Paliperidone reverts Toll-like receptor 3 signaling pathway activation and cognitive deficits in a maternal immune activation mouse model of schizophrenia

Accepted Manuscript Paliperidone reverts Toll-like receptor 3 signaling pathway activation and cognitive deficits in a maternal immune activation mouse model of schizophrenia Karina S. MacDowell, Eva Munarriz-Cuezva, Javier R. Caso, José L.M. Madrigal, Arantzazu Zabala, J. Javier Meana, Borja García-Bueno, Juan C. Leza PII:

S0028-3908(16)30590-1

DOI:

10.1016/j.neuropharm.2016.12.025

Reference:

NP 6548

To appear in:

Neuropharmacology

Received Date: 13 September 2016 Revised Date:

16 December 2016

Accepted Date: 26 December 2016

Please cite this article as: MacDowell, K.S., Munarriz-Cuezva, E., Caso, J.R., Madrigal, J.L.M., Zabala, A., Meana, J.J., García-Bueno, B., Leza, J.C., Paliperidone reverts Toll-like receptor 3 signaling pathway activation and cognitive deficits in a maternal immune activation mouse model of schizophrenia, Neuropharmacology (2017), doi: 10.1016/j.neuropharm.2016.12.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

MacDowell et al

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Paliperidone reverts Toll-like receptor 3 signaling pathway activation and cognitive deficits in a maternal immune activation mouse model of

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schizophrenia.

Karina S. MacDowell, PhDa,d,e, Eva Munarriz-Cuezva, PhD b,d, Javier R. Caso, PhD a,d,e, José L.M. Madrigal, PhD a,d,e, Arantzazu Zabala, PhD c,d,f, J. Javier Meana, PhD, MD b,d,f,

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Borja García-Bueno, PhD a,d,e, Juan C. Leza, PhD, MD a,d,e

Address correspondence:

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Department of Pharmacology, Faculty of Medicine, University Complutense, Madrid (a); Departments of Pharmacology (b) and Neurosciences (c), University of Basque Country UPV/EHU, Bizkaia; Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM) (d); Instituto de Investigación Sanitaria Hospital 12 de Octubre and Instituto de Neuroquímica UCM, Madrid (e); BioCruces Health Research Institute, Bizkaia (f). Spain.

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Juan Carlos Leza: [email protected].

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Department of Pharmacology, Faculty of Medicine, University Complutense, Madrid

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Number of words in abstract: 241 Number of words in manuscript: 4562 Number of references: 58 Number of figures: 6 Number of tables: 1 Supplemental information: Yes (3 figures and 2 tables) Short Title: Paliperidone regulates innate immune system activation

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MacDowell et al

ACCEPTED MANUSCRIPT ABSTRACT

The pathophysiology of psychotic disorders is multifactorial, including alterations in the immune system caused by exogenous or endogenous factors. Epidemiological and experimental studies indicate that infections during the gestational period represent a

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risk factor to develop schizophrenia (SZ) along lifetime. Here, we tested the hypothesis that the antipsychotic paliperidone regulates immune-related brain effects in an experimental model of SZ. A well described prenatal immune activation model of SZ in

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mice by maternal injection of the viral mimetic poly(I:C) during pregnancy was used.

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Young-adult offspring animals (60PND) received paliperidone ip (0.05 mg/kg) for 21 consecutive days. One day after last injection, animals were submitted to a cognitive test and brain frontal cortex (FC) samples were obtained for biochemical determinations. The adults showed an activated innate immune receptor TLR-3

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signaling pathway, oxidative/nitrosative stress and accumulation of pro-inflammatory mediators such as nuclear transcription factors (i.e., NFκB) and inducible enzymes (i.e., iNOS) in FC. Chronic paliperidone blocked this neuroinflammatory response possibly by

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the synergic activation and preservation of endogenous antioxidant/anti-inflammatory

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mechanisms such as NRF2 and PPARγ pathways, respectively. Paliperidone administration also stimulated the alternative polarization of microglia to the M2 antiinflammatory profile. In addition, paliperidone treatment improved spatial working memory deficits of this SZ-like animal model. In conclusion, chronic administration of paliperidone to young-adult mice prenatally exposed to maternal immune (MIA) challenge elicits a general preventive anti-inflammatory/antioxidant effect at both intracellular and cellular polarization (M1/M2) level in FC, as well as ameliorates specific cognitive deficits. 2

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Keywords: Maternal immune activation; TLR3; paliperidone; inflammation 1. Introduction Alterations in the innate immune system, caused by endogenous or exogenous factors, have been proposed as a key component in the pathophysiology of several

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psychotic diseases including schizophrenia (SZ) (revised in Leza et al., 2015). Epidemiological studies indicate that infections during the gestational period represent a risk factor for developing SZ over the course of a lifetime (Brown and Derkits, 2010).

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Although still not conclusive, several original studies and meta-analyses suggest that

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infections could facilitate the occurrence of damage in neurodevelopment, neurotransmission and sensorial information processing, which may play a role in the emergence of SZ (Arias et al., 2012; Khandaker et al., 2015).

Considering this evidence, animal models based on maternal immune activation

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(MIA) are currently used for the study of SZ (Meyer et al., 2005; Zuckerman et al., 2003). The synthetic analogue of the viral double stranded RNA polyinosinicpolycytidilic acid (poly(I:C)) is a Toll-like Receptor 3 (TLR3) agonist that, administered to

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pregnant mice, elicits immune responses analogous to those observed in viral

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infections (Meyer, 2014). Stimulation of the innate immune system through TLR3 triggers several intracellular signaling pathways that induce antiviral and inflammatory responses mediated by the transcription factors interferon regulatory transcription factor 3 (IRF3) and the nuclear factor κB (NFκB), respectively (Matsumoto and Seya, 2008). This acute inflammatory response is mainly characterized by the secretion of pro-inflammatory cytokines (Cunningham et al., 2007). The imbalance of cytokine milieu in accordance with the excessive production of reactive Oxygen and Nitrogen species and pro-inflammatory mediators could compromise the normal course of fetal 3

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neurodevelopment and predispose to long-lasting structural/functional brain abnormalities, leading to the emergence of psychopathological behavior in adulthood (Meyer et al., 2009; Venkatasubramanian and Debnath, 2013). TLR3 activation during gestation inhibits cortical neurogenesis, synaptic transmission and behavioral

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abnormalities in the rodent offspring (De Miranda et al., 2010; Zuckerman et al., 2003). All these changes contribute to yield a neurodevelopmental altered animal model that displays face, construct and predictive validity for schizophrenia studies (Ozawa et al.,

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2006).

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In response to immune activation, not only pro-inflammatory, but also antiinflammatory and antioxidant mechanisms are activated in the central nervous system (CNS). The antioxidant pathway regulated by the nuclear transcription factor (erythroid-derived 2)-like 2, NRF2 is the first example of the stated antioxidant

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mechanism. Under basal conditions, NRF2 is inactivated in the cytoplasm by binding to Kelch-like ECH-associated protein 1 (KEAP1). In presence of oxidative stress signals, NRF2 translocates into the nucleus where it binds to consensus sequences of

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antioxidants response elements (ARE). ARE encode a wide variety of antioxidant

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enzymes including some dedicated to glutathione synthesis and to the elimination of oxygen reactive species (Zhang et al., 2013). On the other hand, activation of the gamma isoform of peroxisome proliferator-activated nuclear receptors (PPARγ) by endogenous/synthetic ligands produces a multifaceted anti-inflammatory/antioxidant and an anti-excitotoxic and pro-energetic response in different CNS pathologies (Garcia-Bueno et al., 2008). Moreover, the polarization of microglia to the antiinflammatory phenotype M2 secretes anti-inflammatory cytokines such as the transforming growth factor beta (TGFβ) and interleukin 10 (IL10) (Hu et al., 2015). 4

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Increased M2 profile could provide neuroprotection under CNS pathological conditions, and its modulation is emerging as a promising target in neuropsychiatric diseases (Reus et al., 2015). The long-term mechanisms of action of antipsychotic drugs remain unclear

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since their therapeutic effects are not fully explained by their action on dopaminergic and serotonin receptors. Previous studies showed that several atypical antipsychotics, including risperidone, present an anti-cytokine effect in in vivo/in vitro settings (Kato et

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al., 2007; MacDowell et al., 2013; Sugino et al., 2009). Some effects of antipsychotics

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on anti-inflammatory/oxidant pathways have also been described (Drzyzga et al., 2006; MacDowell et al., 2016) but their potential direct actions on innate immunity remain unexplored. This study aims to elucidate the effects of paliperidone on MIA-induced cognitive

dysfunctions

and

activation

of

TLR-3,

neuroinflammatory

and

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counterbalancing NRF2 and PPARγ pathways.

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ACCEPTED MANUSCRIPT 2. Material and Methods 2.1 Animals and experimental model:

Pregnant C57BL/6J mice (Harlan Ibérica, Spain) were injected i.p. with either 5 mg/kg poly(I:C) (Sigma-Aldrich, Spain) or the vehicle (saline solution) on gestational

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day 9.5 (Holloway et al., 2013). Animals were maintained under standard temperature and humidity conditions in a 12-h light/dark cycle (lights on at 08:00) with free access to food and water. Experimental protocols were approved by the Animal Welfare

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Committee of the University of Basque Country in accordance with European

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legislation (D2010/63/UE). This prenatal immune activation induces behavioral and brain structural/functional anomalies in adult offspring (Meyer, 2014). See Figure S1 in the supplementary information (SI) for details.

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2.2 Drug administration and experimental designs:

Male and female pups were born from three poly (I:C)-treated and four salinetreated dams and were randomly assigned among four treatment groups with

antipsychotic

paliperidone

(3-[2-[4-(6-Fluoro-1,2-benzisoxazol-3-yl)-1-

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atypical

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variables of pre-treatment (poly(I:C) vs. saline) and drug (paliperidone vs. vehicle). The

piperidinyl]ethyl]-6,7,8,9-tetrahydro-9-hydroxy-2-methyl-4H-pyrido[1,2-a]pyrimidin-4one) (PubChem CID: 115237, Sigma-Aldrich, Spain) was dissolved in a saline solution with 0.26 mM of acetic acid (vehicle, Veh), pH adjusted to 7.4 with NaOH. Young-adult animals (≥60 postnatal days; PND) originated from the seven litter were injected i.p. with either paliperidone (P; 0.05 mg/kg) or the vehicle for 21 consecutive days. Group sample sizes were poly(I:C)/Veh, n=9 (4 female and 5 male); poly(I:C)/P, n=8 (4 female and 4 male); saline/Veh, n=11 (5 female and 6 male); saline/P, n=8 (3 female and 5 6

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male) (see Figure S1 in SI). The dose of paliperidone was selected to be similar to commonly prescribed human dosages for a 50 kg adolescent, and on the basis of previous in vivo determinations of behavioral and brain structural abnormalities in adulthood in poly(I:C) offspring (Piontkewitz et al., 2011; Richtand et al., 2011). No

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differences in body weight between the four animal groups were observed.

2.3 Behavioral test:

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Animals were submitted to an alternation task T-maze test (Holloway et al,

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2013). The full experiment consisted of three parts: habituation, training, and testing. During the three periods, animals were partially food-deprived. Habituation and training were performed whereas animals were under treatment with paliperidone or vehicle. For habituation, animals were placed on the T-maze with food spread. This

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was repeated three times a day for 5 days. During training, animals received six trials a day. Each training trial consisted of two runs, a forced and a free run. In the forced run, mice were forced to obtain a piece of food from goal alley of the T-maze, with the

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other alley blocked by its door. Then, animals were placed back into the start arm for

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10 and 40 s delay periods (three trials for each delay period). At the free run, animals were allowed to choose either goal alley. If the mice chose the same arm into which they had been forced, they did not receive food reward whereas if they chose the opposite arm they received food reward and the choice was considered correct. The sequence of delays and forced-run food locations were randomized each day. Animals received six trials a day with a 5-min intertrial interval. It was considered that animals were trained when controls mice made more than 70% correct choices on two consecutive days. Animals reaching this threshold were maintained under daily 7

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training until chronic paliperidone/vehicle treatment was finished (21 days). The test day (24 h after the last dose of paliperidone or vehicle), mice were tested for their performance in the T-maze recording the number of correct choices in similar

2.4 Preparation of biological samples:

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conditions to those in training period.

After the T-maze test, animals were subjected to cervical dislocation. The brain

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was removed from the skull and, after careful removal of the meninges; frontal

assayed.

2.5 Western Blot Analysis:

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cortical areas (FC) from both brain hemispheres were excised and frozen at -80°C until

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To determine the expression levels of Toll-like Receptor 3 (TLR3), TIR-domaincontaining adapter-inducing interferon-β (TRIF), interferon regulatory factor 3 (IRF3/pIRF3), nuclear factor kappa-b inhibitor alpha (IκBα), nitric oxide synthase

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inducible (iNOS), cyclooxygenase 2 (COX2), protein kinase B (AKT/pAKT),

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phosphoinositide 3-kinase (PI3K), Lipocalin-prostaglandin D synthase (L-PGDS), Arginase I (ArgI) and folate receptor 2 (FOLR2), brain FC samples were homogenized by sonication in PBS (pH=7) mixed with a protease inhibitor cocktail (Complete®, Roche, Spain). Protein levels of PPARγ, NFκB and NRF2 were determined in nuclear extracts and KEAP1 in cytosolic extracts. Nuclear and cytosolic extracts were prepared according to published protocols (MacDowell et al., 2013). All western blots were performed at least three times in separate assays. Protein levels were measured using the Bradford method (see details in SI). 8

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2.6 Real Time-Polymerase Chain Reaction Analysis: Total cytoplasmic RNA was prepared from samples of brain FC using TRIZOL reagent (Invitrogen, New York, USA); aliquots were converted to complementary DNA using

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random hexamer primers. Quantitative changes in mRNA levels of TLR3, TRIF, IRF3, NFκB, IκBα, iNOS, COX2, microsomal prostaglandin E synthase-1 (mPGES1), L-PGDS, AKT, PI3K, KEAP1, NRF2, hemoxygenase-1 (HO1), NADPH dehydrogenase quinone 1

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(NQO1), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT),

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tumour necrosis factor alpha (TNFα), interleukin 1Beta (IL1β), interleukin 6 (IL6), interleukin 10 (IL10), interferon alpha (IFNα), interferon beta (IFNβ), fractalkine (CX3CL1), transforming grown factor beta (TGFβ), signal transducer and activator of transcription 1 (STAT1), ArgI, FOLR2, glyceraldehyde-3-phosphate dehydrogenase

details in SI).

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2.7 Lipid peroxidation

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(GADPH) and tubulin were estimated by real time-polymerase chain reaction (see

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Lipid peroxidation was measured by the thiobarbituric acid test for malondialdehyde (MDA) (see details in SI).

2.8 NRF2 Activity

Activation of nuclear factor NRF2 was measured in nuclear extracts through a commercially ELISA-based kit (Cayman Europe) following the manufacturer’s instructions.

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2.9 Statistical Analyses: Data are expressed as mean±SEM. For comparisons between two groups a two-tailed t-test was employed. For multiple comparisons, a two-way ANOVA was

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used and the Bonferroni post hoc test was applied in case of significant interaction, considering as the first factor the presence or absence of MIA (poly(I:C)) and as the second, the presence or absence of paliperidone treatment. In behavioral

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experiments, a three-way ANOVA (MIA x treatment) with repeated measures (10 s

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and 40 s delays) was applied. All the results of the ANOVA analyses (F values and dfs) are included in Table 1 and Table S2 (SI). A p value < 0.05 was defined as statistically

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significant.

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3. Results 3.1 Effects of paliperidone on TLR-3 signaling pathway activation and neuroinflammation after MIA

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Analysis of in vitro findings showed that TLR3 signaling pathway was increased in animals exposed to MIA. Two-way ANOVA of TLR-3 protein expression in FC samples showed significant effects for poly(I:C), paliperidone treatment and interaction (Table

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1). The pharmacological treatment with paliperidone (PND 60 to 80) inhibited the TLR-

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3 up-regulation induced by MIA (Figure 1A). Two-way ANOVA for other components of TLR-3 pathway, TRIF and phosphorylated-IRF3 (pIRF3), reported a significant protein increase in animals exposed to MIA (Table 1) (Figure 1B,C). The TLR-3 stimulation produces the nuclear translocation of NFκB by the degradation of the inhibitory

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subunit IκBα. Two-way ANOVA of NFκB (p65 nuclear subunit) and IκBα expression values identified significant effects for poly(I:C), treatment and interaction (Table 1). Paliperidone treatment blocked the increase of nuclear content of NFκB and the

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decrease of cytoplasmic protein levels of IκBα in animals exposed to poly(I:C) (Figure

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1D,E). The activation of NFκB in animals exposed to MIA was associated with an increase of the pro-inflammatory enzyme iNOS (Figure 1F) that was opposed by paliperidone, but no differences were observed in COX2 protein expression (Figure 1G). Two-way ANOVA had shown significant effects of poly(I:C), treatment and interaction for iNOS (Table 1). The other enzymatic source of COX2-dependent oxidative/nitrosative stress is the microsomal-PGE2 synthase-1 (m-PGES-1). Both protein (data not shown) and mRNA levels of m-PGES-1 remained unchanged in all

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groups studied (Figure 2SH). These protein expression results were paralleled by mRNA expression levels measured by RT-PCR (Figure S2 and Table S2 in SI).

Finally, Two-way ANOVA showed significant effects for poly(I:C), treatment and

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interaction in the levels of the lipid peroxidation marker malondialdehyde (MDA) (Table 1). An increase of MDA was found in animals exposed to MIA that was fully

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affected by paliperidone treatment (Figure 1H).

antioxidant enzymes after MIA

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3.2 Effects of paliperidone on brain NRF2 regulatory pathway and NRF2-dependent

Two-way ANOVA revealed an effect of treatment for NRF2 expression and a significant effect of poly(I:C), treatment and interaction for NRF2 activity (Table 1).

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There were no significant differences in NRF2 protein expression and activity between control group and animals exposed to MIA, but the analyses demonstrated that paliperidone treatment after MIA increased NRF2 nuclear protein levels and activity

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(Figure 2A,B).

The up-stream regulators of NRF2, PI3K and pAKT/AKT protein expression

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showed an increase in animals exposed to MIA (Table S2) insensitive to paliperidone administration (Figure S3C,E in SI). Post-hoc test after two-way ANOVA demonstration of interaction effect (Table S2) revealed that paliperidone-treated animals exposed to poly(I:C) showed lower KEAP1 mRNA levels than animals without treatment (Figure S3F in SI). As NRF2 is a crucial regulator of the endogenous antioxidant response, we studied whether paliperidone was capable of regulating the expression of the main 12

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antioxidant enzymes in control and MIA conditions. Two-way ANOVA for Hemoxygenase-1 (HO1) and superoxide dismutase (SOD) mRNA expression showed a significant effect for poly(I:C), treatment and interaction (Table 1). Catalase mRNA levels displayed effect for treatment and interaction (Table 1). Post-hoc comparisons

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confirmed the stimulatory effect of paliperidone when compared to vehicle treatment in poly(I:C) offspring (Figure 2C-E). Glutathione peroxidase (GPx) and NADPH dehydrogenase quinone 1 (NQO1) mRNA levels remained unaltered in all groups

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studied (Figure S3H-I in SI).

3.3 Effects of paliperidone on microglia M2 polarization after MIA We analyzed whether paliperidone treatment modulates the cytokine environment to stimulate the transition of microglia from poly(I:C)-induced

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inflammatory M1 to the anti-inflammatory M2 profile. Two-way ANOVA revealed a significant effect of poly(I:C) and treatment for TNFα mRNA levels, a significant effect of treatment and interaction for IL1β mRNA levels, and a significant effect of poly(I:C),

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treatment and interaction in IL6 mRNA levels (Table 1). Thus, MIA induced an

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increment of the main pro-inflammatory cytokines TNFα and IL6 mRNA levels, but no changes were appreciated in IL1β mRNA levels (Figures 3A-C). Paliperidone decreased mRNA levels of these cytokines in animals exposed to poly(I:C) (Figure 3A-C). Also, the cytokines implicated in the anti-viral response IFNα and IFNβ, and the transcription factor STAT1, were evaluated. Two-way ANOVA for IFNα showed a significant effect of poly(I:C), treatment and interaction whereas in the case of IFNβ and STAT1, significant effects of treatment and interaction were identified (Table 1). In animals exposed to

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MIA an increased mRNA expression of IFNα, IFNβ, and STAT1 was observed that was prevented by paliperidone administration (Figure 3D-F). On the other hand, two-way ANOVA for TGFβ mRNA levels showed a significant effect of poly(I:C), treatment and interaction, and for IL10 mRNA levels the difference

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was due to poly(I:C) and interaction (Table 1). In the case of CX3CL1 mRNA expression a significant effect for treatment and interaction was found (Table 1). Paliperidone treatment induced a selective increment on poly(I:C)-exposed offspring of the anti-

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inflammatory cytokines TGFβ, IL10, as well as the chemokine CX3CL1, all of these

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molecules being inducers of a M2 profile (Figure 3G-I).

Subsequently, we studied the effect of MIA on brain M2 phenotype markers Arginase I (ArgI) and Folate receptor 2 (FOLR2) and the potential regulatory mechanism of paliperidone on microglia polarization profile. Two-way ANOVA

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identified significant effects of poly(I:C), treatment and interaction on FOLR2 mRNA expression whereas ArgI mRNA levels remained unaltered (Table 1) (Figure 4A). Paliperidone treatment increased the mRNA FOLR2 expression compared to the rest of

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groups (Figure 4B). Significant effects for poly(I:C), treatment and interaction were

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found for ArgI protein expression. Regarding FOLR2 protein, the significant effects were for treatment and interaction (Table 1). The post-hoc Bonferroni analysis displayed that MIA exposure reduced ArgI and FOLR2 protein levels, and that paliperidone treatment exerted opposite influence on this down-regulation (Figure 4C,D). Another inter-related mechanism implicated in the regulation of the antiinflammatory response is the synthesis of deoxyprostaglandins, such as 15-deoxyProstaglandin J2. Consequently, we studied the expression of its specific enzymatic 14

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source, lipocalin-Prostaglandin D2 synthase (L-PGDS) and its nuclear target, PPARγ. Two-way ANOVA of L-PGDS showed a significant effect of treatment and interaction whereas in the case of PPARγ expression significant effects for poly(I:C), treatment and interaction were found (Table 1). Although animals exposed to MIA showed no

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differences in L-PGDS mRNA (Figure S2I) and protein expression (Figure 4E), paliperidone treatment increased L-PGDS protein levels in these animals (Figure 4E).

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paliperidone administration (Figure 4F).

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PPARγ was decreased in MIA animals and this effect was significantly affected by

3.4 Effects of paliperidone on alternation task T-maze test after MIA The working memory status of the different animal groups was studied in a Tmaze test task with delay periods at 10 and 40 seconds. Three-way ANOVA analysis

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with repeated measures demonstrated the absence of delay influence (F[1,31]=2.18; p=0.15) and identified a significant effect for poly(I:C) and interaction (Figures 5A,B). Bonferroni post-hoc tests showed that paliperidone treatment improved deficits

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induced by MIA in this task.

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ACCEPTED MANUSCRIPT 4. Discussion

The data presented here provide evidence to support a regulatory role of the antipsychotic paliperidone in the activation of the TLR-3 pathway and the subsequent neuroinflammatory response in the FC of the adult offspring of dams that received

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MIA with poly(I:C) administration in pregnancy (see Figure 6 for a detailed diagram of the pathway studied). These results are especially relevant, considering previous studies showing increased TLR3 expression in SZ drug-free patients (Muller et al.,

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2012), and decreased TLR3 expression in patients under neuroleptic treatment (Chang

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et al., 2011). A thorough study of the possible related mechanisms shows that paliperidone treatment activated compensatory NRF2 antioxidant and PPARγ antiinflammatory pathways and stimulated the alternative polarization of microglia to the M2 anti-inflammatory profile. In addition, chronic treatment of paliperidone at a

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therapeutic low dose improves the characteristic spatial working memory deficits of this animal model of SZ.

Besides the well-established cognitive impairment and the considerable

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evidence for low-intensity neuroinflammation in SZ, the causal relationship or just

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association between both conditions is unknown (Ribeiro-Santos et al., 2014; Cabrera et al., 2016) and mechanistic studies based in animal models are necessary. The animal model based on poly(I:C) administration to pregnant dams is

consistent with a MIA and subsequent aberrant neurodevelopment hypothesis of schizophrenia and with the typical emergence of this disorder during adolescence or early adulthood. Behavioral and morpho-functional abnormalities in the offspring of poly(I:C)-treated mothers are absent in juvenile periods but appear after puberty (Meyer, 2014; Meyer and Feldon, 2012; Ozawa et al, 2006; Zuckerman et al, 2003). 16

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However, further investigations are required to assess whether the long-term effects of MIA both at behavioral and molecular level are a direct consequence of the prenatal exposure or resultant of neurodevelopmental alterations produced in response to maternal exposure to poly(I:C) that could render the offspring more vulnerable in

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adulthood. In this vein, several observations suggest that TLR3 activation and high levels of proinflammatory cytokines during fetal development could induce deficits in immature neurons altering neuronal connectivity processes (De Miranda et al., 2010).

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Apart from this neurodevelopmental hypothesis, some authors have indicated that a

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mechanism related to cytokine-induced cognitive deficits could be the modulation of indoleamine 2,3-dioxygenase (IDO) enzyme activity and, consequently, the kynurenine pathway (Khandaker and Dantzer, 2016). Indeed, other possibility is the effects of cytokines and chemokines on BDNF signaling pathway (Stuart and Baune, 2014).

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The effect of atypical antipsychotic drugs on cognitive deficits in SZ is controversial (Lett et al., 2014). In the present study, chronic paliperidone administration was fitted to act when brain and behavioral impairments have been

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established at late adolescence / early adulthood (PND60-80) (Ozawa et al, 2006).

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Alternative preventive strategies based on pharmacological treatments during periadolescence have also been proposed (Meyer and Feldon, 2012). It is well known that chronic risperidone administration up to PND70 prevents structural abnormalities and behavioral deficits in the poly(I:C) rat offspring, including enlargement of ventricular volume, impaired neurogenesis, disturbed micro-vascularization, loss of parvalbuminexpressing hippocampal interneurons and locomotor response to amphetamine (Piontkewitz et al., 2011; Piontkewitz et al., 2012; Richtand et al., 2011). Also, it has been previously described that low doses of risperidone and paliperidone normalize 17

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both basal and MK-801-induced increase in cortical extracellular glutamate (Roenker et al., 2011). Furthermore, acute treatment with clozapine reverses impaired long-rate neuronal synchrony between hippocampus and medial prefrontal cortex (Dickerson and Bilkey, 2013), and chronic clozapine treatment during adulthood improves

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offspring working memory deficits induced by poly(I:C) administration to pregnant mothers (Meyer et al., 2010). However, the potential relationship between all these neurodevelopmental alterations previously described and the inflammatory blocking

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and direct anti-inflammatory/antioxidant effects of paliperidone here shown remains

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still to be elucidated.

Although there is increased evidence of direct effects of poly(I:C) treatment on classic inflammatory mediators (i.e. NFκB, iNOS) (Ribeiro et al., 2013; Song et al., 2011; Volk et al., 2015) in adult animals, the study of TLR3-dependent pathways after MIA

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activation by poly(I:C) deserved investigation. The present results confirmed that this MIA promotes the expression of several elements of the TLR3 signaling pathway, eliciting oxidative/nitrosative stress probably by the accumulation of pro-inflammatory

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mediators such as NFκB and iNOS. Moreover, paliperidone administration at

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therapeutic equivalent doses was able to block these experimentally-induced impairments, leading to potential cytoprotective activity. It is important to mention the absence of effects on the COX-2/m-PGES-1

pathway in the present model. On the contrary, other studies have found increased COX-2 in mice FC submitted to MIA with poly(I:C) (Malkova et al., 2014), and in the hypothalamus of rats peripherally treated with poly(I:C) (Fortier et al., 2004). A thorough study of this pathway is especially relevant in SZ considering that there are several clinical trials using COX-2 inhibitors as adjuvant therapy (Muller et al., 2013). 18

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Regarding oxidative/nitrosative stress, other authors have also found an increase in MDA levels in the FC of Wistar rats receiving poly(I:C) at postnatal day 5–7 (Ribeiro

et

al.,

2013).

Enhanced

MDA

activity

represents

hyperactive

oxidative/nitrosative stress that has been also reported in other LPS administration

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based models of MIA (revised in Boksa, 2010). Oxidative/nitrosative stress could be a consequence of depletion or malfunction of endogenous antioxidant cellular systems, (i.e., the NRF2 signaling pathway) (revised in Leza et al., 2015). In fact, a specific down-

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regulation of the NRF2-dependent enzyme SOD gene expression was found in the

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present study. This result is especially attractive, considering that SOD appears to be a biomarker for SZ that has been described to be decreased in acutely relapsed inpatients, first episode psychosis, and stable outpatients (Coughlin et al., 2013). The global stimulatory effects of paliperidone on HO1, SOD and catalase gene expression

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could account for its ability to counteract inflammation and lipid peroxidation by a mechanism involving NRF2 up-regulation. Further work should determine the functional correlate, beyond mRNA expression, of these increased oxidative signals

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obtained in poly(I:C)-exposed animals.

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The synthesis and release of pro/anti-inflammatory cytokines in the poly(I:C) model of SZ have received more attention than other parameters studied to date. Our results are in general agreement with findings showing and increase in proinflammatory cytokines (Garay et al., 2013). Previous studies have shown that antipsychotic drugs as risperidone, clozapine and haloperidol promote an antiinflammatory effect by decreasing pro-inflammatory and enhancing anti-inflammatory cytokine concentrations (Sugino et al., 2009), as observed in the present study. In fact, overexpression of the anti-inflammatory cytokine IL10 in the brain has been 19

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demonstrated to be sufficient to prevent the development of multiple behavioral dysfunctions in this particular animal model (Meyer et al., 2008). Recently, it has been suggested that microglial activation is closely related to the pathophysiology of SZ (Jhamnani et al., 2013), although behavioral and cognitive

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abnormalities can emerge in absence of microglial alterations (Giovanoli et al., 2016). Increased IL10 levels lead to the M2 phenotype (Orihuela et al., 2016). This polarization is considered responsible for tissue remodeling and repair. A similar

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scenario could be taking place in our conditions, where increased IL10 and the M2

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microglia cellular marker FOLR2 are induced by paliperidone treatment. Furthermore, NRF2 can elicit M2 polarization by secreting Hemoxygenase 1 (HO1), which displays immunomodulatory and anti-inflammatory properties (Naito et al., 2014). Also, fractalkine (CX3CL1) could play a role in microglial polarization. While its primary

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function is the chemo-attraction, it has been found that it also exerts an in vivo regulatory role of microglial neurotoxicity (mediated by M1 phenotype), thereby inducing the M2 phenotype and promoting neuroprotection and neuronal survival in

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murine models of amyotrophic lateral sclerosis and Parkinson's disease (Cardona et al.,

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2006). Interestingly, in the present study, paliperidone treatment stimulated IL10, FOLR2, HO1 and CX3CL1 pro-M2 microglia profile mediators as well as TGF-β, a characteristic cytokine of the M2 microglial response (Zhou et al., 2012). The anti-inflammatory nuclear transcription factor PPARγ has been found to

decrease in patients with first episode psychosis or chronic SZ (Garcia-Bueno et al., 2014; Martinez-Gras et al., 2011). Although the ability of antipsychotic drugs to normalize PPARγ levels in rodent FC has been previously demonstrated for risperidone in a neuroinflammatory model based on the intraperitoneal administration of LPS 20

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(MacDowell et al., 2013), to our knowledge, this is the first evidence of the inhibitory effects of MIA on PPARγ expression in brain. Working memory and cognitive flexibility are some of the behaviors that show dysfunctions in SZ. Dose-dependent response to poly(I:C) administration is an

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important factor when inducing working memory deficits related to SZ (Meyer and Feldon, 2012). Thus, low doses (1 mg/kg) administered at gestational day 9 do not promote cognitive dysfunctions (Canetta et al, 2016) whereas higher doses (5-10

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mg/kg) lead to clear impairments in offspring (Meyer and Feldon, 2012). Also, the

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prenatal timing of poly(I:C) administration generates differential behavior profiles between MIA activation at gestational days 9 and 17, with spatial working memory being affected in both conditions (Meyer and Feldon, 2012). Here, in agreement with previous data in T-maze (Holloway et al., 2013) and Morris water maze (Meyer et al.,

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2010) tests, administration of 5 mg/kg poly(I:C) at gestational day 9.5, induced cognitive deficits in a alternation task T-maze test. The finding adds face validity information to other cognitive dysfunctions described in offspring of poly(I:C)-treated

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mothers and contributes to support the hypothesis that inflammatory hyperactivity

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might be a key factor in the development of cognitive deficits related to SZ (Meyer, 2014). Furthermore, the improvement of the cognitive dysfunctions induced by poly(I:C) after chronic paliperidone treatment provides support for the predictive validity of this animal model for the study of SZ (Young et al., 2012). Finally, some methodological limitations should be considered: first, although a careful removal of the meninges was made, animals were not perfused before brain dissection and there might be remnants of circulatory TLR3 and other inflammatory mediators in the samples. Further studies are needed to elucidate the precise 21

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contribution of possible systemic remnants blood cells to the neuroinflammatory process in the samples here studied; second, in RT-PCR experiments the housekeeping genes β-actin and GADPH have been used. Both genes can exhibit much variability especially in infection models (Kozera and Rapacz, 2013). Although no differences were

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found in GADPH and tubulin levels between all groups studied, the validation of other genes in our particular experimental conditions is needed for the future.

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4.1 Conclusions

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Taken together, the results obtained demonstrate that chronic administration of the atypical antipsychotic paliperidone to young adult mice prenatally exposed to a MIA challenge elicits a general anti-inflammatory/antioxidant effect in FC, as well as improves specific cognitive deficits. The use of adjuvant compounds enhancing these

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pathways could result in a promising new pharmacological mechanism for the better

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management of SZ and other psychotic disorders.

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ACCEPTED MANUSCRIPT Acknowledgements:

This work was supported by the Instituto de Salud Carlos III (FIS 10/00123 & 13/1102), Spanish MINECO (SAF 2013-48586-R), Centro de Investigación en Red de Salud Mental, CIBERSAM, Basque Government (IT 616/13), ERDF Funds and Foundation Santander-

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UCM (GR 58/08). JRC and BGB are Ramón y Cajal post-doctoral fellows (Spanish MEEIC).

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Financial Disclosures:

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None

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ACCEPTED MANUSCRIPT References

Arias, I., Sorlozano, A., Villegas, E., de Dios, L. J., McKenney, K., Cervilla, J., Gutiérrez, B., Gutiérrez, J., 2012. Infectious agents associated with schizophrenia: a meta-analysis. Schizophr. Res 136, 128-136.

RI PT

Boksa, P., 2010. Effects of prenatal infection on brain development and behavior: a review of findings from animal models. Brain Behav. Immun 24, 881-897.

Brown, A. S., Derkits, E. J., 2010. Prenatal infection and schizophrenia: a review of

SC

epidemiologic and translational studies. Am J Psychiatry 167, 261-280.

M AN U

Cabrera, B., Bioque, M., Penadés, R., González-Pinto, A., Parellada, M., Bobes, J., Lobo, A., García-Bueno, B., Leza, J. C., Bernardo, M. 2016. Cognition and psychopathology in first-episode psychosis: are they related to inflammation?. Psychological Med. 46, 2133-2144.

TE D

Canetta, S., Bolkan, S., Padilla-Coreano, N., Song, L. J., Sahn, R., Harrison, N. L., Gordon, J. A., Brown, A., Kellendonk, C., 2016. maternal immune activation leads to selective functional deficits in offspring parvalbumin interneurons. Mol. Psychiatry 21, 956-

EP

968.

AC C

Cardona, A. E., Pioro, E. P., Sasse, M. E., Kostenko, V., Cardona, S. M., Dijkstra, I. M., Huang, D., Kidd, G., Dombrowski, S., Dutta, R., Lee, J. C., Cook, D. N., Jung, S., Lira, S. A., Littman, D. R., Ransohoff, R. M., 2006. Control of microglial neurotoxicity by the fractalkine receptor. Nat. Neurosci 9, 917-924. Coughlin, J. M., Ishizuka, K., Kano, S. I., Edwards, J. A., Seifuddin, F. T., Shimano, M. A., Daley, E. L., Zandi, P. P., Leweke, F. M., Cascella, N. G., Pomper, M. G., Yolken, R. H., Sawa, A., 2013. Marked reduction of soluble superoxide dismutase-1 (SOD1) in

24

MacDowell et al

ACCEPTED MANUSCRIPT

cerebrospinal fluid of patients with recent-onset schizophrenia. Mol. Psychiatry 18, 10-11. Cunningham, C., Campion, S., Teeling, J., Felton, L., Perry, V. H., 2007. The sickness behaviour and CNS inflammatory mediator profile induced by systemic challenge of

RI PT

mice with synthetic double-stranded RNA (poly I:C). Brain Behav. Immun 21. 490502.

Chang, S. H., Chiang, S. Y., Chiu, C. C., Tsai, C. C., Tsai, H. H., Huang, C. Y., Hsu, T. C.,

SC

Tzang, B. S., 2011. Expression of anti-cardiolipin antibodies and inflammatory

M AN U

associated factors in patients with schizophrenia. Psychiatry Res. 187, 341-346. De Miranda, J., Yaddanapudi, K., Hornig, M., Villar, G., Serge, R., Lipkin, W. I., 2010. Induction of Toll-like receptor 3-mediated immunity during gestation inhibits cortical neurogenesis and causes behavioral disturbances. MBio 1 (4).

TE D

Dickerson, D. D., Bilkey, D. K., 2013. Aberrant neural synchrony in the maternal immune activation model: using translatable measures to explore targeted interventions. Front Behav. Neurosci. 7, 217.

EP

Drzyzga, L., Obuchowicz, E., Marcinowska, A., Herman, Z. S., 2006. Cytokines in

AC C

schizophrenia and the effects of antipsychotic drugs. Brain Behav. Immun. 20, 532545.

Fortier, M. E., Kent, S., Ashdown, H., Poole, S., Boksa, P., Luheshi, G. N., 2004. The viral mimic, polyinosinic:polycytidylic acid, induces fever in rats via an interleukin-1dependent mechanism. Am. J. Physiol Regul. Integr. Comp. Physiol. 287, R759-R766. Garay, P. A., Hsiao, E. Y., Patterson, P. H., McAllister, A. K., 2013. Maternal immune activation causes age- and region-specific changes in brain cytokines in offspring throughout development. Brain Behav. Immun. 31, 54-68. 25

MacDowell et al

ACCEPTED MANUSCRIPT

García-Bueno, B., Bioque, M., Mac-Dowell, K. S., Barcones, M. F., MartinezCengotitabengoa, M., Pina-Camacho, L., Rodriguez-Jimenez, R., Saiz, P. A., Castro, C., Lafuente, A., Santabarbara, J., Gonzalez-Pinto, A., Parellada, M., Rubio, G., Garcia-Portilla, M. P., Mico, J. A., Bernardo, M., Leza, J. C., 2014. Pro-/anti-

RI PT

inflammatory dysregulation in patients with first episode of psychosis: toward an integrative inflammatory hypothesis of schizophrenia. Schizophr. Bull. 40, 376-387. García-Bueno, B., Madrigal, J. L., Perez-Nievas, B. G., Leza, J. C., 2008. Stress mediators

SC

regulate brain prostaglandin synthesis and peroxisome proliferator-activated

M AN U

receptor-gamma activation after stress in rats. Endocrinology 149, 1969-1978. Giovanoli, S., Weber-Stadlbauer, U., Schedlowski, M., Meyer, U., Engler, H., 2016. Prenatal immune activation causes hippocampal synaptic deficits in the absence of overt microglia anomalies. Brain Behav. Immun. 55, 25-38.

TE D

Holloway, T., Moreno, J. L., Umali, A., Rayannavar, V., Hodes, G. E., Russo, S. J., Gonzalez-Maeso, J., 2013. Prenatal stress induces schizophrenia-like alterations of serotonin 2A and metabotropic glutamate 2 receptors in the adult offspring: role of

EP

maternal immune system. J. Neurosci. 33, 1088-1098.

AC C

Hu, X., Leak, R. K., Shi, Y., Suenaga, J., Gao, Y., Zheng, P., Chen, J., 2015. Microglial and macrophage polarization-new prospects for brain repair. Nat. Rev. Neurol 11, 56-64. Jhamnani, K., Shivakumar, V., Kalmady, S., Rao, N. P., Venkatasubramanian, G., 2013. Successful use of add-on minocycline for treatment of persistent negative symptoms in schizophrenia. J. Neuropsychiatry Clin. Neurosci. 25, E06-E07. Kato, T., Monji, A., Hashioka, S., Kanba, S., 2007. Risperidone significantly inhibits interferon-gamma-induced microglial activation in vitro. Schizophr. Res 92, 108-115.

26

MacDowell et al

ACCEPTED MANUSCRIPT

Khandaker, G. M., Cousins, L., Deakin, J., Lennox, B. R., Yolken, R., Jones, P. B., 2015. Inflammation and immunity in schizophrenia: implications for pathophysiology and treatment. Lancet Psychiatry 2, 258-270. Khandaker, G.M., Dantzer, R.,

2016.

Is

there

a

role

for

immune-to-brain

RI PT

communication in schizophrenia?. Psychopharmacology (Berl) 233,1559-1573.

Kozera, B., Rapacz, M., 2013. Reference genes in real-time PCR. J Appl Genet. 54, 391– 406.

SC

Lett, T. A., Voineskos, A. N., Kennedy, J. L., Levine, B., Daskalakis, Z. J., 2014. Treating

Psychiatry 75, 361-370.

M AN U

working memory deficits in schizophrenia: a review of the neurobiology. Biol.

Leza, J. C., García-Bueno, B., Bioque, M., Arango, C., Parellada, M., Do, K., O'Donnell, P., Bernardo, M., 2015. Inflammation in schizophrenia: A question of balance.

TE D

Neurosci. Biobehav. Rev. 55, 612-626.

MacDowell, K. S., Caso, J. R., Martin-Hernandez, D., Moreno, B. M., Madrigal, J. L., Mico, J. A., Leza, J. C., Garcia-Bueno, B., 2016. The Atypical Antipsychotic

EP

Paliperidone Regulates Endogenous Antioxidant/Anti-Inflammatory Pathways in Rat

AC C

Models of Acute and Chronic Restraint Stress. Neurotherapeutics. 13:833-843. MacDowell, K. S., García-Bueno, B., Madrigal, J. L., Parellada, M., Arango, C., Micó, J. A., Leza, J. C., 2013. Risperidone normalizes increased inflammatory parameters and restores anti-inflammatory pathways in a model of neuroinflammation. Int. J. Neuropsychopharmacol. 16, 121-135. Malkova, N. V., Gallagher, J. J., Yu, C. Z., Jacobs, R. E., Patterson, P. H., 2014. Manganese-enhanced magnetic resonance imaging reveals increased DOI-induced

27

MacDowell et al

ACCEPTED MANUSCRIPT

brain activity in a mouse model of schizophrenia. Proc. Natl. Acad. Sci. U. S. A. 111, E2492-E2500. Martínez-Gras, I., Pérez-Nievas, B. G., García-Bueno, B., Madrigal, J. L., Andrés-Esteban, E., Rodríguez-Jiménez, R., Hoenicka, J., Palomo, T., Rubio, G., Leza, J. C., 2011. The

decreased in schizophrenia. Schizophr. Res. 128, 15-22.

RI PT

anti-inflammatory prostaglandin 15d-PGJ2 and its nuclear receptor PPARgamma are

Matsumoto, M., Seya, T., 2008. TLR3: interferon induction by double-stranded RNA

SC

including poly(I:C). Adv. Drug Deliv. Rev 60, 805-812.

M AN U

Meyer, U., 2014. Prenatal poly(i:C) exposure and other developmental immune activation models in rodent systems. Biol Psychiatry 75, 307-315. Meyer, U., Feldon, J., 2012. To poly(I:C) or not to poly(I:C): Advancing preclinical schizophrenia research through the use of prenatal immune activation models.

TE D

Neuropharmacology 62, 1308-1321.

Meyer, U., Feldon, J., Schedlowski, M., Yee, B. K., 2005. Towards an immunoprecipitated neurodevelopmental animal model of schizophrenia. Neurosci.

EP

Biobehav. Rev. 29, 913-947.

AC C

Meyer, U., Feldon, J., Yee, B. K., 2009. A review of the fetal brain cytokine imbalance hypothesis of schizophrenia. Schizophr. Bull. 35, 959-972. Meyer, U., Knuesel, I., Nyffeler, M., Feldon, J., 2010. Chronic clozapine treatment improves prenatal infection-induced working memory deficits without influencing adult hippocampal neurogenesis. Psychopharmacology (Berl) 208, 531-543. Meyer, U., Murray, P. J., Urwyler, A., Yee, B. K., Schedlowski, M., Feldon, J., 2008. Adult behavioral and pharmacological dysfunctions following disruption of the fetal brain

28

MacDowell et al

ACCEPTED MANUSCRIPT

balance between pro-inflammatory and IL-10-mediated anti-inflammatory signaling. Mol. Psychiatry 13, 208-221. Muller, N., Myint, A. M., Krause, D., Weidinger, E., Schwarz, M. J., 2013. Antiinflammatory treatment in schizophrenia. Prog. Neuropsychopharmacol. Biol.

RI PT

Psychiatry 42, 146-153.

Muller, N., Wagner, J. K., Krause, D., Weidinger, E., Wildenauer, A., Obermeier, M.,

schizophrenia. Psychiatry Res. 198, 341-346.

SC

Dehning, S., Gruber, R., Schwarz, M. J., 2012. Impaired monocyte activation in

M AN U

Naito, Y., Takagi, T., Higashimura, Y., 2014. Heme oxygenase-1 and anti-inflammatory M2 macrophages. Arch. Biochem. Biophys. 564, 83-88.

Orihuela, R., McPherson, C. A., Harry, G. J., 2016. Microglial M1/M2 polarization and metabolic states. Br. J. Pharmacol. 173, 649-665.

TE D

Ozawa, K., Hashimoto, K., Kishimoto, T., Shimizu, E., Ishikura, H., Iyo, M., 2006. Immune activation during pregnancy in mice leads to dopaminergic hyperfunction and cognitive impairment in the offspring: a neurodevelopmental animal model of

EP

schizophrenia. Biol. Psychiatry 59, 546-554.

AC C

Piontkewitz, Y., Arad, M., Weiner, I., 2011. Risperidone administered during asymptomatic period of adolescence prevents the emergence of brain structural pathology and behavioral abnormalities in an animal model of schizophrenia. Schizophr. Bull. 37, 1257-1269. Piontkewitz, Y., Bernstein, H. G., Dobrowolny, H., Bogerts, B., Weiner, I., Keilhoff, G., 2012. Effects of risperidone treatment in adolescence on hippocampal neurogenesis, parvalbumin expression, and vascularization following prenatal immune activation in rats. Brain Behav. Immun. 26, 353-363. 29

MacDowell et al

ACCEPTED MANUSCRIPT

Reus, G. Z., Fries, G. R., Stertz, L., Badawy, M., Passos, I. C., Barichello, T., Kapczinski, F., Quevedo, J., 2015. The role of inflammation and microglial activation in the pathophysiology of psychiatric disorders. Neuroscience 300, 141-154. Ribeiro, B. M., do Carmo, M. R., Freire, R. S., Rocha, N. F., Borella, V. C., de Menezes, A.

RI PT

T., Monte, A. S., Gomes, P. X., de Sousa, F. C., Vale, M. L., de Lucena, D. F., Gama, C. S., Macedo, D., 2013. Evidences for a progressive microglial activation and increase in iNOS expression in rats submitted to a neurodevelopmental model of

SC

schizophrenia: reversal by clozapine. Schizophr. Res 151, 12-19.

M AN U

Ribeiro-Santos, R., Teixeira, A. L., Salgado, J. V., 2014. Evidence for an immune role on cognition in schizophrenia: A systematic review. Curr. Neuropharmacol. 12, 273280.

Richtand, N. M., Ahlbrand, R., Horn, P., Stanford, K., Bronson, S. L., McNamara, R. K.,

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2011. Effects of risperidone and paliperidone pre-treatment on locomotor response following prenatal immune activation. J. Psychiatr. Res. 45, 1194-1201. Roenker, N. L., Gudelsky, G., Ahlbrand, R., Bronson, S. L., Kern, J. R., Waterman, H.,

EP

Richtand, N. M., 2011. Effect of paliperidone and risperidone on extracellular

AC C

glutamate in the prefrontal cortex of rats exposed to prenatal immune activation or MK-801. Neurosci. Lett. 500, 167-171. Song, X., Li, W., Yang, Y., Zhao, J., Jiang, C., Li, W., Lv, L., 2011. The nuclear factorkappaB inhibitor pyrrolidine dithiocarbamate reduces polyinosinic-polycytidilic acidinduced immune response in pregnant rats and the behavioral defects of their adult offspring. Behav. Brain Funct. 7, 50.

30

MacDowell et al

ACCEPTED MANUSCRIPT

Stuart, M.J., Baune, B.T., 2014. Chemokines and chemokine receptors in mood disorders, schizophrenia, and cognitive impairment: a systematic review of biomarker studies. Neurosci. Biobehav. Rev. 42, 93-115. Sugino, H., Futamura, T., Mitsumoto, Y., Maeda, K., Marunaka, Y., 2009. Atypical

RI PT

antipsychotics suppress production of proinflammatory cytokines and up-regulate interleukin-10 in lipopolysaccharide-treated mice. Prog. Neuropsychopharmacol. Biol. Psychiatry 33, 303-307.

SC

Venkatasubramanian, G., Debnath, M., 2013. The TRIPS (Toll-like receptors in immuno-

M AN U

inflammatory pathogenesis) hypothesis: a novel postulate to understand schizophrenia. Prog. Neuropsychopharmacol. Biol. Psychiatry 44, 301-311. Volk, D. W., Chitrapu, A., Edelson, J. R., Roman, K. M., Moroco, A. E., Lewis, D. A., 2015. Molecular Mechanisms and Timing of Cortical Immune Activation in Schizophrenia.

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Am. J. Psychiatry. 172:1112-1121.

Young, J. W., Powell, S. B., Geyer, M. A., 2012. Mouse pharmacological models of cognitive disruption relevant to schizophrenia. Neuropharmacology 62, 1381-1390.

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Zhang, M., An, C., Gao, Y., Leak, R. K., Chen, J., Zhang, F., 2013. Emerging roles of Nrf2

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and phase II antioxidant enzymes in neuroprotection. Prog Neurobiol 100, 30-47. Zhou, X., Spittau, B., Krieglstein, K., 2012. TGFbeta signalling plays an important role in IL4-induced alternative activation of microglia. J. Neuroinflammation 9, 210. Zuckerman, L., Rehavi, M., Nachman, R., Weiner, I., 2003. Immune activation during pregnancy in rats leads to a postpubertal emergence of disrupted latent inhibition, dopaminergic hyperfunction, and altered limbic morphology in the offspring: a novel neurodevelopmental model of schizophrenia. Neuropsychopharmacology 28, 1778-1789. 31

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ACCEPTED MANUSCRIPT Figure Legends

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Figure 1. Paliperidone effects on brain TLR3 inflammatory pathway and neuroinflammation after MIA exposure. Protein expression levels of TLR3, TRIF, pIRF3, NFκB, IκBα, iNOS, COX2 (A-G) and activity of the lipid peroxidation marker MDA (H) in the frontal cortex of mice treated with vehicle (C, Veh) or paliperidone (P) in control and prenatal MIA (poly(I:C)) conditions. The densitometric data of the respective band of interest were normalized by β-actin or HDAC1 (nuclear extracts). In the D panel blots were cropped (black lines) for improving the clarity and conciseness of the presentation. Bars represent means+SEM. **p<0.01, ***p<0.001 vs Control (C) group; ##p<0.01, ###p<0.001 vs poly(I:C)+Veh group. Two-way ANOVA followed by Bonferroni post-hoc test. For TRIF and pIRF3 comparison, unpaired t-test was used.

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Figure 2. Paliperidone effects on brain NRF2 antioxidant regulatory pathway after MIA exposure. Protein expression levels of NRF2 (A), NRF2 activity (B), and mRNA levels of the antioxidant enzymes HO1, SOD and catalase (C-E) in the frontal cortex of mice treated with vehicle (C, Veh) or paliperidone (P) in control and prenatal MIA (poly(I:C)) conditions. The densitometric data of the respective band of interest were normalized by HDAC1. Bars represent means+SEM. ***p<0.001 vs Control (C) group; &&p<0.01, &&& p<0.001 vs Control+P group; #p<0.05, ##p<0.01, ###p<0.001 vs Poly(I:C)+Veh group. Two-way ANOVA followed by Bonferroni post-hoc test. For NRF2 protein expression comparison, unpaired t-test was used.

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Figure 3. Paliperidone effects on pro/anti-inflammatory cytokine levels and STAT1 transcription factor after MIA exposure. mRNA levels of the pro-inflammatory cytokines TNFα, IL1β, IL6, IFNα, and IFNβ (A-E), the transcription factor STAT1 (F), the anti-inflammatory cytokines TGFβ and IL10 (G, H) and the chemokine CX3CL1 -fractalkine- (I) in the frontal cortex of mice treated with vehicle (C, Veh) or paliperidone (P) in control and prenatal MIA (poly(I:C)) conditions. Bars represent means+SEM. *p<0.05, **p<0.01, ***p<0.001 vs Control (C) group; & p<0.05, &&p<0.01 vs Control+P group; #p<0.05, ##p<0.01, ###p<0.001 vs Poly(I:C)+Veh group. Two-way ANOVA followed by Bonferroni post-hoc test. For TNFα mRNA levels comparison, unpaired t-test was used.

Figure 4. Paliperidone effects on microglia M2 polarization after MIA exposure. mRNA and protein expression levels of microglial M2 phenotype cellular markers ArgI and FOLR2 (A-D), and protein levels of L-PGDS and PPARγ (E, F) in the frontal cortex of mice treated with vehicle (C, Veh) or paliperidone (P) in control and prenatal MIA (poly(I:C)) conditions. The densitometric data of the respective band of interest were normalized by β-actin or HDAC1 (nuclear extracts). In the E panel blots were cropped (black lines) for improving the clarity and conciseness of the presentation. Bars 32

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represent means+SEM. **p<0.01, ***p<0.001 vs Control (C) group; &p<0.05, &&& p<0.001 vs Control+P group; ##p<0.01, ###p<0.001 vs Poly(I:C)+Veh group. Two-way ANOVA followed by Bonferroni post-hoc test.

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Figure 5. Paliperidone effects on a spatial working memory task after MIA exposure. Percentage of correct choices in alternation task T-maze test in mice treated with vehicle (C, Veh) or paliperidone (P) in control and prenatal MIA (poly(I:C)) conditions. Animals were tested at 10 (A) and 40 seconds (B) delay intervals. Bars represent means+SEM. ***p<0.001 vs control (C) group; #p<0.05, ##p<0.01, vs poly(I:C)+Veh group. Three-way ANOVA (MIA x treatment) with repeated measures (delay) followed by post-hoc test.

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Figure 6. Schematic representation of the major TLR3 signaling pathway, inflammatory response and counterbalancing mechanisms. TLR3 is expressed in endosomal compartments and recognizes viral dsRNA and the synthetic analogue poly(I:C). Activation of TLR3 pathway induces phosphorylation of IRF3, which translocates to the nucleus and generates an antiviral response by secreting IFN type 1. On the other hand, it also induces degradation of the inhibitory subunit IκBα by IKK, facilitating NFκB translocation to nucleus where it activates transcription of inflammatory cytokines and enzymes that leads to reactive oxygen and nitrogen species accumulation. Counterbalancing mechanism includes PPARγ and NRF2 activation. Oxidative stress signals induce a conformational change in the inhibitory NRF2 protein, KEAP1, enabling NRF2 translocation to the nucleus and bind to specific DNA sequences called antioxidant response elements (ARE) promoting the gene transcription of antioxidant and anti-inflammatory enzymes. PPARγ decreases the expression and activity of NFκB. Abbreviations: TRIF (TIR-domain-containing adapter-inducing interferon-β), TRAF (TNF receptor-associated factor), IRF3 (interferon regulatory factor 3), IFNs (interferons), STAT (signal-transducing activators of transcription), NFκB (nuclear factor-kappaB), IKKs (inhibitors of NF-kB kinase), IκBα (inhibitor of the NFkB alpha), PPARγ (peroxisome proliferator activated receptor gamma), KEAP1 (Kelch-like ECH-associated protein 1), NRF2 (nuclear factor E2-related factor 2).

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Table 1. ANOVA analyses (F values and dfs).

Interaction F(1,31)=8.90; p=0.0055 F(1,29)=6.72; p=0.0148 F(1,29)=0.72; p=0.4044 F(1,29)=1.61; p=0.2140 F(1,29)=7.43; p=0.0107 F(1,29)=8.37; p=0.0072 F(1,29)=4.32; p=0.0465 F(1,29)=0.02; p=0.8920 F(1,29)=7.24; p=0.0117 F(1,29)=4.16; p=0.0507 F(1,29)=5.86; p=0.0220 F(1,29)=5.58; p=0.0250 F(1,29)=19.78; p=0.0001 F(1,29)=5.41; p=0.0272 F(1,28)=2.91; p=0.0994 F(1,29)=11.33; p=0.0022 F(1,29)=13.18; p=0.0011 F(1,29)=5.23; p=0.0296 F(1,28)=12.88; p=0.0012 F(1,29)=5.56; p=0.0254 F(1,29)=10.41; p=0.0031 F(1,29)=7.11; p=0.0124 F(1,29)=4.71; p=0. 0384 F(1,29)=0.28; p=0.5985 F(1,29)=24.69; p<0.0001 F(1,29)=5.90; p=0.0216 F(1,29)=11.23; p=0.0022 F(1,29)=5.95; p=0.0210 F(1,29)=4.70; p=0.0384

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Treatment F(1,31)=2.46; p=0.1270 F(1,29)=6.30; p=0.0179 F(1,29)=3.75; p=0.0627 F(1,29)=1.23; p=0.2758 F(1,29)=9.03; p=0.0054 F(1,29)=6.57; p= 0.0159 F(1,29)=9.78; p=0.0040 F(1,29)=0.68; p=0.4149 F(1,29)=8.78; p=0.0060 F(1,29)=6.34; p=0.0176 F(1,29)=6.30; p=0.0179 F(1,29)=11.16; p=0.0023 F(1,29)=17.18; p=0.0003 F(1,29)=6.22; p=0.0186 F(1,28)=4.37; p=0.0459 F(1,29)=13.96; p=0.0008 F(1,29)=10.26; p=0.0033 F(1,29)=5.72; p=0.0235 F(1,28)=18.07; p=0.0002 F(1,29)=4.41; p=0.0445 F(1,29)=7.79; p=0.0092 F(1,29)=3.84; p=0.0596 F(1,29)=6.36; p=0.0174 F(1,29)=0.05; p=0.8227 F(1,29)=26.38; p<0.0001 F(1,29)=6.49; p=0.0164 F(1,29)=10.90; p=0.0026 F(1,29)=6.24; p=0.0184 F(1,29)=5.05; p=0.0324

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Poly(I:C) F(1,31)=50.24; p<0.0001 F(1,29)=9.81; p=0.0039 F(1,29)=13.57; p=0.0009 F(1,29)=11.21; p=0.0023 F(1,29)=20.44; p<0. 0001 F(1,29)=16.47; p=0.0003 F(1,29)=13.04; p=0.0011 F(1,29)=0.01; p=0.9276 F(1,29)=6.28; p=0.0181 F(1,29)=0.18; p=0.6737 F(1,29)=5.79; p=0.0227 F(1,29)=16.30; p=0.0004 F(1,29)=6.07; p=0.0199 F(1,29)=0.86; p=0.3613 F(1,28)=8.86; p=0.0060 F(1,29)=2.14; p=0.1541 F(1,29)=9.49; p=0.0045 F(1,29)=11.56; p=0.0020 F(1,28)=0.09; p=0.7695 F(1,29)=2.22; p=0.1474 F(1,29)=4.53; p=0.0420 F(1,29)=4.68; p=0.0388 F(1,29)=2.40; p=0.1326 F(1,29)=0.71; p=0.4060 F(1,29)=5.25; p=0.0294 F(1,29)=14.45; p=0.0007 F(1,29)=0.99; p=0.3275 F(1,29)=1.37; p=0.2506 F(1,29)=11.15; p=0.0023

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Parameter T-Maze WB TLR3 WB TRIF WB pIRF3 WB NFκB WB IκBα WB iNOS WB COX-2 MDA WB NRF2 Act NRF2 mRNA HO1 mRNA SOD mRNA CAT mRNA TNFα mRNA IL1β mRNA IL6 mRNA IFNα mRNA IFNβ mRNA STAT1 mRNA TGFβ mRNA IL10 mRNA CX3CL1 mRNA ArgI mRNA FOLR2 WB ArgI WB FOLR2 WB L-PGDS WB PPARγ

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ACCEPTED MANUSCRIPT Highlights Injection of a viral mimetic in pregnancy was used as animal model of schizophrenia Offspring mice showed activation of innate immune TLR-3 signaling pathway in brain

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The antipsychotic paliperidone elicits anti-inflammatory/antioxidant effects in brain