Prenatal maternal immune activation increases anxiety- and depressive-like behaviors in offspring with experimental autoimmune encephalomyelitis

Accepted Manuscript Prenatal maternal immune activation increases anxiety- and depressive-like behaviors in offspring with experimental autoimmune encephalomyelitis Jafar Majidi-Zolbanin, Mohammad-Hossein Doosti, Morteza Kosari-Nasab, Ali-Akbar Salari PII: DOI: Reference:

S0306-4522(15)00238-9 http://dx.doi.org/10.1016/j.neuroscience.2015.03.016 NSC 16123

To appear in:

Neuroscience

Accepted Date:

7 March 2015

Please cite this article as: J. Majidi-Zolbanin, M-H. Doosti, M. Kosari-Nasab, A-A. Salari, Prenatal maternal immune activation increases anxiety- and depressive-like behaviors in offspring with experimental autoimmune encephalomyelitis, Neuroscience (2015), doi: http://dx.doi.org/10.1016/j.neuroscience.2015.03.016

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.

Prenatal maternal immune activation increases anxiety- and depressive-like behaviors in offspring with experimental autoimmune encephalomyelitis

Jafar Majidi-Zolbanin a, Mohammad-Hossein Doosti a, Morteza Kosari-Nasab b, Ali-Akbar Salari b,c,*

a Immunology

b

c

Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Laboratory of Neuropsychopharmacology and Psychoneuroimmunology, Hayyan Research Institute,

University of Tabriz, Tabriz, Iran

*Corresponding author at: Laboratory of Neuropsychopharmacology and Psychoneuroimmunology, Hayyan Research Institute, University of Tabriz, P.O. Box. 51666-16471, Tabriz, Iran; Tel.: +98-9194099673; Fax: +98-411-3368208; E-mail: [email protected]

Running title: Maternal immune activation and Multiple Sclerosis Abstract: 242 words Body: 5663 words Figures: 6

1

Abstract

Multiple sclerosis (MS) is thought to result from a combination of genetics and environmental factors. Several lines of evidence indicate that significant prevalence of anxiety and depressionrelated disorders in MS patients can influence the progression of the disease. Although we and others have already reported the consequences of prenatal maternal immune activation on anxiety and depression, less is known about the interplay between maternal inflammation, MS and gender. We here investigated the effects of maternal immune activation with Poly I:C during mid-gestation on the progression of clinical symptoms of experimental autoimmune encephalomyelitis (EAE; a mouse model of MS), and then anxiety- and depressive-like behaviors in non-EAE and EAE-induced offspring were evaluated. Stress-induced corticosterone and tumor necrosis factor-alpha (TNF-α) levels in EAE-induced offspring were also measured. Maternal immune activation increased anxiety and depression in male offspring, but not in females. This immune challenge also resulted in an earlier onset of the EAE clinical signs in male offspring and enhanced the severity of the disease in both male and female offspring. Interestingly, the severity of the disease was associated with increased anxiety/depressive-like behaviors and elevated corticosterone or TNF-α levels in both sexes. Overall, these data suggest that maternal immune activation with Poly I:C during midpregnancy increases anxiety- and depressive-like behaviors, and the clinical symptoms of EAE in a sex-dependent manner in non-EAE or EAE-induced offspring. Finally, the progression of EAE in offspring seems to be linked to maternal immune activation-induced dysregulation in neuroimmune-endocrine system.

Key words: Maternal inflammation, Sex differences, Anxiety, Depression, Poly I:C, EAE.

2

1. Introduction

Multiple sclerosis (MS) is known as one of the most common neuroinflammatory diseases in humans that is more prevalent in women (Tomassini and Pozzilli, 2009). In recent years, there has been an increasing interest in investigating the prevalence of affective disorders in MS patients. Although, previous studies have indicated a significant prevalence of anxiety and depression among MS patients, as compared with the general population (Beiske et al., 2008, Brown et al., 2009, Dahl et al., 2009, Arnason, 2010, Feinstein, 2011, Giordano et al., 2011, Wood et al., 2012). However, it has been reported that anxiety is more common than depression in patients with MS (Noy et al., 1995, Feinstein et al., 1999, Wood et al., 2012), and also depression in MS is evident even early on, and absent disability (Arnason, 2010). A number of studies have also suggested that there is a significant association between affective disorders such as depression and anxiety and suicidal intent or completed suicide in patients with MS in comparison with the general population (Stenager and Stenager, 1992, Korostil and Feinstein, 2007, Wood et al., 2012). Although, hypothalamic-pituitary-adrenal (HPA) axis dysregulation is associated with anxiety and depression in non-MS population (Young et al., 2004, Stetler and Miller, 2011) and MS patients (Wallin et al., 2006, Wood et al., 2012), clinical studies have provided evidence that HPA axis hyperactivity in patients with MS may contribute to the progression of disease (Bergh et al., 1999, Heesen et al., 2002, Schumann et al., 2002, Gold et al., 2005) and subtle HPA axis alterations during the earlier phases of MS may be a marker for depressive symptomatology (Gold et al., 2010). It is also important to note that there is a considerable relationship between proinflammatory cytokines and anxiety/depression (Raison et al., 2006, Peruga et al., 2011, Haji et al., 2012, Kaster et al., 2012). Therefore, the importance of evaluating such neuropsychiatric diseases as two of the most

3

important determining factors of the general health and the quality of life in MS patients is unquestionable. Over the past few decades, “fetal programming” has been one of the major interesting research subjects for neuroscientists to investigate the fetal origins of many neurological disorders including schizophrenia, autism, cerebral palsy, epilepsy, Alzheimer, Parkinson, anxiety, depression, and multiple sclerosis (Bale et al., 2010, Del Giudice, 2012, Harvey and Boksa, 2012, Solati et al., 2012, Faa et al., 2014, Zager et al., 2014). In addition, there is now compelling evidence that maternal adverse experiences including stress and infection during pregnancy affect normal development of the brain and behavior in offspring in adulthood. For instance, we and others have demonstrated that prenatal maternal infection/immune activation with Lipopolysaccharide (LPS, a component of gram negative bacteria) or Polyinosinic: Polycytidylic acid (Poly I:C, a synthetic double stranded RNA) enhances the risk for a number of neuropsychiatric-like behaviors such as anxiety, depression, schizophrenia and autism in adult mice offspring (for review see (Harvey and Boksa, 2012, Babri et al., 2014a)). Interestingly, in light of this evidence, some studies indicated that prenatal maternal immune activation results in earlier onset of the clinical symptoms and more severe neurological deficits in experimental autoimmune encephalomyelitis (EAE), an animal model which mimics many aspects of MS (Solati et al., 2012, Mandal et al., 2013, Zager et al., 2014). Considering that, on the one hand, one of the most significant current discussions in this research area is that whether or not interaction between two environmental risk factors during prenatal and postnatal periods is associated with greater vulnerability to neuropsychiatric disorders in offspring in later life. And on the other hand, although there have been strong connections between depression/anxiety and multiple sclerosis in clinical studies, to date there are no studies that directly investigated possible links between the effects of prenatal maternal immune activation and

4

the prevalence of anxiety- and depressive-like behaviors in EAE-induced mice offspring. Therefore, the aim of this study was to examine and validate the presence of these behavioral disorders following prenatal maternal immune activation in mice offspring with EAE, which may provide insights into the fetal potential origins involved in the disease in order to increase our understanding of the effects of environmental risk factors on anxiety and depression in people with multiple sclerosis. 2. Materials and methods 2.1. Subjects, General housing conditions and Ethics Male and female C57BL/6 mice (70–80 days) were obtained from the animal house of Pasteur Institute (Tehran, Iran). Animals were housed in standard polycarbonate cages in a temperaturecontrolled room (23 ± 1°C) with a fixed 12 h light–dark cycle (08:00–20:00) and free access to food and water. These conditions were kept as a standard housing condition in all stages of experiments. All procedures of the study were performed in accordance with the ethical guidelines set by Research and Ethics Committee of Tabriz University of Medical Sciences (GN-90.2-2.5) which completely coincides with the ‘‘National Institutes of Health NIH Guide for the Care and Use of Laboratory Animals (NIH; Publication No. 85-23, revised 1985). 2.2. Breeding The breeding procedure and the verification of gestational day (GD) 0 have previously been described elsewhere (Enayati et al., 2012). Briefly, the breeding began after a 2-week period of acclimatization to the new animal holding room. In order to facilitate of mating, male and female mice were kept together one-by-one in a cage. Successful mating was confirmed next morning (8:00 A.M.) with the presence of vaginal plug, and that day was referred as GD 0. Once a pregnant

5

female was identified, it was removed from the breeding cage and housed individually in a standard cage. 2.3. Prenatal maternal immune activation Mice were given intraperitoneal injections of 20 mg/kg/100µl Poly I:C (Sigma Co, USA; dissolved in sterile PBS) on GD 12, because previous studies found this dose to be most effective with intraperitoneal delivery for inducing a robust immune response in C57BL/6 pregnant mice and behavioral abnormalities in their offspring in adulthood (Shi et al., 2003, Smith et al., 2007, Hsiao and Patterson, 2011, Garay et al., 2013, Khan et al., 2014). Pregnant mothers were returned to their housing immediately following PBS or Poly I:C treatment. In order to perform the behavioral tests, in each sex, mice from both prenatal treatment conditions were divided into 3 clusters (each cluster only used for two behavioral tests or the EAE clinical signs , with a 2-days interval between each test; N=10 or 15/group male or female, see Fig.1). All pregnant animals were allowed to have normal delivery and the first day of birth was considered as PND 0 (MajidiZolbanin et al., 2014). One day after the birth, all litters were culled to 4 pups per mother (2 males and 2 females). On PND 23, litters were weaned by removal of the mother and then were housed with the same sex litter-mates (2 animals per cage). Only one offspring from each litter was randomly assigned for each of the experiments to avoid litter-effects. 2.4. Induction of experimental autoimmune encephalitis For EAE-induction in C57BL/6 offspring on PND 80, animals were anaesthetized by an i.p. injection of ketamine hydrochloride (50 mg/kg; Alfasan, Woerden-Holland) plus Xylazine (5 mg/kg; Alfasan, Woerden-Holland). 300 µg of Myelin oligodendrocyte glycoprotein peptide (MOG35–55; Amino acid sequence-Purity ≥95% (HPLC): Met-Glu-Val-Gly-Trp-Tyr-Arg-Ser-Pro-

6

Phe-Ser-Arg-Val-Val-His-Leu-Tyr-Arg-Asn-Gly-Lys; KJ Ross-Petersen ApS, Copenhagen, Denmark) was emulsified with an equal volume of complete Freund's adjuvant (CFA; Sigma, F5881, USA) containing 500 µg of heat-killed Mycobacterium tuberculosis (100 µl total volume), then this emulsion was injected subcutaneously in both hind flanks of each mouse. Mice also received intraperitoneal injections of pertussis toxin (300 ng in 100 µl PBS; List Biological Lab, Campbell, CA, USA) at the time of immunization and again 48 h later. Each injection was performed with a 1ml insulin syringe needle. The EAE clinical symptoms in the offspring were evaluated daily on a scale from 0 to 7 according to the following criteria: 0 = no symptoms; 1 = distal limp tail; 2 =complete limp tail; 3=one hind limb paralyzed; 4 = both hind limbs paralyzed; 5=Hind limbs and one forelimb paralyzed; 6 =Hind limbs and both forelimbs paralyzed; 7 =moribund/death. 2.5. Behavioral testing We assessed anxiety- and depression-related behaviors at 7 dpi and 9 dpi by means of the elevated plus maze (EPM), light-dark box (LDB), tail suspension test (TST) and forced swim test (FST) based on the onset of clinical signs in EAE-induced offspring (Fig. 2) and previous studies have also measured anxiety-like behavior in EAE-induced mice during 7-9 days post immunization (dpi) before the onset of motor deficits (Haji et al., 2012). Behavioral assessments began at PND 87. All behavioral parameters were recorded by observers blind to the treatment and the observers have recorded all parameters for each of the behavioral tests by using a stopwatch. In order to avoid any confounding effect of motor deficits on the offspring’s behavior in the tests, the locomotor activity was assessed in an open field for 3 min before each behavioral testing session as previously described (de Paiva et al., 2010). In addition, all behavioral tests were conducted in a quiet room during the light period (between 14:00 and 18:00 h) under illumination of 75 lux and the mice were kept in the room for at least 1 h before the assessment. At the end of each test

7

session, the arena was carefully cleaned with 70 % ethanol and after test the cage was transported back to the colony room. In all experiments, each male or female offspring was tested only once in the one test. 2.5.1. Elevated plus-maze The EPM test, which is one of the well-known methods for testing of anxiety, was performed as previously described (Amani et al., 2013). The EPM was a plus-shaped apparatus, constructed from grey wood, elevated to a height of 50 cm above the floor. This apparatus was consisted of a central platform (5×5cm), two open arms (30×5cm), and two equal closed (30×5×15cm) arms opposite to each other with an open roof. The offspring were placed individually at the center of the EPM, facing one of the open arms and allowed 5 min of free exploration. A relatively dark box was used to hold the mice in before placing on the maze in order to increase their exploratory behavior. The observers measured: (a) time spent in the open arms, (b) time spent in the closed arms, (c) number of entries into the open arms, and (d) number of entries into the closed arms during the 5-min test period. An entry was defined as all four paws on the arm. For the purpose of analysis, percent of open-arm time [OAT %: time in open arm/ time in open + closed arm × 100], and percent of open arm entries [OAE %: number of open arm entries/number of open arm + closed arm entries × 100] were defined and employed as indexes of anxiety-like behaviors in rodents. 2.5.2. Light-dark box The light–dark box apparatus consisted of a white-black wooden rectangular box (length 46 cm, width 27 cm, and height 30 cm), which divided into two compartments (light and large: 27cm×27cm, dark and small: 18cm×27cm) by a partition. These areas connected by a small central

8

open door (7.5cm×7.5 cm) located in the center of the partition at floor level. The large compartment was open at the top, illuminated by a 100 W bulb located 90cm above the apparatus and the small compartment had a removable black lid at the top. To start the test, each offspring was placed at the center of the light compartment, facing away from the door and the animal was allowed to explore freely both compartments for 5 min and their behavior was recorded during this time. The following parameters were recorded: light compartment time (LCT), light compartment entries (LCE) and latency to enter the light compartment (LL) after the first entry into the dark division. A decrease in the amount of time spent and numbers of entries into the light compartment were evaluated as indicative of anxiety-like behaviors (Majidi-Zolbanin et al., 2013). 2.5.3. Tail suspension test The TST was performed as previously described (Doosti et al., 2013). At the beginning of the experiment, each mouse was individually suspended by the tail using a clamp, 2 cm from the end, in a grey wooden enclosure (40 cm high, 30 cm wide and 20 cm deep) such that the head of mouse was about 25 cm above the floor. The total duration of immobility was recorded (in seconds) during the 5 min test period. Any animals that did climb their tails were removed from the experimental group, and were not used in the analysis. Immobility was defined as the lack of motion of the whole body, whereas mobility was defined as any movement of the body. 2.5.4. Forced swim test The FST remains one of the most widely used tools for measuring behavioral despair in rodents. To describe this behavioral model in mice, the following procedure was adopted, mice were individually placed into the transparent glass cylinders (Height: 25cm, Diameter: 10cm), filled with water to a height of 15 cm and maintained at 25±1ᵒC. The water was replaced by fresh water

9

between each test. The total duration of immobility was recorded during the last 4 min of the 6 min testing period. At the end of swimming session, the animals were removed from the cylinder, dried with towels, and placed gently near an electric heater for 15–30 min. Each mouse was judged to be immobile when it ceased struggling and remained floating motionless in the water and making only those movements necessary to keep its head above water. An increase in the duration of immobility time is known as an indicative of depressive-like behavior in mice (Babri et al., 2014b). 2.6. Corticosterone and tumor necrosis factor-alpha Stress-induced corticosterone levels or TNF-α concentration in EAE-induced offspring were measured on PND 89, 25 min after the LDB (TNF-α) or FST (corticosterone), respectively. The blood was collected using cardiac puncture method as previously described (Enayati et al., 2012) and circulating levels of TNF-α (BioLegend Co., USA) and corticosterone (Bio-Medical Assay Company, China) in the offspring serum were measured by using cytokine specific quantitative sandwich ELISA kit according to the manufacturer’s instructions. All samples and standards were assayed in duplicate. 2.7. Statistics All data were analyzed based on the analysis of variance (ANOVA) using the statistical package of SPSS (IBM, Version 21). The clinical scores were analyzed by three-way repeated measures ANOVA with treatment and sex as between subject factors and dpi as a within-subject (repeated measure) factor (Fig. 2). The anxiety and depression data in Fig. 3 to 5 were analyzed by three-way ANOVA with sex, EAE induction and treatment as main factors. The corticosterone and TNF-α data in Fig. 6 were analyzed using the two-way ANOVA with sex and treatment as main factors. All data

10

are presented as the mean ± standard error of the mean (S.E.M). A P-value less than 0.05 was considered statistically significant. 3. Results 3.1. Effects of prenatal maternal immune activation on EAE clinical symptoms in offspring Mean clinical scores are shown from day 8 to day 26 post-immunization for female and male offspring in Fig. 2. Three-way ANOVA analysis revealed the main effects of DPI [F

10.475, 586.594

=

379.879, P<0.001], sex [F 1, 56 = 61.79, P<0.001] and treatment [F 1, 56 = 73.65, P<0.001]. Significant interactions existed between DPI × sex [F 10.475, 586.594 = 8.64, P<0.001] and DPI × treatment [F 10.475, 586.594

= 2.63, P=0.003], while there were no significant differences between the interaction of sex ×

treatment, and DPI × sex × treatment. Furthermore, Fig. 2, left panel, illustrates that prenatal immune activation increased the mean clinical scores on days 19 to 24 post-immunization [P<0.05 and P<0.01] in Poly I:C female offspring, as compared to PBS female offspring. The data analysis also revealed that prenatal immune activation significantly increased the mean clinical signs on days 10 to 13 [P<0.05 and P<0.001] and 19 to 21 [P<0.05] post-immunization in Poly I:C male offspring relative to PBS male offspring (Fig. 2, right panel). These results suggest that prenatal maternal immune activation alters the day of onset and severity of clinical signs in a sex-dependent manner in offspring. 3.2. Effects of prenatal maternal immune activation on anxiety-related behaviors in adult offspring with EAE 3.2.1. Elevated plus-maze We assessed the effects of prenatal maternal immune activation on anxiety-related behaviors in adult offspring with EAE on PND 87 in the EPM. The three-way analysis revealed the overall

11

main effects of EAE [F1, 72=46.33, P<0.001] and treatment [F1, 72=62.05, P<0.001] in OAT %, and sex [F1, 72=14.46, P<0.001], EAE [F1, 72=52.72, P<0.001] and treatment [F1, 72=67.96, P<0.001] in OAE %. Significant interactions existed between EAE × treatment in OAT % [F1, 72=4.83, P=0.031] and OAE % [F1,

72=4.53,

P=0.037], and also sex × treatment [F1,

72=7.95,

P=0.006] in OAE %. In the EPM,

prenatal Poly I:C exposure resulted in decreases in OAT % [P=0.037] and OAE % [P=0.001], high levels of anxiety, in male offspring (Fig. 3, right panel) but not in females (Fig. 3, left panel), in comparison with the PBS-treated group. In addition, there were significant decreases in OAT % [P<0.001] and OAE % [P<0.001] in both Poly I:C-treated male and female offspring with EAE, as compared with the PBS-treated animals with EAE. These findings indicate that prenatal maternal immune activation can be a risk factor for increasing anxiety-related behaviors in offspring with EAE. 3.2.2. Light-dark box The LDB was performed two days after EPM and its data are presented in Fig. 4. The three-way analysis showed the overall main effects of sex (LCT: [F1, P=0.015]; LL: [F1,

72=10.13,

P=0.002]), EAE (LCT: [F1,

72=4.49,

72=89.44,

P=0.037]; LCE: [F1,

P<0.001]; LCE:

[F1,

72=6.17,

72=55.61,

P<0.001]; LL: [F1, 72=24.93, P<0.001]) and treatment (LCT: [F1, 72=62, P<0.001]; LCE: [F1, 72=51.16, P<0.001]; LL: [F1, 72=48, P<0.001]) in LCT, LCE and LL. There were significant interactions between sex × treatment (LCT: [F1, 72=4.39, P=0.039]; LL: [F1, 72=10.53, P=0.002]), and EAE × treatment (LCT: [F1, 72=19.09, P<0.001]; LL: [F1, 72=10.13, P=0.002]) in LCT and LL, and also EAE × treatment [F1, 72=5.21,

P=0.025] in LCE. In the LDB, prenatal immune activation decreased LCT [P=0.006] and LCE

[P=0.023], and increased LL [P=0.006], high levels of anxiety, only in male offspring relative to the PBS-treated animals. These data show that prenatal exposure to Poly I:C did not affect anxietyrelated behaviors in female offspring in comparison with the PBS-treated mice. Moreover, there

12

were significant declines in LCT [P<0.001] and LCE [P<0.001], and significant increases in LL [P=0.007 and P<0.001] in both Poly I:C-treated male and female offspring with EAE, as compared with the PBS-treated animals with EAE. These findings confirm the EPM data showing that prenatal immune activation can increase anxiety-related behaviors in offspring with EAE 3.3. Effects of prenatal maternal immune activation on depression-related behaviors in adult offspring with EAE 3.3.1. Tail suspension test In this part of the study, we evaluated the effects of prenatal immune activation on depressionrelated behaviors in adult offspring with EAE on PND 87 in the TST. The three-way analysis showed the overall main effects of sex [F1,

72=14.21,

P<0.001], EAE [F1,

72=48.39,

P<0.001] and

treatment [F1, 72=23.52, P<0.001] on the immobility time in the TST. There were also significant interactions between sex × EAE [F1, 72=4.43, P=0.039] and sex × treatment [F1, 72=4.95, P=0.029] in the TST. However, there was no interaction between EAE × treatment [F1, 72=2.87, P=0.094]. As it can be seen in the Fig. 5A, prenatal Poly I:C exposure increased the immobility time [P=0.043], high levels of depression, only in male offspring in comparison with the PBS-treated group. The data analysis also revealed that prenatal Poly I:C treatment did not affect depression-related behaviors in female offspring relative to the PBS-treated mice. Furthermore, there were significant increases in the immobility time in both Poly I:C-treated male [P=0.02] and female [P<0.001] offspring with EAE, as compared with the PBS-treated animals with EAE. These data demonstrate that Poly I:C exposure during fetal brain development may results in increased depression-related behaviors in offspring with EAE. 3.3.2. Forced swim test

13

The FST was performed two days after TST and its data are shown in Fig. 5B. The three-way analysis revealed the overall main effects of sex [F1, 72=23.84, P<0.001], EAE [F1, 72=49.5, P<0.001] and treatment [F1, 72=42.94, P<0.001] on the immobility time in the FST. There were also significant interactions between sex × treatment [F1,

72=4.56,

P=0.036] and EAE × treatment [F1,

72=6.76,

P=0.011] in the FST. Fig. 5B shows that prenatal Poly I:C treatment increased the immobility time [P=0.003], high levels of depression, only in male offspring, as compared to the PBS-treated mice. However, prenatal immune activation did not affect depression-related behaviors in female offspring in comparison with the PBS-treated group. In addition, there were significant increases in the immobility time in both Poly I:C-treated male [P=0.003] and female [P<0.001] offspring with EAE, as compared with the PBS-treated mice with EAE. Interestingly, these findings also confirm the TST results indicating that prenatal immune activation can elevate depression-related behaviors in offspring with EAE. 3.4. Effects of prenatal maternal immune activation on stress-induced corticosterone and tumor necrosis factor-alpha in adult offspring with EAE Concentration of corticosterone (Fig. 6A) and TNF-α (Fig. 6B) were assayed in the serum of EAE-induced offspring following the exposure to the LDB and FST on the PND 89 respectively. The two-way analyses indicated the overall main effects of sex [F1, 36=6.04, P=0.019] and treatment [F1, 36=40.57,

P<0.001] in COR levels, and treatment [F1,

36=50.68,

P<0.001] in TNF-α concentration.

There was also a significant interaction between sex × treatment [F1, 36=5.13, P=0.03] in COR levels. Moreover, there were significant increases in levels of corticosterone and TNF-α in both Poly I:Cexposed female [P<0.01] and male [P<0.001] offspring with EAE, as compared with the PBS-treated mice with EAE. Therefore, our findings show that prenatal immune activation can affect the development of HPA axis and immune system in adult offspring.

14

4. Discussion We have recently reported that maternal immune activation with LPS during late pregnancy increased anxiety- and depression-related behaviors in NMRI male offspring, but not in C57BL/6 male offspring (Enayati et al., 2012, Babri et al., 2014a). Our findings confirm the findings reported by Khan et al., showing a higher level of depression-related behavior in C57BL/6 male offspring from pregnant dams exposed with PolyI: C during mid-gestation (Khan et al., 2014). Nevertheless, we here demonstrate that maternal immune activation during mid-gestation did not alter anxietyand depression-related behaviors in adult female offspring. In other words, these data show that there is a significant effect of gender in mediating the effects of maternal immune activation on affective disorders in the offspring. However, the literature concerning maternal inflammation is extremely limited to studies focusing on male offspring, in particular, in anxiety and depressionrelated behaviors. For instance, our previous findings indicated that female mouse offspring born to dams treated with lipopolysaccharide (LPS) in late gestation (GD 15, 16 or 17), as compared to male offspring, showed an increased level of severity of anxiety- and depression-like behaviors with increasing age in the EPM and FST respectively (Enayati et al., 2012, Salari et al. unpublished data). Additionally, Wang et al. (2010) found that female mouse offspring of dams that received LPS (GD 8–15) indicate anxiety-like behavior at PND 200 in the open field, as compared to prenatal LPS male offspring (Wang et al., 2010). On the other hand, Schwendener et al (2009) demonstrated that prenatal immune activation with Poly I:C on GD17 did not produce overt changes in anxiety-related behaviors in the open field and EPM in offspring (Schwendener et al., 2009). The results of this study also show that maternal exposure to Poly I:C during mid-pregnancy significantly resulted in an earlier onset of the EAE clinical signs in male offspring and an increase in the severity of the disease in both sexes in offspring. In agreement with this finding, a study

15

conducted by Solati et al., reported that prenatal exposure to LPS in mid-gestation led to an earlier onset and an augmentation of EAE clinical signs in male offspring (Solati et al., 2012). In another study, maternal Poly I:C administration during mid-pregnancy produced female offspring that indicated earlier onset of clinical symptoms of EAE (Mandal et al., 2013). More recently, Zager et al, (2014) also published a paper in which they demonstrated that maternal immune stimulation with LPS in late-gestation resulted in elevated the severity of EAE clinical symptoms in female offspring. Although, our results confirm previous findings that prenatal maternal immune activation significantly alters the day of onset and severity of EAE clinical signs in offspring, these findings may also help in understanding the role of sex as an important factor in several immunological, endocrinological and behavioral disorders induced by maternal immune activation in the offspring. MS is more prevalent in females than males, similar gender differences have been indicated in the animal model of MS (EAE) provoked by myelin basic protein (MBP), proteolipid protein (PLP) and MOG in SJL mice, indeed, greater severity of EAE was observed in the female SJL mice relative to males (Cua et al., 1995, Voskuhl et al., 1996, Dalal et al., 1997, Bebo et al., 1998, Papenfuss et al., 2004). However, no significant difference between female and male C57BL/6 mice with MOGinduced EAE was reported (Okuda et al., 2002, Papenfuss et al., 2004). What can be inferred about these findings is that maternal immune activation as an environmental risk factor may sexdependently affect stress-related behaviors and the severity of EAE symptoms in offspring in later life. While a number of animal and human studies have suggested that sex hormones affect the severity of EAE and MS respectively (Voskuhl, 2011, Voskuhl and Gold, 2012, Harbo et al., 2013), the mechanism(s) by which this occurs is not well understood. Moreover, we observed that maternal inflammation increased the levels of anxiety- and depression-related behaviors as well as corticosterone and TNF-α levels in both male and female

16

offspring with EAE. In line with these findings, clinical studies have shown that anxiety and depression are often considered the most common neuropsychological disorders influencing MS patients (Wallin et al., 2006, Wood et al., 2012). Different neurobiological mechanisms such as neuro-endocrine and neuro-immune interactions are thought to have key roles in the development of anxiety and depression in non-MS people and MS patients or EAE (Young et al., 2004, Raison et al., 2006, Peruga et al., 2011, Stetler and Miller, 2011, Haji et al., 2012, Kaster et al., 2012). Considering that there are multifactorial etiologies including environmental and genetic factors contributing to the pathophysiology of MS, anxiety, and depression disorder (Gross and Hen, 2004, Lesch, 2004, Handel et al., 2010), abnormalities in activities of neuro-immune-endocrine pathways may provide a possible explanation of the elevated prevalence of anxiety and depression in MS patients. For instance, several studies provide evidence for HPA axis hyperactivity in up to 50% of MS patients (Heesen et al., 2007, Ysrraelit et al., 2008, Gold et al., 2011) resulting in increased levels of circulating cortisol in the body. Interestingly, in this regard, a meta-analysis review reported that stress is associated with increased risk of relapses in MS patients (Mohr et al., 2004). On the one hand, with regard to the relationship between dysregulation of the HPA axis and disruption of the pattern of cortisol (in humans) or corticosterone (in rodents) secretion and subsequently increased anxiety- and depression-related behaviors in non-MS population or mice and depression in MS population (Abelson and Curtis, 1996, Young et al., 2004, Wallin et al., 2006, Stetler and Miller, 2011, Amani et al., 2013, Doosti et al., 2013, Babri et al., 2014a) and also increased disease progression in MS patients because of HPA axis hyperactivity, on the other side (Bergh et al., 1999, Heesen et al., 2002, Schumann et al., 2002, Gold et al., 2005). It is conceivable that alterations in HPA axis activities (e.g., an increase in corticosterone levels) during the earlier stages of MS may be an indication or marker for depressive symptomatology in later phases (Gold et al., 2010).

17

We here explain potential mechanisms by which corticosterone and TNF-α could affect the development of anxiety and depression-related behaviors in the offspring with EAE. Previous studies have indicated that prenatal maternal immune activation induces hippocampal abnormalities in offspring (Zuckerman et al., 2003, Makinodan et al., 2008, Cui et al., 2009, Ito et al., 2010, Oh-Nishi et al., 2010, Garay et al., 2013). For instance, Khan et al., (2014) demonstrated that maternal exposure to Poly I:C (20 mg/kg on GD 12) during mid-pregnancy produced adult offspring with depressive-like behavior and deficits in cognition and hippocampal long-term potentiation accompanied by disturbed proliferation of newborn cells in the dentate gyrus and compromised neuronal maturation and survival. Furthermore, experimental data in rodents and primates suggested that smaller hippocampal volumes in major depression disorder may be mediated by neuronal apoptosis or by decreasing neurogenesis (Sapolsky, 2000, Henn and Vollmayr, 2004). For example, hippocampus is vulnerable to effects of increased glucocorticoids level which affects cell proliferation, receptor expression and neuronal structure and function (Murray et al., 2008), and chronic stress has also been indicated to decrease neurogenesis in the hippocampus (McEwen, 1999). Considering that, on the one hand, limbic regions like hippocampus are involved in regulating HPA axis activity (Cullinan et al., 2008) in response to stress, anxietyand depression-related behaviors (Murray et al., 2008, Koolschijn et al., 2009, Solati and Salari, 2011), and on the other hand, corticosterone action is mostly mediated through the high affinity mineralocorticoid receptor (MR) and low affinity glucocorticoid receptors (GR) (Kellner and Wiedemann, 2008). Therefore, it seems reasonable to speculate that deficits in the hippocampus following maternal immune activation may have a crucial role in the pathophysiology of anxietyand depression-related behaviors and HPA axis hyperactivity in PolyI:C-EAE offspring by altering the expression levels of MR and GR receptors which in turn results in altered feedback of the HPA axis at the level of the hypothalamus. Taken together, in our view, one inference to be drawn from

18

previous studies and these findings is that anxiety and depression in MS patients or in PolyI:C-EAE offspring may be mediated by MR dependent HPA axis hyperactivity. Here, we also demonstrate that anxiety- and depression-related behaviors with increasing TNFα levels are evident in Poly I:C-EAE offspring before the appearance of clinical symptoms. Considering the close association between central nervous system and inflammation in the MS and EAE, proinflammatory cytokines such as TNF-α (a key player in MS pathology) can be likely candidates to directly alter neuronal activity and contribute to anxiety and depression independently of HPA axis dysregulation (Haji et al., 2012, Kaster et al., 2012) in offspring. In support of this idea, recent clinical and experimental studies on behavioral disorders led to the development of a novel theory, which links TNF-α and depression or anxiety-related disorders (Mikova et al., 2001, Raison et al., 2006, Peruga et al., 2011, Haji et al., 2012, Kaster et al., 2012, Babri et al., 2014b). For instance, high level of TNF-α has been found in peripheral blood and in cerebrospinal fluid of depressed patients (Zorrilla et al., 2001, Raison et al., 2006). Moreover, previous studies indicated a significant relationship between increased indexes of anxiety and TNFα levels in transgenic mice overexpressing TNF-α in the brain (Fiore et al., 1996, Fiore et al., 1998), and also reduced anxiety-like behavior at the EPM in TNF-α knockout mice (Yamada et al., 2000). An interesting study conducted by Kaster et al., demonstrated that intracerebroventricular administration of TNF-α produced a depressive-like behavior in mice that is completely prevented by anti-TNF-α. It is noteworthy that these findings were confirmed by TNFR1 knockout mice exhibiting an antidepressant-like behavior in the FST and TST tests (Kaster et al., 2012). In accordance with these data, recent studies described that blockade of TNF-α signaling with etanercept is associated with decreased levels of behavioral disorders in both humans and rodents (Tyring et al., 2006, Krishnan et al., 2007, Bercik et al., 2010), and intracerebroventricular injection

19

of etanercept decreased anxiety-like behavior in EAE-induced mice (Haji et al., 2012). On the other side, it has been demonstrated that the drugs used for the treatment of autoimmune or inflammatory diseases targeting TNF-α are effective in reducing symptoms of depression (Soczynska et al., 2009, Haroon et al., 2011). Since the levels of proinflammatory cytokines like TNF-α dramatically increase in the brains of MS patients and of EAE mice (Imitola et al., 2005), the changes in the levels of TNF-α expression during early disease stages may have accounted for affective disorders such as anxiety and depression in the later disease phase (Peruga et al., 2011). There is also evidence that alterations in anxiety and depression-like behaviors in EAE-induced mice are associated with histopathological changes in the hippocampus. In other words, increased levels of TNF-α in the hippocampal tissue is related to neuronal loss (Peruga et al., 2011). Activation of TNF-α receptors results in different cellular responses such as inflammation, proliferation, apoptosis and necrosis in the hippocampus (Golan et al., 2004, Iosif et al., 2006, McCoy and Tansey, 2008, Keohane et al., 2010). Given that the blood brain barrier (BBB) is damaged in EAE mouse model, TNF-α can cross the BBB and may result in a neuronal loss in the hippocampus which in turn leads to increased anxiety and depression-related behaviors in PolyI:CEAE offspring. Collectively, these data are well in line with previous clinical and preclinical studies in MS patients and EAE mice which described hippocampal neurodegeneration and chronic inflammation as two important factors that may be associated with anxiety and depression-related disorders (Miller et al., 2009).

5. Conclusion Taken together, our findings add new evidence to the prior research showing prenatal maternal immune activation may sex-dependently predispose the offspring to the development of anxiety

20

and depression-related behaviors and clinical symptoms of experimental autoimmune encephalomyelitis in adulthood, and these results also support the idea that endocrine and immune pathways are critically important as targets for therapeutic strategies.

Acknowledgments This study was supported by a grant from the Immunology Research Center at Tabriz University of Medical Sciences (GN-90.2-2.5). Conflict of interest The authors declare no conflict of interest.

21

References Abelson JL, Curtis GC (1996) Hypothalamic-pituitary-adrenal axis activity in panic disorder: 24-hour secretion of corticotropin and cortisol. Arch Gen Psychiatry 53:323-331. Amani M, Samadi H, Doosti M-H, Azarfarin M, Bakhtiari A, Majidi-Zolbanin N, Mirza-Rahimi M, Salari A-A (2013) Neonatal NMDA receptor blockade alters anxiety-and depression-related behaviors in a sexdependent manner in mice. Neuropharmacology 73:87-97. Arnason BG (2010) Multiple sclerosis and depression: a neuroimmunological perspective. Neuroimmune Biol 9:269-307. Babri S, Doosti M-H, Salari A-A (2014a) Strain-dependent effects of prenatal maternal immune activation on anxiety-and depression-like behaviors in offspring. Brain Behav Immun 37:164-176. Babri S, Doosti M-H, Salari A-A (2014b) Tumor necrosis factor-alpha during neonatal brain development affects anxiety-and depression-related behaviors in adult male and female mice. Behav Brain Res 261:305-314. Bale TL, Baram TZ, Brown AS, Goldstein JM, Insel TR, McCarthy MM, Nemeroff CB, Reyes TM, Simerly RB, Susser ES (2010) Early life programming and neurodevelopmental disorders. Biol Psychiatry 68:314-319. Bebo BF, Schuster JC, Vandenbark AA, Offner H (1998) Gender differences in experimental autoimmune encephalomyelitis develop during the induction of the immune response to encephalitogenic peptides. J Neurosci Res 52:420-426. Beiske A, Svensson E, Sandanger I, Czujko B, Pedersen E, Aarseth J, Myhr K (2008) Depression and anxiety amongst multiple sclerosis patients. Eur J Neurol 15:239-245. Bercik P, Verdu EF, Foster JA, Macri J, Potter M, Huang X, Malinowski P, Jackson W, Blennerhassett P, Neufeld KA (2010) Chronic gastrointestinal inflammation induces anxiety-like behavior and alters central nervous system biochemistry in mice. Gastroenterology 139:2102-2112. e2101. Bergh FT, Kümpfel T, Trenkwalder C, Rupprecht R, Holsboer F (1999) Dysregulation of the hypothalamopituitary-adrenal axis is related to the clinical course of MS. Neurology 53:772-772. Brown R, Valpiani E, Tennant C, Dunn S, Sharrock M, Hodgkinson S, Pollard J (2009) Longitudinal assessment of anxiety, depression, and fatigue in people with multiple sclerosis. Psychol Psychother 82:41-56. Cua DJ, Hinton DR, Stohlman SA (1995) Self-antigen-induced Th2 responses in experimental allergic encephalomyelitis (EAE)-resistant mice. Th2-mediated suppression of autoimmune disease. J Immunol 155:4052-4059. Cui K, Ashdown H, Luheshi GN, Boksa P (2009) Effects of prenatal immune activation on hippocampal neurogenesis in the rat. Schizophr Res 113:288-297. Cullinan WE, Ziegler DR, Herman JP (2008) Functional role of local GABAergic influences on the HPA axis. Brain Struct Funct 213:63-72. Dahl OP, Stordal E, Lydersen S, Midgard R (2009) Anxiety and depression in multiple sclerosis. A comparative population-based study in Nord-Trøndelag County, Norway. Mult Scler 15:1495-1501. Dalal M, Kim S, Voskuhl RR (1997) Testosterone therapy ameliorates experimental autoimmune encephalomyelitis and induces a T helper 2 bias in the autoantigen-specific T lymphocyte response. J Immunol 159:3-6. de Paiva VN, Lima SN, Fernandes MM, Soncini R, Andrade CA, Giusti-Paiva A (2010) Prostaglandins mediate depressive-like behaviour induced by endotoxin in mice. Behav Brain Res 215:146-151. Del Giudice M (2012) Fetal programming by maternal stress: insights from a conflict perspective. Psychoneuroendocrinology 37:1614-1629. Doosti M-H, Bakhtiari A, Zare P, Amani M, Majidi-Zolbanin N, Babri S, Salari A-A (2013) Impacts of early intervention with fluoxetine following early neonatal immune activation on depression-like behaviors and body weight in mice. Prog Neuropsychopharmacol Biol Psychiatry 43:55-65.

22

Enayati M, Solati J, Hosseini M-H, Shahi H-R, Saki G, Salari A-A (2012) Maternal infection during late pregnancy increases anxiety-and depression-like behaviors with increasing age in male offspring. Brain Res Bull 87:295-302. Faa G, Marcialis M, Ravarino A, Piras M, Pintus M, Fanos V (2014) Fetal Programming of the Human Brain: Is there a Link with Insurgence of Neurodegenerative Disorders in Adulthood? Curr Med Chem 21:3854-3876. Feinstein A (2011) Multiple sclerosis and depression. Mult Scler J 17:1276-1281. Feinstein A, O'connor P, Gray T, Feinstein K (1999) The effects of anxiety on psychiatric morbidity in patients with multiple sclerosis. Mult Scler 5:323-326. Fiore M, Alleva E, Probert L, Kollias G, Angelucci F, Aloe L (1998) Exploratory and displacement behavior in transgenic mice expressing high levels of brain TNF-α. Physiol Behav 63:571-576. Fiore M, Probert L, Kollias G, Akassoglou K, Alleva E, Aloe L (1996) Neurobehavioral alterations in developing transgenic mice expressing TNF-α in the brain. Brain Behav Immun 10:126-138. Garay PA, Hsiao EY, Patterson PH, McAllister A (2013) Maternal immune activation causes age-and regionspecific changes in brain cytokines in offspring throughout development. Brain Behav Immun 31:5468. Giordano A, Granella F, Lugaresi A, Martinelli V, Trojano M, Confalonieri P, Radice D, Solari A (2011) Anxiety and depression in multiple sclerosis patients around diagnosis. J Neurol Sci 307:86-91. Golan H, Levav T, Mendelsohn A, Huleihel M (2004) Involvement of tumor necrosis factor alpha in hippocampal development and function. Cereb Cortex 14:97-105. Gold SM, Kern KC, O'Connor M-F, Montag MJ, Kim A, Yoo YS, Giesser BS, Sicotte NL (2010) Smaller cornu ammonis 2–3/dentate gyrus volumes and elevated cortisol in multiple sclerosis patients with depressive symptoms. Biol Psychiatry 68:553-559. Gold SM, Krüger S, Ziegler KJ, Krieger T, Schulz K-H, Otte C, Heesen C (2011) Endocrine and immune substrates of depressive symptoms and fatigue in multiple sclerosis patients with comorbid major depression. J Neurol Neurosurg Psychiatry 82:814-818. Gold SM, Raji A, Huitinga I, Wiedemann K, Schulz K-H, Heesen C (2005) Hypothalamo–pituitary–adrenal axis activity predicts disease progression in multiple sclerosis. J Neuroimmunol 165:186-191. Gross C, Hen R (2004) The developmental origins of anxiety. Nat Rev Neurosci 5:545-552. Haji N, Mandolesi G, Gentile A, Sacchetti L, Fresegna D, Rossi S, Musella A, Sepman H, Motta C, Studer V (2012) TNF-α-mediated anxiety in a mouse model of multiple sclerosis. Exp Neurol 237:296-303. Handel AE, Giovannoni G, Ebers GC, Ramagopalan SV (2010) Environmental factors and their timing in adult-onset multiple sclerosis. Nat Rev Neurol 6:156-166. Harbo HF, Gold R, Tintoré M (2013) Sex and gender issues in multiple sclerosis. Ther Adv Neurol Disord 6:237-248. Haroon E, Raison CL, Miller AH (2011) Psychoneuroimmunology meets neuropsychopharmacology: translational implications of the impact of inflammation on behavior. Neuropsychopharmacol 37:137-162. Harvey L, Boksa P (2012) Prenatal and postnatal animal models of immune activation: relevance to a range of neurodevelopmental disorders. Dev Neurobiol 72:1335-1348. Heesen C, Gold S, Huitinga I, Reul J (2007) Stress and hypothalamic–pituitary–adrenal axis function in experimental autoimmune encephalomyelitis and multiple sclerosis—a review. Psychoneuroendocrinology 32:604-618. Heesen C, Gold SM, Raji A, Wiedemann K, Schulz K-H (2002) Cognitive impairment correlates with hypothalamo–pituitary–adrenal axis dysregulation in multiple sclerosis. Psychoneuroendocrinology 27:505-517. Henn FA, Vollmayr B (2004) Neurogenesis and depression: etiology or epiphenomenon? Biol Psychiatry 56:146-150. Hsiao EY, Patterson PH (2011) Activation of the maternal immune system induces endocrine changes in the placenta via IL-6. Brain Behav Immun 25:604-615.

23

Imitola J, Chitnis T, Khoury SJ (2005) Cytokines in multiple sclerosis: from bench to bedside. Pharmacol Ther 106:163-177. Iosif RE, Ekdahl CT, Ahlenius H, Pronk CJ, Bonde S, Kokaia Z, Jacobsen S-EW, Lindvall O (2006) Tumor necrosis factor receptor 1 is a negative regulator of progenitor proliferation in adult hippocampal neurogenesis. J Neurosci 26:9703-9712. Ito HT, Smith SE, Hsiao E, Patterson PH (2010) Maternal immune activation alters nonspatial information processing in the hippocampus of the adult offspring. Brain Behav Immun 24:930-941. Kaster MP, Gadotti VM, Calixto JB, Santos AR, Rodrigues ALS (2012) Depressive-like behavior induced by tumor necrosis factor-α in mice. Neuropharmacology 62:419-426. Kellner M, Wiedemann K (2008) Mineralocorticoid receptors in brain, in health and disease: possibilities for new pharmacotherapy. Eur J Pharmacol 583:372-378. Keohane A, Ryan S, Maloney E, Sullivan AM, Nolan YM (2010) Tumour necrosis factor-α impairs neuronal differentiation but not proliferation of hippocampal neural precursor cells: role of Hes1. Mol Cell Neurosci 43:127-135. Khan D, Fernando P, Cicvaric A, Berger A, Pollak A, Monje F, Pollak D (2014) Long-term effects of maternal immune activation on depression-like behavior in the mouse. Transl Psychiatry 4:e363. Koolschijn P, van Haren NE, Lensvelt-Mulders GJ, Pol H, Hilleke E, Kahn RS (2009) Brain volume abnormalities in major depressive disorder: A meta-analysis of magnetic resonance imaging studies. Hum Brain Mapp 30:3719-3735. Korostil M, Feinstein A (2007) Anxiety disorders and their clinical correlates in multiple sclerosis patients. Mult Scler 13:67-72. Krishnan R, Cella D, Leonardi C, Papp K, Gottlieb A, Dunn M, Chiou C, Patel V, Jahreis A (2007) Effects of etanercept therapy on fatigue and symptoms of depression in subjects treated for moderate to severe plaque psoriasis for up to 96 weeks. Br J Dermatol 157:1275-1277. Lesch KP (2004) Gene–environment interaction and the genetics of depression. J Psychiatry Neurosci 29:174-184. Majidi-Zolbanin J, Azarfarin M, Samadi H, Enayati M, Salari A-A (2013) Adolescent fluoxetine treatment decreases the effects of neonatal immune activation on anxiety-like behavior in mice. Behav Brain Res 250:123-132. Majidi-Zolbanin J, Doosti M, Baradaran B, Amani M, Azarfarin M, Salari A (2014) Neonatal immune activation during early and late postnatal brain development differently influences depression-related behaviors in adolescent and adult C57BL/6 mice. Neuroimmunol Neuroinflamma 1:35-39. Makinodan M, Tatsumi K, Manabe T, Yamauchi T, Makinodan E, Matsuyoshi H, Shimoda S, Noriyama Y, Kishimoto T, Wanaka A (2008) Maternal immune activation in mice delays myelination and axonal development in the hippocampus of the offspring. J Neurosci Res 86:2190-2200. Mandal M, Donnelly R, Elkabes S, Zhang P, Davini D, David BT, Ponzio NM (2013) Maternal immune stimulation during pregnancy shapes the immunological phenotype of offspring. Brain Behav Immun 33:33-45. McCoy MK, Tansey MG (2008) TNF signaling inhibition in the CNS: implications for normal brain function and neurodegenerative disease. J Neuroinflammation 5:45. McEwen BS (1999) Stress and hippocampal plasticity. Ann Rev Neurosci 22:105-122. Mikova O, Yakimova R, Bosmans E, Kenis G, Maes M (2001) Increased serum tumor necrosis factor alpha concentrations in major depression and multiple sclerosis. Eur Neuropsychopharmacol 11:203-208. Miller AH, Maletic V, Raison CL (2009) Inflammation and its discontents: the role of cytokines in the pathophysiology of major depression. Biol Psychiatry 65:732-741. Mohr DC, Hart SL, Julian L, Cox D, Pelletier D (2004) Association between stressful life events and exacerbation in multiple sclerosis: a meta-analysis. BMJ 328:731. Murray F, Smith DW, Hutson PH (2008) Chronic low dose corticosterone exposure decreased hippocampal cell proliferation, volume and induced anxiety and depression like behaviours in mice. Eur J Pharmacol 583:115-127.

24

Noy S, Achiron A, Gabbay U, Barak Y, Rotstein Z, Laor N, Sarova-Pinhas I (1995) A new approach to affective symptoms in relapsing-remitting multiple sclerosis. Compr Psychiatry 36:390-395. Oh-Nishi A, Obayashi S, Sugihara I, Minamimoto T, Suhara T (2010) Maternal immune activation by polyriboinosinic-polyribocytidilic acid injection produces synaptic dysfunction but not neuronal loss in the hippocampus of juvenile rat offspring. Brain Res 1363:170-179. Okuda Y, Okuda M, Bernard CC (2002) Gender does not influence the susceptibility of C57BL/6 mice to develop chronic experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein. Immunol Lett 81:25-29. Papenfuss TL, Rogers CJ, Gienapp I, Yurrita M, McClain M, Damico N, Valo J, Song F, Whitacre CC (2004) Sex differences in experimental autoimmune encephalomyelitis in multiple murine strains. J Neuroimmunol 150:59-69. Peruga I, Hartwig S, Thöne J, Hovemann B, Gold R, Juckel G, Linker RA (2011) Inflammation modulates anxiety in an animal model of multiple sclerosis. Behav Brain Res 220:20-29. Raison CL, Capuron L, Miller AH (2006) Cytokines sing the blues: inflammation and the pathogenesis of depression. Trends Immunol 27:24-31. Sapolsky RM (2000) Glucocorticoids and hippocampal atrophy in neuropsychiatric disorders. Arch Gen Psychiatry 57:925-935. Schumann EM, Kümpfel T, Then Bergh F, Trenkwalder C, Holsboer F, Auer DP (2002) Activity of the hypothalamic–pituitary–adrenal axis in multiple sclerosis: Correlations with gadolinium-enhancing lesions and ventricular volume. Ann Neurol 51:763-767. Schwendener S, Meyer U, Feldon J (2009) Deficient maternal care resulting from immunological stress during pregnancy is associated with a sex-dependent enhancement of conditioned fear in the offspring. J Neurodev Disord 1:15-32. Shi L, Fatemi SH, Sidwell RW, Patterson PH (2003) Maternal influenza infection causes marked behavioral and pharmacological changes in the offspring. J Neurosci 23:297-302. Smith SE, Li J, Garbett K, Mirnics K, Patterson PH (2007) Maternal immune activation alters fetal brain development through interleukin-6. J Neurosci 27:10695-10702. Soczynska JK, Kennedy SH, Goldstein BI, Lachowski A, Woldeyohannes HO, McIntyre RS (2009) The effect of tumor necrosis factor antagonists on mood and mental health-associated quality of life: novel hypothesis-driven treatments for bipolar depression? Neurotoxicology 30:497-521. Solati J, Asiaei M, Hoseini MHM (2012) Using experimental autoimmune encephalomyelitis as a model to study the effect of prenatal stress on fetal programming. Neurol Res 34:478-483. Solati J, Salari A (2011) Involvement of dorsal hippocampal NMDA-glutamatergic system in anxiety-related behaviors of rats. Neurochem J 5:194-199. Stenager EN, Stenager E (1992) Suicide and patients with neurologic diseases: methodologic problems. Arch Neurol 49:1296-1303. Stetler C, Miller GE (2011) Depression and hypothalamic-pituitary-adrenal activation: a quantitative summary of four decades of research. Psychosom Med 73:114-126. Tomassini V, Pozzilli C (2009) Sex hormones, brain damage and clinical course of Multiple Sclerosis. J Neurol Sci 286:35-39. Tyring S, Gottlieb A, Papp K, Gordon K, Leonardi C, Wang A, Lalla D, Woolley M, Jahreis A, Zitnik R (2006) Etanercept and clinical outcomes, fatigue, and depression in psoriasis: double-blind placebocontrolled randomised phase III trial. Lancet 367:29-35. Voskuhl R (2011) Sex differences in autoimmune diseases. Biol Sex Differ 2:1. Voskuhl RR, Gold SM (2012) Sex-related factors in multiple sclerosis susceptibility and progression. Nat Rev Neurol 8:255-263. Voskuhl RR, Pitchekian-Halabi H, Mackenzie-Graham A, McFarland HF, Raine CS (1996) Gender differences in autoimmune demyelination in the mouse: implications for multiple sclerosis. Ann Neurol 39:724733. Wallin MT, Wilken JA, Turner AP, Williams RM, Kane R (2006) Depression and multiple sclerosis: review of a lethal combination. J Rehabil Res Dev 43:45.

25

Wang H, Meng X-H, Ning H, Zhao X-F, Wang Q, Liu P, Zhang H, Zhang C, Chen G-H, Xu D-X (2010) Age-and gender-dependent impairments of neurobehaviors in mice whose mothers were exposed to lipopolysaccharide during pregnancy. Toxicol Lett 192:245-251. Wood B, Van der Mei I, Ponsonby A, Pittas F, Quinn S, Dwyer T, Lucas R, Taylor B (2012) Prevalence and concurrence of anxiety, depression and fatigue over time in multiple sclerosis. Mult Scler J 19:217224. Yamada K, Iida R, Miyamoto Y, Saito K, Sekikawa K, Seishima M, Nabeshima T (2000) Neurobehavioral alterations in mice with a targeted deletion of the tumor necrosis factor-α gene: implications for emotional behavior. J Neuroimmunol 111:131-138. Young EA, Abelson JL, Cameron OG (2004) Effect of comorbid anxiety disorders on the hypothalamicpituitary-adrenal axis response to a social stressor in major depression. Biol Psychiatry 56:113-120. Ysrraelit MC, Gaitán MI, Lopez AS, Correale J (2008) Impaired hypothalamic-pituitary-adrenal axis activity in patients with multiple sclerosis. Neurology 71:1948-1954. Zager A, Peron JP, Mennecier G, Rodrigues SC, Aloia TP, Palermo-Neto J (2014) Maternal immune activation in late gestation increases neuroinflammation and aggravates experimental autoimmune encephalomyelitis in the offspring. Brain Behav Immun In Press:Doi:10.1016/j.bbi.2014.1007.1021. Zorrilla EP, Luborsky L, McKay JR, Rosenthal R, Houldin A, Tax A, McCorkle R, Seligman DA, Schmidt K (2001) The relationship of depression and stressors to immunological assays: a meta-analytic review. Brain Behav Immun 15:199-226. 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. Neuropsychopharmacol 28:1778-1789.

26

Figure Legends Fig.1. Experimental design: Effects of maternal immune activation during mid-pregnancy with PolyI:C on anxiety- and depression-related behaviors as well as stress-induced corticosterone and TNF-α levels in male and female offspring with EAE. Fig.2. Clinical course of EAE. The long-lasting effects of prenatal immune activation on the clinical course of EAE in adult female (A) and male (B) offspring. Values are presented as mean ± S.E.M. (N=15). Significant differences: *P < 0.05 and **P < 0.01, compared to the PBS-treated female offspring. +P < 0.05 and +++P < 0.001, compared to the PBS-treated male offspring. Fig.3. Effects of maternal immune activation on anxiety-related behaviors as measured during adulthood in the offspring with EAE in the elevated plus-maze test. Each bar represents mean ± S.E.M. (N=10) of the open arm time % (A), open arm entries % (B) or locomotor activity (C). Significant differences: *P < 0.05, compared to PBS-treated female offspring. compared to PBS-EAE female offspring. +P < 0.05 and

++P

###P

< 0.001,

< 0.01, compared to PBS-treated male

offspring. $$$P < 0.001, compared to PBS-EAE male offspring. Fig.4. Effects of maternal immune activation on anxiety-related behaviors as measured during adulthood in the offspring with EAE in the light-dark box. Each bar represents mean ± S.E.M. (N=10) of the lit compartment time (A), lit compartment entries (B), latency of entry into the dark (C). Significant differences: ##P < 0.01 and ###P < 0.001, compared to PBS-EAE female offspring. +P < 0.05 and

++P

< 0.01, compared to PBS-treated male offspring.

$$$P

< 0.001, compared to PBS-EAE

male offspring. Fig.5. Effects of maternal immune activation on depression-related behaviors (tail suspension test: A and forced swim test: B) in adult offspring with EAE. Values are presented as mean ± S.E.M.

27

(N=10) of total duration of immobility. Significant differences: #P < 0.05 and ##P < 0.01, compared to PBS-EAE female offspring. +P < 0.05 and ++P < 0.01, compared to PBS-treated male offspring. $$$P < 0.001, compared to PBS-EAE male offspring. Fig.6. Effects of prenatal maternal immune activation on stress-induced corticosterone (A) and TNF-α levels (B) in the offspring with EAE. Values are presented as mean ± S.E.M. (N=10) of corticosterone or TNF-α concentrations. Significant differences:

##P

< 0.01, compared to PBS-EAE

female offspring. $$$P < 0.001, compared to PBS-EAE male offspring.

28

Fig. 1

29

Fig. 2

30

Fig. 3 31

Fig. 4 32

Fig. 5

33

Fig. 6 34

Research Highlights •

Early prenatal maternal immune activation increases anxiety sex-dependently in offspring.

•

Early prenatal immune activation alters depression sex-dependently in offspring.

•

Maternal immune activation alters onset and severity of EAE disease in offspring.

•

Maternal immune activation increases anxiety and depression in EAE-induced offspring.

•

Prenatal immune activation alters corticosterone and TNF-α levels in EAE-induced offspring.

35