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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

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-γ–inducible protein 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 removed 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

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

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 · 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 μg/ml of fluoxetine or with 10, 20, or 30 μg/ml of citalopram and then either left unstimulated or stimulated with 10 ng/ml of flagellin, 10 ng/ml of Pam3CSK4, 1 μg/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 μg/ml of fluoxetine or with 10, 20, or 30 μg/ml of citalopram and then either left unstimulated or stimulated with 20 μg/ml of poly(I-C) for 24 hours. Primary human B cells were preincubated for 30 minutes with 1 or 5 μg/ml of fluoxetine or with 10, 20, or 30 μg/ml of citalopram and then either left unstimulated or stimulated with 10 μg/ml of imiquimod or 2.5 μM ODN2006 for 24 hours. Human RA synovial membrane cells were cultured for 24 hours in the presence of media alone or 5 μg/ml of fluoxetine, 30 μg/ml of citalopram, or 10 or 100 μM 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 μM), and 10 ng/ml of M-CSF.

Enzyme-linked immunosorbent assay (ELISA).

Sandwich ELISAs were used to measure human TNF, interferon-γ–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 μg 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 swelling, 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 paraffin-embedded 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 experimental 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

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

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 (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).

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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. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = 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).

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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). Examination of histology sections revealed a tendency toward reduced inflammatory cell infiltration, pannus formation, and loss of joint architecture in the citalopram-treated 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.

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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. ∗ = P < 0.05; ∗∗ = 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.

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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, 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.

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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 colony-stimulating factor (M-CSF)–derived macrophages were preincubated for 30 minutes with 2.5, 5, or 10 μg/ml of fluoxetine (A) or with 10, 20, or 30 μg/ml of citalopram (B) and were then either left unstimulated (US) or were stimulated for 6 hours with 1 μg/ml of R-848, 2 μM 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 μg/ml of citalopram or with 2.5, 5, or 10 μg/ml of fluoxetine and were then either left unstimulated or were stimulated for 24 hours with 20 μg/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. ∗ = P < 0.05; ∗∗∗ = P < 0.001 versus ligand-only response.

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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).

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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 μg/ml of fluoxetine (A) or with 10, 20, or 30 μg/ml of citalopram (B) and were then either left unstimulated (US) or were stimulated for 6 hours with 1 μg/ml of R-848, 10 ng/ml of Pam3Cys-Ser(Lys)4 · 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 μg/ml of fluoxetine or containing 10, 20, or 30 μg/ml of citalopram and were then either left unstimulated or were stimulated for 24 hours with 20 μg/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. ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 versus ligand-only response.

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Macrophages do not induce cytokines in 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 citalopram 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); 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).

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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 μg/ml of fluoxetine (A) or with 10, 20, or 30 μg/ml of citalopram (B) and were then either left unstimulated (US) or were stimulated for 24 hours with 10 μg/ml of imiquimod or 2.5 μM 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. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 versus ligand alone.

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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 μg/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 μg/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).

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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 μg/ml of fluoxetine (n = 10–12 donors) (A), 30 μg/ml of citalopram (n = 9–11 donors) (B), or 10 or 100 μM serotonin (n = 5 donors) (C), and the production of tumor necrosis factor (TNF), interleukin-6 (IL-6), and interferon-γ–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. ∗∗ = P < 0.01 versus unstimulated controls. Values in C and D are the mean and SEM. OD = optical density.

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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 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 μM or 100 μM 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

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

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-γ (IFNγ) 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 antigen-presenting 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 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 both can activate NF-κB 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 μM fluoxetine (2.5–10 μg/ml) and 25–74 μM citalopram (10–30 μg/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 of ∼1 μM 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-κB. 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.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We would like to thank Renee Best for preparation of the human RA synovial tissue and Marc Feldmann for critical reading of the manuscript.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES
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