive disorder
(Wong et al., 2008). Furthermore, it was recently reported that IP-10
levels correlate with the cognitive status of patients with Parkinson
disease, and studies in mice in the context of virus-induced depressive
behavior revealed that IP-10 secretion by endothelial cells directly
conveyed behavioral changes through impairment of synaptic plasticity
(Blank et al., 2016; Rocha et al., 2014). As in our experimental setting
levels of IFN-γ were below the detection limit, it remains unclear
whether the analyzed SGAs directly affected IP-10 release or whether
the observed decrease was due to indirect actions via regulation of IFN-
γ. However, a reduction in IP-10 secretion could contribute to the
therapeutic effect of both olanzapine and aripiprazole. Additionally, we
found that both SGA-treatments resulted in a decrease in MIP-1β
(CCL4) secretion. In comparison to TNF-α or IL-6, the role of MIP-1β is
less well investigated in the context of schizophrenia. However, a recent
study reported significantly increased serum levels of MIP-1β in schi-
zophrenic outpatients when compared to healthy individuals, while a
decrease in serum levels was found in the context of major depressive
disorder (Hong et al., 2017; Lehto et al., 2010).
Finally, treatment with aripiprazole significantly decreased levels of
IL-9 while aripiprazole treatment decreased anti-inflammatory IL-1RA
by trend and olanzapine application resulted in a significant decrease of
IL-1RA. IL-9 was recently found to be increased in serum samples de-
riving from patients with multiple-episode schizophrenia while no
significant differences between first-episode schizophrenic patients and
healthy controls were observed (Frydecka et al., 2018). The same study
described elevated levels of anti-inflammatory IL-1RA in the context of
multiple-episode schizophrenia (Frydecka et al., 2018).
Most previous studies focused on cytokine levels and immune cell
function after in vivo treatment in animal models, healthy volunteers,
or patients with schizophrenia leading to contradicting results (Bilici
et al., 2003; Davey et al., 2012; Handley et al., 2016). Long-term
treatment with different SGAs appears to evoke primarily anti-in-
flammatory effects (Fonseka et al., 2016). However, anti-inflammatory
actions seem to affect state markers that vary dependent on clinical
status and appear to have limited effects on trait markers including
TNF-α (Miller et al., 2011). Furthermore, opposing effects concerning
the influence of antipsychotics on cytokine networks have been re-
ported (Pollmacher et al., 2000) and studies in rats have revealed that
olanzapine treatment resulted in weight gain and simultaneously in-
duced levels of pro-inflammatory cytokines IL-1β and IL-8 in females
(Davey et al., 2012). In vitro studies showed that olanzapine reduced
expression of inflammatory cytokines including IL-10 and TNF-α in
primary rat glia cells under LPS stimulation and aripiprazole similarly
decreased expression of TNF-α in response to IFN-γ in murine microglia
cells (Faour-Nmarne and Azab, 2016; Kato et al., 2008). Contrarily,
both olanzapine and aripiprazole next to other SGAs increased ex-
pression of pro-inflammatory cytokines including IL-1β and TNF-α in
primary human adipose-derived stem cells indicating cell type- and
species-specific effects (Sarvari et al., 2014).
We could show in previous studies that ex vivo culture resulted in
alterations in the composition of PBMC subpopulations (Stapel et al.,
2017). While treatment with either SGA affected culture composition
only marginally when compared to controls, ex vivo culture in itself
resulted in an overrepresentation of CD3+ T cells, while relative
numbers for other lymphocyte subtypes including CD19+ B cells and
CD56+ NK cells as well as CD14+ monocytes were lower than pre-
viously reported for freshly isolated PBMC (Kleiveland, 2015; Stapel
et al., 2017).
As monocytes are potent sources of cytokine secretion, we utilized
100
Journal of Psychiatric Research 105 (2018) 95–102
the human monocytic THP-1 cell line to analyze effects of olanzapine
and aripiprazole in this cell type. Similarly to PBMC both atypical an-
tipsychotics reduced mRNA expression of IL-1β and TNF-α, while IL-6
and IL-10 were not detectable on mRNA level in THP-1 cells. Levels of
secreted IL-1β and TNF-α were found to be decreased, albeit this re-
duction was less pronounced than that observed in PBMC cultures, and
as in PBMC, IL-10 was reduced by trend in response to olanzapine and
decreased significantly with aripiprazole stimulation. Overall results
obtained in THP-1 cells were similar to those from PBMC.
In general, we found that both olanzapine and aripiprazole treat-
ment in PBMC and in monocytic THP-1 cells decrease the expression
and secretion of a variety of cytokines. While we did not perform ex-
periments to investigate potential signaling pathways mediating this
broad effect of SGAs on cytokine expression, one could speculate that
both olanzapine and aripiprazole might target similar downstream
pathways. In this regard, elevated levels of nuclear factor kappa B
(NFκB), which constitutes a key regulator of inflammatory processes
and cytokine expression, has been described in PBMC derived from
patients with schizophrenia, which correlated with expression levels of
TNF-α and IL-1β (Song et al., 2009). Furthermore, both olanzapine and
aripiprazole have been described to suppress NFκB activation and to
diminish inducible nitric oxide synthase (iNOS) expression in experi-
mental models and in both studies SGA treatment was associated with
reduced expression of inflammatory cytokines including TNF-α
(Todorovic et al., 2016; Yoo et al., 2018). Therefore, it is an enticing
possibility that olanzapine and aripiprazole might impact on cytokine
expression by modulating NFκB signaling.
Overall our findings complement results of a meta analysis showing
that while pro-inflammatory cytokines IL-6, INF-γ, and TNF-α are up-
regulated in patients with drug-naïve first-episode psychosis, or an
acute relapse of psychosis, levels of IL-1β, IL-6, and IFN-γ were de-
creased in stable medicated outpatients with treatment-resistant psy-
chosis (Muller, 2017), which might indicate that alterations in cytokine
levels in response to antipsychotic drug treatment could at least in part
be attributed to direct actions on peripheral immune cells. Importantly,
we found a significant decrease in TNF-α mRNA and protein, a cytokine
that remains elevated in stable medicated outpatients and in treatment-
resistant psychosis, which might be due to the contribution of non-
immune cells to the serum cytokine pool in vivo (Miller et al., 2011).
We hypothesize that biologic processes evoked by application of
SGAs might be similar in immune cells of the periphery and of the
brain. Furthermore, as it has previously been shown that peripheral
inflammation and immune responses in the CNS are interconnected, it
appears likely that SGA-mediated immune-modulating effects in the
periphery might also impact on processes in the brain, including neu-
rotransmission and neuronal plasticity (Blank et al., 2016; Engelhardt
and Sorokin, 2009; Kohler et al., 2016). With regards to schizophrenia,
alterations in neuronal plasticity as well as dopaminergic and gluta-
matergic function have been described and dysregulations in neuro-
transmission are well established in the context of depression. Im-
portantly, inflammatory cytokines have been shown to influence the
synaptic availability of monoamines by interfering with tryptophan
metabolism and tetrahydrobiopterin (BH4) level, and via mediation of
SERT expression and activation directly impacting on synaptic avail-
ability of 5-HT (Felger et al., 2013; Maes et al., 2011; Neurauter et al.,
2008; Raison et al., 2010; Zhu et al., 2010). Therefore, one might
speculate that immune modulating effects of SGAs in the periphery as
well as in the CNS might contribute to the therapeutic effect of these
drugs in schizophrenia as well as in major depressive disorder.
Limitations: The present study must be considered a pilot ex-
ploratory study that was conducted in a limited number of subjects. All
analysis were conducted in an ex vivo setting, which allows for analysis
of cytokine expression by PBMC without secondary effects brought
forward by metabolic side effects of SGAs and without serum factors
that impact on half-life of cytokines. However, ex vivo culture results in
an alteration of cell subpopulations as outlined above and PBMC
B. Stapel et al.
isolations also exclude several blood cell types including erythrocytes
and granulocytes that might impact cytokine networks in vivo. In terms
of generalizability of the findings it might be necessary to test the
presented immune-modulating effects of olanzapine and aripiprazole in
an additional in vitro system such as a whole blood assay, since this
would include additional cell types and serum proteins that might in-
fluence cytokine production. Furthermore, we assessed effects of SGAs
on cytokine expression in PBMC from healthy blood donors instead of
schizophrenic patients to obtain results independent of the disease state
and potential medication. Assessing the effects of different anti-
psychotics in blood from patients with schizophrenia should be subject
to further investigations. Therefore, it cannot be excluded that SGAs
might elicit divergent effects in the context of the disease. While we
assured that the SGA concentrations used in this study did not exhibit
cell toxic effects that might influence the observed results, we did not
check the effect of different SGA concentration on cytokine production,
and we therefore cannot exclude that lower concentrations of olanza-
pine and aripiprazole might have divergent effects on cytokine pro-
duction. Lastly, we did not immune challenge PBMC before and during
ex vivo treatment with SGAs, as such stimulation might result in dif-
ferent outcomes that depend on the stimulus and simulates an acute
immune reaction rather than the chronic inflammatory phenotype ob-
served in the context of schizophrenia and depression. While analysis of
spontaneous cytokine secretion avoids unspecific effects by the immune
stimulus and prevents that massive immune reaction resulting from
pyrogen stimulation masks potential effects of SGAs, spontaneous cy-
tokine expression by PBMC is low, resulting in levels below the detec-
tion limit of the respective assays for some cytokines.
Taken together, treatment with olanzapine or aripiprazole resulted
in a decreased expression of multiple pro- and anti-inflammatory cy-
tokines in an ex vivo approach independent of disease status, secondary
metabolic side effects, or interfering serum parameters. Our results
suggest that olanzapine and aripiprazole directly target spontaneous
production and secretion of a wider range of cytokines and chemokines
in PBMC cultures including those that previously have been indicated to
be regulated in the context of schizophrenia and/or major depression.
Atypical neuroleptics have been described effective in treatment of
schizophrenia, and as adjunctive therapy in treatment-resistant major
depression (Turner et al., 2014; Zhou et al., 2015). This may also apply
to other psychiatric disorders such as anorexia nervosa, where in-
creased TNF-α levels have been observed (Kahl et al., 2004; Solmi et al.,
2015), and where olanzapine has been discussed as a treatment option
(Dold et al., 2015). We hypothesize that the beneficial effects of olan-
zapine and aripiprazole might at least in part be mediated via their anti-
inflammatory properties. However, further prospective, long-term stu-
dies in patients with schizophrenia and major depression are needed to
confirm this hypothesis.
Declaration of interest
Kai G. Kahl has received speaker honoraria and a research grant by
Servier, and speaker honoraria by Lundbeck, EliLilly and Otsuka. Stefan
Bleich has received honoraria as a member in advisory board of
Oberberg Group. Britta Stapel, Irina Sieve, Christine Falk and Denise
Hilfiker-Kleiner declare no potential conflict of interest.
Appendix A. Supplementary data
Supplementary data related to this article can be found at https://
doi.org/10.1016/j.jpsychires.2018.08.017.
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