Role of Immune and Inflammatory Mechanisms in Stroke

Microglia

As brain resident cells, microglia are the first responders to ischemia and are engaged in intimate cross-talk with other intrinsic brain cells and infiltrating leukocytes. In the acute stage of ischemia, microglia are attracted to blood vessels and extensively interact with ECs and pericytes, regulating the capillary diameter and cerebral blood flow via altering pericyte or astrocyte coverage, which may contribute to the impaired integrity of the blood vessels [30^, 31]. Activated microglia were also shown to upregulate the level of reactive oxygen species (ROS) and the secretion of inflammatory cytokines such as interferon (IFN)-γ, IL-1β, and tumor necrosis factor (TNF)-α to increase the extent of cytotoxic responses [32]. The redox signal mediated by NADPH oxide 2 was shown to enhance the sensitivity of microglia based on proinflammatory stimulation, thereby increasing the secretion of neurotoxic cytokines [33]. Under the regulatory effect of NADPH oxide 2, microglia can prominently secrete matrix metalloproteinases (MMPs), especially MMP-3 and MMP-9, and further degrade the cytoskeleton protein to exaggerate the deterioration of BBB. In a study, inhibitors of MMPs were found to decrease the cerebral infarct volume, alleviate peri-lesional edema, and ameliorate ICH caused by recombinant tissue plasminogen activators [34]. On the other hand, stimulated microglia have been shown to play a protective role against ischemic injury to a certain extent. In the chronic recovery phase of stroke, microglia contribute to infarct resolution and tissue repair as they have the ability to engulf dead cells, debris, and newborn neurons that fail to integrate into the neural circuit [35^, 36]. At 24 h after ischemic injury, branched and phagocytic microglia form a barrier around the lesion to hinder exaggeration of ischemia. At the same time, globular microglia via expressing Ym1 and CD206 at the core region of the lesion was found to downregulate the inflammatory response and alleviate neurological deficits [37]. The potential mechanisms of the protective effect of microglia in ischemic stroke are (1) Increase in the levels of various neurotrophic factors, including brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor [38]; (2) phagocytosis and clearance of intracerebral neutrophils [39]; (3) elimination of necrotic cells. To achieve therapeutic goals in a clinic, future studies should focus on elucidating the process of subtype-transdifferentiation rather than simple activation or depletion of microglia.

Studies have shown that M1 is the main subtype of microglia in the initial stage of ischemia, and reaches a peak at 3–5 days after ischemic damage. Subsequently, M2 becomes the prominent subtype of microglia and lasts nearly 14 days. The above phenotypic switch from M1 to M2 is more prominent around the ischemic lesion. Moreover, studies have indicated that M1 may exaggerate neuronal death triggered by oxygen-glucose deprivation in cell culture medium, while M2 was shown to inhibit the above progression [40]. The modulators of microglia polarization can be classified into four categories: (1) transcription factors: nuclear transcription factor (NF)-κB and STAT family members are closely related to M1 polarization, whereas nuclear factor erythroid 2-related factor 2 and PPARγ are associated with M2 polarization [41^-44]; (2) receptors: Toll-like receptor (TLR) 4 and sphingosine 1 phosphate receptors are involved in shifting microglia toward M1 phenotype poststroke [45^, 46]; (3) ion channels: the voltage-gated potassium channel Kv1.3 may be one of the main mediators of M1 microglia polarization via modulating Ca²⁺ signaling and induced neuroinflammation [47]; (4) gene modulators: H19 and miRNA-155 have been demonstrated to promote M1 polarization, whereas miRNA-124 and chemokine-like protein TAFA-3 (FAM19A3) are associated with M2 polarization [48].

Neutrophils

Neutrophils, the type of rapid reaction cells, infiltrate into the lesion at 30 min after ischemic stroke. Stimulated by various cytokines and damage-associated molecular patterns, neutrophils migrate from bone marrow, spleen, and peripheral blood into the damaged region, and play a key role in triggering the inflammatory cascade [49]. Whether at the time of ischemia or after reperfusion, neutrophils are considered critical sources of ROS, which could damage neurovascular units and BBB by regulating ECs, smooth muscle cells, perivascular cells, and contribute to the exaggeration of cerebral edema and hemorrhagic transformation. Proteases released by neutrophils such as MMPs and elastase may degrade the basement membrane and extracellular matrix, aggravating the impairment of BBB [50]. However, some authors have also suggested a beneficial effect of neutrophils in the postischemic repair process. The core pathophysiological mechanism of neutrophil-driven repair is angiogenesis. At the site of arterial injury, activated neutrophils deposit cathelicidin antimicrobial peptide, and further promote the release of vascular endothelial growth factor and epidermal growth factor from EC progenitors in a paracrine manner, which stimulates the recovery of ECs [51^, 52].

Lymphoid Cells

Lymphoid cells mainly include T, B, and NK cells. These play an important role in ischemic stroke. T lymphoid cells, which play a central role in ischemic stroke, can upregulate the production of cytotoxic substances and the secretion of oxidative stress-related ingredients such as ROS. Helper T cells (Th) 1 in the vicinity of the infarct area have been shown to stimulate the release of proinflammatory mediators, including IFN-γ, TNF-α, augmenting intercellular adhesion and enhancing vascular permeability. Activation of Th1 cells may cause persistent damage to cortical neurons. Conversely, Th1-defective mice showed significantly reduced volume of infarct area and decreased the number of necrotic neurons. These findings indicated that the pathway by which Th1 promotes the transformation of macrophages into the active state may regulate the inflammation reactions [53]. Another subset of T cells (Tc) that aggravates the progression of ischemia is cytotoxic Tc via killing the pathogens directly and by releasing toxins such as perforins and granzymes. Tc were shown to induce poststroke neuronal apoptosis through the Fas-FasL signaling pathway [54]. Further studies are required to obtain direct evidence of whether Tc can alleviate ischemic injury. While Tc contribute the least to the early dysfunction after stroke, this does not deny the critical effects of Tc on the chronic recovery of ischemic stroke. Shi et al. [55] found that regulatory T (Treg) cell-derived osteopontin could enhance the repair activity of microglia via integrin receptors, thereby facilitating oligodendrocyte regeneration and remyelination during the chronic stages of stroke. Increasing Treg cells with IL-2:IL-2 antibody complex improved long-term stroke recovery [55].

B lymphoid cells, the main undertaker of fluid immune responses, is another type of crucial lymphocytes infiltrating into central nervous system (CNS) after ischemia. Recent studies found a decrease in B cells in the peripheral circulation of patients with acute cerebral ischemia, leading to the suppression of fluid immunity [5]. However, it is not clear whether B cells can immigrate to the CNS from the peripheral circulation like Tc, and the associated mechanisms are not clear.

NK cells are the principal players in natural immunity. These have the ability to spontaneously initiate cytotoxic response and can regulate the activity of B cells, Tc, and bone marrow stem cells. With the significant decrease in peripheral blood, NK cells may mediate low-level innate immunity and increase the risk of infection after ischemia. Therefore, researchers speculated that in the early stage of ischemia and hypoxia, certain inducible factors may promote migration of NK cells into CNS, and kill the targeted cells directly without secondary response or memory effect and regulate the function of other immune cells by secreting cytokines such as IFN-γ [56^-58].

Monocytes

Recruitment of monocytes at the site of lesion is initiated in the early stage after ischemic stroke. Researchers have consistently shown that as early as 24-h post-MCAO, monocytes originating from bone marrow begin to immigrate into the ischemic core and the peri-infarct area, which might transit to microglia by expressing Iba-1 [59]. In the study by Chu et al. [60] Ly6C^hi monocytes showed a 3-fold increase in the blood and 10-fold increase in the CNS after MCAO in C57BL/6J mice and exerted a protective effect via limiting brain injury and neurological deficit. These effects were associated with promoting M2 microglia/macrophage polarization. In addition, CCR2-CCL2 axis is reported to be essential for the immigration of monocytes to the brain [61]. After recruitment, Ly6C^hi monocytes begin to increase the expression of macrophage marker F4/80 [62]. Grosse et al. [63] reported that the proportion of classical monocytes (CD14⁺⁺CD16⁻; Mon1) was higher in patients with moderate/severe stroke, while the proportion of nonclassical monocytes (CD14⁺CD16⁺⁺; Mon3) was higher in patients with mild stroke. They suggested further investigation to elucidate the mechanism of monocyte subsets in human ischemic injury.

Dendritic Cells

As classic antigen presenting cells, DCs form the links and bridges between natural and adaptive immunity. After ischemic injury, granulocyte-macrophage colony stimulating factor may induce the transformation of microglia into DCs with membrane expression of major histocompatibility complexes II [64^, 65]. However, the specific mechanisms by which DCs take part in ischemic stroke are not clear.

Microglia

The most rapidly activated cells after ICH are microglia, with membrane expression of receptors such as TLR [69]. Upon stimulation, microglia can produce proinflammatory cytokines, including IL-1β, IL-6, TNF-α, ROS, and NF-κB; this further exacerbates cerebral water content and aggravates neurological deficit. Yang et al. [70] demonstrated that inhibition of microglial inflammation provided a novel treatment for ICH-induced brain injury via the intervention of ICH-challenged BV2 microglial cells in vitro and male C57BL/6 mice in vivo. Preclinical studies have suggested that M2 microglia/macrophage play a role in brain repair and recovery via engulfing the hematoma and cells’ debris, removing harmful substances, and providing space for tissue regeneration. With increase in the number of M2 microglia, the lesion of the hematoma is eliminated in 7–21 days after ICH [71]. M2 microglia express a series of growth factors and trophic factors, including insulin-like growth factors-1, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, neurotrophin 3, NT-4/5, which could facilitate neurogenesis and neural circuit reframing [72^, 73].

Very little is known about the transition of microglial polarization. Lan et al. [74] suggested that transcription factors, prostaglandin E2 receptors, and sphingosine 1 phosphate receptors may mediate the polarization of microglia. By interacting with various cells, microglia act as guardians of the brain and play a key role after ICH. In a mouse model of blood-induced ICH, Treg cells were found to promote M2-like polarization of microglia and macrophages by activating IL-10-glycogen synthase kinase-3β signaling pathway [75]. Besides, the signaling pathway involving soluble CX3CL1 and CX3C chemokine receptor 1 (CX3CR1) may mediate the interactions between neurons and microglia, associated with microglial activation in the hemorrhagic injury [76]. However, monocyte-specific CX3CR1 deficiency showed no effect on functional improvement after ICH.

The following are the potential mechanisms by which microglia participate in ICH: (1) damage-associated molecular patterns: hemoglobin can induce the release of high-mobility group protein B1 and activate TLR4 of microglia to exacerbate post-ICH inflammation. Studies have shown that inhibiting high-mobility group protein B1 and activating the microglia around the lesion may ameliorate edema and neuronal apoptosis [77]. (2) TLR: hemoglobin may promote the secretion of NF-κB by activating the TLR of microglia, then upregulate the inflammatory cytokines to exacerbate hemorrhagic injury [78]. (3) Inflammatory corpuscle: as the classic pattern recognition receptor of natural immune responses, inflammatory corpuscle is proven to be an active regulatory factor of caspase-1, such as NOD-like receptor protein (NLRP) 3. By increasing the secretion of inflammatory factors such as IL-1β, IL-18, the inflammatory corpuscle expressed by microglia can aggravate the immune and inflammatory damage after ICH [79^, 80].

Neutrophils

Neutrophils infiltrate into the lesion after ICH. In the ICH animal model prepared by collagenase, neutrophils were detected in the vicinity of the lesion at 4 h after ICH; the extent of neutrophilic infiltrate peaked at 3–5 days after hemorrhagic event [81]. Moreover, apoptosis of neutrophils can activate the microglia and further lead to neuroinflammatory injury. Neutrophils are divided into two major subtypes: N1, the proinflammatory subtype, can exacerbate neurological deficit and the level of encephaledema; N2, the anti-inflammatory subtype, can attenuate the inflammatory reaction to preserve the activity of neurocytes. In addition, polymorphonuclear neutrophils (PMN) may exaggerate the deterioration of BBB, resulting in cerebral edema, recurrence of hemorrhage, and neurological deficits [82].

Lymphoid Cells

T lymphoid cells play vital roles in the pathogenesis of ICH. A study illustrated that CD4⁺Tc migrate into hemorrhagic hemisphere at 1 day, peak at 4 days, and gradually decline up to 14 days after ICH. Treg cells likely showed a similar pattern of variation as CD4⁺Tc, but peaked at 7 days after hemorrhagic injury. Treg cells can improve ICH-induced inflammatory damage via regulating microglia/macrophage polarization toward the M2 phenotype, which was related to the activation of IL-10/GSK3/PTEN axis [75].

The effects of B lymphoid cells and NK cells after intracerebral injury may be limited owing to their lesser permeability across the BBB [67^, 81]. A recent research suggested that brain-bound NK cells may contribute to perihematomal edema and neurological deficit via cytotoxicity toward brain ECs and recruitment of neutrophils at the early stages of ICH [67].

Monocytes

Studies have shown that monocytes infiltrate into the brain at 12 h after ICH, and reach peak level on the 5th day [83]. MCP-1 and CC-chemokine receptor (CCr) 2 play an essential role in inducing immigration of monocytes into the CNS; thus, these are potential therapeutic targets for alleviation of inflammatory response after hemorrhagic stroke. Comparing the CCr2^−/− hematopoietic cells with the control, the former exhibited a prominent increase in restoration of neurological function by decreasing the number of downstream monocytes. In addition to the aforementioned experimental studies, clinical prospective studies have also shown a close relation of the level of CCr with the poor prognosis of neurological function at the first week after ICH [84]. In a clinical study of 115 ICH patients, levels of MCP-1 and chemokine ligand (CXCL)-10 showed a negative correlation with modified Ranking Scale (mRS) score, and were independent predictors of prognosis of ICH [85]. Walsh et al. [86] found that the median number of M2 monocyte microparticles was significantly higher in ICH group compared with controls and they suggested that the results represented differences in the chronic inflammatory status in patients susceptible to ICH, such as cellular activation and apoptosis. In addition, the inflammatory monocytes may be responsible for the M1-type macrophage response, characterized by phagocytosis, proteolysis, inflammation, and neurological deficit in the first 3 days after ICH [87].

Recombinant T-Cell Receptor Ligand

RTL551 (I-A^b molecule linked to MOG-35-55 peptide) was shown to decrease cortical and total infarct volume by approximately 50% and inhibit the infiltration of immune cells, including macrophages/activated microglial cells and DCs [92]. Administration of RTL551 4 h after middle cerebral artery occlusion (MCAO) in C57BL/6 mice decreased the infarct size and reduced inflammatory cell infiltration into the CNS, which was related to reduced expressions of T-cell activation (CD44) and CNS homing receptor (CCR5 and CCR7) markers. Similarly, RTL1000 (HLA-DR2 moiety linked to human MOG-35-55 peptide) was also shown to reduce stroke lesion volume in HLA-DR2 transgenic mice [93]. To promote the clinical translation, future studies are underway to determine the optimal duration for which RTL can be administered after ischemia to exert therapeutic benefit.

IL-1 Receptor Antagonist

IL-1Ra is classified into two structural variants, i.e., secreted IL-1Ra and intracellular IL-1Ra (icIL-1Ra). It effectively regulates the activity of IL-1 in the acute phase of ischemic injury. Administration of IL-1Ra was shown to decrease ischemic brain injury in animal models of both transient(t) and permanent(p) MCAO. The specific mechanisms might be related to the neuroprotective effects on microglia at the ischemic lesion [94^-97]. In a study, injecting IL-1Ra-producing bone marrow cells into mice was found to modulate JNK/SAPK signaling pathway resulting in outgrowth of axons and improved neuronal plasticity in the acute phase after ischemic stroke. However, the efficacy and safety of IL-1Ra are yet to be established in clinical studies; in addition, achieving a therapeutic concentration in brain is another challenge as it shows poor permeability across the BBB [98^, 99].

XPro1595, Etanercept, α-Lipoic Acid

In preclinical experiments, topical administration of TNF antagonists, such as XPro1595 (a dominant-negative inhibitor of soluble TNF), to C57BL/6 mice reduced the infarct injury at both 1 and 3 days after ischemia; the effect may be attributable to microglial activation toward a phagocytic phenotype. Compared with the transmembrane TNF, the study showed that soluble TNF may play the main role in relieving ischemic injury by regulating systemic and local inflammation in the CNS [100]. In another study, both XPro1595 and etanercept (inhibitor of both soluble TNF and transmembrane TNF) improved neurological deficit, but without decreasing the infarct volume, via altering microglial responses, modifying liver acute phase response (APR), and changing spleen Tc and microvesicle numbers. Systemically administered XPro1595 was as efficient as etanercept in alleviating infarct damage, improving clinical outcome, and reducing inflammation after focal cerebral ischemia [101]. The therapeutic effects of etanercept and α-lipoic acid were also tested in rats subjected to MCAO. Etanercept and α-lipoic acid, administered either alone or in combination, were found to reduce brain infarct, alleviate BBB disruption and neurological dysfunction by decreasing serum level of TNF-α and downregulating cerebral microglial activation [102]. However, more research about anti-TNF therapy is required to testify its effect on stroke.

Celecoxib

Based on the aforementioned role of pro- and anti-inflammatory cytokines, several therapies targeting these molecules have been investigated on a large scale. The most common anti-inflammatory therapy for ICH is selective COX-2 inhibitor [103]. Injection of celecoxib (a selective COX-2 inhibitor) at concentrations of 10 or 20 mg/kg to adult rats at 20 min, 6 h, and 24 h after hemorrhagic injury, respectively, showed potent effects on neurological function recovery [104]. Compared with the ICH-only group, the group of ICH treated with 10 or 20 mg/kg celecoxib showed a significant decrease of inflammatory cells such as myeloperoxidase-positive cells and OX42-positive cells in the periphery of hematoma. These findings support the theory that COX-2 inhibition may attenuate immune and inflammatory reactions by reducing the production of prostaglandin E2 and decreasing brain edema after ICH [105]. The small size of the study population constrains us from determining the most effective dose of celecoxib for ICH, providing the direction for future investigations.

Minocycline

In a study, minocycline improved the neurobehavioral results and exerted neuroprotective effects on ICH by targeting inflammatory mediators, including TNF, IL-6, MMP-9, and MMP-12 [106]. A wide body of evidence from experimental studies on rodents suggest that minocycline can decrease the level of perihematomal iron overload, iron handling proteins, and further ameliorate the disruption of BBB, brain edema, neuronal apoptosis, and autophagy; this phenomenon may be related with the DKK1-Wnt signaling pathway [107^-109]. Similarly, in piglets, minocycline attenuated ICH-induced white matter damage by activating TGF-β [110^, 111]. The significant effects of minocycline in animal models were also mirrored in patients. A clinical trial demonstrated the safety of high-dose (10 mg/kg) intravenous minocycline in patients with ICH owing to its effect in decreasing the level of MMP-9 in serum [112]. However, larger clinical trials evaluating the efficacy of minocycline in ICH patients with poor prognoses are still needed.

Pinocembrin

Pinocembrin (5,7-dihydroxyflavanone) was shown to be a promising strategy to inhibit post-ICH inflammatory reactions in vivo. It was shown to reduce the lesion volume in a dose-dependent manner and further ameliorate the neurologic deficits. Pinocembrin decreased the level of proinflammatory cytokines related to M1-like microglia such as TNF-α, IL-1β, IL-6, and the expression of TLR4, including its downstream target proteins TRIF and MyD88 in C57BL/6 mice with ICH. In vitro, pinocembrin downregulated the expression of NF-κB in lipopolysaccharide-stimulated BV-2 cells or primary microglia [113]. Although pinocembrin was demonstrated to have an anti-inflammatory effect in the CNS, it is unknown whether it targets microglia. Additional studies of the effects of pinocembrin on microglial polarization are required prior to exploration of its clinical use.

Fingolimod

Fingolimod, a S1P-selective agonist, has shown therapeutic effects on ischemic and hemorrhagic stroke in both animal models and small-scale clinical trials [114]. Intravenous administration of fingolimod (0.5 mg/kg) to C57BL/6 mice attenuated the neurological deficit and reduced lesion size after ischemic stroke. Combination of fingolimod and t-PA improved the outcomes of thrombolytic treatment and decreased the risk of hemorrhagic transformation associated with delayed administration of t-PA [115]. In diabetic mice after MCAO, fingolimod decreased the level of infiltrated inflammatory cells and TNF-α, and increased the ratio of Bcl-2/Bax. However, fingolimod remarkably exacerbated brain edema and the expression of tight junction proteins ZO-1 and occludin, the detrimental effects of which outweighed the beneficial effects leading to the lack of improvement in neurological outcomes at 24 h after tMCAO [116]. In a clinical study, a combination of fingolimod with alteplase ameliorated endpoint outcomes, and attenuated reperfusion injury in patients with acute ischemic stroke was treated in the first 4.5 h after symptom onset [117]. Furthermore, co-administration of fingolimod with alteplase in acute ischemic stroke patients in the 4.5–6 h after symptom onset promoted both anterograde reperfusion and retrograde collateral flow resulting in favorable neurological outcomes [118]. The small sample size, absence of blinded treatment administration, the lack of an independent data safety and monitoring entity, and suboptimal randomization method may have introduced an element of bias. However, the findings indicated that fingolimod extends the time window for alteplase in acute ischemic stroke.

CD-1 mice treated with fingolimod showed improved neurological function and decreased brain edema at 24 and 72 h following experimental ICH. It reduced the number of lymphocytes in blood and CNS and the expressions of ICAM-1, IFN-γ, IL-17 at 72 h after hemorrhagic stroke. Intraperitoneal injection of fingolimod in rats decreased the spatial and motor learning deficit, along with reduced brain atrophy and neuron loss in the basal ganglia [119]. The beneficial impacts of fingolimod were also mirrored in patients with ICH. Administration of oral fingolimod within 72 h of primary supratentorial ICH decreased perihematomal edema and neurological dysfunction. Investigations revealed that fingolimod reduced the circulating immune cells such as CD4⁺T, CD8⁺T, CD19⁺B, NK, NKTc, and plasma ICAM. The level of anti-inflammatory cytokine IL-10 was increased in the fingolimod group. In addition, it helped preserve vascular integrity by reducing the level of plasma MMP9 and rT1% [120]. Whether the administration of fingolimod beyond 3 days can prolong the decrease of edema deserves further investigations, for its effect on an immune-deficient state and compromise the neural repair mechanism.

Ruxolitinib, Fimasartan, Andrographolide

Targeting NLRP3 is a viable strategy for ameliorating neurological injury both in ischemic stroke and ICH. In C57BL/6 mice, ruxolitinib treatment improved neurological scores, attenuated the infarct volume, and improved brain edema 3 days after ischemia attack via activating JAK2/STAT3 pathway, which might be related to the downregulation NLRP3 inflammasome expression. A limitation of this study is that the researchers mainly focused on the anti-inflammatory effect of ruxolitinib rather than its other roles, including angiogenic effect on rescuing cerebral ischemia injury [121].

Pretreatment with a low-dose (0.5 mg/kg) fimasartan to Sprague-Dawley rats alleviated ICH-induced secondary brain damage by inhibiting the NLRP3 [91]. The effects of regular doses of fimasartan in post-ICH treatment still need to be studied further to determine its clinical and translational relevance. Besides, it may be required to validate the translational potential of ICH models in elder animals for future studies. Andrographolide (Andro), the major active constituent of Andrographis paniculata, was tested in male Sprague-Dawley mice after ICH. Researchers investigated the potential effects of Andro on ICH-induced secondary brain injury. Andro was found to decrease the level of IL-1β and lactate dehydrogenase and inhibited microglia pyroptosis triggered by ICH via suppressing the NLRP3. The results suggested that Andro could be a potential candidate for reducing neuronal cell death and degeneration. However, there are a few limitations to the study. Future studies are required to testify the treatment of Andro in female and elder ICH models. In addition, the specific mechanisms by which Andro blocks NK-κB, NLRP3 inflammasome, and other pathways are still not entirely clear [122^, 123].