Abstract
Increasing evidence has shown that the immune system is involved in the schizophrenia development, with alterations in immune cell reactivity being one possible factor contributing to its pathogenesis. The purpose of the study was to evaluate in vitro the capability of peripheral blood mononuclear cells (PBMCs) obtained from subjects with schizophrenia and controls to engage in spontaneous and phytohemagglutinin (PHA)-stimulated cytokine production. The concentrations of various cytokines (interleukin (IL)-1β, IL-17A, tumor necrosis factor (TNF), interferon (IFN)-γ and IL-10) in supernatants from cultured PBMCs were measured using the cytometric bead array. No significant differences in the spontaneous production of IL-1β, IL-17A, IFN-γ and IL-10 by PBMCs were detected between individuals with schizophrenia and controls. TNF synthesis by PBMCs was found to be lower among those with schizophrenia. In all subjects and controls, greater cytokine generation was associated with PBMCs treated with PHA compared with those that were not. The PBMCs from people with schizophrenia displayed considerably higher sensitivity to mitogen stimulation, as the production of IL-17A, TNF and IFN-γ was at least threefold of that observed in healthy subjects, which may be driven by antipsychotics taken by patients with schizophrenia. Correlation was observed between spontaneous production of IFN-γ and Positive and Negative Syndrome Scale G subscore (which measures the general symptoms of schizophrenia) and between PHA-stimulated synthesis of IL–17A and G subscore. Our data confirm that the immune system dysregulation may underlie schizophrenia pathophysiology. There is a potential possibility that immunological tests could be used as a diagnostic, therapeutic and side-effects biomarker for schizophrenia, but further studies are needed.
Significance of this study
What is already known about this subject?
An accumulating body of data indicates that immunological alterations take place in people with schizophrenia.
There is information that dysregulation of cytokine/chemokine activity significantly contributes to the etiopathology of schizophrenia.
There are few ambiguous reports on cytokine production by cultured peripheral blood mononuclear cells (PBMCs) in schizophrenia.
What are the new findings?
We present the first study to analyze in vitro cytokine synthesis of PBMCs from people with schizophrenia and those without, and which takes into account precisely measured anthropometric and body composition markers of obesity, which may have a potent impact on cytokine synthesis.
Our findings indicate that PBMCs from subjects with schizophrenia were significantly more sensitive to mitogen stimulation.
Correlations were found between synthesis of some cytokines and clinical symptoms of schizophrenia.
How might these results change the focus of research or clinical practice?
The present results provide support for the hypothesis that the immune mechanisms are dysregulated in the pathophysiology of schizophrenia and could potentially be used in the future as diagnostic and/or therapeutic biomarkers for schizophrenia.
Introduction
Schizophrenia affects roughly 1% of the global population and is associated with multidomain symptoms (psychotic, negative, cognitive, affective, locomotor and others).1 Its pathophysiology is believed to involve several mechanisms. Dysfunction of signaling in glutamatergic, serotonergic, dopaminergic and gamma-aminobutyric acid underlie the core schizophrenia pathophysiology.2 Although the role of signaling neurotransmitters in schizophrenia has been intensely studied, the precise underlying pathological mechanisms remain unclear. There is some evidence that dysbiosis of the intestinal microbiota may lead to epithelial damage, the development of low-grade inflammation within the intestinal barrier and an increase in gut permeability. This may result in the translocation of microbial components into the circulation, which could play a role in the etiology of schizophrenia.3 Increased serum concentration of soluble CD14, known as intestinal bacterial translocation marker, and of IgG antibodies to Saccharomyces cerevisiae—an intestinal inflammation marker, has been reported in schizophrenia.4 5 There are also suggestions that exposure to Toxoplasma gondii has been associated with the development of schizophrenia.5 6
There is increasing body of evidence that the etiopathogenetic of schizophrenia may be driven by dysregulations in the immune mechanisms, as indicated by reports on altered number and activity of immune cells in schizophrenia, for example, increased the neutrophil-lymphocyte ratio or number of leukocytes and neutrophils in schizophrenia subjects.7 Additionally, higher number of monocytes in people with schizophrenia was noticed.7–9 However, Theodoropoulou et al 10 observed no differences in monocyte percentages between individuals with schizophrenia and healthy volunteers. Studies have suggested that subjects with schizophrenia have an increased mean percentage of B cells.8 9 11 Maino et al 11 reported significantly lower levels of T cells among individuals with schizophrenia; in turn, Theodoropoulou et al 10 found the percentages of T cells not to significantly differ between people with schizophrenia and healthy volunteers. Other studies have also documented elevated numbers of T helper (Th)17 cells and natural killer (NK) cells in subjects with schizophrenia.9 12 In turn, Karpiński et al 13 reported significantly lower levels of CD8 cells and NK cells and higher levels of granulocytes in people with schizophrenia.
A growing body of evidence indicates that the levels of some humoral factors involved in the immune response are also changed in the course of schizophrenia. Serum concentrations of interleukin (IL)-1β, tumor necrosis factor (TNF), IL-6, interferon (IFN)-γ and C reactive protein (CRP) have been found to be significantly higher in individuals with schizophrenia than healthy subjects,10 14–17 while conversely, IL-17 levels have also been found to be significantly lower in those with schizophrenia.12 18 Some studies have reported significant decreases in IL-10 level in people with schizophrenia compared with healthy volunteers.19 20 On the other hand, Frayyeh and Jawad El-Saffar21 observed a significant increase of IL-10 serum levels in individuals with schizophrenia. Our previous observations have indicated that subjects with schizophrenia display significantly lower serum levels of cathelicidin LL-37, which plays a role in antibacterial defense and exerts pro-inflammatory effects.22 Likewise, changes in toll-like receptor (TLR) expression, molecules taking part in pathogen-associated molecular pattern and danger-associated molecular pattern recognition have been noted in people with schizophrenia. Müller et al 23 have shown that expression of TLR3 and TLR4 on monocytes is significantly higher in people with schizophrenia. On the other hand, no changes in TLR1, TLR2, TLR4, TLR6 or TLR9 and downregulation of TLR3 and TLR5 expression in monocytes from people with schizophrenia was also reported.24
Some studies suggest that the reactivity of peripheral blood mononuclear cells (PBMCs) may be altered in the course of schizophrenia.25–30 It is clear that PBMCs represent the main sources of cytokines found in plasma and/or serum. Hence, the present study evaluates the in vitro capability of PBMCs to take part in spontaneous and phytohemagglutinin (PHA)-stimulated cytokine production in individuals with schizophrenia. As the circulating cytokines found in serum or plasma can be produced by a broad range of cells including endothelial and epithelial cells, adipocytes and fibroblasts, as well as immune cells, the present study measures the in vitro secretion of cytokines by PBMCs, which may better reflect the potential of cytokines to influence inflammation.
The idea that schizophrenia may be associated with inflammation is not new. It must be emphasized that measurements of inflammatory and immune parameters in mental disorders must be considered in the context of metabolic parameters and body composition. Obesity is more prevalent in the population of patients with psychiatric disorders, and might change immune parameters. Ferreira-Hermosillo et al 31 indicated no differences in serum levels of IL-6, IL-10 and TNF in subjects with metabolic syndrome; however, the subjects displayed higher serum concentrations of CXCL8. Dicker et al 32 reported that PBMCs from subjects with obesity show increased production of IL-2, TNF-α and IFN-γ and lower amount of IL-10 compared with those from participants with normal weight. Likewise, Sirota et al 33 documented significantly higher secretion of TNF and IL-1β by PBMC among obese individuals than non-obese participants; however, obese individuals produced smaller amounts of IL-2. Concentration of cytokines may be affected by cardiometabolic parameters. Therefore, the analysis included biochemical and anthropometric measurements combined with a body composition measurements.34 This is the first analysis of such parameters in individuals with schizophrenia.
Materials and methods
Subjects
This as was a cross-sectional, prospective study. Twenty-seven adult (aged 18–60 years) European Caucasian outpatients with paranoid schizophrenia (confirmed using structured interview based on ICD-10 criteria). Twenty-seven randomly selected healthy participants were also included as the control group. For the control group, exclusion criteria included personal or familial psychiatric history (both diagnosis and/or treatment). For all subjects, exclusion criteria also included immunological disorders (eg, allergies, asthma, HIV), inflammatory conditions (both acute and chronic) and any systemic diseases or cancer. All study subjects were physically, neurologically and endocrinologically healthy, which was conformed using standard tests: physical examination, complete blood count, CRP, liver and renal tests (aminotransferases, bilirubin, urea, creatinine) and blood electrolytes. All subjects included in the study has been informed about both methods and the aims of the study and they have expressed their informed consent for participation in this study.
Clinical assessments
We have assessed four major domains of schizophrenia symptoms using standard tools: Positive and Negative Syndrome Scale (PANSS; with its three subscores: positive, negative and general symptoms) and Calgary Depression Scale for Schizophrenia (CDSS).35 Each patient was scored by the same, trained rater.
Blood collection and isolation of PBMCs
Between 08:00 and 09:00 hours after at least an 8-hour overnight fast, blood samples were collected. To isolate PBMCs from whole blood by density gradient centrifugation Histopaque-1077 (Sigma-Aldrich, St Louis, Missouri, USA) was used. Centrifuged buffy coat (centrifugation at 400 g for 30 min at room temperature) was transferred into a clean conical centrifuge tube, and washed (three times) in 1x phosphate buffered saline (Sigma-Aldrich). We used trypan blue and a Bürker chamber as a traditional method in order to measure the viability and concentration of isolated PBMCs.
Cell culture
The PBMCs were suspended at a concentration of 106 cells per mL in RPMI-1640 supplemented with 1% penicillin/streptomycin (Sigma-Aldrich), 2 mM L-glutamate (Gibco, Waltham, Massachusetts, USA) and 10% heat-inactivated fetal bovine serum (Gibco) in 48-well sterile (non-pyrogenic) polystyrene flat bottom plates (Corning, Tewksbury, Massachusetts, USA) in the presence or absence of 5 µg/mL PHA (Sigma-Aldrich). PBMCs were cultured in a humidified atmosphere of 5% CO2 at 37°C. After 72 hours cultured, the cell-free supernatants were collected and frozen in aliquots at −80°C.
Cytokine measurements
To measure cytokine concentrations in supernatants from cultured PBMCs, the Cytometric Bead Array kits (BD Biosciences, San Diego, California, USA) according to the manufacturer’s protocol were used. Calibur flow cytometer (BD Biosciences) was used to analyze samples using BD FCAP Array Software (BD Biosciences). Results are expressed as pg/mL.
Anthropometry
Standard methods were used for anthropometric measurements (a wall-mounted height measure, Seca 955 (Seca, UK) digital chair scale, non-stretchable fiber measuring tape). Body mass index (BMI) was calculated as: body weight (kg)/height (m2). Waist-to-hip ratio (WHR) was calculated as WHR circumferences ratio.
Body composition
Dual-energy X-ray absorptiometry (DXA) was used for body composition analysis (Lunar iDXA scanner, GE Healthcare, UK). The procedure was done by a trained operator, prior to anthropometry measurements. Using DXA, we measured total body fat (TBF; expressed in kilograms and as a percentage of total body weight). Fat mass index (FMI) was calculated using standard equation (TBF (kg)/height (m2).
Statistical analysis
Statistical calculations were performed using Statistica V.13.1 (Statsoft, Tulsa, Oklahoma, USA). Simple descriptive statistics (means, SE of the means and SD) were generated for all continuous variables. Normality of distribution was tested with the Shapiro-Wilk test. The Wilcoxon signed-rank test was used to analyze differences in cytokine production in healthy controls and individuals with schizophrenia. The Mann-Whitney U test was performed to examine differences in cytokine production between healthy controls and individuals with schizophrenia. Correlations were tested using Pearson’s coefficient. The level of significance was set at p<0.05 (two-sided).
Results
Clinical and demographic data
In table 1, we detailed demographic and cardiometabolic parameters of the all study subjects.
Demographic, cardiometabolic and anthropometric parameters were comparable for both groups. Subjects with schizophrenia were chronically ill, as indicated by mean treatment duration (16.1±10.8 years), number of hospitalizations (10.3±11.9) and number of acute psychotic episodes (6.1±4.2). Mean time from the last hospitalization was 4.0±7.1 months. All patients were on antipsychotic therapy, with standard or above-standard doses (as indicated by mean defined daily dose for antipsychotics being 2.5±1.3, with an equivalent of 756.8±381.9 mg/day of chlorpromazine). Severity of schizophrenia symptoms was moderate (total PANSS score 65.4±14.6, P sub-score 15.3±5.1, N sub-score 18.0±4.5, G subscore 32.1±7.3), while severity of depression was low (CDSS score 1.9±1.9).
In vitro cytokine production by PBMCs
The in vitro production of IL-1β, IL-17A, TNF, IFN-γ and IL-10 by non-stimulated PBMCs from healthy controls and people with schizophrenia is shown in figure 1. It was found that the mean production of IL-1β, IL-17A, IFN-γ and IL-10 did not differ between subjects with schizophrenia and controls. In addition, individuals with schizophrenia demonstrated lower constitutive TNF production by PBMCs than healthy controls (p<0.05). It should be emphasized that constitutive in vitro synthesis of IL-17A, IFN-γ and TNF by PBMCs was low (up to 54, 24 and 16 pg/mL, respectively). IL-10 production by non-stimulated PBMCs was higher (up to 1500 pg/mL) and IL-1β production was the highest, reaching up to 9000 pg/mL.
Figure 2 shows the levels of IL-1β, IL-17A, TNF, IFN-γ and IL-10 in cell-free supernatants of PHA-stimulated PBMCs from subjects with schizophrenia and healthy volunteers. For IL-1β, IL-17A, TNF and IL-10 production, no differences between the two studied groups were found. The mean in vitro production of IFN-γ by PBMCs in response to mitogenic stimulation was significantly higher (p<0.05) in people with schizophrenia than in healthy patients. It should be noted that the levels of PHA-stimulated synthesis of IL-17A and IFN-γ were low (up to 100 and 110 pg/mL, respectively), and TNF and IL-10 production was found to be 3400 and 3900 pg/mL, respectively. IL-1β was found to demonstrate the highest in vitro secretion by PBMCs after stimulation with PHA (16 000 pg/mL).
Both in healthy controls and in subjects with schizophrenia, the in vitro production of all presented cytokines by PBMCs was significantly higher in cells stimulated with PHA than in non-stimulated cells (table 2). However, the increase in IL-17A, TNF and IFN-γ secretion by mitogen-stimulated PBMCs was at least threefold higher in subjects with schizophrenia than in healthy controls (table 2).
Correlations
A very strong, positive correlation was observed between IL-1β and TNF, as well as strong positive correlations between IL-1β and IL-10, IL-17A and IFN-γ; as well as between IL-10 and TNF production by non-stimulated PBMCs from healthy controls. Likewise, moderate positive correlations were found between IL-1β and TNF, IL-17A and IL-10, as well as between TNF and IL-10 production, by PHA-stimulated PBMCs in healthy subjects (table 3).
Within the schizophrenia group, a very strong positive relationship was observed between the production of IL-1β and IL-10, and strong positive correlations were seen between the production of TNF and IL-1β, as well as between TNF and IL-10, by non-stimulated PBMCs. Moderate positive correlations between IL-1β and IL-17A, IL-1β and IFN-γ, IL-17A and IL-10 and between IFN-γ and IL-10 levels were also noticed. Significant strong positive correlations were observed between IL-1β and IL-10, as well as between IL-1β and TNF production, by PBMCs from subjects with schizophrenia following PHA stimulation. Moderate positive correlations were also observed between IL-1β and IL-17A, IL-17A and IL-10 as well as between TNF and IL-10 (table 4).
Moderate positive correlations were observed between spontaneous in vitro production of IFN-γ and G subscore (one of five domains of the PANSS) (R=0.43, P=0.02) and between PHA-stimulated synthesis of IL-17A and G subscore (R=0.39, P=0.04). No correlations were observed between the production of cytokines and CDSS score.
With three exceptions in the whole study group and in both subgroups, no significant correlations were observed between spontaneous or PHA-stimulated synthesis of any of studied cytokines and anthropometric parameters (BMI, body weight, FMI, WHR, TBF). In the control group, PHA-stimulated production of TNF was negatively correlated with TBF (R=−0.42, P=0.033) and in the schizophrenia group, PHA-stimulated production of IFN-γ was positively correlated with body weight (R=0.43, P=0.03) and with TBF (R=0.40, P=0.04). Considering the lack of other correlations, these seem to be accidental and of no clinical importance.
Linear regression models were created for all studied cytokines and cardiometabolic parameters, all of which included smoking as a co-variable. No model was statistically significant. Finally, no significant differences were found regarding the levels of cytokines (both spontaneous and PHA-stimulated) between smokers versus non-smokers, either in the whole study group or in both subgroups.
Discussion
Studies suggest that dysregulation of immune processes may contribute to the etiopathology of schizophrenia; however, the exact mechanisms are still unknown.36 Without a doubt, the activity of cell populations taking part in immunological mechanisms and their capability to respond to stimulation can be of great importance. In particular, cell ability to produce cytokines and chemokines, humoral factors affecting both physiological and pathological processes, seems to be very relevant. Therefore, the aim of the present study was to provide a comparative evaluation of cytokine production by non-stimulated and mitogen-stimulated PBMCs from individuals with schizophrenia and healthy volunteers. It examines the production of three cytokines acting together to initiate and promote inflammatory responses (IL-1β, IL-17A and TNF), the synthesis of anti-inflammatory cytokine IL-10 and the production of IFN-γ, a cytokine with immunoregulatory properties. It is important to note that the contribution of cytokines in the development of inflammation can be more accurately assessed by measuring their production by PBMCs than by serum cytokine level validation.
Our results indicate significantly lower TNF production by PBMCs in people with schizophrenia than in controls, and that PHA-induced IFN-γ synthesis was significantly higher in cases of schizophrenia than in controls. Interestingly, greater production of cytokines by PBMCs was observed following mitogen stimulation compared with spontaneous synthesis. Additionally, the sensitivity of PBMCs to stimulation was considerably higher among individuals with schizophrenia, with the production of cytokines, except for IL-1β, being many times greater than that observed for PBMCs from healthy subjects.
Until now, only a few reports have compared cytokine production by cultured PBMCs obtained from subjects with schizophrenia with healthy controls, and these have yielded conflicting results. Rapaport and Bresee30 reported that subjects with schizophrenia had significantly higher mitogen-stimulated IL-2 and lower IL-6 levels, but displayed no changes in PHA-stimulated IFN-γ and IL-10 production when compared with healthy controls. Reale et al 29 found significantly higher levels of constitutively and lipopolysaccharide (LPS)-induced monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein 1-alpha (MIP-1α), IL-8 and IL-18, but lower RANTES and IFN-γ levels released by PBMCs of subjects with schizophrenia compared with controls. Similarly, Krause et al 28 also reported a reduced IFN-γ response following LPS stimulation. Cazzullo et al 27 stated that in vitro production of IL-2 and IFN-γ was significantly higher in individuals with schizophrenia than in controls, but no changes in IL-2, IL-4 and IL-10 synthesis were noted. Avgustin et al 25 showed increased production of IFN-γ and IL-4 by LPS-stimulated PBMCs from individuals with schizophrenia.
The capacity of PBMCs to secrete cytokines in vitro in response to stimulation with mitogens was also evaluated in subjects with the major depressive disorder (MDD). Weizman et al 37 documented that LPS-induced IL-1β synthesis by PBMCs in untreated people with MDD was significantly lower when compared with that in normal controls. Also, the ability of PBMCs to produce IL-2 in response to PHA stimulation was significantly lower in the untreated subjects with MDD than control subjects.37 Likewise, after LPS stimulation, the PBMCs from the MDD individuals displayed no differences in IFN-γ secretion to those taken from controls, as well as lower TNF secretion compared with healthy volunteers. People with MDD have also been found to present lower IL-2 and IL-10 synthesis by PBMCs after PHA stimulation than healthy controls.38
This is the first study to analyze the relationship between cytokine production and schizophrenia symptoms while excluding the effect of obesity. In this study, we documented positive correlations between spontaneous in vitro production of IFN-γ and G subscore and PHA-stimulated synthesis of IL-17A and G subscore. Previously, Cazzullo et al 27 indicated a positive significant correlation between PANSS total score and the level of IFN-γ synthesis.
Cytokines, including chemokines, are a large group of signaling molecules that are secreted by a broad range of body cells including immune cells. These factors regulate and control the course of physiological processes such as cell proliferation, differentiation and apoptosis. They are also well known for their significant roles in hematopoiesis, angiogenesis and wound healing. It is essential to emphasize that cytokines strongly affect the immune processes and modulate the course of inflammation to varying degrees, as they may act as pro-inflammatory or anti-inflammatory agents. Moreover, cytokines play key roles in brain development and maturation.39
Finally, a growing body of data indicate that cytokines and chemokines, either directly or indirectly, can play a role in signaling the brain and regulating the cross-talk between the central nervous system and the immune system. Data also indicate that dysregulation of cytokine and chemokine activity significantly contributes to the etiopathology of schizophrenia.40 Our findings suggest that immune mechanism dysregulation is associated with the pathophysiology of schizophrenia, as well. In particular, cytokine dysregulation could be responsible for the neurodegenerative aspects observed in schizophrenia, particularly in patients with a long duration of illness.41 In the future, cytokines dysregulation could potentially be used as a diagnostic, therapeutic and side-effects biomarker for schizophrenia. Therefore, further and more detailed studies are needed, which are currently being conducted in our laboratory.
The result of this study should be interpreted within the context of its limitations. First, the study groups had a relatively low number of subjects. Second, all individuals with schizophrenia in this study were on heterogeneous treatment. It cannot be ruled out that antipsychotic drugs may have an effect on in vitro synthesis of cytokines and some authors indicate that both antipsychotics42 43 and antidepressants44 may influence in vitro cytokine production. However, it is not clear whether this relationship might be associated with drugs or whether it might reflect symptomatic improvements and clinical recovery due to therapy. Third, its cross-sectional design limits our abilities to establish a causal relationship. Wherefore, studies with more homogeneous antipsychotic therapy groups concerning people with schizophrenia are needed.
Footnotes
Contributors EK, AW and EBB conceived and designed the experiments; AW, AŁ and PR collected the sample information; EK and PŻ performed the experiments; EK, AW and AŁ analyzed the data; EK, PŻ, AW and EBB wrote the manuscript.
Funding This research was funded by the Medical University of Lodz (grant number 502-03/6- 164-01/502-64-106, 503/6-164-01/503-61-001, and 503/6-164-01/503-66-001).
Competing interests None declared.
Ethics approval Protocol of the study was accepted by the Bioethics Commission of the Medical University of Lodz (RNN/122/16/KE).
Provenance and peer review Not commissioned; externally peer reviewed.
Patient consent for publication Not required.