Schizophrenia is a chronic and highly disruptive brain disorder that affects approximately 1% of the population worldwide.[1,2] Both heritable and environmental factors, such as maternal starvation and infections, are widely accepted to increase the risk of developing this disease. The immunopathogenesis of schizophrenia has been researched extensively, and this disease is involved with a chronic low-grade inflammation. Impaired T-cell function is a risk factor for schizophrenia. The cytotoxic T lymphocyte antigen 4 (CTLA4) gene located on chromosome 2q33 encodes CTLA4, which is an essential immune checkpoint molecule for T-cell activation and proliferation, and CTLA4 deficiency can lead to immune dysregulation. However, the relationship between CTLA4 and schizophrenia remains unknown.
There are two dominant isoforms of CTLA4: membrane-bound CTLA4 (mCTLA4) and soluble CTLA4 (sCTLA4).[8,9] The mCTLA4 mRNA contains four exons: exon 1 (leader peptide), exon 2 (ligand-binding domain), exon 3 (transmembrane domain), and exon 4 (cytoplasmic tail).[8,10] The sCTLA4 mRNA lacks exon 3, and is therefore deficient in membrane anchoring.[9,11] Several variants within the CTLA4 gene have been demonstrated to be significantly associated with schizophrenia susceptibility.[12,13] However, none of these variants have been implicated in genome-wide association studies.[14,15] Because the low replication rates and the missing heritability are known limitations of genome-wide association studies, further investigation into the relationship of CTLA4 with schizophrenia is warranted.
To evaluate the roles of CTLA4 in schizophrenia etiology, we first investigated the gene structure of CTLA4 and selected tag single nucleotide polymorphisms (SNPs) for further analysis. Then, we evaluated CTLA4 expression quantitative trait loci (eQTLs) and mRNA expression in different regions of normal brains using publicly available gene expression databases (see Additional file 1, http://links.lww.com/JR9/A20 for the information about CTLA4 gene expression databases). Finally, we assessed differential expression of the two CTLA4 isoforms in the peripheral blood mononuclear cells (PBMCs) of schizophrenia patients and healthy controls using quantitative real-time polymerase chain reaction (qRT-PCR) to explore the relationship between the two CTLA4 isoforms and schizophrenia.
Subjects and methods
In this observational study, all subjects were unrelated, were of Chinese Han ethnicity, and provided written informed consent in accordance with the protocol approved by the Bioethics Committee of corresponding research institutes (approval No. 20150016) on March 6, 2015 and the principles of the Declaration of Helsinki. The study location, period of data, and sample size calculation were described in our previous study.
The subjects whose PBMCs were used in the two-stage qRT-PCR analysis were described in our previous study. According to the previous study design protocol, stage 1 included 24 schizophrenia patients and 24 healthy controls, and stage 2 included 41 schizophrenia patients and 37 healthy controls. The characteristics of all subjects are listed in Table 1. Briefly, schizophrenia was diagnosed according to the criteria described by the Diagnostic and Statistical Manual of Mental Disorders Fourth Edition (DSM-IV). All the schizophrenia patients were first-episode and drug-naïve subjects. Subjects who met any of the following criteria were excluded: (1) pregnancy; (2) presence of other psychiatric disorders; or (3) a history of substance abuse or drug addiction. All healthy controls recruited via the health examination were assessed using the Structured Clinical Interview for DSM-IV TR Axis I Disorders (non-patient edition). Healthy controls who met any of the following criteria were excluded: (1) first-degree relative with a history of severe mental disorder; (2) history of autoimmune/inflammatory diseases; (3) infection within the past 3 to 4 weeks; or (4) receiving any treatments including taking any medication.
CTLA4 gene structure
The genotypic data for the SNPs located within the CTLA4 gene region (Chr2: 204,730,700–204,740,300 based on GRCh37.p13) were obtained from the 1000 Genomes project for individuals of European descent from Utah (CEU) and Chinese Han Beijing (CHB). The linkage disequilibrium blocks were constructed according to the default confidence intervals rule based on SNPs meeting the following criteria: Hardy–Weinberg equilibrium P value cutoff of 0.001, minimal genotype percentage >80%, maximal Mendel error of 1 and minor allele frequency > 0.02, using Haploview ver. 4.2.
CTLA4 eQTL and expression analysis
We first explored the CTLA4 mRNA expression pattern in various normal human brain tissues and in whole blood using RNA sequencing–based expression data from the Genotype–Tissue Expression (GTEx) Portal (https://gtexportal.org/home/). To assess the impact of the tag SNPs on mRNA expression, we focused on eQTL data for the tag SNPs in multiple brain tissues utilizing BRAINEAC data. Based on the tag SNP genotyping results (Additional Table 1, http://links.lww.com/JR9/A20) and the expression levels of specific exons (Additional Table 2, http://links.lww.com/JR9/A20) in the BRAINEAC database, we performed an association analysis between the tag SNPs and expression of the two main CTLA4 isoforms (mCTLA4 and total CTLA4) within multiple brain tissues.
BRAINEAC data were used to profile the expression of mCTLA4 and total CTLA4 in 10 human brain regions. Additionally, the temporal dynamics of mCTLA4 and total CTLA4 transcription throughout the lifespan were determined using the BrainCloud database (http://braincloud.jhmi.edu), which is described in detail in the Additional files, http://links.lww.com/JR9/A20.
qRT-PCR analysis of CTLA4 mRNA expression in human PBMCs
Heparinized blood was collected from the vein of participants in the morning after overnight fasting, and PBMCs were isolated from the blood using Lympholyte H (Cedarlane Laboratories, Burlington, Canada). Total RNA was extracted from the PBMCs using an RNA 6000 Nano Kit (Agilent Technologies, Waldbronn, Germany). The RNA integrity value for the extracted RNA was > 7.5. Next, the RNA was reverse transcribed into complementary DNA, which was used as the template for 384-well plate TaqMan real-time PCR (Thermo Fisher Scientific, Cambridge, Boston, MA) to determine the expression of full-length mCTLA4 and sCTLA4 in the test and validation groups. The primers used to perform qRT-PCR were: sCTLA4, 5’-GGC AAC CTA CAT GAT GGG GA-3’, 5’-GGC TTC TTT TCT TTA GCA ATTA CAT A-3’; mCTLA4, 5’-AGG TGA CTG AAG TCT GTG CG-3’, 5’-GCA CGG TTC TGG ATC AAT TAC A-3’. Cycling conditions were 95°C for 15 seconds, 55°C (sCTLA4) or 60°C (mCTLA4) for 30 seconds and 72°C for 30 seconds to ensure that each amplicon was a single product, with all reactions being run in triplicate. The threshold cycle (CT) was defined as the fractional cycle number at which the fluorescence surpassed the fixed threshold. To identify a stable internal standard, the expression levels of seven candidate reference genes, 18S, β-actin, CPT1A, EC1, NUPR1, PPARD, and SCD5, were tested and validated using the mini-software of geNorm 3.5. geNorm calculates the gene expression stability value (M) for each candidate reference gene as the pairwise variation (V) for the gene with all other genes. The pairwise variation between 2 sequential normalization factors, Vn/Vn+1, was used to determine the optimal number of reference genes, with a cutoff value of 0.15. The expression of the target genes was calculated based on that of the suggested internal standard genes, and mRNA expression was calculated using the 2−ΔΔCt method. Furthermore, expression levels were normalized to non-template controls.
Chi-squared and t tests were performed to evaluate differences between the schizophrenia subjects and the healthy controls. The association of each SNP with CTLA4 expression in different brain regions was assessed by the 2-way analysis of variance and unpaired t tests. Differential CTLA4 expression in PBMCs from schizophrenia patients and healthy controls was compared by Mann–Whitney U test. A P value of < 0.05 (2-sided) was considered statistically significant. For the multiple comparisons, P values were corrected using the Bonferroni method, and a corrected P value < 0.05 was considered statistically significant. Statistical analyses were performed using the R software platform (http://www.r-project.org).
CTLA4 tag SNP selection
The CTLA4 gene structure is shown in Figure 1. The variants within the CTLA4 region that exhibited high linkage disequilibrium were located in 1 complete haplotype block in CEU, and in 2 haplotype blocks in CHB. The CEU population exhibited more relative variants in CTL4 than the CHB population. rs733618 and rs231779 were selected as the tag SNPs; and rs231775 and rs3087243, which are commonly tested SNPs, were selected for use as additional tag SNPs. rs733618 is located upstream of CTLA4, rs3087243 is located downstream of CTLA4, rs231775 is located in exon 1, and rs231779 is located in intron 1.
CTLA4 eQTL and spatiotemporal mRNA expression
Using RNA sequencing-based expression data from the GTEx Portal, we found that CTLA4 was widely expressed in various brain tissues (Additional Fig. 1, http://links.lww.com/JR9/A20). CTLA4 expression was lower in the brain compared with whole blood. The specific distribution of CTLA4 expression across diverse brain tissues was similar to the expression profiles in BRAINEAC (Additional Fig. 2, http://links.lww.com/JR9/A20). We were able to identify two main different CTLA4 isoforms, as total CTLA4 (which includes mCTLA4 and sCTLA4) expression was significantly greater than mCTLA4 expression in normal subjects.
Next, the BRAINEAC database was used to identify cis-eQTLs by assessing the impact of the tag SNPs on CTLA4 expression. The genotyping results for the selected tag SNPs and CTLA4 expression data as determined by different probes extracted from the BRAINEAC database are detailed in Additional Tables 1 and 2, http://links.lww.com/JR9/A20. Re-analysis of the BRAINEAC data revealed that rs733618 only affected CLTA4 expression in the hippocampus, and that the minor C allele was significantly associated with increased total CTLA4 (P = 0.0024) and mCTLA4 (P = 0.0144) expression (Fig. 2A). Based on uncorrected P values, the minor T allele of the rs231779 tag SNP was associated with increased expression of mCTLA4 (P = 0.0336), but not total CTLA4 (P = 0.2607) in the hippocampus (Fig. 2B). After correcting the P values for multiple comparisons, rs733618 was only significantly associated with total CTLA4 expression (PBonf. = 0.019), and not with mCTLA4 expression (PBonf. = 0.115). As for the remaining brain regions, no tag SNPs were found to have any significant effect on CTLA4 expression at the mRNA level.
Analysis of temporal CTLA4 expression pattern data from BrainCloud demonstrated that total CTLA4 expression increases gradually after birth and decreases after the age of 10 years, while mCTLA4 levels are stable throughout the lifespan, which suggests that sCTLA4 expression levels vary during human brain development (Additional Fig. 3, http://links.lww.com/JR9/A20).
CTLA4 expression in human PBMCs
mCTLA4 and sCTLA4 mRNA expression in PBMCs was determined by a two-stage association study, which showed that both mCTLA4 and sCTLA4 expression levels were above the limit of detection for the 384-well plate TaqMan real-time PCR method. Pairwise variation analysis conducted using geNorm showed that β-actin and PPARD were the most stable candidate references genes, and they were therefore selected for use as internal standards (Additional Fig. 4, http://links.lww.com/JR9/A20). sCTLA4 expression was significantly reduced in the schizophrenia test group compared with the healthy control group, while mCTLA4 expression was not (P = 0.009 and 0.769 for sCTLA4 and mCTLA4, respectively; fold change = 0.657 for sCTLA4; Fig. 3). These results were subsequently confirmed within the validation group (P = 0.0017 and 0.1333 for sCTLA4 and mCTLA4, respectively; fold change = 0.704 for sCTLA4). The combined two-stage results also suggested that sCTLA4 expression, and not mCTLA4 expression, was significantly reduced in the schizophrenia group compared with the control group (P < 0.0001 and 0.142 for sCTLA4 and mCTLA4, respectively; fold change = 0.67 for sCTLA4).
Schizophrenia is a chronic psychiatric disorder that involves a compromised central nervous system (CNS), aberrant immune function, and disrupted connections between these 2 systems. Patients with schizophrenia exhibit disturbances in T-cell proliferation and cytokine production.[1,28] Moreover, while T cells are typically located in the thymus and peripheral blood, activated T cells have been reported to traverse the blood-brain barrier and infiltrate the brain,[29,30] and tissue-resident memory T cells have been detected in the CNS. CTLA4, which is mainly produced by T cells, is an important inhibitor of T-cell proliferation and has two main isoforms: mCTLA4 and sCTLA4. Here, we evaluated the role of CTLA4 in schizophrenia by studying its mRNA expression.
By analyzing public databases, we found for the first time that human brain tissue contains 2 CTLA4 isoforms that are generated by alternative splicing: sCTLA4 and mCTLA4. Many factors can regulate alternative splicing, such as trans-acting proteins and cis-acting regulatory sites. CpG DNA methylation has also been shown to regulate alternative splicing. eQTL analysis showed that the rs733618 tag SNP located upstream of CTLA4 can function as a cis-eQTL to affect mCTLA4 or total CTLA4 expression in the hippocampus; although we cannot exclude the possibility that other factors also affect mCTLA4 or sCTLA4 expression. However, the hippocampus has been reported to exhibited impaired working memory performance and disrupted functional connectivity in schizophrenia patients, and is thus considered to be one of main brain regions affected by schizophrenia.[34,35] Genetic structural magnetic resonance imaging (sMRI) analysis of the same tag SNPs within another group of schizophrenia patients and healthy controls suggested that rs231779 and rs733618 are significantly associated with altered structural connections in the dentate gyrus, a part of the hippocampus (data not shown). These results indicate that CTLA4 expression may be related to schizophrenia.
Within the inflamed CNS, brain-resident memory T cells are activated to protect against invading antigens. When an aggressive immune response occurs, expression of CTLA4, a key immune checkpoint molecule, is significantly up-regulated to tightly control T-cell activation and the immune response and to re-establish tissue homeostasis in the CNS. In this study, we investigated the roles of the two CTLA4 isoforms in PBMCs. qRT-PCR analysis revealed that sCTLA4 expression was significantly decreased in patients with schizophrenia, while mCTLA4 expression was not. sCTLA4 is secreted into human serum[9,11] and can bind CD80/86 and inhibit T-cell proliferation. Notably, sCTLA4 can infiltrate the brain much more easily than T cells can. It is possible that sCTLA4 may inhibit T-cell over-activation in the brain. The reduction in the level of CTLA4 mRNA may lead to continuous activation of T cells, which may play a role in schizophrenia pathogenesis. Additionally, we found that total CTLA4 expression increases gradually after birth and decreases after the age of 10, while mCTLA4 levels in the brain are stable throughout the lifespan, suggesting a potential regulatory role of sCTLA4 in postnatal brain development. Peak sCTLA4 expression may coincide with the onset of schizophrenia.
This study had several limitations. First, blood samples were used instead of brain samples. However, because the brain and peripheral organs may have cross-organ interactions through vascular and neural networks throughout the whole body, studies based on clinical blood samples can provide useful information about schizophrenia pathophysiology. Second, the sample size used in this study was small because of strict standards for subject recruitment and sample collection. Although the sample size was not sufficient to perform multiple genetic association analyses, we believe it was appropriate for evaluating the expression of a limited number of genetic factors using corrected P values.
Taken together, our findings suggest that sCTLA4 expression levels are associated with schizophrenia, and that lower sCTLA4 expression levels may increase the risk of schizophrenia. Further molecular studies are needed to clarify the detailed role of CTLA4 in schizophrenia pathogenesis.
We are grateful to all the participants for cooperating in this study.
All authors contributed to the study design, data analysis and manuscript preparation, and performed the study, and approved the final version of the manuscript.
This study was supported by the grants from the Ministry of Science and Technology of China (Nos. 2017YFC1001302 and 2017YFC0909200); Natural Science Foundation of Shanghai (Nos. 19ZR1427700 and 19ZR1476100); Shanghai Key Laboratory of Psychotic Disorders (13dz2260500) in Shanghai Mental Health Center, China (No. 19-K02); and Medical Engineering Cross Fund of Shanghai Jiao Tong University (YG2019QNA49 and YG2019QNA52).
Institutional review board statement
This study was conducted in accordance with the Declaration of Helsinki and approved by the Bioethics Committee of corresponding research institutes (approval No. 20150016) on March 6, 2015.
Declaration of participant consent
The authors certify that they have obtained the patient consent forms. In the forms, patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity.
Conflicts of interest
The authors declare that they have no conflicts of interest.
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