Generalized anxiety disorder (GAD) is a highly prevalent and often chronic condition characterised by excessive, uncontrollable and frequent, irrational worries about everyday matters. GAD has a prevalence rate of more than 5% in the general population [1]. Although psychopharmacological and psychological treatments with proven efficacy in GAD exist, 42% of patients with GAD still have symptoms after 12 years, and half of patients who do recover can relapse [2,3]. A review of the etiology of GAD reveals a number of potential factors, including genetics, neurological dysfunction, stress, and life experiences [4]. The number of neurobiological studies aimed at elucidating the aetiology of the disorder has been increasing steadily. One of the biological mechanisms that has recently been the focus of research is that of stress-induced immune system pathologies leading to neuroinflammation and changes in the brain [5]. Chronic stress has been demonstrated to disrupt negative feedback in the hypothalamic-pituitaryadrenal (HPA) axis [5], resulting in increased activation in the sympathetic nervous system and decreased activation in the parasympathetic nervous system activity [6]. Consequently, catecholamine levels, which have been demonstrated to elevate pro-inflammatory cytokines, increase, whereas acetylcholine levels, which have been shown to reduce the levels of proinflammatory cytokines, decrease [5]. This mechanism may result in an increase in proinflammatory cytokine levels [5]. Thus, stress has an impact on the HPA axis, the autonomic nervous system, and, as a consequence, on the immune system. However, the diagnosis of anxiety disorders is based on the presence of specific symptoms, as outlined in the diagnostic criteria, rather than on the presence of objective biomarkers [7]. This results in challenges in both the diagnosis and estimation of severity of the disorder [8].

Oxidative stress has been implicated as one of the contributing factors in the pathophysiology of neuropsychiatric disorders, although the precise mechanisms remain unclear [9,10]. One important pathway may involve glutamate excitotoxicity and the generation of reactive oxygen species (ROS) [11]. Dynamic thiol-disulfide homeostasis (TDH), which reflects the balance between thiol and disulfide groups, serves as a sensitive marker of oxidative stress. Dynamic TDH reflects the balance between thiol groups and their oxidized counterparts, disulfides, and serves as an important marker of oxidative stress [12,13]. Thiols are highly sensitive to oxidation and act as antioxidants by neutralizing ROS. Under oxidative conditions, thiols form disulfide bonds, which are reversible, allowing the system to maintain redox balance. Disruption of this dynamic equilibrium has been associated with reduced antioxidant capacity and has been implicated in various psychiatric disorders [12]. Previous studies have demonstrated that NT and TT levels are decreased in patients with psychiatric disorders such as schizophrenia and bipolar disorder, with lower thiol levels correlating with greater symptom severity. Similarly, in individuals with untreated GAD, disulfide levels and disulfide-to-thiol ratios have been found to be significantly elevated, while the native-to-total thiol ratio was reduced compared to healthy controls (HCs). These findings suggest that oxidative imbalance may be a common feature across multiple psychiatric conditions [14,15].

Chronic stress and associated anxiety initiate proinflammatory changes through the HPA axis, thereby increasing the risk of excessive systemic inflammation [16]. The overall evidence suggests that anxiety disorders can be associated with increased inflammation [17]. A large cohort study reported high C-reactive protein (CRP) levels as a result of interleukin (IL)-6 secretion in patients with GAD [18]. In addition, a meta-analysis reported that CRP, IL-1, IL-1α, IL-2, IL-8, IL-6, IL-12p70, and IL-10 levels were increased in GAD, whereas IL-4 and IL-5 levels did not change [19]. This indicates that inflammation might significantly contribute to the development of anxiety disorders [20]. IL-1 is a cytokine with potent effects on serotonin, acetylcholine, and catecholamines. The majority of the neural, behavioural, and physiological changes triggered by stress also occurred after intracerebroventricular IL-1 administration. The elevation of IL-1 in patients with anxiety disorder lends further support to this information [21]. Mechanistically, increased oxidative stress resulting from the pro-inflammatory responses associated with elevated levels of IL-6 was shown to affect brain function and signalling patterns and therefore was directly implicated in the aetiology of GAD [22]. Many studies have shown that peripheral inflammation also affects brain structures associated with anxiety [16,23,24]. This may be due to the effects of cytokines on monoamines. It has been demonstrated that inflammatory biomarkers, including inflammatory cytokines and acute-phase proteins, are markedly elevated in a considerable number of patients with anxiety disorders. These biomarkers are considered to be potentially involved in the emergence of behavioural symptoms, rather than direct causal agents. This can be attributed to the presence of particular biological mediators between stress and inflammation, including corticotropin-releasing factor [16].

Immunoglobulins, essential components of the immune system, are regulated by the neuroendocrine system. Secretory immunoglobulin A (S-IgA), in particular, has been shown to increase in response to acute stress, while chronic anxiety may impair immune function and reduce immunoglobulin levels [25,26]. Studies have reported a negative correlation between perceived stress and S-IgA concentrations, suggesting that anxiety-related immune alterations may influence humoral immunity [27,28]. IL-40, a novel cytokine first identified in 2017, plays a key role in B cell-mediated immune responses, particularly in IgA production and B cell homeostasis [29,30]. Initially thought to be produced mainly by fetal liver, bone marrow, and activated B cells, IL-40 is now also known to be secreted by T cells, neutrophils, and macrophages [31]. Its involvement in autoimmune and inflammatory conditions—such as Sjögren’s syndrome, systemic lupus erythematosus, and rheumatoid arthritis—suggests that IL-40 may act as both an immune regulator and pro-inflammatory mediator [32]. Moreover, IL-40 has been shown to induce the release of chemokines such as MCP-1 and CXCL8, further implicating it in inflammatory pathways [33].

As with other psychiatric conditions, the diagnosis of GAD currently relies on clinical symptoms rather than objective biological markers. However, symptom overlap with other disorders and heterogeneity within GAD populations can contribute to diagnostic challenges and variable treatment outcomes [34]. Consequently, there is growing interest in exploring potential biomarkers that may help to better understand the pathophysiology of GAD [35]. Given the emerging evidence linking proinflammatory cytokines, oxidative stress markers, and immunoglobulin A to anxiety-related mechanisms, we hypothesized that IL-40 may be associated with GAD. Therefore, in this cross-sectional study, we aimed to investigate serum IL-40 levels in patients with GAD and to examine their relationship with symptom severity, CRP, and TDH, a marker of oxidative stress.

To the best of our knowledge, this study is the first case-control investigation to explore the potential link between the pathophysiology of GAD and the inflammatory cytokine IL-40. Our findings reveal that serum IL-40 levels in patients with GAD were significantly higher compared to the HC group. Furthermore, a substantial positive correlation was identified between IL-40 levels and the severity of anxiety, the duration of anxiety disorder, and CRP. However, although thiol/disulfide parameters were notably elevated in GAD patients compared to healthy individuals, these markers did not show any correlation with IL-40 levels.

The pathophysiology of GAD is complex, involving an intricate interaction between neurological, genetic, and environmental factors [39]. Numerous studies have established a link between changes in inflammatory cytokine levels and anxiety disorders, as well as correlations between cytokine levels and psychiatric disorder severity [40-43]. The correlation between serum IL-40 levels and the severity and duration of GAD observed in the current study may provide insight into potentially important immunological pathways. A significant element of the aetiology of GAD is deregulation in the immune system [17]. The cytokine hypothesis suggests that stress and activation of the sympathetic nervous system may initially trigger cytokine production [44]. Subsequently, neuroinflammatory processes induced by proinflammatory cytokines may lead to alterations in the serotonergic neurotransmitter system within the central nervous system, potentially resulting in decreased serotonin production or increased serotonin reuptake. These processes could contribute to the onset or exacerbation of anxiety symptoms [45]. The findings of the current study, therefore, support the cytokine hypothesis of anxiety disorders. The positive correlation between IL-40 levels and both HAM-A scores and disease duration observed in our study suggests that IL-40 may play a role in the development of GAD. Notably, when we stratified GAD patients based on the duration of the illness, those with long-term illness exhibited significantly higher IL-40 levels compared to those with a shorter illness duration, further supporting this association. The positive correlation between elevated IL-40 levels and the severity and duration of GAD suggests that this cytokine may be involved in the pathophysiology of the disorder. However, this association was modest (r=0.329), and given the exploratory nature of this study, it would be premature to draw conclusions regarding diagnostic utility or therapeutic targeting. Future longitudinal and mechanistic studies are warranted before IL-40 can be considered as a candidate biomarker or treatment target.

A ROC analysis indicated that the IL-40 serum levels had a high predictive value in distinguishing GAD patients from HCs. This finding suggests that IL-40 serum levels could serve as a potential biomarker for diagnosing GAD, monitoring the response to anxiolytic medications, or tracking disease progression. Biomarkers may elucidate the aetiology of mental illnesses, corroborate diagnoses, identify susceptible individuals, and determine the severity of illness. Biomarkers can also be employed to inform treatment decisions and monitor clinical response [46]. Hou et al. [47] (2019) reported that peripheral serum levels of IL-6 can be employed as a means to monitor treatment response to selective serotonin reuptake inhibitors in GAD. Likewise, IL-40 may represent a potential avenue for future investigation as a marker for monitoring therapeutic drug responses in GAD; nevertheless, this remains speculative and requires validation in future longitudinal studies to clarify its relationship with disease severity and pathogenesis.

Several studies have suggested a link between oxidative stress and the mechanisms underlying anxiety disorders [10]. Glutamate levels have been reported to be increased by pro-inflammatory cytokines, which may, in turn, cause excitotoxicity and impair neurogenesis through the activation of N-methyl-d-aspartate receptors. The activation of astrocytes and microglia by pro-inflammatory cytokines has been demonstrated to result in the secretion of reactive oxygen and nitrogen species, which can subsequently cause oxidative damage to neurons [5]. Recently, researchers have turned their attention to TDH as a potential newly identified indicator of oxidative stress. The thiol-DS balance reflects the levels of oxidants and antioxidants in the body. Consequently, current evidence suggests the involvement of the thiol-DS status in the aetiology of an expanding number of illnesses [13]. DS levels and the DS/NT and DS/TT ratios were reported to be significantly elevated in untreated GAD patients compared to HCs. The same study also showed that the ratio of NT to TT was notably reduced [15]. Similarly, another study reported that SS levels and the DS/NT ratio were elevated in drug-free patients with GAD compared to healthy individuals, whereas the NT/TT ratio was reduced [16]. In our study, we observed elevated levels of DS, as well as higher DS/NT and DS/TT ratios while the levels of NT, TT, and the NT/TT ratio were lower in GAD patients versus HCs. These results are consistent with the existing literature, indicating that TDH is disrupted in untreated GAD patients.

Oxidative stress and inflammation are closely related. Elevated ROS production triggers microglial activation, which subsequently causes neuronal death and the release of pro-inflammatory cytokines that inhibit neurogenesis [48]. Our study demonstrated increased levels of IL-40 in GAD patients and an impairment in TDH. However, correlation analyses revealed no significant associations between IL-40 levels and DS, NT, or TT concentrations, nor with the ratios of DS/NT, DS/TT, and NT/TT. The absence of a significant correlation between IL-40 levels and TDH parameters may suggest that these markers operate through distinct pathophysiological mechanisms. It is also possible that IL-40 reflects a more upstream or cytokine-specific immune response, while TDH parameters may be influenced by oxidative stress occurring at a different stage or through different regulatory pathways. Temporal dissociation between cytokine release and redox balance alteration could also contribute to the lack of observed association. Further studies with larger cohorts and longitudinal designs are warranted to explore the potential mechanistic links or independence between these biomarkers.

The serum levels of IL-40 showed good performance in distinguishing GAD patients from HCs in the current study and also revealed a positive correlation with disease severity. These findings indicate that IL-40 may have potential as a biomarker candidate for GAD. However, the diagnostic value of this cytokine should be further evaluated through comprehensive, longitudinal studies with larger and more diverse samples. In conclusion, our findings indicate that IL-40 levels were elevated in GAD patients compared to HCs, supporting the need for further studies to clarify its potential role in the pathophysiology of anxiety disorders. Early diagnosis may also decrease treatment costs and ameliorate the prevalence and morbidity associated with this chronic condition.

Anti-cytokine therapies, currently used to treat various autoimmune and inflammatory disorders [49,50], may have potential in reducing pro-inflammatory responses in GAD. However, understanding the complex immune system balance requires careful evaluation, and the development of targeted treatments depends on a detailed understanding of the specific mechanisms involved in GAD pathogenesis.