Cytokine alterations in CSF and serum samples of patients with a first episode of schizophrenia: results and methodological considerations

The study was performed according to German laws, the Declaration of Helsinki, and local institutional review board guidelines. Participants gave written informed consent. CSF and sera were obtained from 20 FES in-patients diagnosed according to ICD10 and AWMF-S3 guidelines [23] for whom routine differential diagnostic lumbar- and veni-puncture had been performed (Supplementary Table ST1). Samples were collected 8.0 days (median) after admission with acute psychosis. Psychopathology was assessed using the Positive and Negative Syndrome Scale (PANSS) [24]. Exclusion criteria: (a) immunological concomitant diseases, recent/current infections, trauma/systemic diseases and substance abuse, (b) treatment with cortisone or other immunosuppressive/modulating substances. Administered antipsychotics were converted to chlorpromazine (CPZ) units for statistical purposes [25].

Controls (n = 21) had headache (n = 3), pseudotumor cerebri (n = 3) or initially unclear neurological symptoms (n = 15) with no history of psychiatric disorders and underwent lumbar puncture to rule out subarachnoid hemorrhage, infectious or autoimmune central nervous system disease. They were matched for age, gender and body mass index. Routine CSF parameters were within the normal range and showed no significant group differences.

After an investigation of 36 clinical parameters (Supplementary Table ST2, column B), CSF and sera were centrifuged and the supernatants stored at − 80 °C.

Concentrations of 13 cytokines [interferon (IFN)-α2, IFN-γ, interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-18, IL-23, IL-33, monocyte chemoattractant protein (MCP)-1 and tumor necrosis factor (TNF)-α] were determined in triplicate using the LEGENDplex™ Human Inflammation Panel 1 (BioVendor; Brno, Czechia) according to manufacturer instructions. This is a fluorescence–coded microsphere-based multiplex immuoassay for the detection and quantitation of analytes by flow cytometry. Sera were diluted twofold and CSF tested undiluted. Standard curves of each cytokine were analysed in duplicate on two separate occasions. The LOB and LOD were determined for each assay according to CLSI guidelines [16, 22], using the formulas below:

For statistical analysis, we used only those cytokines for which at least 2 of the 3 replicates and > 50% of samples gave readings above the LOD.

Data were analyzed using R (v4.0.5). Chi-square tests were performed to calculate group differences regarding gender and smoking. Corrected PANSS scores were derived by subtraction of minimum from raw scores [26]. Most data were not normally distributed as indicated by Shapiro–Wilk tests. Thus, group differences of continuous variables were calculated by Mann–Whitney U tests. MCP-1 serum levels (p < 0.001) were significantly higher in smokers. Therefore, diagnosis-dependent differences in MCP-1 were calculated by ART (analysis of variance using an aligned rank transformation of the data) with the covariate smoking. Due to the exploratory nature of the study, group statistics were not corrected for multiple comparisons.

We used Spearman rank tests with false discovery rate (FDR)-corrected p-values (q-values) to identify correlations of cytokines with routine blood/CSF, demographic and clinical parameters. Cliff’s delta (δ) was used to assess effect sizes (δ ≥ 0.147 = small, δ ≥ 0.330 = medium, δ ≥ 0.474 = large) [27]. All statistical tests were two-tailed with p < 0.05 considered significant.

According to our criteria, 12 out of 13 cytokine assays could be measured in serum samples with > 50% giving readings above the LOD (Table 1). The excluded cytokine was IL1β, for which only 6 out of the 21 control and 3 of the 20 FES samples had values above the LOD. In contrast to the serum results, only two, namely MCP-1 and IL-8, could be measured in CSF.

Neither of the two measurable CSF cytokines showed significant differences in concentrations between the groups (Table 2). The use of the current stringent approach might explain why we did not find some of the cytokine increases reported in a previous meta-analysis [6]. For the serum analysis, the levels of MCP-1 were significantly higher with a large effect size in FES (n = 20) [327.2 (225; 463.8) pg/mL] compared to controls (n = 19) [220.0 (108.5; 265.9) pg/mL, p = 0.007, δ = 0.495]. This was confirmed by ART with the covariate smoking [p = 0.024, δ = 0.479].

This study was limited as we only detected 2 out of 13 cytokines in CSF samples, obviating comparisons with cytokine readings in serum samples. Also, the small sample size of this exploratory investigation necessitates validation in larger cohorts. Regarding the biological findings, we did not assess the potential links of MCP-1 and IL-18 with metabolism-related disturbances as no measures of insulin resistance or visceral fat accumulation were available. Finally, although we controlled for the potential effects of antipsychotic medication, disease duration and smoking, we could not account for other influences like nutrition and sleep, and the use of headache patients as controls might bias the results as inflammatory causes may play a role in different forms of headache.

To our knowledge, this is the first multiplex cytokine immunoassay study of paired serum/CSF samples from FES patients and controls using criteria based on strictly determined LODs. The main advantage of using the CLSI guideline is a reduction of false positives as this approach takes into account the variance of both the blank and lowest concentration samples¹. The potential disadvantages include the possibility of not detecting low-concentration cytokines close to these variable regions. Using this stringent approach, we detected 12 cytokines in serum and only two in CSF using a 13-plex panel. The undetectable cytokine assays all gave signals that were indistinguishable from the blank readings. This revealed potential limitations in the sensitivity of multiplex cytokine assays of CSF. This calls attention to the need for more sensitive assays that can be used to obtain reliable readings in CSF, such as gold nanoparticle immuno-PCR [41, 42] and single molecule arrays [43, 44]. Both methods can provide sensitivities approximately 100- to 1000-fold greater than conventional multiplexed immunoassay systems.