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Gene expression pattern of CCL2, CCL3, and CXCL8 in patients with bipolar disorder

Mohsen Ghoryani1,2, Farhad Faridhosseini3,4, Ali Talaei3,4, Reza Faridhosseini5,6, Jalil Tavakkol‐Afshari1,6, Maliheh Dadgar Moghaddam7, Parisa Azim4, Zanireh Salimi4, Hadi Zare Marzouni5, Mojgan Mohammadi1,5 1Immunology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran, 2Department of Laboratory Sciences, School of Para‐Medical Sciences, Torbat Heydarieh University of Medical Sciences, Torbat Heydarieh, Iran, 3Psychiatry and Behavioral Sciences Research Center, Mashhad University of Medical Sciences,Mashhad, Iran, 4Department of Psychiatry, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran, 5Allergy Research Center, Mashhad University of Medical Sciences, Mashhad, Iran, 6Department of Immunology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran, 7Neurogenic Inflammation Research Center, Mashhad University of Medical Sciences, Mashhad, Iran


INTRODUCTION
Psychiatric disorders are among the most common diseases in the world, they are a group of disorders associated with the lack of control over mood and mental experience of severe discomfort. [1,2] People with elevated mood (manic) show expansion, hypnosis, sleep deprivation and increased self-confidence and great thoughts. People who develop depression are characterized by reduced energy, feelings of guilt, anorexia, and suicidal ideation. [3,4] Other symptoms include alterations in the cognitive capabilities, level of activity, speech, and other functions such as appetite, pathophysiology of BD. [12,13] Patients with BD have elevated peripheral levels of pro-inflammatory biomarkers such as interleukin 1 (IL-1) β, C-reactive protein, and tumor necrosis factor-α. [14,15] Chemoattractant cytokines known as chemokines are key immune mediators in leukocyte trafficking in inflammatory and normal conditions. Some evidence suggests that chemokines are involved in neurobiological processes like modulation of neurotransmitter system and neuroinflammatory responses, and may be good therapeutic targets in BD. [16,17] CCL2 known as monocyte chemoattractant protein-1 has chemotactic effect on monocyte/macrophages, dendritic cells, and T lymphocytes. Neurons, microglia, and astrocytes up-regulate the expression of CCL2 and CCR2 in inflammatory conditions. [18] CCL3 also known as macrophage inflammatory protein 1-alpha (MIP-1-alpha) is considered as neutrophil chemoattractant. In the central nervous system (CNS), astrocytes express CCL3 and its receptor (CCR1 and CCR5). [17,18] CXCL8 (also IL-8) is a chemotactic factor for granulocytes, B, and T lymphocytes, dendritic cells, and natural killer cells. Astrocytes, neurons, microglia, and endothelial cells of the blood-brain barrier (BBB) express CXCL8 receptors (CXCR1 and CXCR2), constitutively. [17,19] Evaluation of inflammatory biomarkers such as chemokines in patient with BD and compare them with healthy individuals can provide valuable information for future studies to use chemokines for both therapeutic and diagnostic purposes in BD. In this study, we evaluated the gene expression of CCL2, CCL3, and CXCL8 in whole blood samples of patients with BD and compared them with healthy individuals.

Participants
A total of 48 patients with BD and 48 age-and sex-matched healthy controls were enrolled in this study. All patients were included if they had no history of infection, inflammatory, autoimmune, and cancerous diseases. They were recruited from April to August 2016 at Ibn-Sina Psychiatric Hospital, Mashhad University of Medical Sciences, Mashhad, Iran, and were assessed with the Structured Clinical Interview for DSM-IV-Axis I Disorders (SCID-I) to confirm BD. To characterize the severity of manic and depressive symptoms, patients were also examined with the Young Mania Rating Scale and the Hamilton Depression Rating Scale, respectively. The cutoff point for these scales was considered as 7. Healthy controls with no history of psychiatric disorders (evaluated through SCID-I) and also severe medical and inflammatory diseases from the local population were enrolled in this study. This study was approved by the Ethics Committee of Mashhad University of Medical Sciences, and an informed consent was obtained from each patient and healthy control. The ethical code is IR.MUMS.fm. REC.1394.494.

RNA extraction and cDNA synthesis
Total RNA was extracted from the whole blood samples using Total RNA Purification Mini kit for Blood/Cultured Cell/Tissue (Favorgen, Ping-Tung, Taiwan), and then cDNA was synthesized using cDNA Synthesis Kit (Favorgen), following the manufacturer's instructions.

SYBR ® green real-time polymerase chain reaction
Nucleotide database at https://www.ncbi.nlm.nih.gov, and Beacon Designer software (version 7.0, Premier Biosoft, Palo Alto, CA, USA) were used to design specific primers for CCL2, CCL3, CXCL8, as target genes and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as housekeeping gene. Primers were designed and then blasted at https://www.ncbi.nlm.nih. gov/tools/primer-blast for use in the SYBR ® Green real-time polymerase chain reaction (PCR). The primer sequences used in the present study are shown in Table 1. SYBR ® Green real-time PCR was performed on the cDNA samples using SYBR ® Premix EX Taq II (2X) (Takara Bio, Inc., Otsu, Japan) to evaluate CCL2, CCL3, CXCL8, and GAPDH gene expression levels. Real-time PCR was performed in a Rotor-Gene 6000 thermal cycler (QIAGEN, Hilden, Germany).
The SYBR ® Green real-time PCR conditions were initial denaturation at 95°C for 10 min followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 20 s. To compare levels of mRNA expression between patients and healthy controls, delta-CT values were calculated.

Statistical analysis
All statistical analyses were conducted using the IBM SPSS Statistics 21 (SPSS Inc., Chicago, IL, USA). Data were tested for normality of distribution using the Kolmogorov-Smirnov test and all data were normally distributed. The difference of delta-CT values between patients and healthy controls were compared with the independent samples t-tests. Statistical significance was defined as a value of P < 0.05.

RESULTS
The demographic characteristics of the patients and healthy controls are presented in Table 2. CCL2 gene was expressed at higher levels in patients with BD (delta-CT; mean ± standard error of mean [SEM] −1.13 ± 0.26) as compared to healthy controls (delta-CT; Mean ± SEM −1.74 ± 0.25) but not significant (P = 0.097) [ Figure 1a]. Patients with BD showed higher levels of CXCL8 gene expression (delta-CT; mean ± SEM 3.93 ± 0.50) compared to healthy controls (delta-CT; mean ± SEM 3.40 ± 0.49), the difference was not statistically significant (P = 0.450) [ Figure 1b]. On the contrary, the lower expression levels for CCL3 gene in patients with BD (delta-CT; mean ± SEM −1.51 ± 0.80) was observed as compared to healthy controls (delta-CT; mean ± SEM −1.19 ± 0.30) but not statistically significant (P = 0.714) [ Figure 1c].

DISCUSSION
Results of this study revealed that there was no significant difference in the gene expression levels of CCL2, CCL3, and CXCL8 in patients with BD as compared to healthy controls. Previous studies on chemokines in patients with BD often focused on serum levels, and there is limited information available regarding the chemokine gene expression profiles in BD at different phases or stages of the disease. To our knowledge, there is just one report regarding the gene expression of CCL2 in monocytes of patients with BD and their offspring with mood disorders that showed an increasing in the level of CCL2. [20] In addition, Nakatani  et al. indicated that brain tissue from patients with BD had lower levels of CCL3 gene expression in comparison to healthy controls. [21] There are no published data with regard to CXCL8 gene expression in patients with BD. However, there are few studies evaluating the serum levels of CCL2, CCL3, and CXCL8 in patients with BD, Alzheimer's disease (AD) and schizophrenia. Brietzke et al. showed no statistically significant difference in the serum levels of CCL2, CCL3, and CXCL8 between patients with BD and healthy controls. [22] Drexhage et al. demonstrated that patients with BD had elevated level of CCL2 in serum in comparison to healthy controls. [23] O'Brien et al. and Barbosa et al. reported that patients with BD had increased serum levels of CXCL8 compared to healthy controls. [24,25] Expression of CCL2, CCL3, and CXCL8 and their receptors by neurons, astrocytes, and endothelial cells of the BBB may indicate the role of neuroinflammatory processes in the etiology of BD. [17][18][19] Neuroinflammation is considered as one of the potential mechanisms contributing to the pathogenesis of other neuropsychiatric diseases such as AD and schizophrenia. [17,26] High levels of CCL2 and CXCL8 in the serum of patients with AD was reported. [27] Results about the association of serum levels of CCL2 and CXCL8 in patients with schizophrenia are controversial. In some studies, higher serum levels of CCL2 and CXCL8 in patients with schizophrenia compared to healthy controls was reported, whereas other studies showed no significant differences between serum levels of mentioned chemokines in patients with schizophrenia and healthy controls. [17,28] In this study, we extracted total RNA from leukocytes after lysis of erythrocytes and evaluated the expression of CCL2, CCL3, and CXCL8 genes. As previously mentioned, these chemokines in addition to some leukocytes, are also expressed in neurons, astrocytes, and endothelial cells of the BBB. [17][18][19] Chemokine gene expression in CNS-associated cells may show the different patterns from that of leukocytes, and hence we suggest evaluating and comparison of chemokines gene expression in CNS-associated cells with leukocytes in patients with BD and comparison with healthy individuals in future studies. Access to brain tissue may be associated with some limitations. Emerging technologies such as reprogramming and differentiation of somatic peripheral cells of patients into neurons [29] might provide an alternative source of neurons to study of biological and immunological effects of chemokines gene expression in patients with BD.

Limitations
One of the limitations of our study was the loss of follow-up due to the lack of cooperation of the patients. In addition, lack of measurement of the chemokines in the serum levels and comparison with gene expression data not only in mania but also in depression phase of BD made us unable to have a better conclusion regarding the effect of the chemokines on BD.

CONCLUSION
We found no significant differences in the expression levels of CCL2, CCL3, and CXCL8 genes in peripheral blood cells of patients with BD as compared to healthy controls. Our suggestions for future studies are an increase in the sample size, following up the patients in various time points and sample collections from patients with BD in mania and also in depression phases to have a more accurate conclusion regarding the effects of chemokines in BD. To have serum levels of the chemokines of patients with BD and comparing with the gene expression levels would be so valuable too.