Treg Cells in Ischemic Stroke: A Small Key to a Great Orchestrion

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INTRODUCTION

Statistical forecasts show an annual increase in the number of patients with ischemic stroke until 2030. Ischemic stroke is a leading cause of patient disability and the second most common cause of mortality worldwide, following ischemic heart disease [1, 2].

Currently, the main international and Russian therapeutic recommendations for treating patients in the first hours of an ischemic stroke focus on rapidly restoring blood flow and preventing ischemic brain tissue necrosis. These include neuroprotection, thrombolysis, mechanical thromboextraction, and anticoagulant therapy [3]. Difficulties arise when the therapeutic window to restore cerebral blood flow is missed. In this case, the therapeutic options are limited to protecting brain cells from oxidative stress [3] by preventing ferroptosis [4] and providing symptomatic therapy. Several biomolecules (TIM-3, DOR, PD-1, ROCK, ADAMTS-13, S1PR, etc.) that may play a role in stroke pathogenesis [1, 5–7] are being investigated as potential targets for stroke therapy; however, their therapeutic potential remains to be evaluated in randomized clinical trials.

Recent studies [2, 5–7] have highlighted the crucial role of inflammatory response within the ischemic penumbra and associated immune response in determining the course of ischemic perifocal tissue, focal organization, and neuronal plasticity during the recovery of lost function. The role of regulatory T cells (Tregs) in anti-inflammatory signaling cascade regulation and immune response suppression has been widely studied [2, 5, 8–10].

Studies on animals with experimental cerebral ischemia have accumulated considerable data on the potential translational significance of Tregs in stroke. Although some studies have noted a relationship between Tregs and cerebral microvascular dysfunction and the development of microthrombosis [11–13], several studies have reported a positive effect of Tregs in ischemic stroke. Ex vivo-engrafted Treg administration reduces the size of experimental brain infarction [9]. Tregs have been found to reduce the area of inflammatory tissue damage in ischemic stroke, block neuroinflammation, regulate neural tissue regeneration and angiogenesis, improve white matter recovery, inhibit astrogliosis, and reduce the cytotoxic activity of microglia, resulting in favorable effects on the recovery of neurological deficits [10, 13–16].

Pilot clinical trials have been conducted on the therapeutic potential of Tregs in neurology for multiple sclerosis, and the results have been favorable [8]. Additionally, preclinical studies on the safety and efficacy of Tregs in experimental stroke therapy show promise [9]. The next step is to evaluate the clinical relevance and feasibility of introducing Treg therapy for ischemic stroke into clinical practice. The urgency of the problem is highlighted by the lack of effective neuroprotective methods for fatal stroke.

Thus, we analytically reviewed over one hundred Russian and foreign publications that investigate the diagnostic, prognostic, and therapeutic applications of Tregs in stroke and that were published between 2018 and 2023.

IMMUNE RESPONSE TO ISCHEMIC STROKE: ROLE AND THERAPEUTIC POTENTIAL OF T-REGULATORY CELLS

Ischemic stroke can cause nerve tissue damage and increased permeability of the blood–brain barrier, leading to an intense inflammatory reaction and immune response. This response can be either innate or adaptive with a local autoimmune orientation. Maintaining a balanced immune response is crucial because an overexpressed or suppressed response can have harmful consequences to the patient. The course and outcome of ischemic stroke are determined by the features and orientation of the immune response [9, 18–20].

The brain is an immunoprivileged organ, with resident immune cells (microglia), borderline macrophages, and T cells present in the membranes’ structures [21]. In stroke, blood–brain barrier damage allows immune cells to migrate to the zone of ischemic injury [17], accompanied by mitochondrial degradation, oxidative stress, lactoacidosis, metabolic changes, excitotoxicity, and neuroinflammation in the damaged brain area, which can cause different forms of cell death, including apoptotic or non-apoptotic (necroptosis, ferroptosis, cuproptosis, parthanatos, and pyroptosis) [22–25]. The release of alarmins, which are bioactive molecules that act as danger signals and include heat shock proteins, S100B, HMGB1, beta-amyloid, hyaluronan, etc., triggers innate immunity mechanisms. This, in turn, stimulates the pro-inflammatory polarization of microglia and peripheral immune cell migration to the ischemic penumbra. Subpopulations of T- and B-lymphocytes, neutrophils, monocytes, astrocytes, and microglia are involved in different stages of this cascade. In this case, Tregs control the immune response and autoimmune reactions by suppressing the proliferation and/or effector activity of T- and B-cells, natural killer cells (NK-cells), and antigen-presenting cells [14]. We will not discuss the similar immunosuppressive role of regulatory B-cells (Bregs) [28]. Local inflammation is a common occurrence in conditions such as multiple sclerosis, hemorrhagic stroke, brain injury, and demyelinating diseases of the central nervous system [8, 10, 26].

In rodent experiments, Tregs have been found to reduce the degree of damage to the blood–brain barrier in the first hours after stroke development. This effect is observed even when recombinant tissue plasminogen activator, a drug approved by the US Food and Drug Administration for thrombolytic therapy, is administered within the first hours after stroke [15]. In reducing CCL2 expression by vascular endothelial cells, Tregs have a positive effect on the permeability of the blood–brain barrier and survival of oligodendrocytes and their precursors [16] and inhibit or suppress reactive astrogliosis [29]. Simultaneously engaging stem cells in the subventricular niches, spleen, and intestine along the brain–intestine axis [30] determines the characteristics of cellular response in neuroinflammation in the brain in the poststroke period. Tregs play a role in reducing the degree of demyelination [31], such as in multiple sclerosis [8], and in reducing inflammation in different types of nervous system tissue lesions [10].

Tregs perform functions beyond their immunosuppressive role. They participate in the regulation of metabolism and cellular sensitivity to insulin. Additionally, they influence stem cells in various locations, such as the bone marrow or intestine, potentially affecting stem cells in subventricular and subgranular niches [30]. This influence may further impact the cellular response to ischemic stroke.

A therapeutic approach aimed at modulating the immune response through Tregs is an attractive option [5]. Two possible strategies exist:

1) Pharmacological action on Tregs and immune mechanisms using low-molecular-weight drugs, microRNAs, monoclonal antibodies, and cytokines

2) Transplantation of autologous Tregs of a certain phenotype to the patient after ex vivo augmentation [8]

Experimental data showed that Tregs are a potential therapeutic solution for immunoprotection and neural tissue repair on day 1 after stroke development. Implementing Treg-targeted therapy for poststroke ischemia has become a subject of discussion in recent publications [32–34].

In mice with experimental stroke, exogenous Treg infusion has been shown to increase the number of Tregs from the first day to the second week and persists for a month. This reduces stroke volume and long-term functional recovery [16]. Therefore, it is critical to determine the molecular and cellular mechanisms of Treg-cell regulation of the immune response during the development of ischemic stroke [35]. The potential clinical application of autologous Treg transplantation as a therapeutic tool in ischemic stroke is promising, given its successful application in multiple sclerosis [8] and the observed reduction of Treg levels and dysfunction in stroke [36]. This gives hope that they will find clinical application in ischemic stroke [34].

Phenotypic and functional characteristics of Tregs

The human T-lymphocyte population includes CD4⁺ T-helper cells (Th), CD8⁺ cytotoxic T cells (CTL), γδ T cells, and Tregs, with several distinguishable subpopulations (Table 1) [37–43]. Tregs are a functionally distinct subpopulation of CD4+ T cells, with the FoxP3 transcription factor (forkhead box protein 3) considered as its obligatory marker [44]. The immunosuppressive activity of Tregs is closely related to the level of FoxP3 expression, and a decrease in FoxP3 content is associated with its reduction [45]. Autoimmune syndromes associated with the absence of functional Tregs develop because of mutations in the gene encoding FoxP3 [46]. The transcription factor FoxP3 is the determinant in the differentiation of T cells along the immunosuppressor pathway, producing Tregs from nonregulatory T cells (Tconv) [47].

In vivo, FoxP3 is induced by signaling pathways from the T-cell receptor in the thymus [48]. In humans, FoxP3 is represented by three functionally different isoforms [49], and the expressions of the different isoforms affect the development of some diseases. For instance, in allergic rhinitis, the expression of FOXP3∆2 isoform leads to a decrease in the number and functional activity of Tregs [51]. Therefore, the presence of these isoforms should be considered when phenotypically characterizing Tregs.

The FOXP3 gene, which encodes the FoxP3 protein in humans, is in the X chromosome in the centromeric region (region Xq11.3-q13.3). This location may contribute to the higher risk of stroke and lower survival rate after stroke in menopausal women compared with men of the same age group [52]. The immune response differs between men and women, which is partly due to the karyotype and influence of sex hormones. Many of these hormones affect Tregs. For instance, this study examined the variations in CD4⁺CD25⁺ FoxP3⁺Treg levels during different stages of the menstrual cycle in healthy women. However, the effect of these changes on ischemic stroke requires further investigation [53]. Progesterone stimulates the formation of inducible regulatory T cells (iTregs) [54], whereas testosterone promotes the expansion of Tregs and affects chromatin modification in the region encoding FoxP3 [55]. In mice, the gene encoding FoxP3 is also in the X chromosome. An experiment was conducted on transgenic mice in which the SRY gene, necessary for the development of a male-type organism, was transferred to the autosome. The results showed that the difference in the course of stroke, regardless of the level of sex hormones, is primarily determined by the karyotype. Karyotype XX was associated with a larger infarct volume after experimental middle cerebral artery occlusion than XY [56].

There are two primary methods of Treg pool formation. The first involves the development of naive “natural” Tregs (nTreg) in the thymus, which are activated in peripheral tissues. These cells exhibit a phenotype that includes CD4+, Helios⁺, TCR(CD3), CD25^high, CD39, CD73, CD127^low, CD152 (CTLA-4), CD357(GITR), and LAG3 [10, 57]. In the second pathway, CD4+ T-helper cells (Th) differentiate into Th1 cells (under the influence of cytokines IL-12, IFN-γ, IL-27), Th2 cells (under the influence of IL-4), Th17 cells (under the influence of IL-1β, IL-23, IL-6, TGF-β), or iTregs (in the presence of IL-2 and TGF-β) after leaving the thymus. CD4 and FoxP3 are the main markers of iTregs. These cells secrete TGF-β, either IL-10 or IL-35, and can be subdivided into Th3, Tr1, and iTr35 cells, respectively [10, 48]. The T-cell receptor repertoires of nTregs and Tregs, whose precursors are T-helper cells, differ [58]. Furthermore, they differ in the methylation profiles of certain CpG DNA in the enhancer region of the gene encoding FoxP3. In cells formed from T-helper cells, this region is methylated [59].

CD8⁺ cells and CD4⁺ Tregs have similar functions [40]. In patients with inflammatory diseases such as systemic lupus erythematosus, the number of CD4⁺, CD257^low, and FoxP3⁺ subpopulations increases [41]. Additionally, CD4⁺, CD25⁺, CD127^low, and FoxP3⁺ cells can be divided into naive nTregs and memory mTregs, which have distinct functions (Table 1).

 

Table 1. Basic subpopulation of Treg cells

Subpopulation	Description/function	Basic markers	Source
nTregs	Naive “natural” Tregs formed in the thymus	CD4, FoxP3, Helios, CD25high, CD39, CD73, CD127low, CD152, CD357, LAG3	[10, 37]
Induced iTregs, also known as peripheral pTregs Subdivided into Th3, Tr1, and iTr35	Peripheral-induced effector Tregs formed from CD4+ T-helper cells (Th)	CD4, FoxP3, Helios-	[37, 38]
Activated Tregs	Activated Tregs versus Tconv	CD25, CD127, FOXP3, IKZF2, ITGA4, IKFZ, IKZF2, ITGA4, TRIM-	[39]
Cytotoxic Tregs	Tregs providing direct cytotoxicity	CD8 and FoxP3+ and FoxP3- subpopulations	[40]
Subpopulation with low CD25 levels	Treg subpopulation found in systemic autoimmune diseases	CD4, CD25-/low, FoxP3	[41]
Resident brain Tregs	Resident Tregs of the neural tissue	FoxP3, CD69, PD1, KLRG1, 5-HT7, CD103, Neuropeptide Y, Osteopontin	[42]
mTregs	Memory Tregs, tissue-resident	CD4, CD25, FoxP3, CD45RA-, CD45RO+	[43]

 

FoxP3 is detected in some nonregulatory T cells; however, this does not result in an immunosuppressive phenotype [60]. A detailed panel of markers, including IKFZ⁺⁺ and ITGA4⁺, and the absence of the TRIM marker on Tregs, which is highly expressed in Tconv cells, distinguishes between these cell populations [10, 39].

Tregs differ significantly from other T cells regarding metabolism. In effector T cells, the main regulators of metabolism are mTor, HIF-1a, Myc, ROHA, ICOS, and BCL6. However, in Tregs, this role is played by mTor, Myc, and FoxP3 [61]. Recent studies have shown that FoxP3 controls cell metabolism, facilitating cell adaptation to low glucose and high lactate. This adaptation allows Tregs to function effectively in ischemic tissues [62].

The immunoregulatory functions of Tregs are carried out through the secretion of anti-inflammatory cytokines, such as IL-10 and TGF-b, and the expression of immunosuppressive molecules, including CD152, CD39, and PD-1. Additionally, the binding of extracellular IL-2 and increased expression levels of CD25 (IL-2 receptor) can reduce the intensity of the immune response. Further, regulatory molecules expressed on the cell membrane, such as cytotoxic T-lymphocyte glycoprotein 4 (CTLA4, CTLA-4, CD152), interleukin IL-10, and transforming growth factor TGF-b1, modulate the activity of antigen-presenting cells [63]. CD152 plays a role in downregulating the costimulatory molecules CD80 and CD86 on dendritic cells [64]. These molecules are crucial for stimulation to prevent the proliferation and apoptosis of activated T cells. Moreover, Tregs can directly eliminate T- and B-cells involved in inflammation [61] through perforin and granzyme secretion.

Modern methods of single-cell DNA sequencing have revealed a high degree of phenotypic heterogeneity in cell populations involved in the development of ischemic stroke [65]. A transcriptome analysis has shown that Tregs exhibit high phenotypic heterogeneity, and their ability to adapt to the target tissue is manifested differently in various tissues [66]. Depending on the tissue microenvironment, Tregs can express additional phenotypic markers, such as various integrins, including ITGA4 [39, 57]. RNA sequencing of single cells has clarified the phenotypic markers of various Treg subpopulations, including tissue-specific ones, in both mice and humans [66, 67].

The number of Tregs in the normal brain tissue of a healthy person is small [29], and they are classified as resident cells [42]. They differ from peripheral Tregs by expressing Blimp1, Ccr6, Ccr8, AREG, ST2, IL10, CD69, PD1, KLRG1, 5-HT7, CD103, Neuropeptide Y, and Osteopontin genes [42]. CD69⁺ resident Tregs found in the normal brain of mice and humans have specialized protective functions and are rapidly activated under neuroinflammation conditions. These cells can regulate astrogliosis levels by producing amphiregulin, polarize microglia into a neuroprotective state, and suppress tissue inflammation by producing IL-10 [42].

The idea that Tregs are a homogeneous cell population is outdated. Treg subpopulations differ both phenotypically and functionally. This heterogeneity emphasizes the importance of detailed phenotypic and functional characterization and makes us critical of earlier studies that treated Tregs as a single population. Recruiting immune cells into the brain parenchyma of patients with ischemic injury in the poststroke period for future clinical applications should consider the diametrically opposite local and systemic responses upon activation of different Treg subpopulations.

Treg level as a prognostic marker of stroke

Clinical and laboratory studies indicate a decrease in Tregs in peripheral blood on day 1 after an ischemic stroke. This is due to their active migration from the cerebral microcirculatory bed to the zone of ischemia and inflammation in the brain [36, 68]. This process is dynamic and reversible; therefore, a decrease in peripheral Tregs in ischemic stroke may not always be detected [69]. Hence, it is critical to determine whether the dynamics of peripheral Treg levels, either as a whole or as individual subpopulations, can provide additional prognostic data in cases of ischemic stroke.

The level of certain Treg subpopulations may be crucial for recovery from ischemic stroke. Research has demonstrated that certain Treg subpopulations can produce brain-derived neurotrophic factor (BDNF), a secretory protein that supports neuronal viability. A study has shown an increase in the number of Tregs producing BDNF (BDNF⁺Tregs) in the peripheral blood of patients after ischemic stroke. In patients with a more favorable neurological outcome, the number of BDNF⁺Tregs in peripheral blood after 6 months was higher than that in other patients [70].

Treg levels in peripheral blood may depend on neuroinflammation severity and infarction volume. In patients with an infarct volume of 28.6 ml, the Treg level in peripheral blood decreased insignificantly on day 1 after infarction. However, in patients with an infarct volume >28.6 ml, a significant decrease in Treg levels was observed on day 1, followed by an increase on days 3, 7, and 14 [71].

A recent study has found that patients with acute ischemic stroke had increased Treg frequency and mTreg frequency (with CD45RA⁺ Tregs accounting for about 90% of circulating Tregs) and decreased nTreg frequency and nTreg/mTreg ratio. The nTreg/mTreg ratio on the day of hospitalization may independently predict an unfavorable 90-day outcome in acute ischemic stroke. In contrast, patients with high CD4⁺ Tregs among lymphocytes upon hospital admission had a lower risk of adverse outcomes at discharge [74]. The number of circulating Tregs 48 and 72 h after infarction negatively correlates with infarct volume. That is, increased Treg levels decrease infarct volume [69, 75].

In summary, the level of peripheral Treg may serve as a biomarker for predicting stroke outcomes.

APPROACHES TO Treg LEVEL MODULATION FOR ISCHEMIC INSULT THERAPY

The immunomodulatory and reparative properties of Tregs make them an effective immunosuppressive therapy for the “graft versus host” reaction in some autoimmune diseases, including multiple sclerosis [8]. Isolating, ex vivo expanding, and subsequently transplanting CD4⁺CD25⁺CD127^lowFoxp3⁺ Tregs has allowed for long-term remission of the autoimmune process in such patients. The experimental data confirmed that Tregs induce a neuroprotective effect during the acute stage of ischemic stroke and promote brain recovery during the chronic stage. See below for further details. This effect is mediated by various mechanisms, including direct intercellular interactions and secretion of soluble factors [76].

Transplantation of ex vivo-generated Tregs requires licensing of the production site and registration of the cells themselves as a high-tech drug or obtaining permission to use Tregs as a hospital exception. The technologies of mediated modulation of the amount and functional activity of Tregs are much simpler from the viewpoint of regulatory legislation.

Retinoic acid is a Treg modulator that stimulates Tregs via the TGF-β/Smad3 signaling pathway [77]. It is produced in vivo by dendritic cells [78]. Moreover, chorionic gonadotropin, which was patented in 2023 by Russian researchers for autologous induction of Tregs ex vivo, is a known Treg modulator [79]. Additionally, the immunosuppressant rapamycin, which inhibits the mTOR kinase signaling pathway cascade, can stimulate the expansion of human Tregs ex vivo along with retinoic acid [80]. The use of rapamycin as a pharmacological agent to trigger Treg expansion has been proposed. Research has shown that exposure to rapamycin leads to selective expansion of Tregs [81]. Further, signaling through the mTOR pathway has been found crucial for the activation and maintenance of the immunosuppressive activity of Tregs [82]. These findings indicate a need to modify current ideas about the mechanism of action of rapamycin in Treg expansion.

In mouse experiments, low doses of 5-aza-2’-deoxycytidine, an inhibitor of DNMT DNA methyltransferase that leads to DNA demethylation, resulted in increased Treg level. This effect is expected because of the impact of DNA methylation on FoxP3 expression and subsequent increase in FoxP3 levels in cells after treatment with 5-aza-2’-deoxycytidine. When combined with TGF-β exposure, this treatment made iTregs more similar in phenotype to nTregs.

In mice experiments, the administration of the IL-2 complex and an antibody recognizing IL-2 resulted in an increase in Tregs in the peripheral blood, spleen, and lymph nodes. Moreover, it stimulated CD39 and CD73 expression in these cells. Ultimately, this immunotherapy protected the brain from ischemic damage in transient middle cerebral artery occlusion [85].

In vitro and in vivo studies have shown that nerve tissue autoantigens trigger the conversion of Tconv to Tregs [86]. Additionally, exosomes containing TGF-β, Smad2, and Smad4, which are secreted by embryonic stem cells (ESC-sEVs), lead to a significant increase in Tregs after stroke. This is due to TGF-β/Smad signaling pathway activation, which induces Treg expansion. Therefore, ESC-sEVs may be considered candidates for immune modulation [87]. In mice, recombinant IL-33 administration after ischemic stroke increased IL-10-expressing Tregs in the brain. This decreased the infarct size, the amount of activated microglia, and brain-infiltrating cytotoxic T cells. A more than threefold increase was observed in the number of Tregs in the brain 3 days after stroke compared with controls [88].

An experimental model of cerebral occlusion showed that microglia induced the expression of sirtuin 2 (Sirtuin2) after a stroke. This, in turn, suppressed the anti-inflammatory activity of Tregs, resulting in a significant increase in the transcription factor HIF-1α in Tregs. Conversely, inhibition of HIF-1α blocked the increase in the level of sirtuin 2 [89]. Pharmacologic inhibition of HIF-1α in ischemic stroke may prevent Treg activity suppression by microglia. Inhibition of HIF-1α is considered as a possible approach to stroke therapy, regardless of its role in Treg regulation [90].

The relevance of this problem is evidenced by the number of published studies. Remote ischemic conditioning or remote ischemic postconditioning, a method that induces short reversible episodes of ischemia with reperfusion in certain tissues or organs to protect other distant tissues and organs from ischemic/reperfusion damage, was applied to mice, resulting in increased Treg levels and decreased affected area during stroke and, in turn, indicating that Tregs play a causative role in this process. The same effect was induced by nicotinamide adenine dinucleotide hydrate [91]. A study has discussed the function of the receptor for advanced glycation end products (RAGE) in regulating the metabolism of peripheral CD4⁺ T cells. The study demonstrated that neutralizing RAGE action by adding circulating RAGE (sRAGE) to cells stimulates CD4⁺ T-cell polarization toward the Treg phenotype and reduces stroke size [92]. Currently, active research on the therapeutic potential of microRNAs expressed in Tregs or activating their expansion is underway [93–95].

Notably, several biologically active substances used to treat ischemic stroke affect Tregs [96], including melatonin, estrogen, statins, and vitamin D [96]. Melatonin is particularly attractive owing to its well-known neuroprotective properties, such as in the treatment of cerebrovascular pathology [97]. Research has demonstrated that melatonin impacts T cells, including their activation and differentiation. However, the effect of melatonin is context-dependent. In cases of pathological processes characterized by inflammation, melatonin suppresses the immune response. Conversely, in conditions accompanied by immunosuppression, it stimulates the immune response [98]. For instance, melatonin has increased the number of Tregs in peripheral blood in patients with systemic lupus erythematosus [99]. Another drug candidate that affects Tregs in ischemic stroke is the multitarget drug metformin [100]. Studies have shown that the use of metformin, both along with the immunosuppressant tacrolimus and as monotherapy, leads to an increase in the number of Tregs [101].

Studies assessing the potential use of inhibitors of the sphingosine-1-phosphate receptor (S1P receptor) in stroke therapy have been ongoing for over a decade [102]. One such inhibitor, fingolimod, led to increased FoxP3+Tregs in the spleen and peripheral blood and in the brain after ischemia in a mouse model of ischemic stroke [103]. In a small study cohort consisting of 5 healthy volunteers and 12 patients who had experienced acute ischemic stroke 72 h prior, treatment with the poly(ADP-ribose) polymerase 1 (PARP-1) inhibitor JPI-289 resulted in an increase in Tregs. The study showed a potential therapeutic benefit of JPI-289 for treating acute ischemic stroke. The pro-inflammatory cytokines (IFN-γ, TNF-α, and IL-17) decreased, whereas the anti-inflammatory cytokines (IL-4, IL-10, and TGF-β1) increased. Initially, the proportion of Tregs in peripheral blood was significantly higher in healthy subjects [104]. The effect of PARP-1 inhibition on Treg functions can be explained by the fact that PARP-1 performs poly-ADP ribosylation of FohP3 [105].

Modulation of the immune response, including ischemic stroke, by metabolites of commensal or pathogenic microflora of the gastrointestinal tract has recently gained attention. Published data show that metabolites of commensal microflora, such as butyrate, belong to histone deacetylase inhibitors (HDACi) and can stimulate the formation of peripheral Tregs by increasing the level of histone acetylation in the locus where the gene encoding FoxP3 is located [106]. This is particularly interesting given the general immunomodulatory and neuroprotective role of histone deacetylase inhibitors. HDACi increase CTLA-4 expression, stimulating the immunosuppressive function of human Tregs [107].

Recruitment of Tregs to the brain in ischemic stroke

Recruitment of immune cells to the brain parenchyma during ischemic injury begins with an increase in the permeability of the blood–brain barrier. This increase occurs early in ischemic stroke, within 10 min after reperfusion in rodents and within 2–6 h (average: 3.8 hours) from the onset of stroke in humans [108]. From approximately 24 h, there is a permanent pathological increase in the permeability of the blood–brain barrier, which continues for several weeks [109]. Simultaneously, mediators released in the inflammatory focus stimulate the production of chemokines, such as CXCL8 in humans and CXCL1 and CXCL2 in rodents. This enables peripheral monocytes, neutrophils, NK-cells, and lymphocytes to penetrate the blood–brain barrier and trigger the inflammatory cascade [110]. Tregs infiltrate the brain parenchyma later than other T cells [111]. Although immunophenotypically naive Tregs stay briefly, activated Tregs can migrate to the ischemic hemisphere of the brain and remain there for up to 30 days, particularly in mice [112]. Additionally, local and systemic immunologic mechanisms contribute to the pathogenesis of ischemic stroke, in which peripheral Tregs are likely involved [113].

COVID-19, Tregs, and stroke

The relationship between systemic inflammation, Tregs, and ischemic stroke has become prominent because of the COVID-19 pandemic. Immunopathological mechanisms triggered by SARS-CoV-2 infection, especially in long COVID-19, alter the Treg system, increasing the risk of ischemic stroke and other complications [114, 115]. Previously, we discussed the neurological consequences of COVID-19, including cerebral circulatory disorders [116], and the pathogenetic mechanisms responsible for the increased risk of stroke during and after COVID-19 [26]. In this section, we summarize the main immunopathological links between COVID-19 and increased stroke risk, specifically focusing on the role of Tregs.

Four key pathogenetic mechanisms of COVID-19 damage to the nervous system have been identified. First, SARS-CoV-2 is neurotropic and can directly infect the neuroepithelial cells of the olfactory analyzer, causing damage to the first pair of cranial nerves, allowing the virus to penetrate and spread in the brain. Second, SARS-CoV-2-induced systemic inflammation leads to hypercoagulability and an increased risk of thrombosis, including in cerebral vessels. Third, hypoxemia caused by partial lung damage inevitably affects the central nervous system, leading to a range of complications. The most severe manifestation of these complications is critical-state encephalopathy. SARS-CoV-2 infection can cause immune-mediated damage to nervous tissue. This is due to the production of pro-inflammatory cytokines by resident macrophages and Th17⁺ cells and a shift in the immune balance toward systemic inflammation in response to central nervous system cell infection [114–116]. Tregs play a key role in this mechanism, which increases the risk of stroke.

A 2021 meta-analysis has found that COVID-19 survivors have at least a threefold increased risk of stroke compared to non-survivors [117]. Moreover, all COVID-19 patients experience decreased Treg levels and impaired function [118], which is a key mechanism of the immunopathologic manifestations of the disease [119, 120]. A decrease in Treg levels leads to a Treg/Th17 imbalance, promoting the development of systemic inflammation. This worsens both the prognosis of COVID-19 and comorbid disease outcomes and coronavirus infection complications [120, 121]. The most significant and prolonged decrease in Treg levels was observed in long COVID-19 [121, 122].

Transcriptome analysis of over 100,000 viral antigen-recognizing CD4+ T cells from 40 COVID-19 patients revealed that hospitalized patients, as opposed to outpatients with mild forms of the disease, exhibit a significant increase in cytotoxic T-helper cells (CD4-CTL) compared with SARS-CoV-2-reactive Tregs [123]. These findings reveal a shift in the immune balance toward systemic inflammation, which is associated with the severity of coronavirus infection. Transcriptome analysis has revealed that the severity of COVID-19 is positively correlated with Treg functional impairment, as evidenced by decreased expressions of FoxP3 and immunosuppressive cytokines IL-10 and TGF-β [121]. Conversely, in COVID-19 reconvalescents, the pool of long-lived Tregs with high HLA-DRA expression levels is restored after ≥4 months, and a unique cluster of Tregs overexpressing TGF-β appear [124].

The present study examines the relationship between COVID-19, vaccination, and the risk of stroke. This retrospective study analyzed a cohort of 466 patients and found that those who received the SARS-CoV-2 vaccine had a more favorable course of ischemic stroke compared with the control group. The authors believe that vaccination, which affects the pathogen, may trigger a sanogenetic mechanism by increasing the Treg level in cerebral vessels that provide microcirculation in the area of cerebral ischemia or infarction [125].

CONCLUSIONS

The role of Tregs in the modulation of ischemic stroke is widely acknowledged. A comprehensive set of markers for all Treg subpopulations will enable a more accurate assessment of the functioning of individual Treg populations at different stages of ischemic stroke, from the initial acute phase to its long-term consequences. Available data indicate that Tregs have a protective role in ischemic stroke by affecting the size of the ischemic penumbra and limiting the size of brain infarction, promoting the recovery of neurological deficits. Therapeutic approaches aimed at increasing the number and activity of Tregs may be the optimal solution to protect ischemic neural tissue from immune inflammatory responses, prevent astrogliosis, provide systemic neuroprotection, and facilitate more efficient recovery of neurological impairment after a stroke.

ADDITIONAL INFORMATION

Funding source. This study was supported by the grant of Federal Medical and Biological Agency of Russia (The project “TILs-Glioblastoma”).

Competing interests. The authors declare that they have no competing interests.

Authors’ contribution. O.A. Zhukova, D.A. Chudakova, V.V. Belopasov — analysis of the literature, writing a draft version of the manuscript; E.V. Shirshova, V.P. Baklaushev — manuscript editing; G.M. Yusubalieva — conceptualization of the manuscript, literature review, manuscript editing. The authors made a substantial contribution to the conception of the work, acquisition, analysis of literature, drafting and revising the work, final approval of the version to be published and agree to be accountable for all aspects of the work.

ДОПОЛНИТЕЛЬНАЯ ИНФОРМАЦИЯ

Источник финансирования. Работа выполнена в рамках государственного задания ФМБА России (НИР «TILs-Глиобластома»).

Конфликт интересов. Авторы декларируют отсутствие явных и потенциальных конфликтов интересов, связанных с публикацией настоящей статьи.

Вклад авторов. О.А. Жукова, Д.А. Чудакова, В.В. Белопасов — анализ литературы, написание чернового варианта рукописи; Е.В. Ширшова, В.П. Баклаушев — редактирование текста рукописи; Г.М. Юсубалиева — идея обзора, анализ литературы, редактирование рукописи. Авторы подтверждают соответствие своего авторства международным критериям ICMJE (все авторы внесли существенный вклад в разработку концепции, проведение исследования и подготовку статьи, прочли и одобрили финальную версию перед публикацией).

About the authors

Oksana A. Zhukova

Federal Scientific and Clinical Center for Specialized Medical Assistance and Medical Technologies of the Federal Medical Biological Agency

Author for correspondence.
Email: Oksana.saprikina82@mail.ru
ORCID iD: 0000-0002-0907-0078

Research Associate

Russian Federation, Moscow

Daria A. Chudakova

Federal Center of Brain Research and Neurotechnologies

Email: daria.chudakova.bio@yandex.ru
ORCID iD: 0000-0002-9354-6824
SPIN-code: 1410-9581

PhD, Senior Researcher

Russian Federation, Moscow

Vladimir V. Belopasov

Astrakhan State Medical University

Email: belopasov@yandex.ru
ORCID iD: 0000-0003-0458-0703
SPIN-code: 6098-1321

MD, PhD, Dr. Sci. (Med.), Professor

Russian Federation, Astrakhan

Еlena V. Shirshova

Federal Scientific and Clinical Center for Specialized Medical Assistance and Medical Technologies of the Federal Medical Biological Agency

Email: shirshova.ev@fnkc-fmba.ru
ORCID iD: 0000-0002-3557-5424
SPIN-code: 7491-0434

MD, PhD, Dr. Sci. (Med.), Professor

Russian Federation, Moscow

Vladimir P. Baklaushev

Federal Scientific and Clinical Center for Specialized Medical Assistance and Medical Technologies of the Federal Medical Biological Agency; Federal Center of Brain Research and Neurotechnologies; Pulmonology Scientific Research Institute under Federal Medical and Biological Agency of Russsian Federation

Email: baklaushev.vp@fnkc-fmba.ru
ORCID iD: 0000-0003-1039-4245
SPIN-code: 3968-2971

MD, PhD, Dr. Sci. (Med.), Assistant Professor

Russian Federation, Moscow; Moscow; Moscow

Gaukhar M. Yusubalieva

Federal Scientific and Clinical Center for Specialized Medical Assistance and Medical Technologies of the Federal Medical Biological Agency; Federal Center of Brain Research and Neurotechnologies; Pulmonology Scientific Research Institute under Federal Medical and Biological Agency of Russsian Federation

Email: gaukhar@gaukhar.org
ORCID iD: 0000-0003-3056-4889
SPIN-code: 1559-5866

MD, PhD

Russian Federation, Moscow; Moscow; Moscow

References

Supplementary files