A dichotomy has been proposed for macrophage activation: classic vs. alternative, also M1 and M2, respectively. Like helper T cells, immunological stimulation favors the M1 polarization of male macrophages and the M2 polarization of female macrophages (70, 71). he immunological response elicited in males and females varies in intensity and nature. Macrophages are reportedly polarized toward M1 under the influence of IFN-γ and LPS and toward M2 under the influence of IL-4 or IL-13 and engaged in symbiotic relationships with other cell types to increase diversity (72, 73). These cell types include fibroblasts, mesenchymal stem cells, endothelial cells, T cells, B cells and NK cells. In this respect, mice infected with coxsackievirus B3 exhibited sex-specific differences in macrophage polarization and myocarditis severity (74). Female macrophages are associated with M2 phenotypes, while M1 activation markers are more prevalent in infected male macrophages (74). Macrophage polarization appears gender-specific in viral myocarditis, with M1 macrophages potentially damaging the host and M2 macrophages protecting against infection. In addition, the M2 phenotype macrophage has been associated to the increased susceptibility of female mice to asthma compared to male mice (75, 76). It has been shown that macrophage abundance and TLR2, TLR3 and TLR4 expression are higher, and phagocytosis is increased in females compared to males (77).

T-cell activation in the immunological response exhibits clear sexual dimorphism. The ‘classical’ Th1 and ‘surrogate’ Th2 activation states are two of the many possible activation states of helper T cells. Th1 cell activity and secretion of related cytokines, such as IFN-γ and Interleukin-2(IL-2), are more prevalent in males, while ‘surrogate’ Th2 cell activity and the secretion of IL-4 and IL-10 are more common in females (52, 78). Mice have been found to exhibit similar sex-dependent effects, with males displaying more active Th1 responses and females displaying more active Th2 cell activity (79). Another example is the anti-inflammatory cytokine IL-10, which controls the release of T cells and Th2 cells and is associated with gender. An elevated IL-10 level is associated with a poor prognosis and immunosuppression in females, whereas this relationship is less clear in males. Stroke patients who produce high levels of IL-10 may be more susceptible to infection (80, 81). Lower levels of IL-10 production by cytotoxic T cells after stroke have been documented in males compared to females (82).

Reduced abundance of B cells throughout the body due to age and gender is associated with a loss of neurotrophic signals produced by B cells, an increase in the deleterious effects of B cell antibody production, and a decline in mental acuity (83). Besides, the protein B cell maturation antigen (BCMA) controls the division of B cells and the development of plasma cells (84). Moreover, demyelination, infiltration by inflammatory T cells and macrophages, and the severity of neuroinflammation are reportedly worse in males who lack BCMA than in females (85). Conclusions drawn from these studies suggest that the immunogenicity of the neuroinflammatory milieu is strongly influenced by gender in terms of the mechanisms governing the proliferation, survival, and differentiation of B cells. Although the overall abundance of B cells increased dramatically in women after stroke, the number of regulatory B cells decreased in the spleen compared to males (86). This discrepancy may be attributed to differences in migration to the brain. After experimental autoimmune encephalomyelitis, estrogen was found to increase the abundance of regulatory B cells in the female brain (87).

Microglia are macrophage-like cells in the CNS (88), making up around 5% - 12% of all brain cells (89). When the brain is injured, such as during a stroke, the first line of defense is microglia and activated macrophages, which release cytokines to entice even more immune cells to the injury site (90, 91). Microglia, when activated, can either promote inflammation by becoming M1-like or suppress it by becoming M2. Following an ischemic stroke, M1-like microglia release proinflammatory cytokines such as IL-1β, IL-6, and TNF-α, as well as nitric oxide synthase (92, 93), activating nuclear NF-κB and causing subsequent brain injury (94–96). Inhibiting immunity, releasing anti-inflammatory substances like IL-4 and IL-10, clearing away cell debris and misfolded proteins, encouraging extracellular matrix and tissue repair, and releasing neurotrophic factors account for the efficacy of M2-like substances in reducing inflammation (88, 92). These findings suggest that M1 and M2 microglia phenotypes contribute to the inflammatory response after a stroke (97).

Depending on the activation signals they receive, microglia can either promote injury or repair (98, 99). Different M2 subsets, such as M2a, M2b, M2c, and M2d, each have respective physiological features and biological roles (100). Strong anti-inflammatory and weak phagocytic capabilities, the ability to attract Th2 cells and drive tissue repair, etc., are all hallmarks of M2a induced by IL-4/IL-13 and mannose-CD206 receptors (100, 101). Those with an M2b phenotype exhibit increased expression of CD206, TGF-β, and CD163 and are involved in the immunological memory response and can both trigger inflammation and quell it. The M2c phenotype, associated with tissue remodeling, is triggered by glucocorticoids, IL-10 or apoptotic cells (100–102).

Although both M1 proinflammatory and M2 anti-inflammatory microglia undergo physical changes, their molecular signaling routes and activities evolve differently (93). Current evidence suggests that the early stages of ischemic stroke are characterized by predominant M2 polarization of microglia. The M1 type becomes more prevalent after a few days, particularly in the peri-infarct area (93), ultimately leading to blood-brain barrier breakdown and infiltration of peripheral immune cells (103). These multifaceted impacts of microglia/macrophages raise doubts about the efficacy of simply suppressing microglia/macrophages as a stroke treatment. Instead, rebalancing the ratio of good to negative responses by microglia/macrophages may be more effective.

Dendritic cells are professional antigen-presenting cells that express major histocompatibility complex(MHC) II and are essential for bridging the gap between the innate and adaptive arms of the immune system (104). By presenting antigens, dendritic cells can stimulate T cell-mediated immunological responses. Resting dendritic cells have been documented close to the blood-brain barrier (105). Dendritic cells influence the migration and maturation of neighboring dendritic cells as time passes after a stroke. Most of these cells enter the ischemic area through the bone marrow (106).

Antigen presentation and antibody synthesis are functions of B cells, which are effector cells. The cerebrospinal fluid of human stroke survivors contains immunoglobulin (107, 108). Reducing the abundance of B cells in the body causes an increase in infarct size, which is associated with a poor prognosis and a high overall death rate (109). It has been found that while the early infiltration of B lymphocytes after a stroke may have a short-term favorable effect by increasing immunosuppression and the synthesis of neurotrophic factors, the long-term effects are detrimental due to an increase in autoantibodies (110). Importantly, after an ischemic stroke, IL-10-producing regulatory B cells play a protective effect (111).

As incompletely differentiated cells, monocytes have potent phagocytosis abilities and the ability to mount an appropriate immune response based on their surroundings (112, 113). They can be classified into proinflammatory and anti-inflammatory fractions based on the expression of surface-specific markers. Chemotactic protein-1 (MCP-1, CCL2) and its receptor CCR2 are involved in the inflammatory response (114), and proinflammatory monocytes constitute the primary monocyte subgroups following brain injury (115),CCR2 is expressed high on proinflammatory monocytes while CX3CR1 is expressed low or not at all. As monocytes penetrate the damaged brain, they must express CCR2 in order to differentiate into macrophages. In stable environments, anti-inflammatory monocytes check blood arteries and engage in in situ phagocytosis (116), but they do not express CCR2. There are two main subpopulations of monocytes in rodents, distinguished by their expression of chemokine receptors and Ly-6C (Gr1). The inflammatory response is facilitated by the short half-life and active absorption of Ly6C high proinflammatory factor by the inflammatory tissue. It has been found that Ly6Clow has a long anti-inflammatory half-life and aids in vascular homeostasis (117, 118).

Blood-derived mononuclear cells undergo phenotypic and functional changes in response to the varying inflammatory conditions present during an acute ischemic stroke. The function of monocytes shifts from being proinflammatory M1 type cells to becoming anti-inflammatory M2 type cells on day 3 post-stroke (119). Through the activation of inflammasomes, M1 monocytes release reactive oxygen species (ROS), cytokines, and chemokines, and disrupt the tight connections between endothelial cells that protects the brain’s blood-brain barrier. Receptor P2X4 (P2X4R) activation enhances M1 proinflammatory phenotype polarization (120). When blood-derived mononuclear cells and other innate immune receptors collaborate, secondary inflammatory damage to the blood-brain barrier is exacerbated (121), since blood-derived mononuclear cells upregulate the triggering receptor expressed in bone marrow cells. Importantly, M2 macrophages are generated from monocytes and may shield the blood-brain barrier potentially preventing ischemia injury to the brain by vascular remodeling, physical attachment, and reduced inflammation (122, 123).

There is a favorable relationship between infarct size, stroke severity, and long-term prognosis after an ischemic stroke (124, 125). Neutrophils are white blood cells that rush to an area of illness or damage, where they consume dead cells and release inflammatory signals to draw in additional leukocytes (126). Ischemia-induced neutrophils release ROS, proteases (MMPs, protease 3, elastase), lipocalin-2(LCN-2), and NETs, all of which contribute to the breakdown of the blood-brain barrier. Connexins (primarily cadherin/β-catenin complex, occludin, ZO-1, and claudin-5) are degraded by high permeability-associated signaling pathways (e.g. MLCK, PKC, MAPK, and Rho GTPass) when there is an excess of ROS such as superoxide anion, peroxynitrite, and hydrogen peroxide (127). When it comes to the immunological response to ischemic brain injury, neutrophil adherence is a crucial first step (128). Immune cells are transported to the site of ischemic brain injury via the vascular wall, where they attach to adhesion molecules such as ICAM-1, MAC-1 (CD11b/CD18) and selectin (128). Within a few hours after a stroke, ICAM-1 expression rises in the proximal endothelial tissue of the brain damage, peaking at around 12–48 hours (129). In experimental stroke, infarct size and brain leukocyte infiltration have been found to be decreased in adhesion molecule deficient animals when ICAM-1 was blocked (130, 131). CD11b/CD18, also known as MAC-1, is expressed on the plasma membrane of neutrophils and binds to the intercellular adhesion molecule 1 (ICAM-1) on endothelial cells. Reduced infarct size, survival, and neutrophil infiltration into the ischemic brain are all observed in MAC-1-deficient transgenic mice after an ischemic stroke (132). P-selectin and E-selectin contribute to initial neutrophil recruitment (133), L-selectin induces recruitment of unstimulated neutrophils to activated endothelial regions (134), and selectin itself is a calcium-dependent transmembrane glycoprotein that is responsible for transporting neutrophils after cerebral ischemia (133). P- and E-selectin upregulation is positively linked with post-ischemic inflammatory response enhancement and injury severity in all experimental stroke models (135, 136).

T-lymphocyte activation plays a role in both innate and adaptive immunity, with the ability to promote or suppress inflammation (137). It has been established that 30% of T-lymphocytes are cytotoxic T cells (CD8⁺T cells), which destroy infected cells by cytotoxic processes, while 40% are helper T cells (CD4⁺T cells), which release cytokines to modulate adaptive and innate immune responses (138, 139). Acute cerebral ischemia causes neuroinflammation, activated and infiltrated microglia/macrophages may stimulate activated CD4⁺T cells to develop into Th1 or Th2 cells, generate proinflammatory or anti-inflammatory cytokines, and either harm or protect the brain (138). Proinflammatory cytokines such as IL-2, IL-12, and IFN-γ are released by Th1 cells, which may exacerbate brain injury. Anti-inflammatory cytokines such as IL-4, Interleukin-5(IL-5), IL-10, and IL-13 may be secreted by Th2 cells, which may have a neuroprotective effect on the wounded brain (140). The earliest T cells found after a stroke are CD8⁺T cells, which detected within hours of a stroke (141). Neuronal death and exacerbated brain injury are caused by CD8⁺ T lymphocytes after they come into contact with other cells and become antigen-dependently activated, releasing perforin/granzyme (142).

To mount an immune response to an inflammatory setting, certain naive T cells can independently generate ROS and inflammatory cytokines (143). Naive CD4⁺ T cells differentiate into specific T helper cells (Th), namely Th1, Th2, Th17 and induced T regulatory cells (iTregs) (144). Maintaining immunological homeostasis and suppressing effector T cells are the functions of naturally induced Tregs (nTregs) found in the thymus and the iTregs (145). By recognizing autoantigens and foreign antigens, iTregs can suppress an overactive immune response. The post-stroke neuroinflammatory response is mitigated by Foxp3⁺Tregs (146). Toll-like receptors (TLRs) and T cell receptors (TCRs) are two examples of immunological receptors that T cells express, accounting for their immune features (147). The proinflammatory cytokine IL-17, secreted by T cells, works in tandem with IL-23 to entice monocytes and neutrophils to the site of inflammation (148).

As one of the early cytokines in the inflammatory response to ischemic brain injury (149), TNF-α, is released and generated by monocytes, T cells, mast cells, macrophages, neutrophils, keratinocytes, and fibroblasts (88). Pericerebral cells are stem cells that line the surface of capillaries that TNF-α stimulates to enhance IL-6 production via activation of NF-κB (150, 151). Transmembrane (tmTNF-α) regulates local inflammation by cell-to-cell interaction, and soluble bioactive (sTNF-α) is created by tumor necrosis factor-converting enzyme (TACE) (88). sTNF-α acts systemically and locally in the central nervous system, promoting phagocytosis and cytotoxic activity of macrophages and enhancing the production of IL-6 and IL-1, mediated by binding of TNF-α to the receptors TNFR-1 and TNFR-2. Although TNFR1 mediates sTNF-α, TNFR-2 and TNFR-1 mediate tmTNF-α (149). The neurotoxic and neuroprotective actions of TNF-α in the ischemic brain highlight the central role of TNF-α in the neuroimmune genesis of stroke (152–154).

Microglia, astrocytes, leukocytes, and endothelial cells reportedly contribute to the brain injury response by releasing IL-6. There is an increasing consensus that this multi-functional proinflammatory cytokine increases leukocyte migration, regulates the production of chemokines and the expression of adhesion molecules, and activates acute phase proteins (150, 151, 155). IL-6 released after a stroke can worsen cerebral vascular damage by activating NMDI-Rs and upregulating ET-1 and JNK (156). IL-6 enhances local inflammatory responses by activating and recruiting neutrophils and monocytes and stimulating vascular endothelial cells to produce adhesion molecules and other inflammatory mediators (157). Although IL-6 is a proinflammatory cytokine, it plays a crucial role in cerebral ischemia by acting as a carrier of the inflammatory process during the early phase of stroke and as a neurotrophic factor during the late development of cerebral ischemia (158). Similar neurotrophic factors, including leukemia inhibitory factor (LIF) and ciliary neurotrophic factors, share a common receptor component, gp130 (159). Importantly, the severity of cerebral ischemia injury following an ischemic stroke may be mitigated by administering these cytokines directly into brain tissue following a stroke. Neurogenesis, angiogenesis, and neuronal differentiation are all aided by IL-6 produced by astrocytes, which also promotes Th1 polarization to Th2 and causes an immunosuppressive microenvironment (160). Additionally, IL-6 aids CNS post-traumatic recovery via endothelial cell repair, which may improve vascular reconstruction or angiogenesis following ischemic stroke (161). IL-6 protects neurons from apoptosis, enhances CNS neuron survival, and decreases N-methyl D-aspartate (NMDA) -mediated excitatory toxic neuron injury (158).

The proinflammatory cytokine IL-1 is synthesized by monocytes, macrophages, and epithelial cells (149), and the IL-1 family comprises IL-1α, IL- 1β, and IL-1RN (162). During the early stages of stroke, IL-1 mediates harmful inflammatory processes, such as the upregulation of IL-6, TNF-α, Matrix metallopeptidase 9 (MMP-9), and chemokines in astrocytes, inhibition of neurogenesis (163). IL-1 may also act on the vascular endothelium to encourage the recruitment of white blood cells (164). However, during the subacute and chronic phases of stroke, IL-1 may bring some benefits. It is widely thought that IL-1 may aid recovery from an ischemic stroke since it encourages scar formation from glial cells and boosts angiogenesis (165).

IL-1β is a cytokine that helps keep the immune system in check, and it can increase inflammation influencing nearly all the cytotypes (88). IL-1β is widely acknowledged to promote microglial activation. Indeed, microglia are pivotal in the neuroinflammatory response as effector cells. By producing potentially neurotoxic molecules like TNF-α and iNOS, they exacerbate the inflammatory response and cause secondary brain injury. The IL-1β-mediated phosphorylation and ubiquitination of inhibitor of NF-κB-a (IκB-a) by IRAK activates IκB kinase via the IRAK pathway, which promotes nuclear NF-κB expression and the transcription of target genes, including IL-8 and TNF-α (166). To further exacerbate injury caused by ischemia, IL-1β modulates the PI3K/AKT pathway, promotes IL-6 and other cytokines, and operates synergistically in the ischemic region (167). Growing evidence suggests that phosphorylation of JAK2/STAT3 is stimulated when IL-6 and other proinflammatory cytokines are upregulated (168, 169). Once within the nucleus, phosphorylated STAT3 (P-STAT3) upregulates the IL-1β, IL-6, and TNF-α genes by binding to certain DNA sequence features in the promoter region of the target gene (169). When brain cells are injured, it can be extremely challenging to repair the damage caused by the vicious cycle of inflammation that results.

IL-10 is an anti-inflammatory protein released mostly by monocytes but can also be secreted by other cell types, including Th2 lymphocytes (170). By reducing the body’s inflammatory response, IL-10 reduces the risk of stroke. By activating PI3K and STAT3, IL-10 inhibits the synthesis and activity of Th1 cells (171), reducing the expression and activity of proinflammatory cytokines such as IFN-γ, IL-1β, and TNF-α (172). Ischemic stroke is protected against thanks to IL-10 treatment’s ability to effectively down-regulate the upregulated proinflammatory signals in acute ischemic lesions (173). A study reported that transduction of the IL-10 gene prior to cerebral artery ischemia protected rat brains from ischemic and reperfused injuries by boosting heme oxygenase expression (174). Inhibiting NF-κB has been reported as another anti-inflammatory function of IL-10 (175). In a mouse model of focal cerebral ischemia (MCAO), transgenic mice overexpressing IL-32A showed decreased ischemic neuronal cell death and increased secretion of anti-neuroinflammatory factors (IL-10) by decreasing the release of neuroinflammatory factors (IL-6, IL-1β, TNF-α), thereby reducing astrocyte activation, indicating a cross-talk between IL-32 A and these other cytokines (176). Importantly, taking myelin oligodendrocyte glycoprotein (MOG) through the nose triggers Il-10 secretion from CD4⁺ T cells, which helps reduce stroke-related disability. The neuroprotective effect of oligodendrocyte glycoprotein treatment in MCAO mice (177, 178) may be due to IL-10 released by CD4⁺ T cells. Secondary infarct growth via the nitric oxide route may be facilitated by an increase in IL-10, which reduces the amount of CD11b⁺ cells (177).

The strong anti-inflammatory properties of IL-4 play an important role in determining the prognosis of stroke. By causing naive T cells to differentiate into Th2 cells that secrete anti-inflammatory cytokines, including IL-4, IL-10, and IL13, IL-4 inhibits the activity of Th1 inflammatory effector cells (179). T-cell differentiation and non-specific B-cell transformation are two immunological responses that IL-4 controls (180). Microglia/macrophage M2 polarization is promoted by IL-4, the most notable M2 macrophage polarization promoter. IL-4 is widely thought to be crucial during the acute stage of stroke (181), with dramatically increased serum levels hours after stroke onset (182). A lack of IL-4 causes brain damage and neurological impairment 24 hours after transient MCAO (183). Long-term healing after an ischemic stroke and microglia/macrophage M2 polarization depend heavily on IL-4. After cerebral ischemia, IL-4-deficient mice showed increased populations of M1-polarized microglia/macrophages and greater infarct sizes that caused neurological damage. Importantly, IL-4 recovery could reverse these effects (184). The neuroprotective effects of IL-4 are mediated by activating IL-4/STAT6 signal transduction and suppressing proinflammatory cytokines. Consistently, more proinflammatory cytokines, such as IL-1β and TNF-α, were produced by IL-4 knockout mice (185).

IL-23 in the brain following ischemic stroke is produced mostly by CD^172a+/IRF4⁺ 2 dendritic cells (CDC2s), which regulate IL-17 expression in γδ T cells (186). Antigen-stimulated dendritic cells and macrophages boost Th17 cell growth during persistent inflammation by releasing interleukin-23 (186). The IL-17 produced by Th17 cells accounts for a robust inflammatory response by inducing the expression of many inflammatory cytokines. Dendritic cells migrate to the perivascular infarct area after a stroke, and cDC2s cells stimulate γδ T cells to release IL-17, which recruits neutrophils to the ischemic side of the brain (187). The synergistic effects of Vgamma4 T cell-derived IL-17A and IL-1β/IL-23 in the infarct hemisphere exacerbate the inflammatory cascade and ischemic tissue damage (188).