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Fluoxetine and citalopram exhibit potent antiinflammatory activity in human and murine models of rheumatoid arthritis and inhibit toll-like receptors.

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ARTHRITIS & RHEUMATISM
Vol. 62, No. 3, March 2010, pp 683–693
DOI 10.1002/art.27304
© 2010, American College of Rheumatology
Fluoxetine and Citalopram Exhibit Potent Antiinflammatory
Activity in Human and Murine Models of
Rheumatoid Arthritis and Inhibit Toll-like Receptors
Sandra Sacre, Mino Medghalchi, Bernard Gregory, Fionula Brennan, and Richard Williams
Objective. Selective serotonin reuptake inhibitors
(SSRIs), in addition to their antidepressant effects,
have been reported to have antiinflammatory effects.
The aim of this study was to assess the antiarthritic
potential of 2 SSRIs, fluoxetine and citalopram, in
murine collagen-induced arthritis (CIA) and in a human ex vivo disease model of rheumatoid arthritis (RA).
Methods. Following therapeutic administration of
SSRIs, paw swelling was assessed and clinical scores
were determined daily in DBA/1 mice with CIA. Joint
architecture was examined histologically at the end of
the treatment period. Cultures of human RA synovial
membranes were treated with SSRIs, and cytokine production was measured. Toll-like receptor (TLR) function was examined in murine and human macrophages,
human B cells, and human fibroblast-like synovial cells
treated with SSRIs.
Results. Both SSRIs significantly inhibited disease
progression in mice with CIA, with fluoxetine showing
the greatest degree of efficacy at the clinical and histologic levels. In addition, both drugs significantly inhibited the spontaneous production of tumor necrosis
factor, interleukin-6, and interferon-␥–inducible pro-
tein 10 in human RA synovial membrane cultures.
Fluoxetine and citalopram treatment also inhibited the
signaling of TLRs 3, 7, 8, and 9, providing a potential
mechanism for their antiinflammatory action.
Conclusion. Fluoxetine and citalopram treatment
selectively inhibit endosomal TLR signaling, ameliorate
disease in CIA, and suppress inflammatory cytokine
production in human RA tissue. These data highlight
the antiarthritic potential of the SSRI drug family and
provide further evidence of the involvement of TLRs in
the pathogenesis of RA. The SSRIs may provide a
template for potential antiarthritic drug development.
Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease affecting 1% of the population worldwide. It is characterized by a destructive
inflammation of the joints, leading to progressive disability and reduced life expectancy. The synovial membrane is infiltrated by immune cells, including macrophages and T cells, resulting in the chronic production of
proinflammatory cytokines and matrix metalloproteinases (MMPs), leading to cartilage and bone degradation
(1). The mechanisms responsible for the perpetuation of
the chronic inflammation associated within the RA joint
are currently unknown. However, a family of evolutionary conserved innate immune receptors, the Toll-like
receptors (TLRs), has been suggested to contribute to
this process (2).
TLRs recognize and respond to pathogens and
endogenous molecules that are potentially released
during tissue inflammation and damage (3,4). They are
type I membrane receptors that are mainly found at the
cell surface, except for TLRs 3, 7, 8, and 9, which are
localized to the endosome (5). Most data have indicated
a role of TLR-4 in murine models of experimental
arthritis (6–8). In human studies, most TLRs have been
detected in rheumatoid tissue (9–13) in addition to
potential TLR ligands (14,15). RA synovial tissue re-
Supported by the Arthritis Research Campaign, the Kennedy
Institute of Rheumatology Trustees, the European Commission Collaborative Masterswitch grant, and by the Biomedical Research Centre
funding program of the National Institute for Health Research.
Sandra Sacre, PhD (current address: Brighton and Sussex
Medical School, University of Sussex, Brighton, UK), Mino Medghalchi, MSc, Bernard Gregory, PhD, Fionula Brennan, PhD, Richard
Williams, PhD: Kennedy Institute of Rheumatology, Imperial College
of Science, Technology, and Medicine, London, UK.
Dr. Sacre has filed a patent application for the use of selective
serotonin reuptake inhibitors in the treatment of arthritis (no financial
benefit to declare). Dr. Brennan has received consulting fees, speaking
fees, and/or honoraria from Wyeth (less than $10,000).
Address correspondence and reprint requests to Sandra
Sacre, PhD, Brighton and Sussex Medical School, University of Sussex,
Falmer, Brighton BN1 9RY, UK. E-mail: s.sacre@bsms.ac.uk.
Submitted for publication August 11, 2009; accepted in
revised form November 15, 2009.
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SACRE ET AL
moved during elective surgery produces high levels of
multiple cytokines and MMPs in cultures (11). Using
this tissue, we have previously demonstrated that the
spontaneous production of cytokines is partly dependent
on TLR signaling (11). In subsequent studies, we identified a role of TLR-8 in inducing the production of
tumor necrosis factor (TNF). That study also showed
that a serotonin receptor antagonist, mianserin, inhibited TLRs 3, 7, 8, and 9 in primary human cells and
significantly decreased the production of TNF and
interleukin-6 (IL-6) in human RA synovial cell cultures
(10), suggesting a potential role of 1 or more of these
TLRs in the pathogenesis of RA.
We had initially become interested in selective
serotonin reuptake inhibitors (SSRIs) because of their
reported antiinflammatory effects (16). Increasing evidence has highlighted a link between the immune system
and the symptoms of depression. This was originally
referred to as the macrophage theory of depression,
suggesting an association with inflammatory cytokines
(17). It has since been shown that patients with depression have elevated blood levels of cytokines, as compared with healthy controls, and that these levels are
reduced upon treatment with SSRIs (18,19). In patients
who failed to respond to SSRIs, no reduction in cytokine
levels was observed (20), which suggests a connection
between SSRIs, depression, and the immune system.
In the present study, our aim was to investigate
whether the antiinflammatory properties of 2 SSRIs,
fluoxetine and citalopram, would be beneficial in murine
and human disease models of RA and to determine
whether these drugs could potentially work by a mechanism similar to that of mianserin, through inhibition of
TLR-induced cytokine production.
MATERIALS AND METHODS
Reagents. Cell culture reagents used were penicillin/
streptomycin, RPMI 1640, and Dulbecco’s modified Eagle’s
medium (DMEM) obtained from Cambrex (Verviers, Belgium), indomethacin from Sigma (St. Louis, MO), and fetal
bovine serum (FBS) from PAA Laboratories (Linz, Austria).
The TLR ligands used were chloroform-extracted Escherichia
coli lipopolysaccharide (LPS), resiquimod (R-848), poly(I-C),
CpG-containing oligonucleotide (ODN; 2006 and 1668), and
imiquimod from InvivoGen (San Diego, CA). Flagellin (purified) and Pam3Cys-Ser(Lys)4 䡠 3HCl (Pam3CSK4) were from
Alexis (Nottingham, UK). Fluoxetine hydrochloride and citalopram hydrobromide were purchased from Sigma (Poole,
UK). Macrophage colony-stimulating factor (M-CSF) was
purchased from PeproTech (London, UK). Human CD19
microbeads were purchased from MACS Miltenyi Biotec
(Bisley, UK). All reagents were tested for LPS using the
Limulus amebocyte lysate assay from Cambrex (Walkersville,
MD) (21) and found to have no detectable levels of LPS.
Cell culture. RA synovial membrane cells were isolated from samples obtained from patients undergoing joint
replacement surgery, as previously described (22,23). Immediately after isolation, cells were cultured at 1 ⫻ 105 cells/well in
96-well tissue culture plates (Falcon, Oxford, UK) in RPMI
1640 containing 10% (volume/volume) FBS and 100 units/ml
of penicillin/streptomycin. All patients gave written informed
consent, and the study was approved by the Local Ethics
Committee.
Human macrophages were preincubated for 30 minutes with 1 or 5 ␮g/ml of fluoxetine or with 10, 20, or 30 ␮g/ml
of citalopram and then either left unstimulated or stimulated
with 10 ng/ml of flagellin, 10 ng/ml of Pam3CSK4, 1 ␮g/ml of
R-848 or 10 ng/ml of LPS for 6 hours. Human RA synovial
fibroblasts were preincubated with media alone or containing
1 or 5 ␮g/ml of fluoxetine or with 10, 20, or 30 ␮g/ml of
citalopram and then either left unstimulated or stimulated with
20 ␮g/ml of poly(I-C) for 24 hours. Primary human B cells
were preincubated for 30 minutes with 1 or 5 ␮g/ml of
fluoxetine or with 10, 20, or 30 ␮g/ml of citalopram and then
either left unstimulated or stimulated with 10 ␮g/ml of imiquimod or 2.5 ␮M ODN2006 for 24 hours. Human RA
synovial membrane cells were cultured for 24 hours in the
presence of media alone or 5 ␮g/ml of fluoxetine, 30 ␮g/ml of
citalopram, or 10 or 100 ␮M serotonin.
Primary human synovial fibroblasts and peripheral blood
monocytes were isolated and cultured as previously described
(24–26). Macrophages were derived from monocytes after differentiation for 4 days in the presence of 100 ng/ml of M-CSF. B
lymphocytes were obtained by using CD19 microbeads according to the manufacturer’s instructions. Immediately after isolation, cells were cultured at 2 ⫻ 105 cells/well in 96-well tissue
culture plates in RPMI 1640 containing 5% (v/v) FBS and 100
units/ml of penicillin/streptomycin.
Murine bone marrow–derived macrophages were derived from the femurs of male DBA/1 mice. Macrophages were
cultured for 6 days with DMEM containing FCS (20% v/v),
100 units/ml of penicillin/streptomycin, 2-mercaptoethanol
(50 ␮M), and 10 ng/ml of M-CSF.
Enzyme-linked immunosorbent assay (ELISA). Sandwich ELISAs were used to measure human TNF, interferon␥–inducible protein 10 (IP-10), and IL-6 (R&D Systems,
London, UK). Sandwich ELISAs were also used to measure
murine TNF, RANTES, IL-12, and anticollagen IgG1 and
IgG2a (BD PharMingen, San Diego, CA). Absorbance was
read on a spectrophotometric ELISA plate reader (Labsystems
Multiscan Biochromic) and was analyzed using Ascent software V2.6 (both from Thermo Labsystems, Cambridge, UK).
Cell viability was not significantly affected over this time
period, as evaluated by MTT assay (Sigma, Poole, UK) (27).
CIA model. Male DBA/1 mice (8–12 weeks of age)
were immunized subcutaneously at the base of the tail with
200 ␮g of type II collagen emulsified in Freund’s complete
adjuvant (Difco, West Molesey, UK). The onset of arthritis
was considered to be the day that erythema and/or swelling was
first observed, and each limb of the arthritic mice was given a
clinical score on a scale of 0–3, where 0 ⫽ normal, 1 ⫽ slight
erythema and/or swelling, 2 ⫽ pronounced edematous swell-
ANTIARTHRITIC POTENTIAL OF TWO SSRIs IN HUMAN AND MURINE MODELS OF RA
685
Figure 1. Fluoxetine inhibition of disease progression and paw swelling in the mouse model of collagen-induced arthritis (CIA). Mice with
CIA were given an intraperitoneal injection of vehicle (phosphate buffered saline [PBS]; controls) or fluoxetine (10 mg/kg or 25 mg/kg) once
a day for 7 days. A and B, Mice were assessed for clinical score (n ⫽ 14–15 mice per group) (A) and paw thickness (n ⫽ 11–13 mice per group)
(B) on a daily basis. C, Mice were bled on day 7, and serum levels of IgG1 and IgG2a anticollagen antibodies (n ⫽ 11 mice per group) (left)
and interleukin-12 (IL-12) (n ⫽ 6–7 mice per group) (right) were measured by enzyme-linked immunosorbent assay. D, Arthritis was scored
histologically as described in Materials and Methods (n ⫽ 11 mice per group). Values in A–D are the mean ⫾ SEM of pooled data from 2
separate experiments. Values for IL-12 represent individual mice; horizontal lines show the mean. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍
0.001 for fluoxetine 25 mg/kg versus controls. E, Histologic features of arthritic joints from control mice and mice treated with 25 mg/kg
fluoxetine on day 7 of treatment. Photomicrographs of tarsometatarsal joints (top) and proximal interphalangeal joints (bottom) show
protection from inflammation and joint erosion in fluoxetine-treated mice versus control mice (original magnification ⫻ 5 in top panels; ⫻ 10
in bottom panels).
ing, and 3 ⫽ joint deformity with ankylosis (maximum score 12
per animal).
Fluoxetine (10 or 25 mg/kg) and citalopram (25 mg/kg)
treatments were administered daily for 7 days starting on the
day of arthritis onset. Paw swelling was assessed daily by
measuring hindpaw thickness using calipers. All measurements
were recorded in a blinded manner.
Histologic assessment of arthritis. On completion of
the experiment, the first mouse limb that was observed to show
evidence of arthritis was processed for histologic assessment.
The limb was removed, fixed, decalcified, and paraffinembedded before sectioning and staining with hematoxylin and
eosin. Sagittal sections of the proximal interphalangeal joint of
the middle digit were examined by microscopy in a blinded
manner. The histologic severity of arthritis was graded on a
scale of 0–3, where 0 ⫽ normal, 1 ⫽ minimal synovitis,
cartilage loss, and bone erosions limited to discrete foci, 2 ⫽
synovitis and erosions present, but normal joint architecture
intact, and 3 ⫽ synovitis, extensive erosions, and disrupted
joint architecture.
Statistical analysis. The mean, SD, SEM, and statistical significance were calculated using GraphPad version 3
software (GraphPad Software, San Diego, CA). For statistical
analysis, a 1-tailed t-test of paired data was used with a 95%
confidence interval. The SEM was used for pooled experimen-
tal data, whereas the SD was used in graphs showing representative experiments. A 1-tailed Mann-Whitney test was used
with a 95% confidence interval for the CIA data. P values less
than 0.05 were considered significant.
RESULTS
Fluoxetine halts disease progression in the murine CIA model. To investigate whether the reported
antiinflammatory effect of fluoxetine would be beneficial in a murine model of experimental arthritis, we
decided to use the CIA model. This model was chosen
because of its histologic similarities to human RA, with
comparable synovitis, bone erosion, and pannus formation, and because it responds similarly to anti-TNF
therapy (28). Administration of fluoxetine was given
therapeutically after the onset of clinical arthritis (day 1
of arthritis).
Mice given 10 mg/kg of fluoxetine showed a small
reduction in the clinical score and a slower increase in
paw swelling (Figures 1A and B). At the higher dose
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SACRE ET AL
Figure 2. Citalopram inhibition of disease progression and paw swelling in the mouse
model of collagen-induced arthritis (CIA). Mice with CIA were given an intraperitoneal
injection of vehicle (phosphate buffered saline [PBS]; controls) or citalopram (25 mg/kg)
once a day for 7 days. A and B, Mice were assessed for clinical score (n ⫽ 7 mice per group)
(A) and paw thickness (n ⫽ 7 mice per group) (B) on a daily basis. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍
0.01 for citalopram 25 mg/kg versus controls. C, Mice were bled on day 7, and serum levels
of IgG1 and IgG2a anticollagen antibodies (n ⫽ 5 mice per group) were measured by
enzyme-linked immunosorbent assay. D, Arthritis was scored histologically as described in
Materials and Methods (n ⫽ 4–6 mice per group). Values are the mean ⫾ SEM.
(25 mg/kg), fluoxetine profoundly halted disease progression, with no further elevation in the clinical score or
paw swelling (Figures 1A and B). Neither dose had a
significant effect on the serum anticollagen IgG1 and
IgG2a levels, but at the 25 mg/kg dose, serum IL-12 was
inhibited by 83.5 ⫾ 9.9% (P ⫽ 0.0256) (Figure 1C). The
higher dose of fluoxetine (25 mg/kg) showed a significant reduction in the mean histology score, inhibiting
it by 67 ⫾ 11.4% (P ⫽ 0.0011) (Figure 1D). Vehicle
control sections showed extensive inflammatory cell
infiltration, bone erosion accompanied by pannus formation, and degradation of cartilage. In contrast, in mice
treated with fluoxetine, particularly at the 25 mg/kg
dose, there was reduced inflammation, reduced cartilage
and bone erosion, and the joint architecture was largely
maintained (Figure 1E).
Citalopram treatment confers partial protection
from CIA. Citalopram also reduced the severity of CIA
but was not as effective as fluoxetine. Thus, mice given
25 mg/kg of citalopram showed a significant benefit in
the clinical score from day 2 onward, but there was no
improvement in paw swelling (Figures 2A and B).
Citalopram had no appreciable effect on serum anticollagen IgG1 and IgG2a levels (Figure 2C). Examina-
tion of histology sections revealed a tendency toward
reduced inflammatory cell infiltration, pannus formation, and loss of joint architecture in the citalopramtreated group as compared with the control group.
However, the reduction in the mean histology score (by
46.2 ⫾ 11.9%) did not reach statistical significance (P ⫽
0.0857) (Figure 2D), largely due to the low numbers of
animals treated.
Fluoxetine and citalopram inhibit cytokine production in murine bone marrow–derived macrophages
induced by TLRs 3, 7, and 9. Next, we sought to identify
the mechanism by which SSRIs could suppress inflammation. We had previously shown that mianserin, a
serotonin receptor antagonist, inhibited TLRs 3, 7, 8,
and 9, but not TLRs 2, 4, and 5, in primary human cells
(10). Given the structural and functional similarity of
this class of molecule with the SSRIs, we decided to
investigate whether fluoxetine and citalopram could also
inhibit these TLRs. TLR-8 is not thought to be functional in the murine system since it does not respond to
single-stranded RNA (ssRNA) or other TLR-8 ligands
(29). For this reason, we chose to investigate TLRs 3, 7,
and 9 using activation of TLR-4 as a control.
In murine bone marrow–derived macrophages,
ANTIARTHRITIC POTENTIAL OF TWO SSRIs IN HUMAN AND MURINE MODELS OF RA
687
Figure 3. Fluoxetine and citalopram inhibition of Toll-like receptor 3 (TLR-3), TLR-7, and TLR-9 induction of
cytokines from murine bone marrow–derived macrophages. A and B, Primary murine macrophage colonystimulating factor (M-CSF)–derived macrophages were preincubated for 30 minutes with 2.5, 5, or 10 ␮g/ml of
fluoxetine (A) or with 10, 20, or 30 ␮g/ml of citalopram (B) and were then either left unstimulated (US) or were
stimulated for 6 hours with 1 ␮g/ml of R-848, 2 ␮M CpG-containing oligonucleotide (ODN; 1668), or 100 ng/ml of
lipopolysaccharide (LPS). The dose reponse of tumor necrosis factor (TNF) inhibition (left and middle) was
measured, and the data from the highest dose of each selective serotonin reuptake inhibitor (SSRI) were pooled
from 3 separate experiments (right). C, Primary murine M-CSF–derived macrophages were preincubated for 30
minutes with 10, 20, or 30 ␮g/ml of citalopram or with 2.5, 5, or 10 ␮g/ml of fluoxetine and were then either left
unstimulated or were stimulated for 24 hours with 20 ␮g/ml of poly(I-C). The dose response of RANTES inhibition
(left and middle) was measured, and the data from the highest dose of each SSRI were pooled from 3 separate
experiments (right). Dose response data are the mean and SD of triplicate cultures and are representative of
3 separate experiments. Pooled data are the mean and SEM percentage of the ligand-only response. ⴱ ⫽ P ⬍ 0.05;
ⴱⴱⴱ ⫽ P ⬍ 0.001 versus ligand-only response.
both fluoxetine and citalopram were able to inhibit in a
dose-dependent manner cytokine production induced by
poly(I-C) (TLR-3), R-848 (TLR-7), and CpG (TLR-9)
(Figure 3), whereas LPS (TLR-4) activation was unaffected (Figures 3A and B). Fluoxetine inhibited TLR-7–
and TLR-9–induced TNF production in murine macrophages by 83 ⫾ 2.894% (P ⫽ 0.0006) and 53.6 ⫾ 16.7%
(P ⫽ 0.0425), respectively (Figure 3A). Citalopram
reduced the amount of TNF released by 70 ⫾ 19% (P ⫽
0.0333) for TLR-7 and by 97 ⫾ 0.7% (P ⬍ 0.0001)
for TLR-9 (Figure 3B). TLR-3 activation with poly(I-C)
did not induce TNF production, but it did stimulate
RANTES production in these cells. This was inhibited
by 84 ⫾ 5% (P ⫽ 0.0002) with citalopram treatment and
by 96.4 ⫾ 2% with fluoxetine treatment (P ⬍ 0.0001)
(Figure 3C). No reduction in cell viability was observed.
Fluoxetine and citalopram inhibit cytokine production induced by TLR-3 and TLR-8 in primary human M-CSF macrophages and RA synovial fibroblasts.
To establish the relevance of these findings in human
disease, we further examined whether fluoxetine and
citalopram were also able to inhibit endosomal TLR
signaling in primary human cells. Neither fluoxetine nor
citalopram had any appreciable effect on Pam3CSK4
(TLR-1/2), LPS (TLR-4), or flagellin (TLR-5) activity
(Figures 4A and B), but both treatments selectively
inhibited (by 55.7 ⫾ 4.8% [P ⫽ 0.0007] for fluoxetine
and by 80.2 ⫾ 7.3% [P ⫽ 0.0041] for citalopram) the
R-848 (TLR-7/8 ligand)–induced production of TNF in
human macrophages in a dose-dependent manner (Figures 4A and B).
Macrophages do not induce cytokines in response
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SACRE ET AL
Figure 4. Fluoxetine and citalopram inhibition of Toll-like receptor 3 (TLR-3) and TLR-8
induction of cytokines from primary human macrophages. A and B, Primary human
macrophages were preincubated for 30 minutes with 1 or 5 ␮g/ml of fluoxetine (A) or with
10, 20, or 30 ␮g/ml of citalopram (B) and were then either left unstimulated (US) or were
stimulated for 6 hours with 1 ␮g/ml of R-848, 10 ng/ml of Pam3Cys-Ser(Lys)4 䡠 3HCl
(Pam3CSK4), 10 ng/ml of flagellin, or 10 ng/ml of lipopolysaccharide (LPS). The dose
response of tumor necrosis factor (TNF) inhibition (left) was measured, and the data from
the highest dose of each selective serotonin reuptake inhibitor (SSRI) were pooled from 3
separate experiments (right). C, Rheumatoid arthritis synovial fibroblasts were preincubated with media alone or with media containing 1 or 5 ␮g/ml of fluoxetine or containing
10, 20, or 30 ␮g/ml of citalopram and were then either left unstimulated or were stimulated
for 24 hours with 20 ␮g/ml of poly(I-C). The dose response of interleukin-6 (IL-6) inhibition
(left) was measured, and the data from the highest dose of each SSRI were pooled from 3
separate experiments (right). Dose response data are the mean and SD of triplicate cultures
and are representative of 3 separate experiments using cells from 3 unrelated donors.
Pooled data from these donors are the mean and SEM percentage of the ligand-only
response. ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001 versus ligand-only response.
to TLR-7 ligands; thus, in these cells, R-848 is activating
TLR-8 signaling. Human macrophages do not produce
TNF in response to activation of TLR-3; however,
poly(I-C) activation of TLR-3 on RA synovial fibroblasts
induces a strong IL-6 response (30). Fluoxetine and cita-
lopram inhibited TLR-3–induced IL-6 production in RA
synovial fibroblasts by 56.8 ⫾ 6% (P ⫽ 0.0012) and by
92.7 ⫾ 4% (P ⫽ 0.0009), respectively (Figure 4C).
Primary human macrophages and RA synovial
fibroblasts do not respond to TLR-7 or TLR-9 ligands (31);
ANTIARTHRITIC POTENTIAL OF TWO SSRIs IN HUMAN AND MURINE MODELS OF RA
689
Figure 5. Fluoxetine and citalopram inhibition of Toll-like receptor 7 (TLR-7) and TLR-9
production of interleukin-6 (IL-6) from primary human B cells. A and B, Primary human B
cells were preincubated for 30 minutes with 1 or 5 ␮g/ml of fluoxetine (A) or with 10, 20, or
30 ␮g/ml of citalopram (B) and were then either left unstimulated (US) or were stimulated
for 24 hours with 10 ␮g/ml of imiquimod or 2.5 ␮M CpG-containing oligonucleotide (ODN;
2006). Dose responses (left) are the mean and SD of triplicate cultures and are representative of 3 separate experiments using cells from 3 unrelated donors. Pooled data from these
donors (right) are the mean and SEM percentage of the ligand-only response. Values are
the mean and SD. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001 versus ligand alone.
thus, to investigate these TLRs, we used B cells that
produce IL-6 in response to imiquimod (TLR-7) and CpG
DNA (TLR-9) (32). Fluoxetine and citalopram significantly inhibited TLR-7– and TLR-9–induced production
of IL-6 from B cells in a dose dependent manner (Figure
5). Fluoxetine inhibited TLR-7–induced IL-6 production
by 80 ⫾ 7.2% (P ⫽ 0.004) and inhibited TLR-9–induced
IL-6 production by 92 ⫾ 4% (P ⫽ 0.001) (Figure 5A).
Citalopram inhibited TLR-7–induced IL-6 production by
72 ⫾ 11.8 (P ⫽ 0.0044) and inhibited TLR-9–induced IL-6
production by 81 ⫾ 15.3% (P ⫽ 0.017) (Figure 5B). Cell
viability was measured after all experiments by MTT assay,
and no toxicity was observed (data not shown).
Fluoxetine and citalopram inhibit spontaneous
cytokine production in human RA synovial membrane
cultures. The success of citalopram and, in particular,
fluoxetine in the murine CIA model, in addition to the
ability of these drugs to inhibit endosomal TLR activity
in both murine and human cells, made them attractive
candidates for testing in human RA synovial membrane
cultures. We have previously identified a potential role
of the endosomal TLRs and, in particular, TLR-8 in the
production of TNF and IL-6 in RA synovial membrane
cultures (10). These cultures are produced from RA
tissues removed during elective surgery and are composed of a mixed population of cells that spontaneously
release cytokines without the need for exogenous stimulation (22). This is considered an accepted model of
human disease, and this model was used for the initial
studies that identified the importance of TNF in RA
(22). Fluoxetine at 5 ␮g/ml inhibited TNF by 50.23 ⫾ 9.7%
(P ⫽ 0.0032), IL-6 by 18.4 ⫾ 22.3% (P ⫽ 0.0043),
and IP-10 by 54 ⫾ 15.5% (P ⫽ 0.0035) (Figure 6A).
Citalopram at 30 ␮g/ml was more effective at inhibiting
TNF by 69.6 ⫾ 5.6% (P ⫽ 0.0032), IL-6 by 33 ⫾ 21.2%
(P ⫽ 0.0067), and IP-10 by 63.6 ⫾ 7.7% (P ⫽ 0.0043)
(Figure 6B).
Fluoxetine and citalopram are both SSRIs, and
we therefore wished to ensure that any inhibition observed was not due to the effects of serotonin on the
cells. In addition to the important function of serotonin
in the central nervous system, there is also a role for this
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SACRE ET AL
Figure 6. Fluoxetine and citalopram inhibition of spontaneous cytokine production in human rheumatoid
synovial membrane cultures. A–C, Rheumatoid synovial membrane cells were cultured for 24 hours in the
presence of media alone, 5 ␮g/ml of fluoxetine (n ⫽ 10–12 donors) (A), 30 ␮g/ml of citalopram (n ⫽ 9–11
donors) (B), or 10 or 100 ␮M serotonin (n ⫽ 5 donors) (C), and the production of tumor necrosis factor
(TNF), interleukin-6 (IL-6), and interferon-␥–inducible protein 10 (IP-10) was determined. D, Cell
viability was measured by MTT assay. Results are representative of all experiments performed in A and
B. Each data point in A and B represents an individual donor. ⴱⴱ ⫽ P ⬍ 0.01 versus unstimulated controls.
Values in C and D are the mean and SEM. OD ⫽ optical density.
molecule in the periphery. Serotonin receptors and
serotonin reuptake receptors are expressed on many of
the cells within the immune system, including macrophages, B cells, and T cells (33). Addition of 10 ␮M or
100 ␮M serotonin to the RA synovial membrane cultures had no appreciable effect on the production of
TNF or IL-6 (Figure 6C). Cell viability was measured
after all experiments by MTT assay, and no toxicity was
observed (Figure 6D).
DISCUSSION
In this study of 2 licensed SSRIs, fluoxetine and
citalopram, we provide data demonstrating a therapeutic
benefit of these drugs, both in the murine CIA model
and in a validated human RA disease tissue model. In
addition, we suggest a potential mechanism for at least
part of the antiinflammatory action observed, perhaps
through inhibition of the endosomal TLRs. Fluoxetine
powerfully halted any further increase in the clinical and
histopathologic severity of established disease in the
CIA model, demonstrating a remarkable inhibition of
inflammation. Citalopram, although effective, was not as
successful as fluoxetine at inhibiting disease pathogenesis in this model. However, this may be due to the
reported, distinctly longer half-life of fluoxetine in vivo
(34). Citalopram affected the clinical score to a greater
extent than paw swelling. This is probably because the
clinical score takes into account the number of paws.
Therefore, it is a more sensitive measure of overall
disease severity than is paw swelling.
The precise mechanism for the antiinflammatory
action of the SSRIs is unknown. However, there have
been reports of fluoxetine suppressing T cell proliferation (35) and inhibiting interferon-␥ (IFN␥) production
in human whole blood cultures (36). In the CIA model,
the levels of serum IL-12 were significantly inhibited by
fluoxetine treatment, suggesting that in addition to the
reported ability to influence T cell proliferation, fluoxetine may also be inhibiting the response of antigenpresenting cells. TLRs are strong activators of inflammatory cytokines and have been suggested to play a role
in RA (37), with most studies indicating a role of TLR-4
in models of experimental arthritis (6–8). Upon investigation, fluoxetine and citalopram were found to inhibit
ANTIARTHRITIC POTENTIAL OF TWO SSRIs IN HUMAN AND MURINE MODELS OF RA
the activation of endosomal TLRs 3, 7, and 9, but not
TLR-4, in murine bone marrow–derived macrophages.
Production of type I IFN by activation of TLRs 3 and 7,
however, has been reported to enhance TLR-4 cytokine
production in monocyte-derived dendritic cells (38).
Thus, inhibition of the endosomal TLRs in the CIA
model by fluoxetine and citalopram treatment may, in
turn, affect TLR-4–induced cytokine production in vivo.
Similar to the observation in the CIA model, fluoxetine
was more effective than citalopram at inhibiting endosomal TLR activation. This would be expected if inhibition of endosomal TLRs forms part of the mechanism by
which these drugs suppressed the disease activity in the
CIA model.
Inhibition of the endosomal TLRs was also evident in primary human cells, where both drugs inhibited
TLRs 3, 7, 8, and 9, but not TLRs 1/2, 4, or 5. The lack
of inhibition of TLR-4 is consistent with a separate study
performed in whole blood (35). In a previous study, we
discovered that mianserin, a serotonin receptor antagonist, is also an inhibitor of TLRs 3, 7, 8, and 9, and
suppressed the spontaneous production of TNF and
IL-6 in human RA synovial membrane cultures (10).
Here, we demonstrated that fluoxetine and citalopram
also inhibit TNF, IL-6, and IP-10 production in these
cultures, supporting a concept of activation of 1 or more
of these TLRs in the pathogenesis of RA. This inhibition
of cytokines was not due to effects of increased serotonin, since the addition of serotonin to the RA cultures
had no effect. Furthermore, the percentages of inhibition of TNF and IL-6 were similar to the percentage
inhibition observed in our previous study with mianserin.
This may suggest a common mechanism that is independent of the serotonergic system, since SSRIs and serotonin receptor antagonists act on different receptors.
Other studies have also suggested that the antiinflammatory mechanism of SSRIs is independent of monoamine transporter blockade (35).
There is increasing evidence in support of a
potential role of 1 or more of these TLRs in RA. It has
recently been demonstrated that TLR-3 can be activated
on synovial fibroblasts by necrotic cells, as would be
found in the RA joint (39). More recently, activation of
TLR-3 on RA fibroblasts was reported to promote
osteoclastogenesis (40). We have previously identified
TLR-8 as a contributor to the TNF production in human
RA cultures, although TLR-8 may not play a role in the
CIA model because murine TLR-8 is not considered to
be functional (29). However, murine TLR-7 responds in
a similar way to human TLR-8; both receptors are
activated by the same natural ligand ssRNA (29) and
691
both can activate NF-␬B and produce TNF in macrophages. Human TLR-7 is not active on macrophages,
but is instead expressed on dendritic cells, where it
induces IFN production (41). It is therefore conceivable
that TLR-7 may have more importance in the CIA
model. It has recently been reported that cross-tolerance
of TLRs 2, 7, and 9 induced by daily administration of
low doses of a TLR-7 ligand has proved beneficial in a
murine serum-transfer model of arthritis, reducing cell
infiltration and bone erosion (42). A role of TLR-9 may
be more predominant on B cells, where activation by
DNA-containing immune complexes has been shown
to stimulate rheumatoid factor–positive autoreactive B
cells (43).
More clinically relevant support for a role of
TLRs in RA has come from the therapeutic use of
chloroquine and from studies of patients deficient in
endosomal TLR function. Chloroquine has been used
for many years in combination with other therapies in a
clinical setting as a treatment for RA (44). It is an
inhibitor of endosomal TLR function, and we have
previously demonstrated that chloroquine significantly
inhibits cytokine production in human RA synovial
membrane cultures (10). It has been suggested that the
benefit observed clinically may be mediated through the
inhibition of these TLRs (45). Additional support for a
role of endosomal TLRs in autoimmunity has emerged
from the identification of patients deficient in UNC93B1
(46), a protein required for TLR-3, TLR-7, TLR-8, and
TLR-9 signaling (47). These patients show an unexpectedly mild phenotype (48,49), but have an increased
number of polyreactive and autoreactive B cells in the
periphery (46), similar to that observed in RA patients
(50). Intriguingly, none have progressed to develop
autoimmunity (46), which suggests that activation of 1 or
more of these TLRs may be required to induce an
autoimmune phenotype.
The mechanism by which fluoxetine and citalopram inhibit endosomal TLRs seems unlikely to be
linked to the antidepressant mechanism of SSRIs. Fluoxetine and citalopram have a high affinity for the
serotonin transporter, with a Kd of 0.81 nM and 1.16 nM,
respectively (51). In the present study, TLR-induced
cytokine production was inhibited in vitro by concentrations equivalent to 7.5–30 ␮M fluoxetine (2.5–10 ␮g/ml)
and 25–74 ␮M citalopram (10–30 ␮g/ml), which are
much higher than the concentrations required for SSRI
activity or the concentrations that would be achieved in
the serum of patients. Fluoxetine and citalopram are
normally prescribed for depression at a maximum dosage of 80 mg/day, which produces a serum concentration
692
SACRE ET AL
of ⬃1 ␮M and ⬃250 nM for the 2 SSRIs, respectively
(52). In the CIA model, suppression of disease progression also required much higher doses (25 mg/kg, as
compared with ⬃1 mg/kg used to treat depression in
humans). Moreover, the addition of serotonin to the
human RA cultures had no effect on the spontaneous
release of TNF. It would therefore seem more likely that
the mechanism is an off-target effect of these drugs.
A possible mechanism by which fluoxetine and
citalopram inhibit endosomal TLR–induced cytokine
production could be through the inhibition of cellular
signaling molecules shared by the endosomal TLRs. All
of the TLRs (except TLR-3) use the myeloid differentiation factor 88–dependent pathway to activate NF-␬B. It
is doubtful, however, that these drugs inhibit a shared
signaling molecule on this pathway, since there was no
inhibition of TLR-2, TLR-4, or TLR-5. In addition,
TLR-3 signals through a different pathway, using TRIF
to induce IFN (30). An alternative mechanism could be
through direct interaction with the receptors, or possibly, a shared accessory molecule required for their
activation, for example UNC93B1. This protein has been
shown to be required for trafficking of the endosomal
TLRs from the endoplasmic reticulum to endosomes,
where they are then activated (53).
The data presented herein demonstrate the ability of fluoxetine and citalopram to selectively inhibit
endosomal TLRs, to decrease inflammation in the murine CIA model, and to decrease inflammatory cytokine
production in human RA tissue. Although these drugs
will undoubtedly have multiple effects in vivo, the data
suggest that targeting the endosomal TLRs may provide
therapeutic benefit in RA. Fluoxetine and citalopram
are not ideal candidates to be progressed into clinical
trials, since our in vitro data suggest that effective
inhibition would require levels above their safe therapeutic dosages. Accordingly, there does not appear to be
any published anecdotal evidence of a clinical benefit in
disease pathogenesis in RA patients prescribed SSRIs
for depression. Nevertheless, these data in conjunction
with our previous study support the concept of a contribution from endosomal TLRs to the chronic inflammation associated with the pathogenesis of RA and demonstrate the potential to selectively target these
receptors with small molecules in the future for the
effective treatment of RA.
ACKNOWLEDGMENTS
We would like to thank Renee Best for preparation of
the human RA synovial tissue and Marc Feldmann for critical
reading of the manuscript.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Sacre had full access to all of the
data in the study and takes responsibility for the integrity of the data
and the accuracy of the data analysis.
Study conception and design. Sacre, Medghalchi, Gregory, Brennan,
Williams.
Acquisition of data. Sacre, Medghalchi.
Analysis and interpretation of data. Sacre, Medghalchi, Gregory,
Williams.
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