Antidepressant effects of selective serotonin reuptakeinhibitors (SSRIs) are attenuated by antiinflammatorydrugs in mice and humansJennifer L. Warner-Schmidta,1, Kimberly E. Vanoverb, Emily Y. Chena, John J. Marshalla, and Paul Greengarda,1

aLaboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY 10065; and bIntra-Cellular Therapies, New York, NY 10032

Contributed by Paul Greengard, March 28, 2011 (sent for review March 10, 2011)

Antiinflammatory drugs achieve their therapeutic actions at leastin part by regulation of cytokine formation. A “cytokine hypothe-sis” of depression is supported by the observation that depressedindividuals have elevated plasma levels of certain cytokines com-pared with healthy controls. Here we investigated a possible in-teraction between antidepressant agents and antiinflammatoryagents on antidepressant-induced behaviors and on p11, a bio-chemical marker of depressive-like states and antidepressantresponses. We found that widely used antiinflammatory drugsantagonize both biochemical and behavioral responses to selec-tive serotonin reuptake inhibitors (SSRIs). In contrast to the levelsdetected in serum, we found that frontal cortical levels of certaincytokines (e.g., TNFα and IFNγ) were increased by serotonergicantidepressants and that these effects were inhibited by antiin-flammatory agents. The antagonistic effect of antiinflammatoryagents on antidepressant-induced behaviors was confirmed byanalysis of a dataset from a large-scale real-world human study,“sequenced treatment alternatives to relieve depression” (STAR*D),underscoring the clinical significance of our findings. Our data in-dicate that clinicians should carefully balance the therapeutic bene-fits of antiinflammatory agents versus the potentially negativeconsequences of antagonizing the therapeutic efficacy of antide-pressant agents in patients suffering from depression.

citalopram | fluoxetine | S100A10

Mood disorders including major depressive disorder (MDD)affect as many as one in five individuals and are the most

prevalent psychiatric conditions (1). Approximately one-third ofpatients suffering from MDD are refractory to any kind of antide-pressant treatment including selective serotonin reuptake inhibitors(SSRIs), tricyclic antidepressants (TCAs), monoamine oxidaseinhibitors (MAOIs), and electroconvulsive therapy (ECT) (2).The hypothesis that cytokines play a role in depression is based

in part on the observations that (i) many patients undergoingtreatment involving IFNα or interleukin-2 develop depressivesymptoms; (ii) sickness behavior induced by the endotoxin li-popolysaccharide (LPS) or interleukin-1 shares some commonfeatures with depression; (iii) several cytokines can activate thehypothalamic–pituitary–adrenal (HPA) axis, which is commonlyactivated in depressed individuals; and (iv) some cytokines regu-late brain norepinephrine or serotonin systems, which are linkedto MDD and its treatment (3–6).p11, a member of the S100 family of proteins, is a key regu-

lator of depressive-like states and antidepressant responses inrodent models (7–9). p11, also called S100A10, is a small acidicprotein that interacts with specific serotonin receptors to regu-late their trafficking and influence their localization at the cellsurface (7, 9). This action affects the excitability of the cells andleads to profound behavioral responses. p11 knockout (KO)mice display a depressive-like phenotype and p11 overexpressingmice display antidepressant-like responses in classical behavioralparadigms, including the tail suspension and forced swim tests(7). Genetically “helpless”mice, which exhibit some symptoms ofdepression, as well as humans suffering with MDD, have reduced

levels of p11 mRNA and protein in the cerebral cortex andstriatum (7, 10). In rodents, three classes of antidepressants(SSRIs, TCAs, and ECT) increase p11 levels in the cerebralcortex and hippocampus (7, 8). Here we report an investigationof antiinflammatory drugs and of antidepressant agents on p11expression and antidepressant behavior.The “sequenced treatment alternatives to relieve depression”

(STAR*D) clinical trial was a large, real-world study of treatment-resistant depression that evaluated a series of treatments andclinical outcomes. Many patients continue to suffer residualsymptoms after weeks of treatment with a single agent and evenafter trying many different antidepressants and combinationtherapies. The STAR*D study found that only 36.8% of patientsexhibited remission after treatment with the SSRI, citalopram(CIT), and found a cumulative remission rate of 67% after mul-tiple treatments were attempted (11). The underlying factorscontributing to treatment resistance remain unclear. We used theSTAR*D dataset to determine whether nonsteroidal antiin-flammatory drugs (NSAIDs) could play a role in the treatmentoutcome of depressed individuals taking SSRIs.Here we test a model in which SSRI antidepressants increase

brain levels of certain cytokines, which in turn regulate p11 levelsand ultimately control the behavioral response to an SSRI an-tidepressant (Fig. 1). Importantly, we have identified an antag-onism by NSAIDs of SSRI responsiveness, which is likelymediated through the action of certain cytokines and p11 in thebrain. We believe that this antagonism contributes, in part, to thehigh resistance rates to SSRIs seen in MDD. We believe thatreduced use of NSAIDs by physicians in severely depressedpatients being treated with SSRIs would significantly improvepositive outcomes from this major class of antidepressant.

ResultsEffects of SSRIs and NSAIDs on Certain Cytokines and p11. Wemeasured mouse brain levels of cytokines using a bead-basedELISA following chronic treatment with the SSRI citalopram inthe presence or absence of ibuprofen (IBU) cotreatment (SIMaterials and Methods). We focus on the frontal cortex, a brainarea that is strongly linked to antidepressant responses in miceand humans (12, 13). Results identified cytokines that fell intoone of three major categories: (i) cytokines that were increasedby citalopram, the effect of which was abolished by IBUcotreatment (Fig. 2A); (ii) cytokines that were increased by cit-alopram, the effect of which was not affected by IBU cotreat-ment (Fig. 2A); or (iii) cytokines that were not changed by either

Author contributions: J.L.W.-S., K.E.V., and P.G. designed research; J.L.W.-S., K.E.V., E.Y.C.,and J.J.M. performed research; J.L.W.-S., K.E.V., E.Y.C., and J.J.M. analyzed data; and J.L.W.-S.,K.E.V., and P.G. wrote the paper.

The authors declare no conflict of interest.

See Commentary on page 8923.1To whom correspondence may be addressed. E-mail: jschmidt@rockefeller.edu orgreengard@rockefeller.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1104836108/-/DCSupplemental.

9262–9267 | PNAS | May 31, 2011 | vol. 108 | no. 22 www.pnas.org/cgi/doi/10.1073/pnas.1104836108

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citalopram or IBU (IL-1a, IL-4, and IL-13). IBU reduced plasmalevels of both citalopram and its metabolite didesmethyl cit-alopram (ddCIT) compared with mice that received citalopramalone (CIT: 1508.36 ± 282.7 versus 537.03 ± 65.8 pg/μL, P <0.05; ddCIT: 128.0 ± 21.0 versus 34.5 ± 10.8 pg/μL, P < 0.01).We have shown previously that p11 is increased in the mouse

frontal cortex by multiple classes of antidepressants (7, 8). Herewe investigated whether antiinflammatory agents alone or incombination with antidepressants regulated p11 levels. Inter-estingly, coadministration of either ibuprofen (IBU) or anotherNSAID, acetylsalicylic acid (ASA), with antidepressants for 14 dblocked the increase in p11 caused by two different SSRIs, cit-alopram or fluoxetine (Fig. 2B). The tricyclic antidepressant(TCA) desipramine induced a small increase in p11, and thisincrease was not significantly affected by IBU or ASA [Fig. 2B,interaction NSAID × antidepressant: F(6, 52) = 4.48, P < 0.01].Taken together, these experiments show that SSRI anti-depressants increase brain levels of certain cytokines and p11,the effect of which is abolished by NSAID cotreatment.

Antidepressant-Induced Increases in p11 Levels Are Cytokine De-pendent. Two of the cytokines that were regulated by both cit-alopram and ibuprofen, namely, IFNγ and TNFα, were furtherstudied as possible mediators of the inhibitory effects of NSAIDson SSRI-induced p11 levels described above. First, we in-vestigated whether IFNγ or TNFα signaling is required for an-tidepressant-induced increases in p11, using IFNGR1 or TNFR1KO mice. Western blotting analysis of frontal cortex fromIFNGR1 KO, TNFR1 KO, or WT control mice treated withchronic citalopram revealed that both IFNGR1 and TNFR1signaling are necessary for the increase in p11 by citalopram.Citalopram significantly increased p11 in WT, but not IFNGR1KO or TNFR1 KO mice [Fig. 3A, interaction genotype ×treatment F(1, 22) = 6.22, P < 0.05].To examine whether IFNγ or TNFα was sufficient to increase

p11 in vivo, mice were injected with recombinant IFNγ, TNFα,

or vehicle and euthanized 4 h later. Western blot analysis re-vealed that both cytokines significantly increased p11 protein inthe frontal cortex compared with vehicle injected controls [Fig.3B, F(2, 16) = 8.519, P < 0.01].Immunohistochemical detection of IFNγ receptor 1

(IFNGR1), p11, and the neuronal marker NeuN demonstratedthat neurons in layer 5 of the mouse cortex express both p11 andIFNGR1 (Fig. S1). TNF receptor 1 (TNFR1) was also coex-pressed with p11 in cortical neurons (Fig. S1). These data sup-port the idea that these cytokines may regulate p11 levels.

NSAIDs Prevent the Antidepressant-Like Effect of SSRIs in ClassicalBehavioral Paradigms. Because p11 has been shown to be bothnecessary and sufficient for behavioral antidepressant responses(7–9) and IBU potently inhibited antidepressant-inducedincreases in p11 (Fig. 2B), we examined the possibility that IBUmight inhibit the behavioral response to antidepressant drugs.We tested various classes of antidepressants including SSRIs(citalopram and fluoxetine), TCAs (imipramine and de-sipramine), a MAOI (tranylcypromine), and an atypical antide-pressant (bupropion) in two well-established mouse models ofdepression: the tail suspension test (TST) and the forced swimtest (FST). All antidepressants tested significantly reduced im-mobility time in both the TST [Fig. 4A, interaction antidepres-sant × NSAID: F(4, 95) = 6.11, P < 0.001] and the FST [Fig. 4B,interaction antidepressant × NSAID: F(6, 106) = 5.07, P < 0.001].IBU significantly attenuated the antidepressant-like effects ofSSRIs in both tests (Fig. 4 A and B). IBU was less effective inaltering the behavioral response to TCAs and failed to alter thebehavioral response to other classes of antidepressant drugs.To examine the specificity of the effect of IBU on SSRI-

induced behavioral changes, we tested the effect of three dif-ferent NSAIDs and an analgesic on the behavioral response tocitalopram. Mice were treated with IBU (1 mg/mL), naproxen (2mg/mL), acetylsalicylic acid (3 mg/mL), or acetaminophen (3 mg/mL) in their drinking water for 5–7 d and received a single in-jection of citalopram (20 mg/kg, i.p.) or saline before testing inthe TST or FST. All of the drugs tested significantly blocked theantidepressant effect of citalopram on immobility time in both tests(Fig. 4 C and D) compared with mice that received citalopramalone [TST interaction antidepressant × NSAID: F(6, 129) = 3.25,P < 0.01; FST interaction antidepressant ×NSAID: F(6, 106) = 5.07,P < 0.005].To determine whether ibuprofen inhibits the behavioral

response to chronic SSRI administration, we coadministeredcitalopram and ibuprofen for 2 wk before testing mice in the

Antidepressants Cytokines p11 behavioral response

NSAIDs

Fig. 1. Schematic representation of the model tested in the current study.Here we suggest that antidepressants, specifically SSRI antidepressants, in-crease brain levels of certain cytokines, which in turn increase levels of p11,the effect of which produces behavioral antidepressant responses. Each stepin this pathway can be antagonized by NSAID coadministration.

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Fig. 2. Effect of antidepressants and NSAIDs on cytokine levels and p11 in the mouse frontal cortex. (A) Mouse frontal cortex samples from animals treatedwith vehicle (VEH), citalopram (CIT), ibuprofen (IBU), or both IBU and CIT were analyzed for levels of cytokines. Group 1 cytokines were increased by cit-alopram, the effect of which was abolished by ibuprofen cotreatment. Group 2 cytokines were increased by citalopram, the effect of which was not affectedby ibuprofen. (B) Western blot analysis of p11 protein or actin loading control in the frontal cortex of mice receiving a chronic selective serotonin reuptakeinhibitor (citalopram or fluoxetine) or a tricyclic antidepressant (desipramine), alone or in combination with ibuprofen or acetylsalicylic acid. Representativeblots (Upper); quantification of five to six mice per group (Lower). All data are presented as means ± SEM. Statistically significant effects of antidepressants(#P < 0.05) or NSAIDs (*P < 0.05) are noted.

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novelty suppressed feeding (NSF) test or TST. In both tests,ibuprofen blocked the behavioral antidepressant-like response tocitalopram [Fig. 4 E and F, NSF interaction antidepressant ×NSAID: F(1, 56) = 4.23, P < 0.05; TST interaction antidepressant ×NSAID: F(1, 56) = 4.23, P < 0.05]. There were no differencesamong groups tested in home cage feeding (vehicle 0.08 g ± 0.024;citalopram 0.1 ± 0.033; ibuprofen 0.08 ± 0.02; ibuprofen andcitalopram 0.08 ± 0.02). Citalopram or ibuprofen and citalopram

significantly increased bodyweight (vehicle 23.3g ± 0.27; cit-alopram 24.7 ± 0.23; ibuprofen 23.4 ± 0.35; ibuprofen and cit-alopram 25.0 ± 0.20, P < 0.01).

Effects of Cytokines and p11 on Behavioral Responses. Cytokines,their receptors, and p11 are expressed by neurons, glia, andendothelial cells. To determine whether the effects of anti-depressants, IFNγ, or TNFα on immobility required neuronal

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Fig. 4. Effects of antidepressants and NSAIDs on behavioral responses. NSAIDs and other analgesics attenuate the behavioral response to SSRIs. IBU (5–7 d)diminished the behavioral response to the selective serotonin reuptake inhibitors citalopram (CIT) and fluoxetine (FLX), was less effective in altering be-havioral responses to the tricyclic antidepressants imipramine (IMI) and desipramine (DMI), and did not affect responses to other classes of antidepressants,including the monoamine oxidase inhibitor tranylcipromine (TCP) and the atypical antidepressant bupropion (BUP) in two tests of antidepressant activity, thetail suspension test (A) and forced swim test (B). Mice receiving 5–7 d of nonsteroidal antiinflammatory drugs ibuprofen (IBU), naproxen (NPX), acetylsalicylicacid (ASA), or the analgesic acetaminophen (ACE) showed diminished response to citalopram (CIT) in the tail suspension test (C) and forced swim test (D).There was no response to chronic citalopram treatment when ibuprofen was coadministered before testing in the tail suspension test (E) or the noveltysuppressed feeding test (F). All data are presented as means ± SEM. Statistically significant effects of antidepressants (#P < 0.05) or NSAIDs/analgesics(*P < 0.05, **P < 0.01) are noted. n = 8–16 per group.

9264 | www.pnas.org/cgi/doi/10.1073/pnas.1104836108 Warner-Schmidt et al.

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p11 expression, we generated a conditional p11 KO mouseby crossing a mouse with lox-P sites flanking exon 2 of the p11gene with a mouse expressing the Cre recombinase under theCAMK2α promoter (14, 15). The CAMK2α promoter was cho-sen because it is expressed only in neurons of the forebrain. Theresulting p11 KO mouse lacked p11 only in cells that expressedthe CAMK2α-Cre. Immunohistochemical analysis of the p11 KOmice showed a complete lack of p11 protein expression in theneurons of the cortex and hippocampus, and lessened expressionin the striatum (Fig. S2). Citalopram had no effect on TST im-mobility in p11 KO mice (Fig. 5A). In contrast, p11 KO miceresponded normally to a tricyclic antidepressant, desipramine,underscoring the specificity of our results for serotonergic anti-depressants (Fig. 5A).Acute injection of IFNγ (10 μg/kg) or TNFα (10 μg/kg) before

testing in the TST significantly reduced immobility in wild-typemice, consistent with an antidepressant-like effect [Fig. 5B, in-teraction genotype × treatment F(2, 64) = 3.55, P < 0.05]. A lowerdose of IFNγ (1 μg/kg) or TNFα (1 μg/kg) had no effect on TSTimmobility (IFNγ 107.9% ± 9.6, n = 6; TNFα 98.6% ± 7.3, n =5). IFNγ or TNFα had no effect on TST immobility in p11 KOmice [Fig. 5B, interaction genotype × treatment F(2, 77) = 5.18,P < 0.01], suggesting that the antidepressant-like actions of IFNγand TNFα are mediated by p11 in neurons of the cortex.We tested whether behavioral responses to chronic SSRI

treatment were absent in IFNGR1 KO or TNFR1 KO mice.The effect of citalopram on TST immobility was abolished inIFNGR1 KO mice, but not in TNFR1 KO mice [Fig. 5C, in-teraction genotype × treatment F(2, 71) = 93.32, P < 0.0001].Citalopram significantly reduced the latency to feed in WT micebut not in IFNGR1 KO or TNFR1 KO mice in the NSF test [Fig.5D, interaction genotype × treatment F(4, 107) = 8.39, P < 0.001].Both KO mouse lines responded normally to desipramine in theTST (WT: 61.03% ± 9.9; IFNGR1 KO: 71.56% ± 9.3; TNFR1KO: 65.4% ± 8.1, percentage of vehicle control group ± SEM),demonstrating the specificity for serotonergic antidepressants.

NSAIDs and Other Analgesics Inhibit SSRI Efficacy in a Clinical Pop-ulation. The TST and FST are two well-established rodent be-havioral paradigms that predict antidepressant efficacy in humans(16, 17). To determine whether NSAIDs influence SSRI efficacyin clinically depressed individuals, we took advantage of theSTAR*D dataset. The first level of this large scale clinical trialinvestigated remission rates in depressed patients taking cit-alopram for 12 wk (Materials and Methods). Contingency tablesand analyses were performed on 1,546 human subjects for whichthere were concomitant medication data and a data point atweek 12 for presence/absence of clinical “remission” from de-pressive symptoms (Table 1). These data show that 182 subjects

were in remission at the end of 12 wk of treatment with cit-alopram and had taken an NSAID at least once during those 12wk. There were 628 subjects in remission who had not taken anNSAID. There were 227 subjects who were treatment resistant(i.e., did not experience remission) and had taken an NSAID atleast once during the 12 wk of treatment. Finally, there were509 subjects who were treatment resistant and had not takenany NSAID.Of those subjects who took an NSAID, 45% were in remission,

and 55% were treatment resistant. Of those subjects who did nottake anNSAID, the reverse relationship was observedwith 55% inremission and 45% being treatment resistant. In other words,a higher percentage of patients were treatment resistant to cit-alopram if they had taken an NSAID than if they had not taken anNSAID.This relationship was statistically significant (P=0.0002).Similar analyses were conducted for other analgesics and

similar results were found. Subjects taking other analgesics wereless likely to undergo remission (37% in remission) comparedwith those not taking analgesics (54% in remission, P = 0.0002).Another analysis was conducted to determine whether the

relationship between remission and concomitant medication wasstrongest for subjects who were taking both NSAIDS and otheranalgesics. Despite the relatively fewer number of subjects takingboth NSAIDs and other analgesics, the relationship appeared tobe quite strong with 63% of subjects who were taking both types

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Fig. 5. Effects of cytokines and p11 on behavioral responses. (A) CAMK2α conditional p11 KO mice show reduced immobility in response to desipramine, butdo not respond to citalopram in the tail suspension test. (B) Immobility is significantly reduced by acute injection of IFNγ or TNFα in wild-type mice, but not inCAMK2α-conditional p11 KO mice (n = 13–24 per group). All data are presented as means ± SEM. *P < 0.05, **P < 0.01. (C and D) Behavioral analysis of WT,IFNGR1 KO, or TNFR1 KO mice treated for 14 d with citalopram in the tail suspension test (C) or the novelty suppressed feeding test (D). All data are presentedas means ± SEM. Statistically significant effects of citalopram (#P < 0.05) or genotype (*P < 0.05) are noted.

Table 1. Effects of NSAIDs and other analgesics on treatmentresponse to citalopram in humans

RemissionNo Remission

P valueremission rate (%)

NSAID 182 227 44.5 P = 0.0002No NSAID 628 509 55.2Analgesic 52 88 37.1 P = 0.0002No analgesic 758 648 53.9Both (NSAID and

analgesic)23 40 36.5 P = 0.0138

None 787 696 53.1Either (NSAID or

analgesic)211 275 43.4 P < 0.0001

None 599 461 56.5Vitamins 44 31 58.7 P = 0.2874No vitamins 766 705 52.1

Number of patients in remission or not in remission at the end of 12 wk oftreatment with citalopram shown as a function of presence or absence ofvarious concomitant treatments. Data were analyzed using Fisher’s exacttest; P < 0.05 indicated a statistically significant relationship between theconcomitant medication and the treatment outcome.

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of medication failing to show remission (P = 0.0138) in contrastto 47% failure for patients taking neither.Moreover, a contingency table was set up for subjects who had

taken either NSAIDs or analgesics. The relationship betweentaking either of these medications and being more likely to showtreatment resistancewashighly statistically significant (P< 0.0001).Importantly, there seemed to be specificity in the relationship

between the presence of an NSAID and/or other analgesic withtreatment resistance (lack of remission) because there was norelationship between treatment remission and other concomitantmedications such as vitamins.

DiscussionPrevious work from our laboratory indicated that p11 is a de-terminant of depressive-like states and antidepressant responses(7–9). Here we provide evidence that antidepressants increasebrain levels of certain cytokines, which increase p11 levels, whichthen induce antidepressant-like behavioral responses (Fig. 1).We have further shown that IFNγ and TNFα are two cytokinesthat may be involved in this process. Antiinflammatory drugsantagonized both the induction of p11 by and the behavioralresponse to SSRI antidepressants. We used the STAR*D datasetto confirm our results in a clinical population. Consistent withour mouse studies, we found that human patients reportingconcomitant NSAID or other analgesic treatments showed a re-duced therapeutic response to citalopram. Concomitant use ofNSAIDs may be an important reason for high SSRI treatmentresistance rates. We suggest that NSAIDs and other analgesicsmay potentially interfere with the therapeutic efficacy of SSRIs.P11 expression is detected in various brain areas including the

frontal cortex, hippocampus, striatum, amygdala, and dorsal ra-phe nucleus (7). Overexpression of p11 in the forebrain mimicsthe action of an antidepressant (7). Here we show that p11 inforebrain neurons is necessary for the action of an SSRI anti-depressant, but not a tricyclic antidepressant, suggesting thatSSRI and noradrenergic antidepressants might act through dif-ferent mechanisms and that p11 is selectively involved in path-ways related to SSRI activity.The antidepressant-like effects of TNFα on p11 and behavior

could be mediated by direct effects of TNFα on neurons or in-directly through neurotrophic factors. TNFα increases pro-duction by astrocytes of neurotrophic factors, including nervegrowth factor (NGF), glia-derived neurotrophic factor (GDNF),and brain-derived neurotrophic factor (BDNF) (18, 19). TNFαalso increases BDNF in human cerebral endothelial cells (20),and BDNF (i) increases p11 in the mouse cerebral cortex and (ii)has antidepressant-like and neurogenic effects that require p11(8, 21). The effects of TNFα on levels of neurotrophins may bean important component of the mechanism by which TNFαproduces an antidepressant-like response in rodent models ofdepression.Our results further suggest that, like TNFα, IFNγ mediates its

antidepressant activity through its action on p11. IFNγ increasesp11 through a direct interaction with IFNγ binding sites onthe p11 promoter (22). IFNγ increases neurite outgrowth (23),promotes neuronal differentiation (24), and enhances neuro-genesis (25), all of which are consistent with antidepressant-likeactivity (26). However, it has been suggested that IFNγ mediatesdepression-behaviors caused by immune activation (27). Othershave suggested that IFNγ plays a role in depression-like behav-iors, but find that IFNγ KO mice respond normally to chronicunpredictable stress in the FST (28). These results are not in-consistent with our findings. It is well established that there is anincrease in the level of plasma cytokines in depressed subjects(4). We speculate that the production of these cytokines andtheir actions in the periphery may be distinct from those localeffects observed in brain areas like the frontal cortex. It is alsopossible that the increased levels of certain cytokines in the pe-

riphery of depressed individuals are involved in efforts by thebrain to compensate for depression.Future studies will be necessary to determine the mechanism

by which NSAIDs inhibit SSRI efficacy. Acetaminophen is notgenerally thought to be antiinflammatory, so perhaps the anti-pyretic actions of the NSAIDs and analgesics is more related totheir antagonism of SSRI efficacy. The two drugs could interferewith each other in the periphery, because most NSAIDs do notreadily cross the blood brain barrier and because blood levelsof citalopram and its metabolite were decreased in mice thatalso received ibuprofen. We cannot exclude the possibility thatNSAIDs are having a direct effect on the interaction betweenSSRIs and the serotonin transporter, even though the in-volvement of p11 in antidepressant activity is mediated by neu-rons in the forebrain.Analysis of our clinical data strongly suggests that remission

rates among depressed individuals may be improved by avoidingcertain common over-the-counter medications such as ibupro-fen, aspirin, and acetaminophen. Depressed patients treatedfor 12 wk with citalopram are significantly less likely to showfull remission if they are concomitantly taking NSAIDs and/orother analgesics.The data suggest that treatment with NSAIDs prevents clinical

responses to antidepressants. However, it is possible that un-derlying condition(s) contribute to treatment resistance ratherthan any one particular mechanism of action of concomitantmedication. Indeed, it has been reported that depressed patientswith painful physical symptoms took longer to achieve remissionfrom depressive symptoms and were less likely to achieve re-mission than patients without pain (29). We cannot exclude thepossibility that severity of depression and accompanying painsymptoms could be associated with antidepressant treatmentresistance. However, in one study, Leuchter and colleagues (29)adjusted statistically for potential confounding factors such asrace, sex, ethnicity, and severity of depression at baseline, andreport that the statistical significance of the relationship betweenpain and remission from depression was lost. They concludedthat the presence and severity of physical pain are not predictorsof poor antidepressant treatment outcome, but that physical painis associated with some factors that are predictors. Our presentdata suggest that at least one of the factors associated withphysical pain that is a predictor of SSRI treatment outcome isconcomitant therapy with NSAIDs and other analgesics. Despitethe lack of understanding of causality associated with our clinicaldata analysis, our animal data strongly suggest that treatmentwith NSAIDs and/or other analgesics prevents antidepressantaction of SSRIs.Because the clinical analyses were conducted as post hoc

analyses, it would be informative to evaluate the effects ofNSAIDs and other analgesics on SSRI antidepressant responsein a prospective, double-blind, randomized clinical study. Spe-cifically, it will be important to standardize medications to betterevaluate their role in determining treatment outcome. In thepresent study, no adjustments were made for multiple compar-isons in the analyses. However, the effects were highly statisti-cally significant such that small statistical adjustments would notaffect the overall statistical significance or interpretation of thedata. Moreover, the lack of a significant relationship betweenvitamins and clinical response suggests specificity to the un-derlying mechanisms by which NSAIDs and other analgesicsprevent clinical remission. In addition, medical coding for con-comitant medication in the database may not have been consis-tent across subjects or medications. Individuals taking manyprescription medications may have been less likely to reportover-the-counter medications, so reports of over-the-counterNSAIDs and analgesics may be underrepresented in theSTAR*D dataset. Also, there are no data regarding dose ofconcomitant medications in the database and there is little in-formation regarding duration of use (i.e., duration is only

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available for some medications). The utility of the data shown inTable 1 may be limited by not being able to differentiate betweensubjects taking concomitant medications for a single adminis-tration or only rarely and subjects taking the medicationschronically. The magnitude of effect for the relationship betweenmedication and treatment resistance may be even greater forthose subjects who chronically take NSAIDs and/or other anal-gesics. Lastly, any subjects who discontinued before week 12 mayhave discontinued due to lack of efficacy. The percentage ofsubjects in remission may be overrepresented at week 12.However, many subjects (26%) dropped out of the acute phaseof the trial due to nonmedical reasons (2). Therefore, subjectdiscontinuation may or may not have affected the outcome of thepresent results. Despite these weaknesses, our findings indicatethat many common over-the-counter medications greatly affecttreatment response to SSRI antidepressants.NSAIDs have been reported to increase the efficacy of some

antidepressant treatments, but those reports focus on tricyclic ornoradrenergic antidepressants (30) and not SSRIs. Our dataindicate that the antagonism by NSAIDs of antidepressantresponses is specific for serotonergic antidepressants. It is im-portant to emphasize that the interaction between antidepres-sants and antiinflammatory agents appears to be specific to theefficacy of SSRIs and not a general effect on all classes of anti-depressants. Furthermore, there is no evidence from our studiesthat NSAID administration alone has any effect on depressive-like states.We report here a robust inhibitory effect of NSAIDs on SSRI-

induced increases in p11 and on antidepressant-like behaviorsin rodents. We confirmed the association in a dataset froma large-scale real-world human study (STAR*D), underscoringthe clinical significance of these results. Work is ongoing to un-derstand the cellular and molecular mechanism(s) underlyingthese effects, but the clinical implications of our findings areclear. With that, we urge the medical community to consider

these findings when designing treatment strategies for theirpatients that include SSRIs.

Materials and MethodsAnimals. Eight- to 10-wk-old male mice were used for all experiments, andwere housed four to five per cage with ad libitum access to food and water.C57BL/6 mice were purchased from Charles River Laboratories. p11 KO mice(derived and maintained at The Rockefeller University) were also used (7).CAMK2α−p11 conditional knockout mice were generated for these studies(SI Materials and Methods ). Animal use and procedures were in accordancewith the National Institutes of Health guidelines and approved by the in-stitutional animal care and use committees.

Sample Preparation and Western Blotting. Western blotting was performedusing standard procedures as described (9) (SI Materials and Methods).

Behavioral Assays. The TST, FST, and open field locomotor activity wereperformed as described (9). Novelty suppressed feeding was performed asdescribed (21).

Clinical Data Analyses. Please see SI Materials and Methods.

Statistical Analyses. All comparisons were made by ANOVA using Prism 5software (GraphPad). In experiments composed of more than two groups,data were first analyzed by two-way ANOVA followed by a post hoc Bon-ferroni test. Statistical significance was set at P < 0.05.

ACKNOWLEDGMENTS. This work has been supported by The SkirballFoundation, USAMRAA Grants W81XWH-08-1-0111 and W81XWH-09-1-0401, National Institutes of Health (NIH)/National Institute of Mental Health(NIMH) Grant MH074866, NIH/National Institute on Aging Grant AG09464.Plasma analyses were carried out in the MS/Proteomics Resource of theW. M. Keck Foundation Biotechnology Laboratory at Yale University. Theclinical data analysis was made possible by limited access datasets distributedfrom “Sequenced Treatment Alternatives to Relieve Depression” (STAR*D),supported by NIMH Contract N01MH90003 to the University of Texas South-western Medical Center. The clinicaltrials.gov identifier is NCT00021528.

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Corrections

CELL BIOLOGYCorrection for “Spinster is required for autophagic lysosomereformation and mTOR reactivation following starvation,”by Yueguang Rong, Christina McPhee, Shuangshen Deng, LeiHuang, Lilian Chen, Mei Liu, Kirsten Tracy, Eric H. Baehreck,Li Yu, and Michael J. Lenardo, which appeared in issue 19, May10, 2011, of Proc Natl Acad Sci USA (108:7826–7831; first pub-lished April 25, 2011; 10.1073/pnas.1013800108).The authors note that, due to a printer’s error, the author name

Eric H. Baehreck should have appeared as Eric H. Baehrecke.Additionally, the author name Christina McPhee should haveappeared as Christina K. McPhee. The corrected author lineappears below. The online version has been corrected.

Yueguang Rong, Christina K. McPhee, ShuangshenDeng, Lei Huang, Lilian Chen, Mei Liu, Kirsten Tracy,Eric H. Baehrecke, Li Yu, and Michael J. Lenardo

www.pnas.org/cgi/doi/10.1073/pnas.1108410108

DEVELOPMENTAL BIOLOGYCorrection for “Tissue-specific roles of Axin2 in the inhibitionand activation of Wnt signaling in the mouse embryo,” by LihuiQian, James P. Mahaffey, Heather L. Alcorn, and KathrynV. Anderson, which appeared in issue 21, May 24, 2011 of ProcNatl Acad Sci USA (108:8692–8697; first published May 9,2011; 10.1073/pnas.1100328108).The authors note that on page 8693, left column, first para-

graph, line 9, “T-to-C” should instead appear as “T-to-A.” Theauthors note that on page 8693, right column, first paragraph,lines 3 and 4, “Axin2canp/Axinnull mice (n=15)” should insteadappear as “Axin2canp/Axin2null mice (n=15).”

www.pnas.org/cgi/doi/10.1073/pnas.1108478108

NEUROSCIENCECorrection for “Antidepressant effects of selective serotoninreuptake inhibitors (SSRIs) are attenuated by antiinflammatorydrugs in mice and humans,” by Jennifer L. Warner-Schmidt,Kimberly E. Vanover, Emily Y. Chen, John J. Marshall, and PaulGreengard, which appeared in issue 22, May 31, 2011, of ProcNatl Acad Sci USA (108:9262–9267; first published April 25,2011; 10.1073/pnas.1104836108).The authors note that the following acknowledgment was

omitted from the article: “This work was supported by NIHGrant MH090963.”

www.pnas.org/cgi/doi/10.1073/pnas.1109215108

www.pnas.org PNAS | July 5, 2011 | vol. 108 | no. 27 | 11297–11298

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NEUROSCIENCECorrection for “Functional agonism of insect odorant receptorion channels,” by Patrick L. Jones, Gregory M. Pask, DavidC. Rinker, and Laurence J. Zwiebel, which appeared in issue 21,May, 24, 2011, of Proc Natl Acad Sci USA (108:8821–8825; firstpublished May 9, 2011; 10.1073/pnas.1102425108).The authors note that Figures 2, 3, and 4 appeared incorrectly.

The corrected figures and their legends appear below.

www.pnas.org/cgi/doi/10.1073/pnas.1108343108

Fig. 4. 8-Br-cAMP and 8-Br-cGMP did not elicit currents in AgOrco orAgOrco+AgOr10 cells. (A) Representative trace from whole-cell recordingsfrom cells expressing AgOrco with application of 8-Br-cAMP, 8-Br-cGMP, andVUAA1. (B) Representative trace from cells expressing AgOrco+AgOr10 withapplication of 8-Br-cAMP, 8-Br-cGMP, BA, and VUAA1. (C) Representativetrace from cells expressing rCNGA2 with application of 8-Br-cGMP. Holdingpotentials for all recordings were −60 mV. (D) Histogram of normalizedcurrents from cyclic nucleotide and control responses (n = 4). All currentsnormalized to VUAA1 responses.

Fig. 2. RR blocks inward currents of AgOrco alone and in complex. (A–C)Representative traces of RR-blocked inward currents in AgOrco (A) andAgOrco+AgOr10 (B and C) cells. Holding potential was −60 mV for A–C. (D)Analysis of RR blockage of VUAA1 and BA-induced currents from A (n = 5),B (n = 5), and C (n = 4).

Fig. 3. AgOrco is a functional channel and responds to VUAA1 in outside-out membrane patches. (A) Single-channel recording from an outside-outexcised patch pulled from a cell-expressing AgOrco. (B–D) Expansions oftrace A before (B), during (C), and after (D) a 5-s application of −4.0 logMVUAA1. All-point current histograms of trace expansions are on the rightsides of B–D. Excised membrane patch was held at −60 mV.

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