Schizophrenia (SZ) is a severe chronic mental illness that affects approximately 1% of the world population of both sexes (1, 2). Its symptoms are classified into positive symptoms (delusions, hallucinations general alterations, and cognitive and sensorial perceptions), negative symptoms (blunted affect, poor hygiene, depressed mood, and anhedonia), and cognitive impairment (3). Its causes and pathophysiology are not fully known yet.

This illness shows a complex multifactorial profile, involving several environmental, genetic, and psychosocial factors (2, 4, 5). The disease onset is frequently preceded by a subclinical symptoms phase (prodromal phase), associated with increased dopamine synthesis. However this association is observed only in patients who have developed mental illness later in life (5). The most accepted pathophysiological theory of SZ is the dopaminergic hypothesis (5, 6). This hypothesis is supported by the fact that all drugs used in SZ treatment have some action on the inhibition of dopaminergic receptors (7, 8). On the other hand, dopaminergic agonists, such as amphetamines, show an ability to precipitate psychotic symptoms (7).

The pathophysiology of SZ, according to this hypothesis, is related to a dopaminergic hypofunction on the prefrontal cortex (negative symptoms) and a secondary hyperfunction on the striated cortex (positive symptoms) (9). This theory has some inconsistencies because there is a gap of 2–4 weeks between the peak of D₂ receptors blocked by the drug and the clinical response (10). Approximately one-third of patients show low or no improvement of symptoms with antipsychotic (AP) use, which motivates the search for new therapeutics targets (11).

Previously, it was believed that the central nervous system (CNS) was relatively protected from peripheric inflammatory processes due to the blood–brain barrier. However, this is not true because inflammatory processes increase the permeability of the blood–brain barrier and possibilities the leukocytes invasion in brain. Inflammatory processes have more importance in the acute phase, when invasions may occur to the CNS by macrophages and lymphocytes, while in the chronic phase, there a (12, 13). Taking this into account, a hypothesis is that the patient’s inflammatory profile may predict the response to clozapine and be useful in classifying the subtypes of SZ.

There is a growing number of recent studies that correlate inflammatory markers with psychiatric conditions such as mood disorders and suicidal behavior. Inflammatory processes in the hypothalamus–pituitary–adrenal (HPA) axis have been reported as involved in negative clinical outcomes regardless of the presence/absence of psychiatric conditions. Increased levels of stress are linked to HPA alterations and dysregulation of the serotoninergic system, including 5-HTTA receptors in individuals with mood disorders and suicidal attempts (14). Although well known, the origin and extent of this process are not well understood.

Moreover, subjects with abnormally elevated inflammatory markers may even be at higher risk of developing treatment resistance. Importantly, compared to non-suicidal subjects, higher mean concentrations of inflammatory mediators for treatment resistance have been found in both the periphery and brain of individuals who present with suicidal behaviors (15).

Considering that psychiatric practice today is still based on subjective diagnostic criteria, the discovery of biological markers as neutrophil-lymphocyte ratio (NLR) and immune inflammatory systemic index (SII), would play a great role in the credibility of diagnosis. It would aim to estimate the metabolic changes that can be caused by mental illness, clinical response, and tolerance to drugs such as clozapine. In this study, we evaluate the correlation between inflammatory markers [neutrophil, lymphocyte, platelets, IIS, NLR, and Monocyte-Linphocyte Ratio (MLR)], daily doses of clozapine, and the severity of psychopathology in SZ.

This research work is a cross-sectional study with a control group (non-clozapine patients) and a trial group (clozapine patients). Patients were recruited from March 2022 to February 2023 from the Schizophrenia Program (PRODESQ) of a university hospital [Clinical Hospital of Porto Alegre (HCPA)], of a psychosocial care center [Centro de Atendimento Psicossocial (CAPS)] of the same hospital, and of a non-governmental mental health institution [Associação Gaúcha dos Familiares de Pacientes com Esquizofrenia (AGAFAPE)], all located in the city of Porto Alegre-RS, Brazil. The inclusion criteria were as follows:

Agreement to participate in the research study.

For the trial group, besides these criteria, patients were required to be taking clozapine at a stable dose for at least 15 days.

The exclusion criteria were as follows:

We initially assessed 116 patients’ records, with 12 being excluded because of diagnoses of overlapping brain injuries, chronic inflammatory processes, etc., as shown in Figure 1. After exclusions, 104 patients remained (78 men and 26 women).

The epidemiologic characteristics of the study’s sample (sex, age, literacy level, smoking, comorbidities, drug use, hospitalization, BMI, age of onset, and clozapine dosage) are described in Table 1.

The Brief Psychiatric Rating Scale (BPRS) was used to evaluate the severity of psychiatric symptoms. The highest score was registered for the items “somatic concern” (median: 1.5; interval: 0–3) and “anxiety” (median: 3; interval: 2–4). The BPRS total score median was 20 (interval:12–30).

The clozapine users showed a significantly higher neutrophil count (mean ± DP: 5.03 ± 2.07) in comparison to non-clozapine users (mean ± SD: 3.48 ± 1.27; p = 0.031) as shown in Table 2.

Although there were no significant differences related to other parameters, clozapine users seem to present a tendency to have higher inflammatory markers, except for monocytes, which was equal, and for MLR, which showed a lower count in clozapine users. These results are presented in Table 3.

The correlation between inflammatory markers and daily dose of clozapine (DDC) was evaluated, but no statistical significance was found for any evaluated parameters (Table 4).

Furthermore, no correlation was found between inflammatory markers and the severity of symptoms.

Patients using APs other than clozapine showed significantly higher BPRS scores than those who did not use any AP medication (p = 0,036). Furthermore, the use of clonazepam and amitriptyline was associated with significantly higher BPRS scores (p < 0,001 e p = 0,047, respectively).

Treatment with clozapine showed a significantly higher neutrophil count among clozapine users than non-clozapine users (p = 0,031). These findings are in agreement with an experimental study in animal models. In this study, an increase in neutrophils and a decrease in lymphocyte counts were observed a few hours after a single dose of clozapine (20).

Our study also showed that patients who use amitriptyline and/or other antipsychotic drugs have higher BPRS scores than patients who do not use these drugs. This finding is probably due to the ultra-refractory profile of these patients, indicating that the symptomatic manifestation remains resistant to treatment, including with clozapine use. The majority of patients included in this study were men (75%). Despite epidemiological studies showing a higher incidence of schizophrenia in male individuals, and the male/female ratio being 1:4 without significant difference in prevalence, these conclusions could not be verified because this is a cross-sectional study (21). A shown hypothesis in a related study suggests that the discrepancy between incidence and prevalence of schizophrenia could be due to differences in treatment adherence between the sexes and higher suicide rates associated with the male sex (22). This factor can affect the prevalence of schizophrenia up to five times, as shown in a study that assigned these variations to applied methodology, different risk factors, and the predominance of some regional genetic profiles (23).

It was also observed that schizophrenia tends to manifest a bit earlier in men than in women and that refusal to participate in treatment, which tends to be higher in men than women, leads to a worse prognosis. Despite women (median of 19 years) having a lower age of onset than men (median of 20 years), this difference was not significant (p = 0,4,844). The lack of statistical significance could be explained by the small sample size, as the sample does not show a normal distribution for age of onset (Shapiro–Wilk: W = 0.93155, p < 0,005). In addition, the BPRS score also did not show a significant difference between men and women (p = 0,172).

These findings suggest a need to consider sample asymmetry and a possible selection bias because patients with severe cognitive impairment were excluded from the research due to the impossibility of applying the BPRS, as well as refusal to participate in a significant proportion of patients, which probably indicates a worse psychopathological condition.

When classified in early-onset (less than 40 years) and late-onset (more than 40 years) (24), it was observed that almost all patients had early onset (less than 40 years), with only one patient having a late onset of the disease. This fact is according to several studies that show that schizophrenia onset is more frequent in early adulthood and rare in old age. These studies also suggest that schizophrenia could have distinct courses depending on the age of onset, supporting the hypothesis that late-onset schizophrenia could be considered a subtype of the disease (19).

This study has shown an association between clozapine use and neutrophil increase, such as an increase of SII and NLR, considering control of SAH and dyslipidemia. These results agree with previous studies that showed an inflammatory response due to clozapine administration. Furthermore, amitriptyline shows a decrease in SII and NLR, corroborating research that suggested an anti-inflammatory effect of tricyclic antidepressants.

An expressive neutrophil increase was observed and a trend in the ratio may indicate significance in multivariate analysis in subgroups of patients. Therefore, such is the importance of these aspects to future research, which should aim to better understand the role of inflammatory markers in SZ and their treatment.

The increase in BPRS scores in patients in treatment with other AP drugs and/or clonazepam may indicate the severity of symptoms in these patients. However, according to preview studies, no correlation was observed between inflammatory markers and BPRS score. Countering the lack of correlation between inflammatory markers and the clinical state of patients with SZ, our results suggest that such markers are relevant in the preview of clinical responses to clozapine. It becomes evident, however, that there is a need to expand the sample and stratify the patients according to different comorbidities in future studies. Despite this, the inflammatory profile has been shown to be an important factor that must be considered by clinicians in prescribing clozapine. Measuring the serum dosage of clozapine and cohort studies are needed to confirm the findings and assess the causality between inflammatory markers, disease course, and response to clozapine.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

The studies involving humans were approved by Committee of Ethics in Research at Hospital de Clínicas de Porto Alegre. OHRP/EUA (Office for Human Research Protections): IORG0000588. IRB 00000921 Federalwide Assurance (FWA) HHS (U.S. Department of Health & Human Services) Protection of Human Subjects: FWA00002409. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

VHSC: Conceptualization, Investigation, Methodology, Project administration, Writing – original draft, Writing – review & editing. ADT: Conceptualization, Data curation, Investigation, Visualization, Writing – review & editing. APA: Methodology, Visualization, Writing – review & editing. RM: Visualization, Writing – review & editing. PSB-d-A: Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing – review & editing.