Serum cytokine profile in schizophrenic patients

Abstract

Schizophrenia is a chronic severe mental disorder in which immunologic imbalances have been reported. Some researchers have demonstrated dysregulation of cytokine levels in this mental disorder. Considering the possible role of cytokines in the pathogenesis or clinical course of schizophrenia, we assessed serum levels of IL-4, IL-10, IL-17A and IFN- $\gamma$ in a homogenous sample of Iranian patients who were in remission under treatment with Clozapine compared with sex and age matched controls. There was a statistically significant difference in IL-4 levels between patients and healthy subjects ( $P=$ 0.023). IL-4 levels were 83% higher in cases than in control group. No significant group*gender interaction was detected suggesting the similar IL-4 levels between males and females in each study group. There was no statistically significant difference in IFN- $\gamma$ levels between patients and healthy subjects, but IFN- $\gamma$ levels were 70% higher in male compared with female group. No significant differences have been found in serum levels of other cytokines between cases and controls or between males and females. The higher levels of IL-4 in patients treated with Clozapine might be due to effects of this antipsychotic drug on immune responses. Future studies are needed to elaborate the exact underlying mechanism.

Keywords

Schizophrenia IL-4 IL-10 IL-17A IFN-γ

1. Introduction

Schizophrenia is a chronic severe mental disorder with immense global burden [1]. There are several studies indicating the role of prenatal events such as prenatal maternal infections, low birth weight, and high paternal age in the susceptibility to schizophrenia which totally imply the presence of an extensive defect in schizophrenic patients rather than the existence of the sole psychosis [2]. This speculation has been supported by the observation of global cognitive defects through all performance domains in schizophrenic patients [3]. Consistent with the rapid growth of knowledge about the role of immunologic factors in the development of chronic neurologic disorders [4, 5, 6], the contribution of immune dysregulation in the pathogenesis of schizophrenia is being elaborated [7]. Considering the role of serum cytokines in regulation of different aspects of immune responses, researchers have focused on evaluation of serum cytokine levels in schizophrenic patients compared with healthy subjects. Maes et al. have reported elevated IL-6 levels in schizophrenic patients compared with healthy subjects regardless of their response to typical antipsychotics and elevated levels of serum IL-1RA in the treatment-resistant schizophrenia (TRS) patients compared with controls so suggested induction of the monocytic arm of cell-mediated immunity as a feature of schizophrenia especially TRS patients [8]. Besides, Lee et al. showed higher levels of TNF- $\alpha$ and IL-6 but not IFN- $\gamma$ in schizophrenic patients compared with normal controls [9]. However, Simsek et al. did not find any significant difference between adolescent schizophrenic patients and healthy subjects in terms of IL-2, IL-4, IL-6, IL-10, IL-17A, TNF- $\alpha$ , and IFN- $\gamma$ levels [10]. Considering the possible role of cytokines in the pathogenesis or clinical course of schizophrenia, we assessed serum levels of IL-4, IL-10, IL-17A and IFN- $\gamma$ in a homogenous sample of Iranian patients who were in remission under treatment with Clozapine™ compared with sex and age matched controls.

2. Material and methods

2.1 Study participants

This case-control study included 38 schizophrenic patients and 38 age and sex matched healthy subjects. Patients were selected from psychology department of Hamadan University of Medical Sciences. Patients were diagnosed based on the Diagnostic and Statistical Manual of Mental Disorders, fifth edition (DSM-V) criteria for schizophrenia [11] and were under treatment with Clozapine™. A structured psychiatric interview was used for evaluation of control subjects to rule out the presence of any psychiatric disorder. All persons with cancer, recent or persistent infectious disorder, autoimmune disorders, nerve muscle coupling disorders and pregnancy were excluded from the study. Ethics approval for the study was obtained from Ethical Committee of Hamadan University of Medical Sciences. Informed written consents were obtained from all study participants.

2.2 Cytokine detection

Peripheral venous blood samples were collected from schizophrenic patients and normal controls. Serum samples were collected through centrifugation for 10 min at 3000 r/min at 4 ${}^{\circ}$ C. Commercially available ELISA kits (Boster Biological Technology, Ltd, Pleasanton, CA, USA) were used for determination of serum cytokine levels (IL-4, IL-10, IL-17A and IFN- $\gamma$ ) based on the manufacturer’s protocol on Infinite M200 (Tecan, Männedorf, Switzerland) microplate reader. Assessments of each cytokine level in every sample were performed in duplicate as stated in the protocol. The smallest measurable cytokine levels in each ELISA kit was 1.5, 0.5, 10 and 2 pg/mL for IL-4, IL-10, IL-17A and IFN- $\gamma$ respectively with intrassay and interassay coefficients of variation being less than 10% in each analysis.

2.3 Statistical analysis

To describe the data, we presented median and Interquartile [IQR] for the continuous variables and frequencies and percentages for the categorical variables. The Quantile regression was used to compare non-normal dependent variables between the case and control groups with adjusting the effect of gender. For Quantile regression analysis ‘quantreg’ package was used. All statistical analyses were conducted in R 3.4.3 Statistical Software.

Table 1
All patients were in remission. None of the study participants were smoker

Variables	Schizophrenia patients	Controls
Female/Male [No. (%)]	14 (37%)/24 (63%)	13 (35%)/25 (65%)
Age (mean $\pm$ SD, Y)	49.4 $\pm$ 3.6	47.6 $\pm$ 3.5
Age range (Y)	30–64	29–59
Age at onset (Y)	34 $\pm$ 1.6	–
Years of illness	9 $\pm$ 0.04	–

3. Results

The study included 38 out-patients with schizophrenia (24 males and 14 females, mean age $\pm$ SD: 49.4 $\pm$ 3.6 years) and 38 healthy subject (25 males and 13 females, mean age $\pm$ SD: 47.6 $\pm$ 3.5 years). Demographic and clinical characteristics of study participants are described in Table 1. All patients were in remission. None of the study participants were smoker.

Table 2
Descriptive statistics results of serum IL-4 levels in schizophrenic patients and healthy subjects

	Number	Mean	SD	Median	[IQR]	$P$ -value ${}^{\text{a}}$
		(pg/mL)
Total
Case	38	81.39	113.41	56.5	[29, 86.7]	0.081
Control	38	122.76	259.7	31	[22, 93.75]
Male
Case	24	93.75	140.02	56.5	[31.5, 106.25]	0.192
Control	25	145.48	313.94	28	[22, 94.5]
Female
Case	14	60.21	33.9	51	[28.75, 86.75]	0.514
Control	13	70.08	87.84	38	[25, 121.5]

${}^{\text{a}}$ Based on univariate median test between two independent group.

3.1 Comparison of IL-4 levels between schizophrenic patients and healthy subjects

We assessed Il-4 levels between cases and controls and within sex-based subgroups (Table 2 and Fig. 1).

Table 3
The median regression results for comparison of Ln (IL-4) between case and control groups

Variable	Coefficient	SE	$t$	$P$ -value	95% confidence
					interval ${}^{\text{a}}$
Group	0.606	0.26	2.32	0.023	[0.09, 1.13]
Gender	0.095	0.27	0.35	0.728	[ $-$ 0.45, 0.64]
Group*Gender	$-$ 0.288	0.57	$-$ 0.51	0.612	[ $-$ 1.41, 0.83]

${}^{\text{a}}$ Estimations are based on main effects, the group*gender interaction removed from final model.

Table 4

Descriptive statistics results of serum IL-10 levels in schizophrenic patients and healthy subjects

	Number	Mean	SD	Median	[IQR]	$P$ value
		(pg/mL)
Total
Case	38	81.39	113.41	56.5	[20, 40.25]	0.54
Control	38	122.76	259.7	31	[23.75, 95.5]
Male
Case	24	93.75	140.02	56.5	[18, 37.25]	0.648
Control	25	145.48	313.94	28	[21.5, 97]
Female
Case	14	60.21	33.9	51	[20.7, 50]	0.896
Control	13	70.08	87.84	38	[25.5, 84]

Figure 1.

Box-and-scatter plot of IL-4 level by group and gender.

Figure 2.

Box-and-scatter plot of IL-10 level by group and gender.

After using Quantile regression to control the effect of sex on relative IL-4 levels, there was a statistically significant difference in IL-4 levels between patients and healthy subjects ( $P=$ 0.023). The expected median difference in IL-4 level in case and control groups after adjusting for sex was $\exp$ (0.606) $=$ 1.833 which means IL-4 levels were 83% higher in cases than in control group. No significant group*gender interaction was detected suggesting the similar IL-4 levels between males and females in each study group (Table 3).

3.2 Comparison of IL-10 levels between schizophrenic patients and healthy subjects

We assessed IL-10 levels between cases and controls and within sex-based subgroups (Table 4 and Fig. 2).

Table 5
The Quantile regression results for comparison of Ln (IL-10) between case and control groups

Variable	Coefficient	SE	$t$	$P$ -value	95% confidence
					interval
Group	$-$ 0.223	0.32	$-$ 0.70	0.489	[ $-$ 0.86, 0.42]
Gender	0.0001	0.38	0	$>$ 0.999	[ $-$ 0.77, 0.77]
Group*Gender	0.154	0.54	0.29	0.776	[ $-$ 0.92, 1.23]

Table 6

Descriptive statistics results of serum IL-17 levels in schizophrenic patients and healthy subjects

	Number	Mean	SD	Median	[IQR]	$P$ value
		(pg/mL)
Total
Case	38	81.39	113.41	56.5	[93.2, 213.2]	0.447
Control	38	122.76	259.7	31	[72.7, 198.7]
Male
Case	24	93.75	140.02	56.5	[98.7, 248]	0.443
Control	25	145.48	313.94	28	[73.5, 213.5]
Female
Case	14	60.21	33.9	51	[89.7, 164.5]	0.481
Control	13	70.08	87.84	38	[54, 165.5]

Table 7

The Quantile regression results for comparison of Ln (IL-17) between case and control groups

Variable	Coefficient	SE	$t$	$P$ -value	95% confidence
					interval
Group	0.255	0.195	1.31	0.195	[ $-$ 0.13, 0.64]
Gender	$-$ 0.262	0.204	$-$ 1.29	0.202	[ $-$ 0.67, 0.14]
Group*Gender	0.033	0.424	0.08	0.938	[ $-$ 0.81, 0.87]

${}^{\text{a}}$ Estimations are based on main effects, the group*gender interaction removed from final model.

After using Quantile regression to examine the effect of age and sex on relative IL-10 levels, there was no statistically significant difference in IL-10 levels between patients and healthy subjects or between males and females (Table 5).

Table 8

Descriptive statistics results of serum IFN- $\gamma$ levels in schizophrenic patients and healthy subjects

	Number	Mean	SD	Median	[IQR]	$P$ value
		(pg/mL)
Total
Case	38	81.39	113.41	56.5	[24, 57.2]	0.862
Control	38	122.76	259.7	31	[22, 49.5]
Male
Case	24	93.75	140.02	56.5	[24, 57.7]	0.856
Control	25	145.48	313.94	28	[21.5, 33.5]
Female
Case	14	60.21	33.9	51	[24.5, 53.2]	0.401
Control	13	70.08	87.84	38	[25, 102.5]

${}^{\text{a}}$ Estimations are based on main effects, the group*gender interaction removed from final model.

Table 9

The Quantile regression results for comparison of Ln (IFN- $\gamma$ ) between case and control groups

Variable	Coefficient	SE	$t$	$P$ -value	95% confidence
					interval
Group	0.071	0.219	0.330	0.746	[ $-$ 0.36, 0.5]
Gender	0.533	0.263	2.030	0.046	[0.009, 1.05]
Group*Gender	$-$ 0.604	0.368	$-$ 1.640	0.105	[ $-$ 1.33, 0.13]

${}^{\text{a}}$ Estimations are based on main effects, the group*gender interaction removed from final model.

Figure 3.

Box-and-scatter plot of IL17 level by group and gender.

Figure 4.

Box-and-scatter plot of IFN- $\gamma$ level by group and gender.

3.3 Comparison of IL-17 levels between schizophrenic patients and healthy subjects

We assessed IL-17 levels between cases and controls and within sex-based subgroups (Table 6 and Fig. 3).

After using Quantile regression to examine the effect of age and sex on relative IL-17 levels, there was no statistically significant difference in IL-17 levels between patients and healthy subjects or between males and females (Table 7).

3.4 Comparison of IFN- $\gamma$ levels between schizophrenic patients and healthy subjects

We assessed IFN- $\gamma$ levels between cases and controls and within sex-based subgroups (Table 8 and Fig. 4).

Figure 5.

Correlation analysis between IL-4 and IL-17 levels.

Figure 6.

Correlation analysis between IL-4 and IL-10 levels.

Figure 7.

Correlation analysis between IL-4 and IFN- $\gamma$ levels.

Figure 8.

Correlation analysis between IL-10 and IL-17 levels.

After using Quantile regression to examine the effect of age and sex on relative IFN- $\gamma$ levels, there was no statistically significant difference in IFN- $\gamma$ levels between patients and healthy subjects. But a significant difference has been found between IFN- $\gamma$ levels in male and female subjects ( $P=$ 0.046). The expected median difference in IFN- $\gamma$ level for males and females after adjustment of other variables was $\exp$ (0.533) $=$ 1.704 which means IFN- $\gamma$ levels were 70% higher in male compared with female group (Table 9).

3.5 Correlation analysis between the serum levels of cytokines

No correlation was found between Ln values of cytokine levels after application of Spearman correlation analysis (Figs 5–10).

4. Discussion

Cytokines as essential signaling proteins of the immune system participate in immune regulation both in the periphery and the brain [2]. So evaluation of their serum levels is helpful in identification of their role in the pathogenesis of psychiatric disorders as well. Significant associations have been found between cytokine aberrations and acute worsening of schizophrenia in an independent manner from antipsychotic treatment modalities. IL-1 $\beta$ , IL-6, and TGF- $\beta$ have been suggested as indicators of acute exacerbations, while IL-12, IFN- $\gamma$ , TNF- $\alpha$ , and sIL-2R might predict disease-associated symptoms and signs [2].

Figure 9.

Correlation analysis between IL-17 and IFN- $\gamma$ levels.

Figure 10.

Correlation analysis between IL-10 and IFN- $\gamma$ levels.

In the present study we evaluated cytokine levels in schizophrenic out-patients who were responsive to Clozapine. To control the effects of smoking on cytokine levels which is one of the main pitfalls of the previous studies [2], none of our study cohort smoke during the recent 6 months prior to sampling. We detected significantly higher levels of IL-4 in patients compared with healthy subjects. Serum IL-4 levels have been correlated with negative symptoms of schizophrenia in a population of drug-naive juveniles with first episode early onset disease [10]. Another study which assessed the serum concentrations of cytokines in drug-naive patients with first episode psychosis (FEP) and patients with schizophrenia relapse detected higher levels of IL-4 in serum of patients in relapse compared to controls and patients with FEP [12]. Our result is in agreement with the latter study.

As all patients assessed in the current study were treated with Clozapine™, we cannot exclude the effects of this antipsychotic treatment on cytokine levels. In vitro treatment of peripheral blood mononuclear cell (PBMC) obtained from patients with FEP clozapine has previously shown the effects of this drug on elevation of the IL-4 and IL-10 levels while reduction of IFN- $\gamma$ levels [13]. However, in the present study, we did not detect any significant difference in the levels of cytokines other than IL-4 between cases and controls to evaluate the results of the mentioned in vitro study. Although in vitro studies are valuable in identification of the underlying mechanisms of observed clinical or biochemical abnormalities in patients, they inevitably suffer from pitfalls resulting from unappreciation of several unknown factors which are present in whole body and contribute in disease process.

We also detected higher IFN- $\gamma$ levels in female subjects compared with male subjects. The gender-dependent expression pattern of IFN- $\gamma$ has been described previously. IFN- $\gamma$ promoter is the subject of regulation by 17 $\beta$ -estradiol (E2) [14]. Moreover, animal studies have shown higher IFN- $\gamma$ expression and release in female animals compared with males during infection [15]. Besides, evidences support the role of IFN- $\gamma$ in the etiology of the gender-specific dissimilarities in the immune system [16].

We failed to detect any significant correlation between the levels of assessed cytokines in our cohort. Considering the relative number of study participants this negative result should be interpreted with caution.

In brief, in the present study we detected higher levels of IL-4 in schizophrenic patients treated with Clozapine compared with healthy subjects. However, none of the other assessed cytokines showed different expression pattern between cases and controls which might be due to the effects of Clozapine in returning aberrant cytokine levels to their normal values. The observed similar levels of these cytokines between cases and controls in our study might also been explained by the stable status of patients assessed in the current study. A meta-analysis of data obtained from stable medicated schizophrenic out-patients has previously revealed no significant differences in blood IL-6 levels between these patients and control subjects in spite of significantly higher levels of this cytokine in acutely relapsed patients as well as patients in FEP compared with healthy subjects [2]. Future studies are needed to compare cytokine levels in a large cohort of patients including FEP, acutely relapsed in-patients and stable medicated schizophrenic out-patients with controlling confounding parameters such as smoking to elaborate the effects of cytokine imbalances in the development of schizophrenia or its clinical course.

Footnotes

Acknowledgments

The current study was supported by a grant from Hamadan University of Medical Sciences (Grant Number: 9507134243).

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Serum cytokine profile in schizophrenic patients

Abstract

Keywords

1. Introduction

2. Material and methods

2.1 Study participants

2.2 Cytokine detection

2.3 Statistical analysis

Table 1 All patients were in remission. None of the study participants were smoker

Table 2 Descriptive statistics results of serum IL-4 levels in schizophrenic patients and healthy subjects

Table 3 The median regression results for comparison of Ln (IL-4) between case and control groups

Table 5 The Quantile regression results for comparison of Ln (IL-10) between case and control groups

3.4 Comparison of IFN- γ levels between schizophrenic patients and healthy subjects

4. Discussion

Footnotes

Acknowledgments

References

Table 1
All patients were in remission. None of the study participants were smoker

Table 2
Descriptive statistics results of serum IL-4 levels in schizophrenic patients and healthy subjects

Table 3
The median regression results for comparison of Ln (IL-4) between case and control groups

Table 5
The Quantile regression results for comparison of Ln (IL-10) between case and control groups

3.4 Comparison of IFN- $\gamma$ levels between schizophrenic patients and healthy subjects