Generalized anxiety disorder (GAD) is a chronic neuropsychiatric disorder characterized by persistent and excessive uncontrollable fear or worry (occurs for at least 6 months) about various aspects/activities of daily life, affecting the educational, occupational, or social lives of the affected people [1]. If a person is excessively worried about anything for most days over at least 6 months, he/she is considered to have GAD. Though currently the prevalence rate of GAD is 3–6% worldwide [1,2,3], the prevalence is increasing day by day due to the complexity of modern lifestyles and thus warrants attention from national and international authorities to take interventions for mitigating and managing this disorder properly. If it remains undiagnosed or untreated, the uncontrollable and persistently intense anxiety can lead to a marked reduction in cognitive functions or a reduced capacity to work properly in all spheres of life, including educational, family, social, and individual routine work. As such, chronic GAD leads to a reduced quality of life and thereby poses a significant mental health concern globally.

Despite its high prevalence, significant morbidity, and socioeconomic burden, GAD remains poorly characterized in terms of its pathophysiology or effective treatment options. Though the precise cause and mechanism of pathogenesis are still unknown, evidence suggests that multiple factors, including disrupted serotonergic, dopaminergic, and GABAergic neurotransmission and excessive glutamatergic neurotransmission in the brain, genetic factors, family or environmental stress, chronic diseases, hyperthyroidism, childhood trauma, and special personality traits, are linked to GAD. Alterations in monoaminergic neurotransmissions in limbic systems (cingulate gyrus, hippocampus, amygdala, thalamus, and hypothalamus) due to the lower synaptic availability of serotonin, norepinephrine, and dopamine are thought to be associated with anxiety symptoms. Besides, decreased GABA-mediated inhibitory neurotransmission in the amygdala or excessive activation of excitatory glutamatergic neurotransmission are also suggested to be involved in GAD pathology.

Currently, available pharmacotherapies for GAD include selective serotonin reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors (SNRIs), pregabalin, and benzodiazepines, which act by reversing these altered monoaminergic neurotransmitter systems. Alongside these drug treatments, non-pharmacological therapies such as several psychological interventions, including cognitive-behavioral therapy, and the acquisition and application of stress management skills, including relaxation and mindfulness skills are also widely used for the management of GAD. However, currently, available pharmacotherapies (SSRIs, SNRIs, pregabalin, and benzodiazepines) have failed to demonstrate the required efficacy in treating anxiety disorders, as 50% of patients failed to respond to these drugs, and at least in 30% of cases, there is a recurrence of the disease following the pharmacological treatment [1, 4, 5]. Moreover, studies reported a higher rate of discontinuity from these pharmacotherapies with low patient adherence or compliance due to the adverse effects, including sexual dysfunction for SSRIs and SNRIs, nausea and dizziness for pregabalin, demonstrating an urgent need for searching for novel anxiolytics [3]. These findings raised questions about the validity of the currently available mechanism of pathogenesis and suggested that the altered monoaminergic neurotransmitter system might not fully explain the molecular mechanism of GAD development, suggesting other pathophysiological factors might be involved in GAD. Recently, dysregulated immune systems have attracted great interest as an important pathophysiological factor for the development of GAD [4, 6,7,8]. Several clinical and preclinical studies suggest a link between the altered immune system and GAD pathology. Preclinical studies in mice also demonstrated that administration of pro-inflammatory cytokines (including IL-1β, TNF-α, and IL-6) in mice resulted in anxiety-like behaviors that were attenuated or normalized after injecting either anti-inflammatory cytokines or antagonists for the concerned cytokines [9,10,11,12,13]. A recent prospective cohort study conducted by Hou et al., (2019) demonstrated that administration of selective serotonin reuptake inhibitors (escitalopram or sertraline) resulted in a significant reduction in peripheral pro-inflammatory cytokines, and the authors suggested that the anxiolytic effects of these SSRIs might partly be based on their acute anti-inflammatory activities [14], implicating a significant association between dysregulated peripheral immune systems and GAD development. The development of anxiety-like symptoms in IL-4 gene knock-out mice, reduced levels of IL-4 in anxious mice, and the significant attenuation of anxiety-like behaviors following IL-4 injection demonstrated a positive association between anti-inflammatory cytokines, IL-4 levels, and anxiety pathology [15,16,17,18]. This immune hypothesis of GAD development is further potentiated by findings from several clinical studies that reported that GAD patients showed significantly higher levels of pro-inflammatory cytokines ( IL-1Ra, IL-1, IL-6, TNF-α, etc.) compared to healthy controls (HCs) [19,20,21,22,23,24,25,26,27,28] along with decreased levels of anti-inflammatory cytokines, including IL-4 and IL-10 [25]. Besides, pro-inflammatory cytokines such as TNF-α, and IL-6 were significantly associated with anxiety scores [29]. Consistent with this, a randomized clinical trial in humans demonstrated that LPS administration resulted in enhanced anxiety scores, and the authors suggested a significant correlation between pro-inflammatory cytokine levels and anxiety severity [30]. LPS-mediated microglia activation causes enhanced release of excessive pro-inflammatory cytokines in the basolateral amygdala, which ultimately leads to neuroinflammation in mice, resulting in the development of anxiety and depression-like behaviors by modulating neuronal plasticity. The authors found that anxiety pathogenesis was due to the excessive release of excitatory neurotransmitter glutamate from presynaptic axonal terminals of the prefrontal cortex, leading to neuroplasticity [31]. However, some studies reported either no significant variation in pro-inflammatory or anti-inflammatory cytokine serum levels between GAD patients and HCs [32] or that pro-inflammatory cytokines including IL-1, IL-2, and IL-6 were significantly reduced in GAD patients than HCs [33, 34]. This discrepancy in altered levels of inflammatory cytokines across clinical studies necessitates a further examination of the role of these cytokines in GAD pathophysiology.

Interleukin-2 (IL-2) is one of the major pro-inflammatory cytokines implicated in T cell activation, proliferation, and differentiation and is thus linked to excessive neuro-inflammatory processes [35]. IL-2 has been shown to impair synaptic plasticity and cause neuroinflammation, which ultimately leads to neuronal damage in neurocircuits associated with fear and anxiety signal transduction. IL-2 was also reported to act as a potent modulator of NMDA and kainite-mediated excitability in mesolimbic or mesostriatal systems [36,37,38] and thus affect neuroplasticity. As IL-2 was found to be positively associated with major depressive disorder [38, 39], probably, IL-2 might also be correlated with anxiety disorders like GAD, as MDD and GAD are highly co-morbid themselves and thus might share common pathophysiological factors. Recently, a preclinical study conducted by Gilio et al., (2022) observed that IL-2 administration in experimentally healthy mice triggered marked anxiety and depression-like behaviors, and the authors suggested that inhibition of GABA-mediated synaptic inhibitory neurotransmission was involved in the pathology of anxiety [40].

Interleukin-10 (IL-10) is one of the major anti-inflammatory cytokines that is secreted from Treg cells, Th2 cells, CD4 + T cells, CD8 + T cells, monocytes, macrophages, dendritic cells, B cells, neutrophils in the peripheral nervous system, and from microglia, astrocytes in the central nervous system (CNS) [41]. IL-10 signaling triggers anti-inflammatory, immunosuppressive, and immunoregulatory activities, including downregulating the production and secretion of pro-inflammatory cytokines and chemokines from activated macrophages, neutrophils, mast cells, Th1 cells, and DCS, decreasing the expression of MHC class II and co-stimulatory molecules on macrophages, and thereby suppressing the antigen presentation capacity of APCS [42,43,44,45,46]. In the CNS, it inhibits the production of such cytokines and chemokines by activated microglia and thereby counteracts cellular and tissue damage in response to excessive neuroinflammation [47, 48]. IL-10 has also been shown to stimulate axonal regeneration and activate wound healing through tissue repair [48]. Research also indicates its role as an inhibitor for microglial hyperactivation in response to LPS-induced inflammatory stimulus [49]. Based on its anti-inflammatory and immunoregulatory functions, researchers suggested an intricate role for IL-10 in several auto-immune and neuropsychiatric disorders. For example, Mesquita et al., (2008) observed that IL-10 KO mice developed markedly enhanced depressive-like behavior, which was attenuated after IL-10 administration, and that overexpression of IL-10 resulted in reduced depressive behaviors in mice [50]. Moreover, administration of IL-10 into rats attenuated the pro-inflammatory cytokine IL-1β-induced anxiety-like symptoms in male rats [10], demonstrating that IL-10 possesses anxiolytic activities. Preclinical research using an experimental animal model also suggests that the observed anxiolytic effect of several anti-anxiety drugs, including 3’-deoxyadenosine (3’-dA), imipramine, fluoxetine, and chlordiazepoxide, stems from their ability to upregulate anti-inflammatory cytokine (IL-4, IL-10) expression in the prefrontal cortex and locus coeruleus and simultaneous down-regulation of proinflammatory cytokine gene expression, leading to a correction of the imbalance between proinflammatory and anti-inflammatory states [51, 52]. Though several preclinical studies suggest a potential link between IL-10 levels and anxiety disorder, there is a scarcity of clinical studies aimed at evaluating such an association between IL-10 and GAD development [10].

Currently, there is no objective and cost-effective diagnostic or prognostic biomarker for GAD, which poses challenges in early diagnosis or risk prediction and leads to misdiagnosis or underdiagnosis, hampering the proper management of the disease. Currently available diagnostic tools, including self-reported symptoms and scoring severity based on the patient’s response to the 7-item questionnaire (GAD-7 scores), are subjective. Though neuroimaging techniques such as positron emission tomography (PET) and functional MRI can be used for the proper and objective diagnosis of GAD, due to their high cost and sophistication or complexities, these diagnostic tools are not suitable for either mass-level screening or are not easy to conduct multiple times to monitor the disease progression or therapeutic drug response. As such, the investigation of cost-effective objective biomarkers for GAD is one of the major focuses of current research on GAD. Finding a suitable biomarker is essential for early diagnosis and initiating psychotherapy and pharmacotherapy as early as possible [3]. Several studies were performed investigating the potential association between altered pro-inflammatory cytokines or anti-inflammatory cytokines and the pathogenesis of GAD. However, the actual role of inflammatory cytokines in GAD patients is not well explained. Therefore, the present study aims to explore the role of pro-inflammatory cytokines (IL-2) and anti-inflammatory cytokines (IL-10) in the pathophysiology and development of GAD. Also, we aim to find the potential associations of IL-2 and IL-10 with the severity of GAD patients. We believe the present study results would help to understand the pathophysiology and development of GAD.