The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes coronavirus disease-2019 (COVID-19), led to more than 7 million deaths worldwide since 2019. The COVID-19 causes generalized damage to many organs and systems of the body. After the end of the acute phase of the disease, 10–30% of patients develop post-acute sequelae from SARS-CoV-2 infection, which is called long-COVID or post-COVID syndrome (PCS) [1, 2]. With PCS, the symptoms of COVID infection last for at least three months and this can happen after severe, moderate or mild, or even asymptomatic COVID-19 [3]. Common clinical symptoms of PCS include weakness, fatigue, cough, sub-febrile temperature, headache, loss of smell or taste, sleep disturbances, muscle and joint pain. PCS also involves a variety of pulmonary, neurological, cardiovascular, renal, gastrointestinal, endocrine, and thromboembolic complications [4–9].

The most important consequences of the COVID-19 infection include neurological symptoms, which represent a serious global problem. The risk of neurological and psychiatric diagnoses associated with COVID-19 is thought to have increased following the emergence of the Delta variant [10]. The SARS-CoV-2 virus has a direct neurotropic effect on the brain, which is one of the target organs for coronaviruses. SARS-CoV-2 RNA and anti-SARS-CoV-2 antibodies were detected in the cerebrospinal fluid of patients with acute encephalopathy caused by COVID-19 [11, 12]. Autopsies of the brains of patients who died from COVID-19 have revealed pathological features associated with the SARS-CoV-2 in cortical neurons [13, 14].

SARS-CoV-2 can damage neurons by directly entering the central nervous system (CNS), but even if the virus does not directly enter the CNS, peripheral cytokines involved in the host antiviral response, especially interleukin 6 (IL-6), IL-8, interferon (IFN) and tumor necrosis factor (TNF) can trigger an inflammatory response by increasing the permeability of the blood-brain barrier and participate in neuroinflammation [15, 16]. Studies have shown that psychiatric disorders, including mood disorders and schizophrenia, are closely associated with inflammation. Proinflammatory cytokines can potentially influence emotional behavior and cognitive function by decreasing brain monoamine levels, activating neuroendocrine responses, enhancing excitotoxicity such as increased glutamate levels, and impairing brain plasticity [17]. This may be one of the potential mechanisms for the development of mental disorders in patients with COVID-19 [18, 19].

SARS-CoV-2 virus can trigger a cytokine storm and lead to autoimmune dysregulation [20, 21]. When SARS-CoV-2 replicates in the body, an immune response is activated, which is mediated by membrane-bound immune receptors and downstream signaling pathways [22]. This response includes massive infiltration of monocytes and macrophages that secrete cytokines, chemokines and other inflammatory mediators that contribute to the cytokine storm which can lead to significant tissue damage, organ dysfunction, and respiratory failure during the acute phase of COVID-19 [22, 23]. The results of meta-analyses revealed an increase in the level of cytokines in the blood of patients with first-episode psychosis and exacerbation of schizophrenia, including IL-6, IL-17, IL-8, IFN-γ, transforming growth factor-β (TGF-β) [24].

After the acute phase of inflammation when the pathogen has been eliminated, the phase of inflammation suppression begins. This includes such processes as decrease in pro-inflammatory cytokine production and increase in anti-inflammatory cytokine production, cessation of neutrophil infiltration at the site of inflammation, induction of neutrophil apoptosis and their absorption by macrophages, reprogramming of macrophages from M-1 (pro-inflammatory) activation type to M-2 (proliferative), return of immune cells, particularly dendritic cells and macrophages that have not undergone apoptosis, to the lymphatic and blood vessels, development of immunosuppressive cells at the previous site of inflammation and tissue damage repair [25]. Molecules that stimulate an anti-inflammatory response include IL-10, TGF-α, and other specialized pro-resolving mediators [26–29]. In SARS-COVID-19, along with the development of a pro-inflammatory response, a compensatory anti-inflammatory response also occurs [30]. In particular, the expression of anti-inflammatory cytokines, such as IL-4 and IL-10 increases during and after the acute period of COVID infection [30, 31]. As a result of insufficient suppression by anti-inflammatory cytokines, most patients with long-COVID experience unbalanced long-term production of a number of proinflammatory cytokines [30]. By analyzing cytokine profiles in people who have recovered from COVID-19 and are experiencing neurological symptoms, specific biomarkers may be identified for early detection or prediction of the course of neurological manifestations. This may help tailor treatment plans and monitor recovery or disease progression over time.

The relationship between viral clearance and antibody response in long-COVID is also an area of interest [32–34]. In the study, it was shown that patients with long-COVID have a high specific T-cell response to SARS-CoV-2, which did not decrease for 8 months, in the absence of naive T- and B-lymphocytes [34]. The reason for this stable level of specific immune response in patients with PCS may be the ongoing persistence of SARS-CoV-2 virus. As has been shown, the persistence of the Spike protein and SARS-CoV-2 viral RNA fragments which can be detected in extracellular vesicles may be related to PCS [33]. Thus, the presence and action of antibodies in long-COVID may have implications for treatment strategies and patient management. For example, this may help to guide the decision to prescribe immunomodulatory therapy or other measures to limit persistent symptoms.

The aim of the study was to evaluate the levels of serum cytokines and indicators of the humoral immune response to SARS-CoV-2 in patients with PCS with identified neurological symptoms.

The late effects of COVID-19 (PCS) among which neurological symptoms are the most common long-term clinical manifestations have become a major global health problem [37]. There are various research methods for studying cognitive functions that allow for a comprehensive assessment of aspects of cognitive processes. One of the main methods for studying cognitive functions in clinical practice is neuropsychological testing. These are specialized tests designed to assess cognitive functions taking into account their neurological basis. Neuropsychological tests may include assessment of various aspects of memory, attention, executive functions, spatial and gnostic skills, and other cognitive processes [38]. These tests may also include assessment of deficits in cognitive functions associated with specific neurological conditions or diseases [37]. An important feature of using this method for diagnosing cognitive impairment is its ease of use, speed, and assessment of a wide range of cognitive indicators. The MOCA and the Mini-Mental State Examination (MMSE) are most often used in clinical practice. Due to its lower sensitivity, it was decided not to use the MMSE scale for the study, and the SAGE test was used. The peculiarity of this test is that it is completed independently by the patient. When assessing cognitive functions using the MOCA and SAGE scales in patients in the post-COVID period, a moderate decline in cognitive functions was detected in only two patients out of the total number of patients in the group. At the same time, no correlation was found with the severity of the disease, which is at odds with the data of some studies [39].

When assessing affective disorders, anxiety disorders were detected according to the HADS scale in 5 patients from the PCS group. Depressive disorders were found in 7 patients from the post-COVID-19 group, while 2 patients had moderate depressive disorder. An increase in the level of asthenia was detected in all patients in the post-COVID period according to the MFI-20 scale. This is consistent with the data available in the literature [40, 41]. A history of mild to moderate COVID-19 infection primarily increases the risk of developing affective disorders and asthenia and, to a lesser extent, the development of cognitive impairment.

Neuroinflammation may play a role in the various neurological symptoms and cognitive impairments observed in post-COVID patients. Neurofilament light chain (NFL) protein and glial fibrillary acidic proteins (GFAPs) that maintain the stability of neuronal axons and astrocytes may serve as circulating biomarkers associated with neuronal degeneration and injury [42]. Long-COVID patients with elevated blood NFL and GFAP levels had increased headaches and persistent neuropathic pain [43]. Peluso et al. [34] reported that serum levels of NFL and GFAP in acute COVID-19 patients were positively correlated with IL-6, TNF-α, and CCL2, which can induce immune cell activation and neuroinflammation. This indirect mechanism suggests that proinflammatory cytokines/chemokines may exacerbate significant neuronal injury. Previous studies have identified specific changes in cytokine profiles in long-COVID-19 patients. For example, some studies noted ongoing increases in cytokines such as IL-6 and IL-8. These markers indicate ongoing inflammation and may correlate with the severity and type of long-term COVID symptoms [44]. Prolonged action of IL-6, TNF-α, and C-reactive protein (CRP) was also involved in the systemic and neurological long-term sequelae of COVID-19 [45]. The present study found that IL-6 and IFN-α levels exceeded reference values ​​and were similar to those of acute COVID-19 patients (Table 1), indicating ongoing inflammatory processes. The decrease in these indicators in convalescents without PCS was explained by their lack of neurological symptoms or longer time since the onset of the symptoms. If neurological disorders are detected in the post-COVID period, this data should be considered when prescribing anti-inflammatory therapy.

Our study showed that the severity of cognitive and neurological impairment in most cases was weakly or moderately positively correlated with IL-1 levels. IL-1 is considered a key mediator of immunity and inflammation [46]. In COVID-19, the hyperinflammatory IL-1 family of cytokines, including IL-1α, IL-1β, IL-18, IL-33, and IL-36, have been shown to be the main cause of inflammatory activity, and high levels of these cytokines are directly correlated with disease severity. Therefore, IL-1 family blockers can be considered as effective therapeutic agents for the treatment of COVID-19 [47]. Elevated levels of IL-1 in patients with PCS may reflect ongoing immune activation. It has been shown that chronic inflammation mediated by TNF-α release also contributes to cognitive dysfunction [48].

Another mechanism for PCS development involves chronic inflammation induced by type I IFN [49]. Our data on the increased levels of IFN-α in the most severe cases of COVID-19 and persistent elevations in IFN-α levels in patients with PCS and neurological disorders may support the theory of chronic inflammation caused by type I IFN in PCS. Some studies suggest that there may be a decrease in serotonin levels, which can impair the activity of the vagus nerve and worsen hippocampal reactions and memory [49], but in our study, we did not observe a decrease in serotonin levels in patients with PCS (Figure 5). The highest levels of serotonin were found in acute COVID-19 patients, and the lowest levels were in pre-COVID-19 samples from healthy patients collected in 2018 (Figure S1).

IL-8, also known as CXCL8, is a chemokine that plays an important role in the immune response by recruiting and activating neutrophils, which are critical for inflammation and infection-fighting, and may have a prognostic role in COVID-19 disease [50]. IL-8 has been linked to chronic inflammatory demyelinating polyneuropathy, thus it has been proposed as a possible biomarker of acute immune responses against the nervous system [51].

Our study found a tendency towards lower levels of IL-10 levels in PCS after moderate to severe COVID-19, which could indicate a decreased anti-inflammatory cytokine profile in more severe COVID-19. In the past, individuals with persistent post-COVID symptoms have exhibited lower levels of IL-4 and IL-10, which may indicate that these cytokines could serve as diagnostic markers for long-COVID, a condition characterized by an inflammatory response with reduced anti-inflammatory response mediated by IL-4 and IL-10 [52]. From another perspective, IL-4 contributes to neuroinflammation by recruiting immune cells into the CNS [53]. In our study, both IL-4 and IL-10 levels were higher in patients with PCS than in convalescent patients without PCS, although IL-4 levels were within normal ranges and IL-10 was above normal levels. These findings should be further explored with larger sample sizes.

In addition to the cytokine profile, IgM may provide insight into the immune response during and after infection and understanding the dynamics of viral clearance. Our study showed that several PCS patients had IgM antibodies to the SARS-CoV-2, and overall, the level of IgM seropositivity was higher among these patients (Figure 6). IgM is one of the first antibodies produced by the immune system in response to infection. In the context of COVID-19, it typically appears within days to weeks of symptom onset [54]. The presence of IgM is commonly indicated in recent infections [55]. In our study, IgM was detected on average 2 months after COVID-19, both after mild (in 2 people) and after moderate and severe (in 4 people). Whether the presence of IgM in the post-COVID period among patients diagnosed with neurological disorders for more than 2 months indicates the persistence of the virus is still unclear.

IgG antibodies are associated with long-term immunity [56]. Understanding the specific IgG subclasses produced in response to COVID-19 is critical to assessing the duration of immunity following natural infection or vaccination. The fact that in the present study the levels of IgG, but not IgA, negatively correlated with the Sheehan Anxiety Scale scores may be due to IgG exhibiting neutralizing properties to the SARS-CoV-2 virus. Also, in acute COVID-19, levels of IgM and IgG antibodies correlated negatively with levels of IL-6 and IFN-α (Figure 2B). All this together may indicate a positive role of antibody response in the pathogenesis of COVID-19. This does not allow us to exclude a positive role for individual classes and subclasses of antibodies in PCS.

Different IgG antibody subclasses (e.g., IgG1, IgG2, IgG3, and IgG4) have different functions and properties. Studying these antibody subclasses can provide insight into strength and persistence of immune memory following infection or vaccination. During the COVID-19 pandemic, some studies have shown that certain COVID-19 patients developed IgG4-associated diseases including parenchymal organs, lymph nodes, and pleural lesions [57]. The present study showed that after moderate and severe COVID-19, IgG2 and IgG4 were predominant in the blood of examined patients with PCS and neurological symptoms. The positive correlations between IgA, IgG2, and IgG4 levels with a cytokine-like IL-8 suggest further investigation to the role of IgG4 in post-COVID with neurological features. In summary, this study emphasizes the importance of addressing innate and adaptive immune responses in long-COVID patients with neuroinflammation.

The main limitation of the study is the relatively small sample size, which limits the statistical analyses. In addition, we studied only a limited number of cytokines. However, the study does include a control group of convalescent patients without PCS or neurologic manifestations.

Data on the increase in IFN-α during the most severe course of COVID-19 and the persistence of high levels of IFN-α in the blood during PCS, as well as the detection of IgM antibodies to SARS-CoV-2 in post-COVID serum, were obtained. Elevated levels of cytokines, especially early ones, were higher in patients with PCS compared to those who recovered from COVID-19 without PCS. In PCS patients, IgM antibodies were detected, which could indicate the persistence of the virus. No increase in serotonin was detected in PCS patients compared to convalescent controls.