The link between immune system dysfunction and psychotic disorders is increasingly recognized. Pre- and perinatal infections are established risk factors, potentially influencing both immune system development and brain development. Genetic studies have also identified immune loci associated with psychosis. Numerous studies have reported altered inflammatory markers and cytokines in the blood and cerebrospinal fluid of psychotic patients, although the specific profile of these mediators varies considerably across studies. This inconsistency may be attributed to factors such as sample size, age, smoking, BMI, medication, and the analysis of only a limited set of inflammatory proteins. The human immune response is a complex, coordinated process involving numerous proteins with potentially opposing functions. Therefore, evaluating a broad range of immune-related proteins is crucial for identifying potential biomarkers of psychotic disorders. This study aimed to assess alterations in plasma immune markers in drug-naive FEP patients compared to healthy controls by analyzing 92 immune-related proteins using multiplex proximity extension assay technology. Multivariate statistical analysis was employed to identify key alterations in cytokines.

Existing literature demonstrates a correlation between altered immune function and the pathophysiology of psychotic disorders. Prenatal and perinatal infections are recognized risk factors, impacting both immune system development and brain maturation. Genetic studies have identified immune system loci associated with psychosis. While several studies have shown changes in inflammatory markers and cytokines in patients with psychotic disorders, the findings are inconsistent across studies. These inconsistencies might stem from differences in sample sizes, age ranges, smoking habits, body mass index, medication status, and the analysis of limited numbers of inflammatory proteins. The complexity of the human immune system, which involves an array of proteins with sometimes opposing effects, necessitates a more comprehensive approach. Previous research, therefore, suggested analyzing a wide range of proteins to identify potential biomarkers for psychotic disorders.

This study included 60 drug-naive FEP patients recruited from the Tartu University Hospital, Estonia, and 50 age-matched healthy controls recruited through advertisements. The study period was from 2009 to 2013. Patients were clinically assessed for eligibility by two psychiatrists based on ICD-10 criteria for psychotic disorders (F20-F29). Inclusion criteria for FEP patients included no prior psychiatric care, age between 18 and 45, no antipsychotic medication, and onset of psychotic episode within the past three years. Exclusion criteria for both groups included diabetes, neurological or immune disorders, and (for controls) current or past psychopathology or a family history of psychotic disorders. Demographic data, including age, sex, BMI, waist circumference, smoking, drinking, and cannabis use, were collected. Symptom severity was assessed using the Brief Psychiatric Rating Scale, Global Assessment of Functioning scale, and Clinical Global Impression scale. Overnight fasting blood samples (5 ml) were collected, centrifuged, and the plasma fraction was aliquoted and stored at -80°C. Protein analysis was conducted in two runs using Olink Proteomics' Inflammation panel (92 proteins), employing multiplex proximity extension assay (PEA) technology. Data were normalized using bridge samples, and proteins with >40% values below the limit of detection were excluded (20 proteins). Statistical analysis involved Shapiro-Wilk tests for normality, followed by univariate non-parametric tests (Wilcoxon and Chi-square). Multivariate data analysis used SIMCA version 17, employing principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Protein pathway analysis used the STRING v11.5 database.

Demographic and clinical data showed significant differences between FEP patients and controls in cannabis use and smoking habits. OPLS-DA analysis revealed a significant model (F(6,103) = 15.16, p = 2.25e-12, R² = 0.76, Q² = 0.47) differentiating FEP and healthy control groups. Eleven proteins showed significant discriminatory power (VIPpred ≥ 1.0 and p(corr) ≥ 0.3): Flt3L, EN-RAGE, OSM, TNFβ, TGFα, HGF, IL-6, CCL23, 4E-BP1, FGF-21, and TNFSF14. Nine of these proteins were elevated in FEP patients, while two (Flt3L and TNFβ) were decreased. Protein pathway analysis revealed a network centered around IL-6, highlighting interactions between the significant proteins, with EN-RAGE and OSM potentially stimulating IL-6 production. The increase in EN-RAGE was notably larger compared to other cytokines, including IL-6.

This study identified several proteins with altered levels in FEP patients compared to controls, many associated with immune activation, inflammation, and the acute phase response. Elevated IL-6, OSM, TGFα, and other pro-inflammatory cytokines align with previous studies on acute schizophrenia. The significant increase in plasma EN-RAGE is particularly noteworthy. Previous studies have also reported elevated EN-RAGE in other inflammatory conditions and cardiovascular disease. The neutrophil-derived EN-RAGE, through its interaction with RAGE on target cells, could trigger an inflammatory response potentially affecting brain vasculature. This finding aligns with studies showing increased neutrophil extracellular traps (NETs) in early-stage schizophrenia. The observed alterations in cytokines and EN-RAGE could contribute to the increased cardiovascular risk seen in severe mental disorders. While this study demonstrates a significant association, further research with larger, diverse cohorts and longitudinal studies is needed to confirm these findings and investigate the potential of EN-RAGE as a biomarker for psychosis and its role in cardiovascular risk.

This study demonstrates elevated levels of pro-inflammatory cytokines and, notably, EN-RAGE in the plasma of FEP patients. The increase in EN-RAGE, a protein primarily expressed by neutrophils, suggests its potential as a novel, readily accessible blood-based marker for acute psychosis. Further studies using larger cohorts and longitudinal designs are warranted to validate these findings and assess EN-RAGE's potential as a biomarker. Additional research is also needed to fully understand the roles of RAGE and its various ligands in neuropsychiatric disorders.

This study has some limitations. The sample size, while substantial, is relatively modest, especially for the healthy control group. While factors like age, sex, smoking, and BMI were controlled for, other confounding variables could influence the results. Further, the cross-sectional nature of the study prevents causal inferences. Future research should address these limitations by employing larger, more diverse participant groups and longitudinal study designs.