Increased Interleukin-17 and decreased BAFF serum levels in drug-free acute schizophrenia

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Increased interleukin-17 and decreased BAFF serum levels in drug-free acute schizophrenia Yousri El Kissi, Samar Samoud, Ahlem Mtiraoui, Leila Letaief, Neila Hannachi, Mouna Ayachi, Bechir Ben Hadj Ali, Jalel Boukadida

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Cite this article as: Yousri El Kissi, Samar Samoud, Ahlem Mtiraoui, Leila Letaief, Neila Hannachi, Mouna Ayachi, Bechir Ben Hadj Ali, Jalel Boukadida, Increased interleukin-17 and decreased BAFF serum levels in drug-free acute schizophrenia, Psychiatry Research, http://dx.doi.org/10.1016/j.psychres.2014.10.007 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 galley proof before it is published in its final citable 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.

Increased interleukin-17 and decreased BAFF serum levels in drug-free acute schizophrenia

Authors:Yousri El Kissia, Samar Samoudb,c, Ahlem Mtiraouia, Leila Letaiefa, Neila Hannachib,c, Mouna Ayachia, Bechir Ben Hadj Ali a, Jalel Boukadidab,c.

a

Department of psychiatry, FarhatHached University Hospital, Ibn El Jazzar Street, Sousse 4000,

Tunisia b

Department of Microbiology and Immunology, FarhatHached University Hospital, Ibn El Jazzar

Street, Sousse 4000, Tunisia c

Research Unit (UR02SP13), FarhatHached University Hospital, Ibn El Jazzar Street, Sousse

4000, Tunisia

Corresponding and reprints author Yousri El Kissi Postal address: Department of psychiatry, FarhatHached University Hospital, Ibn El Jazzar Street, Sousse 4000, Tunisia E-mail: [email protected] Tel: (216) 73 221411; Fax: (216) 73 223702

Abstract Hypotheses regarding an immune-cytokine basis of schizophrenia have been postulated with controversial findings and a lack of data related to many cytokines. The aim of this study was to assess serum levels of Interferon-γ (IFN-γ), Interleukin-4 (IL-4), Transforming Growth Factor-β (TGF-β), Interleukin-17 (IL-17) and B-cell Activating Factor (BAFF) in schizophrenic patients and to determine correlations between cytokine levels and clinical parameters. Serum cytokine levels were measured with ELISA techniques in 60 neuroleptic-free patients on acute phase of the disease (BPRS≥40) and 28 healthy controls matched for age and sex. Current symptoms were assessed with Brief Psychiatric Rating Scale (BPRS), Positive and Negative Syndrome Scale (PANSS), Scale for the Assessment of Positive Symptoms (SAPS) and Scale for the Assessment of Negative Symptoms (SANS). No significant difference was found between patients and controls regarding IFN-γ serum levels. IL-4 was not detected in both groups. Patients exhibited significantly higher IL-17 and lower BAFF serum levels. IL-17 and BAFF levels were negatively correlated in schizophrenic patients. SANS global score was negatively correlated with IL-17 and positively correlated with IFN-γ serum levels. These results argue against the involvement of Th1 or Th2 population cells in schizophrenia. IL-17 and BAFF could be valuable markers for schizophrenia. Keywords: Schizophrenia; Immune; Cytokines; IL-17; BAFF

1. Introduction Despite decades of research, the underlying pathological mechanisms of schizophrenia remain to be elucidated. Along with the neurodeveloppemental hypothesis, the immune system dysregulation has widely been discussed as a major factor of the pathogenesis of the disease. The “cytokine hypothesis” stipulates that disturbed cytokine signaling plays a pivotal role in the pathophysiology of schizophrenia (Watanabe et al., 2010; Reale et al., 2011).The hallmarks of type1 and type 2 immune responses such as Interleukin-2 (IL-2), Tumor Necrosis factor-α (TNFα), IL-6, IL-4 and IL-10 have been extensively investigated but results are still controversial (Potvin et al, 2008; Watanabe et al., 2010; Miller et al., 2011). Some authors supported the role of Th1/Th2 imbalance in the development of schizophrenia (Muller et al., 1999; Shwarz et al., 2001). Others claimed the insufficiency of this model and incriminated a novel Th-cells category in the disease (Drexhage et al., 2011; Miller et al., 2011). Thus, it has been suggested that type-3 T-helper cells (Th3), through the production of a transforming growth factor TGF-β1, could suppress Th1 cytokines production such as Interferon-γ (IFN-γ), TNF-α, IL2 and IL2R, modulate the immune response and thus play a crucial role in schizophrenia (Tato and O’Shea, 2006; Gonzalez-Garcia et al.,2009). Most of the previous studies related to inflammation in schizophrenia highlighted the role of proinflammatory cytokines in the disease pathogenesis (Saetre et al., 2007; Soderlund et al., 2009). One of the leading pro-inflammatory cytokines, IL-17 has been proved to induce inflammatory mediators such as cytokines, chemokines, adhesion molecules and growth factors (Park et al., 2005; Gabay and McInnes, 2009; Hemdan et al., 2010) and considered as a mean mediator of the pathogenesis of several autoimmune and chronic inflammatory diseases (Steinman et al., 2007; Tzartos et al., 2008; Murdaca et al. 2011) mainly those with a neurological component such multiple sclerosis (Park et al.,2005). A close relationship between Th17 cells and B cellactivating factor belonging to the TNF family (BAFF) has been supported. BAFF may increase IL-6 receptor expression on CD4+ T cells, enhance IL6-IL-6R/p-STAT3 signaling pathway and promote the dendritic cell maturation and function (Lai et al., 2008). It has been reported that IL6/IL-6R signal pathway is crucial for Th17 cell differentiation (Zheng et al., 2008; Zhou et al., 2010) and would play a crucial role in the expansion of Th17 cells. IL-17 is also required to exert

its proinflammatory effects (Lai et al., 2008). As Th17 cells, BAFF has been directly implicated in several autoimmune and inflammatory conditions with neurological component such as Behcet's disease and systemic lupus (Krumbholz et al., 2005; George-Chandy et al., 2008; Hamzaoui et al., 2008). To the best of our knowledge, data related to cytokine profile in the acute phase of schizophrenia are still inconsistent. The role of Th17 cells and IL17 in the disease has poorly been investigated (Drexhage et al., 2011; Brovocanin et al., 2012; Dimitrov et al., 2013) and the possible implication of BAFF in the pathogenesis of the disease has never been considered. The mechanism by which immune system dysregulation may contribute to the development of schizophrenia symptoms is not yet elucidated. All together, these considerations enhance the importance of examining cytokine profile in acute schizophrenia as well as its probable impact on psychotic symptoms presentation. Since the duration of the disease and the therapeutic profile proved to exert different effects on cytokine production (Song et al. 2000; Hinze-Selch and Pollmacher, 2001), we proposed in our study to control for these confounding factors. The aims of our study were (1) to assess serum levels respectively of IFN-γ, IL-4, TGF-β and IL17 as type-1, type-2, type-3 and type-17 cytokines as well as BAFF levels in drug-free chronic schizophrenic patients in an acute phase of the disease and (2) to determine potential correlations between cytokine levels and clinical and psychopathological parameters. 2. Methods 2.1. Participants Sixty patients meeting the DSM-IV criteria of schizophrenia (APA, 1994) recently admitted in closed wards of the Department of Psychiatry, Farhat Hached Hospital of Sousse, Tunisia, were recruited. At the time of the enrollment, all of them were in an acute phase of the disease as defined by Brief Psychiatric Rating Scale scores (BPRS) ≥ 40. They were either medication naive or medication-free for at least three months. All subjects had a complete medical history, physical examination and laboratory testing if necessary. Patients having had a history of an autoimmune disease were excluded.

Patients were compared to 28 controls free from any psychotic or autoimmune disorder, matched for age and sex, enrolled among volunteer blood donors at Sahloul General Hospital of Sousse. Demographic and clinical characteristics of the two groups are listed in Table 1 and Table 2. The Institutional Review Hospital Board Committee approved the protocol. All participants gave informed consent after the procedure had been fully explained. 2.2. Assessment All participants were assessed by a trained clinical psychiatrist. Schizophrenia diagnosis was established by the Structured Clinical Interview for DSM-IV Axis I Disorders (SCID-I) (First et al., 2002).The Mini International Neuropsychiatric Interview (MINI-Plus) was used to rule out subjects with a history of psychotic disorders among controls (Sheehan et al., 1998). Demographic data, medical history and axis I comorbid disorders were collected. Physical examination and laboratory tests were obtained for all participants. Patients were assessed for psychopathology, severity of illness and global functioning using respectively the Brief Psychiatric Rating Scale (BPRS) (Overall and Gorham, 1962), the Positive and Negative Syndrome Scale (PANSS) (Daneluzzo et al., 2002), the Scale for the Assessment of Negative Symptoms (SANS) (Andreasen, 1983), the Scale for the Assessment of positive Symptoms (SAPS) (Andreasen, 1984) and the Global Assessment of Functioning (GAF). 2.3. Blood sampling and immunological measures For each study participant, blood sample was collected between 8.00 and 11.00 a.m. and was forwarded to the Department of Immunology of Farhat Hached Hospital for immunological analysis. Ten milliliters of fasting blood were withdrawn with an anticoagulant free vacuum tube and immediately centrifuged at 3800 rpm for 10 min. Serum was stored at - 80¢ªC until thawed for assay. The cytokine assays for human IL-17, IFN-γ, IL-12, TGF-β1 and BAFF were done using Duo Set ELISA Development System from R&D Systems, UK. IL-4 ELISA was established with the detection range from 3.0 to 1000 pg/ml using recommended buffers, diluents and substrates. The intra- and inter-assay coefficients of variation were 5.6% and 7.08%, respectively. IFN-γ ELISA

was established with the detection range from 2 to 500pg/ml in the same manner. The intra- and interassay coefficients of variation were 6.7% and 9.2%. TGF-β ELISA was established with the detection range of 50-2500 pg/ml and assay was performed on the 12 times diluted activated plasma samples. Activation of plasma samples with acidic solution is necessary since TGF-β1 is secreted mainly in latent forms (Miyazono et al., 1990). The intra- and inter-assay coefficients of variation were 5.3% and 9.8%. The concentrations of the samples in each plate were calculated according to each standard curve and the dilution factor. 2.4. Statistical analysis Study groups were compared for continuous covariates by a two-tailed t-test. For discrete covariates, study groups were compared by a chi-square test. The pre-treatment IL-4, IL-17, IFN-γ, TGF-β1, BAFF values of patient and control groups were compared by non parametric Mann-Whitney testing. Pearson’s product moment correlation coefficients were calculated to examine the relationships between the cytokine levels and clinical variables. The null hypothesis was rejected at p<0.05. The statistical package used for the analysis was SPSS 11.1. 3. Results 3.1. Serum measures 3.1.1. Circulating cytokine serum levels Results for assays of cytokines are summarized in Table 3. Levels of IL-4 were too low for detection and, for the purpose of the analysis, were given the value of 0. IFN-γ were detected in 40 schizophrenic patients (80%) and 21 controls (75%).TGFβ1 were detected in all patients and all control subjects. No difference was found between the two groups concerning these cytokine levels. IL-17 were detected in 46 schizophrenic patients (76.7%) and 15 controls (53.6%). Schizophrenic patients exhibited significantly higher levels compared to controls (p<10-3). BAFF were detected in all patients and controls. Serum levels were significantly lower in patients compared to controls (p<10-3).

3.1.2. Ratio of serum cytokines Owing to the lack of detection of IL-4, the ratio IFN-γ / IL-4 could not be calculated neither in schizophrenic patients nor in controls. There was no difference between patients and controls concerning IFN-γ / TGF-β ratio. 3.2. Correlations among cytokine serum levels Correlation analysis in schizophrenic patients’ group showed negative and significant relationship between BAFF and IL-17 (r=-0.27; p=0.03). No other correlations were found between cytokines levels neither in schizophrenic patients nor in controls. Data are shown in Table 4. 3.3. Correlations between cytokine serum levels and clinical data 3.3.1. Correlations between cytokine serum levels and clinical features There were no significant correlations between cytokine levels and any clinical variable including patients’ present age, age of illness onset, duration of the illness, duration of untreated psychosis, drug discontinuation period for previously treated patients and clinical presentation of the disease. There were no significant differences of cytokine levels among subgroups of patients with different disease subtypes or taking different medications. No difference was found between smokers and non-smokers. The storage time prior the assay was not correlated with the cytokine concentrations. 3.3.2. Correlations between cytokine serum levels and psychopathology scores The IL-17 level was negatively correlated with the SANS score (r=-0.27, p=0.03) (Fig.1) while the IFN-γ level was positively correlated with the SANS score (r=0.33, p=0.01) (Fig.2). No other significant associations were found between cytokines serum levels and psychopathology, severity of the illness and global functioning as measured by BPRS, PANSS, SAPS and SANS. 4. Discussion

In our study, levels of IL-4 were too low for detection in both groups of patients and controls enrolled. This cannot be explained by inadequate serum storage conditions or cytokine assays since the calibration curve using the results of the ELISA was valid and that other cytokines have been

detected

with

success.

Failure

to

detect

IL-4

would

argue

against

Th2

lymphocytes’activation in the untreated acute schizophrenia. In line with our results, previous researches had shown that IL-4 is less detectable in the acute phase of schizophrenia (Kim et al., 2009) and in case of treatment discontinuation (Avgustin et al., 2005; O’Brien et al., 2008) with a significant increase of serum levels after treatment administration (Avgustin et al., 2005; O’Brien et al. 2008; Kim et al, 2009). However, other studies did not replicate these findings. Some of them found no difference between schizophrenic patients and controls in type-2 immune response (Ruthermundt et al., 1998; Dimitrov et al., 2013). Increased IL-4 levels were shown in cerebrospinal fluid of juvenile schizophrenic patients (Mitteleman et al., 1997) and in serum of schizophrenic drug-free patients compared to first episode psychosis and healthy controls (Borovcanin et al., 2012). It is worth mentioning that in the latter study duration of treatment discontinuation was not specified. There was no difference between patients and controls regarding levels of IFN-γ in our study. Data in the literature are controversial. While some authors found higher levels of IFN-γ in drugnaive first-episode schizophrenia patients compared to healthy control subjects (Ding et al., 2014), others, similarly to us, failed to replicate this result in schizophrenic untreated patients in the acute phase of the disease (Kim et al., 2004). The Th1 hypothesis of schizophrenia may then be questioned. To better explore the cytokine imbalance in schizophrenia patients, it would have been interesting to examine the IFN-γ and IL-4 ratio that is Th1/Th2 ratio. A number of investigators have assessed this balance (Hornberg et al.,1995; Rothermundt et al., 1998; Cazzullo et al.,1998; 2001; Kim et al., 2000). Whether the shift towards Th1 or Th2 side is not confirmed yet, Schwarz et al. (2001) have hypothesized that Th2 overweighs in unmedicated schizophrenic patients. Owing to the lack of detection of IL-4 in our study, this ratio could not be calculated neither in schizophrenic patients nor in controls. Kim et al. (2004) showed that although no significant

difference between INF-. and IL-4 serum levels was found between the two groups, the INFγ/IL-4 ratio was significantly higher among schizophrenics compared to controls suggesting an increased inflammatory response associated with the acute phase of the illness. This result is consistent with O'Brien et al. (2008) findings of elevated TNF-α/IL-4 ratio. We found no difference between patients and controls neither in TGF-β serum levels nor in Th1/Th3 ratio. TGF-β does not seem to be a valuable marker of schizophrenia as stated previously (Kalkman, 2009; Borovcanin et al., 2012). A main finding of our study was the significant increase of IL-17 levels in schizophrenic patients compared to controls. To the best of our knowledge, only four researches managed to investigate the role of Th17 population cell and IL-17 in the pathogenesis of schizophrenia and results were inconsistent. Some authors reported as we did increased IL-17 levels in recent onset schizophrenia patients (Drexhage et al. 2011) as well as significantly higher proportion of Th17 cells in drug-free first-episode psychosis patients (Ding et al., 2013). Others failed to replicate these findings (Borovcanin et al., 2012; Dimitrov et al., 2013). Discrepancies may be essentially due to the variety of inclusion criteria. In fact, Dimitrov et al. (2013) assessed a group of chronic paranoid schizophrenia patients but did not control for antipsychotic treatment. Since it has been shown that antipsychotic drugs can modulate cytokines production activity (Maes et al., 1995; Song et al., 2000), the latter results may be considered cautiously. Borovcanin et al. (2012) considered a heterogeneous sample of drug-naive patients with first episode psychosis (n=88) or in an acute exacerbation of schizophrenia (n=45) and did not specify duration of treatment discontinuation before the essay. They concluded that type-17 immune response is blunted in schizophrenia. The same authors, based on the results of the experimental study of Himmerich et al. (2011), which showed a significant increase of IL-17 levels after Antipsychotics were added to a stimulated serum of healthy female subjects, suggested that antipsychotic drugs are responsible for the increase of IL- 17 concentrations. The finding of a higher IL-17 serum levels in drug-free schizophrenic patients confirms that this elevation is unlikely due to antipsychotic medication. We support that it might be related to the intrinsic profile of the disease. Increased IL-17 serum levels found in our study along with previous reports of increased IL-6 and TNF-α serum levels in schizophrenic patients (Vojdani, 2009) highlight the role of Th17

cells in the inflammatory process of schizophrenia. In fact, these lymphocytes seem to be the one cell population able to produce the three cytokines at once. The classic incrimination of Th17 cells in the genesis of an autoimmune response in addition to its pro-inflammatory actions would strongly support the hypothesis of an auto-inflammatory mechanism in schizophrenia. A close relationship between Th17 cells and Th3 regulatory cells that are rich in factor Foxp3 has been suggested (Gonzalez-Garcia et al., 2009).TGF-β would regulate the IL-17 synthesis by Th17 factor. Low levels of TGF-β and presence of IL-6 allow the differentiation to Th17 while over expression of TGF-β or absence of IL-6 lead to Th3 cells (Tato and O’Shea, 2006). In our study no correlations were found between IL-17 and TGF-β serum levels. As BAFF has been widely incriminated in neurological autoimmune diseases, it was of interest to study it in schizophrenia. Serum levels were significantly decreased in patients compared to control subjects. This result seems intriguing since Lai et al. (2008) postulated that BAFF cells intervenes in Th17 cells expansion and had demonstrated the importance of IL-17 as a key effect or of pro-inflammatory effects of BAFF. A negative correlation between IL-17 and BAFF serum levels has been established in our study. Such correlation has never been reported in vivo. Nevertheless, experimental studies focusing on the signal transduction pathways of IL-17 have already mentioned the existence of a negative correlation between BAFF and IL-17 through the incrimination of Act1 a transcription activator (Li et al., 2008).The Act1 is involved in the negative regulation of humoral immune response via the inhibition of the signaling pathway mediated by BAFF and CD40L. It promotes inflammatory immune response by stimulating signal transduction of IL-17. Thus the negative correlation between these two cytokines found in our study argues for a coordinated regulation probably spawned by the Act1. A further finding of the current study is the significant associations between SANS score and IL17 and IFN-γ serum levels. Data on associations between cytokine levels and psychopathology in the literature are scarce and controversial (Kim et al. 2000; Zhang et al., 2002; Xiu et al. 2012; Dimitrov et al., 2013; Hope et al., 2013). Even if they fail to provide clear pathophysiological explanations, they support the role of cytokines in the pathogenesis of schizophrenia. It has been suggested that structural abnormalities prevailed in the deficient form of schizophrenia (Andreasen, 1982; Ross et al., 2006) whereas dopaminergic dysfunction predominated in other

forms (Ross et al., 2006; Snyder, 2006). In our study, higher IL-17 levels were associated with less marked negative symptoms. In their recent study, Dimitrov et al. (2013) found an association between IL-17 pathway cytokines with positive but not negative symptoms. These findings support the assumption that increased levels of pro-inflammatory cytokines impair brain function (Miller et al., 2009). In our study, we also found that higher IFN-γ levels were associated with more pronounced negative symptoms. Inglot et al. (1994) observed that the patients with high IFN response to lipopolysaccharide or phytohemagglutinin stimulation had dominant positive symptoms whereas in patients with low IFN response, the negative symptoms prevailed. Our result appears to contradict previous assumptions. It can be explained by the pleiotropic role of inflammatory cytokines in the CNS which depends on the complex signals within their microenvironment (Na et al., 2012). The major strengths of this study include the use of restrictive inclusion criteria to limit confounding factors. A relative large sample size of schizophrenic patients in the acute phase of the disease was considered and the effect of antipsychotic medications was controlled. Some limitations have to be mentioned. We did not control for the body mass index, obesity being associated with a sub-clinical inflammation (Hotamisligil, 2006) and we did not collect the number of daily consumed cigarettes. A large variation in measured interleukin levels was observed in both groups reflecting the heterogeneity of study subjects. This could be due to the possible interactions of some confounding factors such as body mass index, current infections preceding the acute phase of the disease, ongoing treatment that could interfere with cytokine network such as anti-inflammatory or hormonal treatment. In summary, we showed blunted type-1, type-2 and type-3 immune response in drug-free acute schizophrenia. We provided evidence of increased production of IL-17 and decreased BAFF serum level with an inverse correlation between the two cytokines. These results may suggest that IL-17 and CD40-BAFF are implicated in schizophrenia. Their signaling pathways seem to be orchestrated differently. Further prospective studies are needed to investigate the role of these cytokines in schizophrenia.

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Correlation between IL-17 serum level and the SANS score

Correlation between Ln IL-17 serum levels and the SANS score

r= - 0.27 p= 0.03

90 80 70

SANS

60 50 40 30 20 10 0 0

1

2

3

4 Ln IL- 17

5

6

7

8

Ln: Cytokines concentrations have been log-transformed before being compared between patients and controls.

Fig.1. Schizophrenic patients showed significant negative associations between IL-17 and the SANS Score

Correlation between INF-
serum level and the SANS score

Correlation between Ln IFN-Gamma serum levels and the SANS score

90 80 70

r=0.33

60 SANS

p=0.01 50 40 30 20 10 0 0

1

2

3 Ln IFN-gamma

4

5

6

Ln: Cytokines concentrations have been log-transformed before being compared between patients and controls.

Fig.2. Schizophrenic patients showed significant positive associations between INF-
and the SANS Score

Highlights

Compared to healthy controls, drug-free schizophrenic patients in acute phase of the disease exhibited significantly higher IL-17 and lower BAFF serum levels. No significant difference was found between patients and controls regarding IFN-γ serum levels. IL-4 was not detected in both groups. IL-17 and BAFF levels were negatively correlated in schizophrenic patients. SANS global score was negatively correlated with IL-17 and positively correlated with IFN-γ serum levels. These results argue against the involvement of Th1 or Th2 population cells in schizophrenia. IL-17 and BAFF could be valuable markers for schizophrenia.

Table 1.Demographic data in schizophrenic patients and controls

Demographic characteristics Age(years) Sex: male (n, %)

Schizophrenic patients (n=60)

Control subjects (n=28)

Test value

p value

37.2 ± 11.6

35.7 ± 11.3

0.36

0.71

34 (56.7)

16 (57.1)

0.28

0.45

Instruction level (n, %) Illiterate /Primary

32 (53.3)

10 (35.7) 3.65

Secondary/ University

28 (46.7)

0.30

18 (64.3)

Marital status, n (%) Married/have been married Single Smoking habits, n (%)

21 (35.0)

15 (53.6)

39 (65.0)

13 (46.4)

19 (31.7)

6 (21.4)

15.65

< 10-3

8,72

0,18

Table 2.Clinical characteristics and Psychopathology measures in schizophrenic patients Clinical characteristics

Schizophrenic patients (n=60)

Age of onset of the disease (years)

24.4 ± 6.7

Duration of illness (years)

12.7 ± 9.8

Subtype, n (%) Undifferentiated subtype Paranoid subtype Disorganized subtype

35 (58.3) 9 (15) 16 (26.7)

Medication status on admission, n(%) Medication-naive

10 (16.7)

Medication-free

50 (83.3)

Data of standardized assessment BPRS

53.42 ± 6.14

PANSS Global score

78.08 ± 13.52

Positive subscale

21.88 ± 5.41

Negative subscale

18.80 ± 6.35

General psychopathology

37.40 ± 4.97

SAPS

36.93 ± 13.21

SANS

36.57 ± 15.94

GAF

36.67 ± 5.49

Table 3.IL-4, IFN-γ, TGF-β1, IL-17 and BAFF serum levels in schizophrenic patients and controls Cytokine

p

Schizophrenic patients

Control subjects

(n=60)

(n=28)

Mean ± SD

Mean ± SD

not detected

not detected

-

-

IFN-γ (pg/ml)

24.55 ± 20.38

23.03 ± 16.85

0.34

0.73

BAFF (pg/ml)

743.66 ± 253.39

1037.14 ± 339.74

- 4.52

<10-3

Median (25th, 75th quartile)

Median (25th, 75th quartile)

7140.00 (3601.00, 10821.00)

4580.00 (1590.00, 10590.00)

0.47

0.22

IL-17 (pg/ml)

110,00 (52.50, 215.00)

40.00 (0.00, 53,75)

2.89

<10-3

IFN-γ/ TGF- β1

0.0031 (0.0014, 0.0076)

0.0051 (0.0010, 0.0159)

-1.51

0.58

concentrations

IL-4 (pg/ml)

TGF- β1 (ng/ml)

t or U

value

Kolmogorov-Smirnov non-parametric test was used to find out whether data is normally distributed or not. Statistical analysis proved that IL-4 and IFN-γ follow a normal distribution while TGF-β, IL-17 and IFN-γ/TGF-β do not. The latter variables were compared by non parametric Mann-Whitney U test.

Table 4.Correlations between cytokine serum levels

Cytokines

INF-γγ

TGF-β β

IL-17

BAFF

INF-γγ

r=1

r = -0,12 p = 0,25

r = -0,19 p = 0,06

r = -0,05 p = 0,61

TGF-β β

r = -0,12 p = 0,25

r=1

r = 0,08 p = 0,42

r = 0,06 p = 0,56

IL-17

r = -0,19 p = 0,06

r = 0,08 p = 0,42

r=1

r = -0,27 p = 0,03

BAFF

r = -0,05 p = 0,61

r = 0,06 p = 0,56

r = -0,27 p = 0,03

r=1