Interaction of BDNF with cytokines in chronic schizophrenia

Brain, Behavior, and Immunity xxx (2015) xxx–xxx

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Interaction of BDNF with cytokines in chronic schizophrenia Xiang Yang Zhang a,b,⇑, Yun-Long Tan a, Da-Chun Chen a, Shu-Ping Tan a, Fu-De Yang a, Hanjing Emily Wu b, Giovana B. Zunta-Soares b, Xu-Feng Huang c, Thomas R. Kosten d, Jair C. Soares b a

Psychiatry Research Center, Beijing HuiLongGuan Hospital, Peking University, Beijing, China Department of Psychiatry and Behavioral Sciences, The University of Texas Health Science Center at Houston, Houston, TX, USA c Centre for Translational Neuroscience, School of Medicine, University of Wollongong, and Illawarra Health and Medical Research Institute, NSW, Australia d Department of Psychiatry and Behavioral Sciences, Baylor College of Medicine, Houston, TX, USA b

a r t i c l e

i n f o

Article history: Received 23 March 2015 Received in revised form 21 September 2015 Accepted 22 September 2015 Available online xxxx Keywords: Schizophrenia Cytokine BDNF Interaction Symptom Cognition

a b s t r a c t Brain-derived neurotrophic factor (BDNF) interacts with cytokines. Although both BDNF and cytokines occur at abnormal levels in schizophrenia patients, their interactions have not yet been examined. We therefore compared serum BDNF, TNF-a, interleukin (IL)-2, IL-6, and IL-8 levels in 92 chronically medicated schizophrenia patients and 60 healthy controls. We correlated these serum levels within these subject groups with each other and with clinical symptoms assessed according to the Positive and Negative Syndrome Scale (PANSS). Compared to the control group, the schizophrenia patients had significantly lower BDNF and TNF-a levels, and higher IL-2, IL-6, and IL-8 levels. The patients also showed a significant positive correlation between BDNF and both IL-2 and IL-8 levels, and low BDNF and TNF-a levels together were associated with poor performance on the PANSS cognitive factor. Thus, an interaction between cytokines and neurotrophic factors may be implicated in the pathophysiology of chronic schizophrenia. In particular, the cytokine TNF-a may interact with BNDF causing cognitive impairment. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction The neurodevelopmental model of schizophrenia suggests a ‘‘2hit’’ model (Keshavan, 1999; Rapoport et al., 2012), in which a first hit may be a prenatal viral or bacterial infection that activates microglia and immuno-inflammatory pathways (Canetta and Brown, 2012; Altamura et al., 2013), followed by secondary inflammatory hits during adolescence with consequent autoimmune responses (Anderson and Maes, 2013). These autoimmune responses in the brain can act through cytokines, and abnormal cytokine levels are associated with various neurological, neurodegenerative, and neurotoxic conditions, including schizophrenia (Altamura et al., 2013; Miller et al., 2013; Na et al., 2014; Müller, 2014; Upthegrove et al., 2014; Zakharyan and Boyajyan, 2014; Sperner-Unterweger and Fuchs, 2015). Two recent meta-analyses have shown cytokine alterations in schizophrenia patients. The first meta-analysis by Potvin et al. (2008) found increased levels of in vivo peripheral interleukin (IL)-1RA, sIL-2R, and IL-6 in schizophrenia patients. The second meta-analysis by Miller et al. (2011) found significant increases in macrophage-derived cytoki⇑ Corresponding author at: UT Center of Excellence on Mood Disorders (UTCEMD), Biomedical and Behavioral Sciences Building (BBSB), 1941 East Road, TX 77054, USA. E-mail address: [email protected] (X.Y. Zhang).

nes IL-1b, IL-6, and TNF-a, as well as the Th1-derived cytokines interferon-c and IL-12 in first-episode psychosis. Low levels of brain-derived neurotrophic factor (BDNF) are associated with schizophrenia (Buckley et al., 2011; Favalli et al., 2012; Nieto et al., 2013) in both chronic antipsychotic-treated patients and antipsychotic-free or naïve, first-episode patients with schizophrenia (Chen et al., 2009; Xiu et al., 2009; Pillai et al., 2010; Rizos et al., 2010; Nurjono et al., 2012). Although some authors have failed to replicate these findings in both medicated and antipsychotic-naïve patients (Green et al., 2011), a recent meta-analysis supports reduced peripheral BDNF levels in both medicated and drug-naïve patients with schizophrenia (Green et al., 2011). Furthermore, BDNF has been associated with positive symptoms (Buckley et al., 2007; Xiu et al., 2009), negative symptoms (Rizos et al., 2008; Chen et al., 2009), and tardive dyskinesia (TD) (Yang et al., 2011; Zhang et al., 2012a,b). Taken together, these findings demonstrate the relevance of BDNF to the pathophysiology of schizophrenia (Nurjono et al., 2012). BDNF and cytokines are well-known to cross-regulate each other (Nawa et al., 2000; Calabrese et al., 2014). For example, IL1b and transforming growth factor (TGF)-a suppress the normal expression of BDNF (Xiong et al., 1999), while IL-1b up-regulates the expression of nerve growth factor (NGF) (Heese et al., 1998). BDNF levels are also positively associated with IL-6 levels in major depressive disorder patients, but not in non-depressed controls

http://dx.doi.org/10.1016/j.bbi.2015.09.014 0889-1591/Ó 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: Zhang, X.Y., et al. Interaction of BDNF with cytokines in chronic schizophrenia. Brain Behav. Immun. (2015), http://dx.doi. org/10.1016/j.bbi.2015.09.014

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(Patas et al., 2014). Among the sources of BDNF are human peripheral blood mononuclear cells (i.e. T-cells, B-cells, and monocytes), which upon stimulation release neurotrophins such as BDNF offering neuroprotection (Schulte-Herbrüggen et al., 2005). Studies in humans and animals have explored the effects of antipsychotics on BDNF and cytokine levels (Nurjono et al., 2012). For example, some studies have found a differential regulation of BDNF mRNA expression in the rat hippocampus and neocortex by typical and atypical antipsychotic administration (Bai et al., 2003), with the atypical antipsychotics olanzapine and quetiapine enhancing BDNF expression (Rizos et al., 2008). Furthermore, serum BDNF levels were higher in chronic schizophrenia patients on clozapine or typical antipsychotics than risperidone (Xiu et al., 2009), and BDNF levels were positively associated with daily clozapine doses in patients with schizophrenia (Pedrini et al., 2011). However, some longitudinal studies have reported that lower serum BDNF levels did not increase after several weeks of antipsychotic treatment (Pirildar et al., 2004; Yoshimura et al., 2007). Alterations in cytokine levels have been repeatedly described in schizophrenia (Potvin et al., 2008; Miller et al., 2011), but the effect of antipsychotics on cytokine levels remains incompletely explored (Tourjman et al., 2013). Antipsychotics have anti-inflammatory effects in schizophrenia (Drzyzga et al., 2006; Tourjman et al., 2013) with significant increases in plasma levels of soluble IL-2 receptor and reductions in plasma levels of IL-1b and interferonc (Tourjman et al., 2013). Therefore, the effects of antipsychotics on BDNF or cytokine levels in schizophrenia deserve further examination. Nawa et al. (2000) have suggested that the interaction between cytokines and neurotrophic factors may contribute to the etiology of schizophrenia, but this association has not been directly examined. We therefore tested this hypothesis by investigating whether: (1) serum BDNF and cytokine levels differ from healthy controls; (2) BDNF and cytokine levels are correlated in schizophrenia patients or healthy controls; (3) BDNF and cytokine levels are independently or interactively associated with the psychopathology of schizophrenia; and (4) BDNF and cytokine levels differ in patients taking typical versus atypical antipsychotic medications.

tion files among those aged 35–75 years who lived in the Haidian District of Beijing, and sent each subject a letter explaining the purpose of the study. Local officials and health centers arranged for the interviews and measurements to take place at the center office at times convenient to the participants. All participants were interviewed by trained investigators. A clinical interview was used to exclude potential controls with Axis I disorders by a research psychiatrist. This psychiatrist used the standardized SCID diagnostic assessment to exclude participants with mental disorders such as common anxiety disorders and mood disorders. All subjects provided signed, informed consent to participate in this study, which was approved by the Institutional Review Board, Beijing Hui-Long-Guan Hospital. They were Han Chinese recruited at the same time from the Beijing area. The inclusion of Han Chinese alone was an a priori criterion. The patients and the healthy controls had a similar socioeconomic status and dietary patterns. We obtained a complete medical history and physical examination from all subjects. We excluded pregnant or breast-feeding females and subjects with medical abnormalities, including central nervous system diseases, acute, unstable or significant medical illnesses (e. g. cancer, infection, lung disease, diabetes, hypertension, or cerebrovascular disease), and a history of severe allergies. Neither the patients nor the healthy controls had any history of alcohol or substance dependence (aside from tobacco). In addition, the use of alcohol or other recreational drugs in the week prior to the current study was an additional exclusion criterion. Finally, we also excluded any control subjects taking psychotropic medications (e.g. antidepressant, anti-anxiety, antipsychotic, or mood stabilizing drugs), hormonal agents, anti-inflammatory agents, antihypertensives, anti-hyperlipidemics, and anti-diabetics. Apart from the antipsychotic medications, these same medication exclusions were applied to the patients. In summary, we had initially planned to recruit 220 people (110 patients vs 110 controls). After screening, 68 subjects (18 patients and 50 controls) were excluded due to documented medical abnormalities (8 patients and 6 controls), substance dependence other than tobacco (2 patients and 11 controls), taking non-psychotic medications (2 patients and 12 controls), comorbid other psychiatric disorders (3 patients and 8 controls), and refusal to participate in the study/inability to provide consent (3 patients and 13 controls).

2. Methods 2.1. Subjects

2.2. Clinical assessment

Ninety-two physically healthy patients (male/female = 75/17) were recruited from the inpatients of Beijing Hui-Long-Guan Hospital, a Beijing City owned psychiatric hospital. No subjects in the present sample were included in previous reports (Zhang et al., 2002). The current study was conducted from December 2006 to May 2008. All patients met the DSM-IV diagnosis of schizophrenia, which was confirmed by two independent experienced psychiatrists based on the Structured Clinical Interview for DSM-IV (SCID). No patients with schizophrenia had comorbid psychiatric disorders. Their clinical subtypes were: 35 paranoid (38.0%); 11 disorganized (12.0%), 11 undifferentiated (12.0%), and 35 residual (38.0%). Patients were aged 35–75 years with mean illness duration of 23.2 ± 7.3 years. All patients had been receiving stable doses of oral antipsychotic drugs for at least 12 months before entering this study. Antipsychotic treatment consisted of monotherapy with either an atypical antipsychotic, including clozapine (n = 49) or risperidone (n = 12), or a typical antipsychotic (n = 31), including haloperidol (n = 11), perphenazine (n = 9), chlorpromazine (n = 6), or sulpiride (n = 5). The mean antipsychotic dose (as chlorpromazine equivalents) was 434.1 ± 369.0 mg/day. For comparison, healthy controls were recruited from the local community. We drew a random sample from the resident registra-

Four psychiatrists who had simultaneously attended a training session in the use of the Positive and Negative Syndrome Scale (PANSS) rated patients on this scale. After training, repeated assessment showed that the inter-observer correlation coefficient was maintained at >0.8 for the PANSS total score. The PANSS introduced by Kay et al. (1987) has been separated into five-factor components labeled as ‘positive’, ‘negative’, ‘cognitive’, ‘depression’, and ‘excitement’ (Wallwork et al., 2012; RodriguezJimenez et al., 2013). The cognitive factor (sometimes called ‘disorga nization’) refers to the patient’s cognitive function, and consists of three PANSS items: ‘Conceptual disorganization’ (P2), ‘Difficulty in abstract thinking’ (N5), and ‘Poor attention’ (G11) (Wallwork et al., 2012). This cognitive factor was later confirmed in the clinical assessment of patients with schizophrenia (Rodriguez-Jimenez et al., 2013), suggesting that the cognitive component of the PANSS is a valid measure of cognitive deficits in schizophrenia.

2.3. Blood sampling We obtained blood samples to assess BDNF and cytokine parameters at the time of the PANSS ratings. Venous blood from

Please cite this article in press as: Zhang, X.Y., et al. Interaction of BDNF with cytokines in chronic schizophrenia. Brain Behav. Immun. (2015), http://dx.doi. org/10.1016/j.bbi.2015.09.014

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a forearm vein was collected between 7 and 9 am following an overnight fast from healthy controls and schizophrenia patients. The serum was separated, aliquoted, and stored at 70 °C before use. All of the samples were assayed in the same assay batches. To maintain the blind status, the code numbers identifying the subjects were held by the investigator until all of the biochemical analyses were completed. Each evaluated parameter was assayed in duplicate for all samples. 2.4. Determination of serum BDNF levels We measured fasting serum BDNF levels by sandwich enzymelinked immunosorbent assay (ELISA) using a commercially available kit as described in our previous report (Chen et al., 2009; Xiu et al., 2009). A research assistant who was blind to the clinical data assayed all of the samples. Inter- and intra-assay variation coefficients were 7% and 5%, respectively. 2.5. Determination of IL-2, IL-6, IL-8, and TNF-a levels in serum Serum IL-2, IL-6, IL-8, and TNF-a were analyzed in duplicate using established procedures. A full description of the assays has been given in our previous report (Zhang et al., 2002). Briefly, these cytokine serum levels were measured by quantitative ELISA using a commercially available kit (NeoBioscience Technology, Beijing, China). The sensitivities for IL-2, IL-6, IL-8, and TNF-a were 0.01 ng/ml, 0.2 ng/ml, 0.4 ng/ml, and 0.1 ng/ml, respectively, with intra-assay coefficients from 3% to 7%, and inter-assay coefficients from 4% to 9%, respectively. A single investigator who was blind to the clinical status of the samples assayed all of the samples. 2.6. Statistical analysis To compare the demographic and clinical variables, we used the chi-squared test for categorical variables and the Student’s t-tests or analysis of variance (ANOVA) for the continuous variables. To compare the BDNF and cytokine levels in the schizophrenia patient and control groups, we first tested these biomarker differences using ANOVA. Whenever the ANOVA was significant, we tested the effects of gender, age, education, smoking, and body mass index (BMI) by adding these variables to the analysis model as covariates. To investigate the interaction between BDNF and cytokines, we correlated each cytokine and BDNF level in the combined group, as well as in the patient and control groups separately. We applied Bonferroni corrections to adjust for multiple testing. To further explore the interaction between the schizophrenia patients and the control groups among all of the cytokines and

BDNF, we used binary logistic regression analysis while controlling for gender, age, education, smoking, and BMI. Lastly, exploratory regression analyses were used to examine the relationships between clinical phenotypes and these serum levels and their interaction effects in patients. Stepwise multiple regression analysis used the PANSS total or cognitive score as dependent variables with BDNF and cytokines, as well as their interaction term as the independent variables. Covariates in these stepwise forward entry models included age, gender, education, smoking, illness duration, medication type (typical vs atypical antipsychotics), dosage and duration of antipsychotic treatment. Two-tailed significance values were used and significance levels were set at 0.05. 3. Results 3.1. Demographic data Table 1 shows no significant difference in the demographic parameters between the patient and control groups. We did not find any significant associations between the demographic parameters and the BDNF or cytokine levels in either the patient or control groups. 3.2. Cytokines and BDNF levels in schizophrenia patients and controls Table 2 shows that IL-2, IL-6, and IL-8 levels were significantly higher, but TNF-a and BDNF levels were significantly lower in the serum of patients than controls (all p < 0.05). When the effects of age, sex, education, smoking, and BMI were examined by adding them to the ANOVA as covariates, a significant difference between patients and controls was still observed in IL-2, IL-6, IL-8, TNF-a (all p < 0.05), and BDNF (p < 0.01). There was no difference in the cytokine or BDNF levels based on antipsychotic type, dose, or duration of antipsychotic drug (all p > 0.05) in the schizophrenia patients. In particular, 49 patients (53.3%) were taking clozapine, and although cytokine profiles may differ in patients treated with clozapine (Miller et al., 2011), we found no significant differences in IL-2, IL-6, IL-8, TNF-a, and BDNF between patients on clozapine vs non-clozapine or clozapine vs typical antipsychotics (all p > 0.05). 3.3. Interaction of BDNF and IL in schizophrenia patients and controls A binary logistic regression analysis showed significant interaction terms for both BDNF  IL-2 (b = 0.45, Wald v2 = 6.89, df = 1, p = 0.009) and BDNF  IL-8 (b = 0.37, Wald v2 = 4.91, df = 1, p = 0.03), but not for BDNF  IL-6 (b = 0.12, Wald v2 = 1.60,

Table 1 Demographics of patients and healthy control subjects.

a

Schizophrenia (n = 92)

Control subjects (n = 60)

F or X2

dfa

p

Sex, M/F Age (years) Education Smokers/non-smokers Body mass index (kg/m2) Age of onset (years) Duration of illness (years) Number of hospitalizations

75/17 47.5 ± 4.4 9.6 ± 2.2 59/33 24.7 ± 3.7 24.2 ± 6.3 23.2 ± 7.3 3.5 ± 2.0

44/16 47.7 ± 4.5 10.2 ± 3.1 33/27 24.5 ± 3.9

1.43 0.08 0.91 1.27 0.06

1 1,151 1,148 1 1,139

0.23 0.78 0.33 0.26 0.84

PANSS Positive symptoms Negative symptoms General psychopathology Total

15.5 ± 6.1 23.9 ± 6.1 34.1 ± 9.2 73.5 ± 17.2

Indicates that there have been some missing data, with inconsistent df value.

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Table 2 Cytokine and BDNF levels in schizophrenia patients and controls. Markers

Schizophrenia

Controls

F

dfa

p

pb

IL-2 (ng/ml) IL-6 (ng/ml) IL-8 (ng/ml) TNFa (ng/ml) BDNF (ng/ml)

7.0 ± 2.9 0.35 ± 0.11 0.97 ± 0.30 10.0 ± 2.0 6.9 ± 2.4

4.2 ± 2.6 0.29 ± 0.19 0.88 ± 0.57 37.2 ± 5.1 9.7 ± 4.5

24.4 5.90 4.74 1963.9 22.1

1,148 1,148 1,148 1,147 1,147

0.00 0.008 0.014 0.00 0.00

0.00 0.016 0.031 0.00 0.00

Note: a Indicates that there have been some missing data, with inconsistent df value. b Indicates adjusted p, with adjustments made for age, gender, education, smoking, and body mass index (BMI).

df = 1, p = 0.21) nor BDNF  TNF-a (b = 0.09, Wald v2 = 2.37, df = 1, p = 0.12). Further correlation analysis explored these significant interactions of BDNF and the cytokine levels in patients and controls. While there were no significant associations between BNDF and any of the cytokines (all p > 0.05) in the combined group nor the healthy control group, the patient only group showed a significant positive association between BDNF and IL-2 (r = 0.34, df = 88, p < 0.001) levels and between BDNF and IL-8 (r = 0.34, df = 87, p < 0.001) (Fig. 1), but not between BDNF and IL-6 (r = 0.02, df = 88, p = 0.86), nor between BDNF and TNF-a (r = 0.19, df = 87, p = 0.07). The IL-6 and IL-8 levels were also correlated (r = 0.37, df = 88, p < 0.001). 3.4. Association between BDNF, cytokine levels and clinical symptoms in schizophrenia patients

IL-8 level (ng/ml)

IL-2 level (ng/ml)

Bonferroni corrected correlation analyses showed a significantly negative association between IL-2 and the PANSS positive

subscore (p < 0.05). Stepwise multiple regression analyses identified the BDNF  TNF-a interaction term (b = 0.29, t = 2.10, p < 0.05) as the influencing factor for the PANSS cognitive factor. 4. Discussion 4.1. Decreased BDNF and altered cytokines in schizophrenia Our finding of decreased BDNF in schizophrenia is consistent with previous studies in serum or plasma (Pillai et al., 2010; Rizos et al., 2010; Nurjono et al., 2012). A recent animal study has demonstrated that blood and plasma BDNF levels reflect brain-tissue BDNF levels (Klein et al., 2011). It has also been found that the brains of schizophrenia patients have reduced BDNF mRNA and protein expression (Ray et al., 2014). Moreover, antipsychotic-naïve schizophrenia patients show a significant correlation between BDNF protein levels in plasma and cerebrospinal fluid (Pillai et al., 2010). These studies suggest that the level of

r=-0.06 df=60 P=0.66

r=0.344 df=88 P<0.001

r=0.338 df=87 P<0.001

r=0.01 df=60 P=0.96

BDNF level (ng/ml)

BDNF level (ng/ml)

Schizophrenia

Healthy controls

Fig. 1. Correlation analysis revealed a significantly positive correlation between BDNF and IL-2 (r = 0.344, df = 88, p < 0.001) or IL-8 (r = 0.338, df = 87, p < 0.001) in patients with schizophrenia, which passed Bonferroni test, but no significant correlation between BDNF and IL-2 (r = 0.06, df = 60, p = 0.66) or IL-8 (r = 0.01, df = 60, p = 0.96) in healthy controls.

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BDNF in the blood may reflect the level of BDNF in the brain. Together, these findings confirm that BDNF is involved in schizophrenia (Nagahara and Tuszynski, 2011). A further finding of our present study is the increased IL-2, IL-6, and IL-8 levels but decreased TNF-a levels in chronic schizophrenia patients. Many authors have observed the lack of consistent results regarding cytokines in schizophrenia (Potvin et al., 2008; Miller et al., 2011; Na et al., 2014). For example, although there is evidence of increased IL-2 (Potvin et al., 2008; Miller et al., 2011), IL-6 (Kubistova et al., 2012; Al-Asmari and Khan, 2014; DunjicKostic et al., 2013; Kalmady et al., 2014; Sasayama et al., 2013; Song et al., 2014), and IL-8 (Maes et al., 2002; Brown et al., 2004; Zhang et al., 2004) levels in schizophrenia, some studies have not found a significant difference from healthy controls in IL-2 (Na et al., 2014), IL-6 (Baker et al., 1996), and IL-8 (O’Brien et al., 2008; Kubistova et al., 2012), or even a decrease in IL-2 (Miller et al., 2011; Na et al., 2014) and IL-6 levels compared to controls (Singh et al., 2009). The decrease in the TNF-a level in schizophrenia patients in this study is consistent with a recent study (Francesconi et al., 2011), although previous studies have observed significantly greater TNF-a levels (O’Brien et al., 2008; Kim et al., 2009), or no difference from healthy controls (Borovcanin et al., 2012, 2013). These differences in immune responses across schizophrenia studies may reflect the many confounding factors including demographics, diet, disease state, medications and cytokine assays. A critical issue is whether the differences in cytokines and BDNF between patients and controls can be attributed to schizophrenia itself or whether they are attributable to confounding factors, particularly the possible effects of chronic antipsychotic medication. In our present study, the patients were all chronically ill and had been taking antipsychotic drugs for a long-term period. Consideration of possible differences between typical and atypical antipsychotics with respect to BDNF and cytokines as indicated in the Introduction, the possible effects of antipsychotics on these biomarkers in the current study should be considered. Recent meta-analysis shows elevated levels of IL-1b, sIL-2R, IL-6, and TNF-a (Upthegrove et al., 2014) or decreased BDNF (Nurjono et al., 2012) in the serum of patients with medication-naïve first episode psychosis (Upthegrove et al., 2014). Moreover, we did not find a correlation between BDNF or these cytokine levels and antipsychotic type, dose, or duration of treatment in the patient group in our current study. These results suggest that decreased BDNF and altered cytokine levels in our current study may be associated with the pathology of schizophrenia, but not with antipsychotic medications. However, due to the cross-sectional design of our current study, the primary effect of antipsychotic medication cannot be ruled out and we still could not answer the question of whether the observed effects are a function of medication rather than illness. Further studies are needed to assess the effects of antipsychotic medicines on BDNF and cytokines in first-episode and drug-naïve patients with schizophrenia using a longitudinal design. 4.2. Interaction of BDNF with IL-2 and IL-8 in schizophrenia We found a positive correlation between BDNF and IL-2 and IL8 levels in schizophrenia patients, despite lower levels of BDNF and higher levels of IL-2 and IL-8 compared to the healthy controls. This is similar to a recent study which showed a positive correlation between BDNF and IL-6 levels in major depressive disorder patients (Patas et al., 2014). Neurotrophins and their respective receptors have been found in human peripheral blood mononuclear cells, and upon stimulation, these cells release significant amounts of neurotrophin, which may be neuroprotective (Schulte-Herbrüggen et al., 2005). The

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observation that immune cells in the injured nervous system produce BDNF lends credence to the concept of neuroprotection from auto-immune reactions (Calabrese et al., 2014). Moreover, previous studies have found that IL-6 and TNF-a were able to enhance extracellular BDNF levels in human monocytes (SchulteHerbrüggen et al., 2005; Patas et al., 2014). A recent animal study has shown that mice lacking BDNF in their immune cells display an attenuated immune response, whereas the injection of BDNFoverexpressed T cells led to a less severe course of experimental autoimmune encephalomyelitis, supporting the concept that BDNF provides neuroprotection from autoimmune reactions (Linker et al., 2010). Another recent animal study has shown that subacute IL-1 administration (8 days) in rats decreased the expression of BDNF, whereas an up-regulation of BDNF and nerve growth factor (NGF) were found after acute IL-1 administration (1 day), suggesting that neuro-inflammation may trigger neuroprotection through the neurotrophic system (Song et al., 2013). Positive BDNFimmune interactions could also reflect an immunotrophic function of BDNF (Patas et al., 2014). Indeed, it has been shown that the conditional deletion of BDNF in T-cells and macrophages reduces T-cell activation and cytokine production (Linker et al., 2010). Impaired B-cell development has also been observed in BDNFdeficient mice (Schuhmann et al., 2005). Taken together, these findings support our positive association of BDNF with IL-2 and IL-8 in patients with schizophrenia. The schizophrenia patients in our study had lower levels of BDNF and higher levels of IL-2 and IL-8 compared to the healthy controls. This is consistent with the activation of the immune system from autoimmune diseases and bacterial or viral infections (Proost et al., 1996; Fineberg and Ellman, 2013; Na et al., 2014), and secondary brain injuries (Cannella and Raine, 2004; Felderhoff-Mueser et al., 2005). Since BDNF plays a critical role in the regeneration, survival, and maintenance of neuronal function (Adachi et al., 2014), this relative increase in BDNF may act as a compensatory mechanism. However, schizophrenia patients cannot produce sufficient BDNF to counteract the inflammatory damage of IL-2 and IL-8. 4.3. Interaction of BDNF and cytokines with clinical phenotypes of schizophrenia We found that low BDNF and TNF-a levels were associated with impairment on the PANSS cognitive factor. This association is consistent with the results by Yogeetha et al. (2013), who reported a significant interactive effect on spatial memory retention in healthy individuals by the polymorphisms BDNF rs6265 (Val66Met), which lowers BDNF levels, and TNF-a rs113325588, which lowers TNF-a levels. Although these associations do not offer a causal pathophysiology, it has been shown that the disrupted TNF-a signaling impairs the development of the hippocampus and causes cognitive dysfunction (Garay and McAllister, 2010; Baune et al., 2012). Low BDNF levels have also been associated with cognitive impairment, especially immediate memory, in schizophrenia (Zhang et al., 2012a,b). Thus, an interaction between neurotrophins (BDNF) and the immune response may be involved in the mechanism of cognitive impairment in schizophrenia including a pathological mechanism of inflammatory damage from IL-2 and IL-8 elevation and insufficient neurotrophin and TNF-a compensation. In summary, both altered cytokines and decreased BDNF levels may be implicated in the pathophysiology of chronic schizophrenia. The positive association between BDNF and IL-2 and IL-8 in schizophrenia patients may reflect a pathological mechanism involving an interaction between inflammatory damage and insufficient neurotrophin compensation. Moreover, inflammatory damage may interact with BNDF to influence clinical symptoms

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and cognitive impairment, which is consistent with the neurodevelopmental hypothesis of schizophrenia. However, due to the cross-sectional design of the current study, the causal relationships among immune dysfunction, the BDNF system, clinical symptoms, and cognitive impairment in schizophrenia are yet to be elucidated. Acknowledgments This study was funded by grants from the National Natural Science Foundation of China (81371477), the Beijing Municipal Natural Science Foundation (7132063 and 7072035), the NARSAD Independent Investigator Grant (20314), and the Stanley Medical Research Institute (03T-459 and 05T-726). These sources had no role in study design, the collection, analysis, and interpretation of data, the writing of the report, or the decision to submit the paper for publication. The authors would like to thank Ms Linda Cohen for her editorial revision of this manuscript. References Adachi, N., Numakawa, T., Richards, M., Nakajima, S., Kunugi, H., 2014. 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