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The spleen may be an important target of stem cell therapy for stroke
Journal of Neuroinflammation volume 16, Article number: 20 (2019)
Stroke is the most common cerebrovascular disease, the second leading cause of death behind heart disease and is a major cause of long-term disability worldwide. Currently, systemic immunomodulatory therapy based on intravenous cells is attracting attention. The immune response to acute stroke is a major factor in cerebral ischaemia (CI) pathobiology and outcomes. Over the past decade, the significant contribution of the spleen to ischaemic stroke has gained considerable attention in stroke research. The changes in the spleen after stroke are mainly reflected in morphology, immune cells and cytokines, and these changes are closely related to the stroke outcomes. Autonomic nervous system (ANS) activation, release of central nervous system (CNS) antigens and chemokine/chemokine receptor interactions have been documented to be essential for efficient brain-spleen cross-talk after stroke. In various experimental models, human umbilical cord blood cells (hUCBs), haematopoietic stem cells (HSCs), bone marrow stem cells (BMSCs), human amnion epithelial cells (hAECs), neural stem cells (NSCs) and multipotent adult progenitor cells (MAPCs) have been shown to reduce the neurological damage caused by stroke. The different effects of these cell types on the interleukin (IL)-10, interferon (IFN), and cholinergic anti-inflammatory pathways in the spleen after stroke may promote the development of new cell therapy targets and strategies. The spleen will become a potential target of various stem cell therapies for stroke represented by MAPC treatment.
Stroke is the most common cerebrovascular disease and the second leading cause of death behind heart disease and is a major cause of long-term disability worldwide . Our understanding of the pathophysiological cascade following ischaemic injury to the brain has greatly improved over the past few decades. Cell therapy, as a new strategy addition to traditional surgery and thrombolytic therapy, has attracted increasing attention . The therapeutic options for stroke are limited, especially after the acute phase. Cell therapies offer a wider therapeutic time window, may be available for a larger number of patients and allow combinations with other rehabilitative strategies.
The immune response to acute stroke is a major factor in cerebral ischaemia (CI) pathobiology and outcomes . In addition to the significant increase in inflammatory levels in the brain lesion area, the immune status of other peripheral immune organs (PIOs, such as the bone marrow, thymus, cervical lymph nodes, intestine and spleen) also change to varying degrees following CI, especially in the spleen . Over the past decade, the significant contribution of the spleen to ischaemic stroke has gained considerable attention in stroke research. At present, the spleen is becoming a potential target in the field of stroke therapy for various stem cell treatments represented by multipotent adult progenitor cells (MAPCs).
Two cell therapy strategies
Two distinct cell therapy strategies have emerged from clinical data and animal experiments (Fig. 1). The first is the nerve repair strategy, which uses different types of stem cells with the ability to differentiate into cells that make up nerve tissue and thus can replace damaged nerves to promote recovery during the later stages after stroke [5,6,7,8,9,10,11]. This strategy usually involves cell delivery to the injury site by intraparenchymal brain implantation and stereotaxic injection into unaffected deep brain structures adjacent to the injury site. The main problem with this strategy is that we should not only ensure the efficient delivery of cells to the injury site but also try to reduce the invasive damage caused by the mode of delivery. Moreover, evaluation of the extent to which cells survive over the long term, the differentiation fates of the surviving cells and whether survival results in functional engraftment is difficult. This strategy mainly includes intracerebral [12,13,14,15], intrathecal  and intranasal administration  (Fig. 2).
The second strategy is an immunoregulatory strategy (typically therapeutic cells are injected intravenously), which takes advantage of the release of trophic factors to promote endogenous stem cell (NSC/neural progenitor cell (NPC)) mobilisation and anti-apoptotic effects in addition to the anti-inflammatory and immunomodulatory effects encountered after systemic cell delivery. The mechanism of action appears to be reliant on “bystander” effects; these effects are likely to include immunomodulatory and anti-inflammatory effects mediated by the systemic release of trophic factors [18, 19], since neither animal nor human data have found any signs of actual engraftment of intravenously delivered cells in the brain [20,21,22]. In addition, many therapeutic stem cells have been found to migrate to PIOs, such as the spleen, following brain injury to play an immunoregulatory role, thus providing a good environment for nerve and vascular regeneration in vivo. This strategy mainly includes intra-arterial [23,24,25,26], intraperitoneal  and intravenous administration [28,29,30,31] (Fig. 2). Currently, systemic immunomodulatory therapy based on intravenous cells is attracting increasing attention .
Immunoregulation may be a better strategy
Further insight into the role of the two strategies has been provided by studies using cellular therapies in experimental models of brain ischaemia. All cells are more efficacious when administered systemically than when delivered via intracerebral administration [32,33,34,35,36,37], probably because intracerebral administration does not guarantee the extent to which cells can migrate from their implantation site in human subjects. Placing cells within the cystic space left as a long-term consequence of ischaemic damage in the absence of some type of bio-scaffold will be unlikely to promote cell adherence or persistence. Moreover, gliosis on the margins of the damaged region may impede cell migration or axonal outgrowth in the same manner as encountered after spinal cord injury.
The pathological progression of stroke is a complex systemic process, and changes in state occur in tissues besides intracranial tissues. Studies have shown that the immune response/regulation after stroke plays an important role in the pathological progression of stroke. The immune response is an important endogenous mechanism of post-stroke activation. Although the immune response following stroke, including cytokine production and inflammatory cell infiltration into damaged brain tissues, has been known for many years [38,39,40], the complexity of the mechanisms involved in post-stroke immune activation, inflammatory damage and tissue repair are unknown. In the future, immunomodulation will be an important potential therapeutic strategy for stroke. Moreover, finding the most appropriate therapeutic target for therapeutic cells may further improve the effectiveness of immunomodulatory treatment.
Stem cells and immunoregulation after stroke
At present, most cells used for immunoregulation therapy after stroke are various types of stem cells. However, animal experiments have shown that anti-inflammatory immune cells (such as regulatory T cells (Tregs), helper T (Th)-2 cells and regulatory B cells (Bregs)) can also alleviate brain damage [41,42,43,44]. In addition, some immune cells are activated after stroke, such as monocytes in some PIOs or astrocytes and have been shown to have protective effects in experimental animals [45,46,47].
Stem cell therapy has received considerable attention and application because of the easy access, strong proliferation and low immunogenicity of the cells. Treatments based on different types of stem cells have been studied for years and even decades in animal models of stroke. Included in the following subsections are specific examples of cell therapies that have been extensively studied in animal models and taken forward to clinical trials. For instance, human umbilical cord blood cells (hUCBs) [32,33,34], haematopoietic stem cells (HSCs) , bone marrow stem cells (BMSCs) , human amnion epithelial cells (hAECs)  and neural stem cells (NSCs)  have all been shown to reduce neural injury in experimental models of stroke.
Interestingly, almost all studies have found that when administered systemically, stem cells migrate to the injured brain and PIOs and in some cases have been shown to modulate the immune response to stroke [32, 35,36,37, 48], which may be one reason that this injection route is more efficacious. Studies have also shown that only a small number of stem cells injected intravenously after a stroke can be transported through the blood-brain barrier to damaged brain tissue . This finding suggests that regulation of the peripheral immune status after stroke may be a potentially important therapeutic strategy, especially for improvement of the long-term prognosis in stroke patients.
In addition to stem cells themselves, exosomes derived from some stem cells have been found to have therapeutic effects on haemorrhagic stroke . For instance, transplantation of pluripotent mesenchymal stem cell (MSC)-derived exosomes promoted functional recovery in an experimental intracerebral haemorrhage (ICH) rat model . MSC-derived exosomes can amplify endogenous brain repair mechanisms and induce neurorestorative effects after CI . Exosomes carry a concentrated group of functional molecules (DNA, ribosomal RNA, circular RNA, long noncoding RNA, microRNA, proteins and lipids) that serve as intercellular communicators not only locally but also systemically. These molecules may be part of the long-distance cell-to-cell communication that operates by paracrine function through secretory factors in the extracellular environment and is responsible for the long-distance effects during cell therapy.
Stroke and inflammation
The pathophysiological process of stroke is very complex and involves energy metabolism disorders, acidosis, loss of cellular homeostasis, excitotoxicity, activation of neurons and glial cells, blood-brain barrier (BBB) destruction and accompanying leukocyte infiltration . Evidence suggests that the immune system is involved in the various pathological stages of stroke . CI initiates an inhibitory effect on lymphatic organs through the autonomic nervous system (ANS), which increases the risk of infection after stroke. Infection after stroke is a major cause of disability and death after stroke . On the other hand, the innate immune system also contributes to repair of brain tissue  (Fig. 3).
Inflammatory cell infiltration and tissue damage
The inflammatory response to stroke starts immediately in the lacuna after arterial occlusion, and production of reactive oxygen species (ROS) increases rapidly in the coagulation-promoting state, accompanied by activation of complement, platelets and endothelial cells [56, 57]. Increased cyclooxygenase-2 (COX-2) activity in inflammatory cells and neurons may lead to increased ROS production in the injured tissues and severe prostaglandin toxicity [58, 59]. ROS also help reduce nitric oxide (NO) activity, leading to platelet aggregation and leukocyte adhesion and thus aggravation of ischaemic injury . After a few minutes of arterial occlusion, the relevant intracellular and extracellular regulation begins immediately. Acute local injury is sensed by pattern recognition receptors (PRRs) by interaction with pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) [61,62,63]. These factors are released by stressed cells in the blood cascade, and PRRs in neurons and glial cells can activate intracellular signal transduction pathways to increase the expression of different pro-inflammatory genes [64, 65]. This mechanism activates immune system factors that cause mast cells to release vasoactive mediators, such as histamine, protease and tumour necrosis factor (TNF), whereas macrophages release pro-inflammatory factors . After the rapid production of inflammatory signals, the interaction between adhesion molecules and integrins is mediated by adhesion receptors to facilitate leukocyte infiltration into the brain parenchyma [67, 68]. After ischaemia, these cells rapidly release pro-inflammatory mediators into the area, and these cytokines contribute to leukocyte infiltration into damaged tissues and activate antigen presentation between dendritic cells (DCs) and T cells [69, 70]. T cells cause tissue damage through IFN-γ and ROS. IL-23 released by microglia and macrophages activates T cells to produce IL-17, which aggravates the acute ischaemic brain injury . Ultimately, this neuroimmune imbalance leads to an early downregulation of systemic cellular immune responses, resulting in functional deactivation of monocytes, Helper T (Th) cells and invariable natural killer T cells (iNKTs) . This stage is often accompanied by increased lymphocyte apoptosis, inhibition of peripheral cytokine release and helper Th1 cells and changes in the Th1/Th2 ratio. Stroke-induced immunosuppression helps to increase the risk of infection, leading to adverse functional outcomes .
Phenotypic and spatial distributions of microglia
Microglia can be regarded as resident immune cells in the central nervous system (CNS) that are activated by local and systemic infections, neurodegenerative diseases and tissue damage. Microglia respond quickly to stroke injury. Microglia enter the ischaemic centre within 60 min after focal ischaemia, and the number of activated microglia increases significantly for up to 24 h. Pro-inflammatory M1 microglia (which release TNF-α, IL-1β, IL-6 and IL-18)  can be observed in the ischaemic core within 24 h after CI, and the number of M1 microglia increases gradually within 2 weeks of CI . Inhibitory M2 microglia (which participate in neuroprotection and promote repair of damaged cells through production of transforming growth factor (TGF)-β, nerve growth factor (NGF) and IL-4)  begin to appear 24 h after injury, and their number gradually increases over time for up to 1 week after ischaemia . The phenotypes and spatial distributions of microglia change with the expansion of damaged brain tissue [76, 77].
Astrocytic proliferation and activation
Astrocytes are the most abundant cell type in the CNS and perform multiple functions that are both detrimental and beneficial for neuronal survival from the acute phase to the recovery phase after ischaemic stroke . IL-15 expression is increased in astrocytes in mouse and human brains after CI, which elevates the level and activation of CD8+ T cells and natural killer cells (NKs), resulting in aggravation of brain tissue damage [79, 80]. IL-15 blockade reduces the effects of NKs, CD8+ T cells and CD4+ T cells in the brains of mice after ischaemia/reperfusion (I/R), resulting in a reduction of the infarct size and improvement in motor and locomotor activity . During the recovery phase, IFN-α is mainly involved in regulation of astrocytic proliferation through blocking and activation . Astrocytes regulate the formation and maintenance of synapses, cerebral blood flow and BBB integrity . Astrocytes also indirectly regulate inflammation by affecting neuronal survival during acute injury and axonal regrowth . Activated astrocytes are beneficial for the recovery of neurological function after stroke . Recent studies have suggested that this endogenous protective mechanism may involve mitochondrial transport from astrocytes to neurons after brain injury, which is mediated by a calcium-dependent mechanism involving CD38 and cyclic ADP ribose signalling .
Mast cells and BBB breakdown
Mast cells, which are located in the perivascular space surrounding the brain parenchymal vessels and in the dura mater of the meninges, are activated during the early stage after stroke and contribute to BBB breakdown and brain oedema by releasing gelatinase [84, 85]. Pharmacological mast cell stabilisation with cromoglycate reduces haemorrhage formation and mortality after administration of thrombolytics in experimental ischaemic stroke , which may involve promotion of BBB breakdown and neutrophil infiltration by mast cells .
Inflammatory reactions lead to the production of inflammatory cytokines and the death of neurons and glial cells, which are regulated by a multiprotein complex called the inflammasome . Nod-like receptors (NLR) in neurons and glial cells may mediate production of the inflammasome, which participates in the inflammatory response to aseptic tissue damage during CI . The inflammasome in damaged brain tissue produces IL-1β and IL-18 after activation, which can cause specific cell death called inflammatory necrosis . In this way, the inflammasome not only helps activate and support innate immunity but also aggravates tissue damage.
Inflammation relief and tissue repair
Inflammation after stroke is also inhibited by auto-suppression, and its remission is regulated by many immunosuppressive factors. The termination of inflammation also triggers structural and functional remodelling of damaged brain tissue. The first mechanism involved in this stage is the clearance of dead cells and is accomplished by microglia and infiltrating macrophages, which are mainly composed of phagocytes [76, 89]. Immunoglobulins targeting CNS antigens may promote the release of IL-10 and TGF-β, thereby inhibiting the immune response and the production of adhesion molecules and inflammatory cytokines . These multipotent immunoregulatory factors can inhibit inflammation and contribute to tissue repair, and their protective effects are conducive to cell survival in ischaemic areas . These growth factors are released by inflammatory cells, neurons and astrocytes and support cell budding, neuronal growth, angiogenesis and even tissue remodelling after ischaemic injury . Insulin-like growth factor (IGF)-1 plays a key role in the neurogenesis process after ischaemic injury, and the astrocyte response is also necessary for the functional recovery of damaged tissues . The roles of vascular endothelial growth factor (VEGF) and neutrophil metalloproteinase are also required for angiogenesis; together, they support the joint activity of inflammatory cells and astrocytes .
Changes in peripheral immune organs after stroke
The pathological process after stroke is a complex systemic immune state change. In addition to severe inflammation in brain tissues (including inflammatory chemokine production, inflammatory cell infiltration, microglial activation and inflammasome production) , the state of PIOs also changes significantly after stroke  (Fig. 4).
CD34+ HSCs/ haematopoietic progenitor cells (HPCs) in bone marrow are mobilised rapidly into the peripheral blood circulation under post-stroke pathological stress and play an important protective role in the pathological process of CI . The prognosis can be effectively improved by accelerating the mobilisation of protective cells in bone marrow or increasing their levels in peripheral circulation after stroke [97, 98]. Clinical trials have also shown that intra-arterial injection of bone marrow-derived CD34+ haematopoietic stem cells/progenitor cells can significantly improve the prognosis of acute ischaemic stroke patients and greatly reduce their mortality and disability rates . In addition, CI regulates the elevation of CD4+CD25+FoxP3+ Tregs from bone marrow via the sympathetic nervous system (SNS) . Stroke reduces C-X-C chemokine ligand (CXCL) 12 expression in bone marrow but increases C-X-C chemokine receptor (CXCR) 4 expression in Tregs and other bone marrow cells. Destruction of the CXCR4-CXCL12 axis in bone marrow promotes mobilisation of Tregs and other CXCR4+ cells into peripheral circulation and eventually migration to damaged brain tissues to facilitate tissue repair .
Animal data have shown that the thymus exhibits loss of a large number of lymphocytes within 12 h after ischaemia/reperfusion (I/R). Cytokine production also changes from the Th1 to the Th2 phenotype [99, 100]. Lymphocytes, such as B cells, T cells and natural killer cells (NKs), were found to be highly apoptotic , and the thymic morphology of the tested mice exhibited significant atrophy after I/R . The non-toxic apoptosis inhibitor Q-VD-OPH significantly reduced programmed death of thymocytes after I/R, effectively reducing the incidence of bacteraemia after CI injury and improving the survival rate of the mice .
Cervical lymph nodes
Treg levels in brain tissue and cervical lymph nodes increase significantly after CI . This increase may be due to changes in BBB permeability after stroke as well as other pathological causes, resulting in a large number of efflux cells and soluble proteins migrating from the brain tissue to the cervical lymph nodes. These cells and proteins migrate to the cervical lymph nodes and play an important role in regulating the pathological immune response after stroke [105, 106]. Many brain-derived antigens that migrate to the cervical lymph nodes after stroke may promote autoimmunity and Treg-based immunomodulation . In addition, antigen-specific T lymphocytes may circulate from other parts of the body to the cervical lymph nodes, where they enter targeted cells via integrin expression on their surfaces and are transported to the damaged hemisphere .
Experimental and clinical evidence has shown that temporary impairment of the immune response is an important factor in the high post-stroke infection rate [53, 109]. The intestine is often exposed to a large number of microorganisms and thus provides potential access to pathogens. Therefore, intestinal barrier dysfunction may be an important risk factor for bacterial translocation and endogenous infection. The numbers of T and B cells in aggregated lymph nodes have been shown to decrease significantly after CI, whereas the numbers of NKs and macrophages do not differ significantly. Compared with that of the control group, no significant change occurred in the lymphocyte subsets of the intestinal epithelium and lamina propria in rats with CI . Stroke may have different effects on the immune cell composition in the intestinal lymphoid tissue, and this change may increase the susceptibility to infection after stroke .
In addition to the immune cell structure, the intestinal flora also plays an important role in the stroke prognosis. The interaction between the immune system and intestinal epithelial surface symbiotic microorganisms is essential for the development, maintenance and functionalization of immune cells [111, 112]. Intestinal symbiotic microorganisms are the most abundant symbiotic chambers in the human body and have potential as a method to regulate the levels of lymphocytes, including Tregs and γδT cells, which play key roles in the pathological process of stroke . Altering the intestinal symbiotic microbial structure of mice using amoxicillin-clavulanic acid compound antibiotics induces tolerance and protection of the mice against I/R injury . This protective effect can be transferred directly between mice through faecal feeding behaviour. Other antibiotics, such as vancomycin, can play a similar role in altering the structure of the intestinal flora and inducing tolerance to I/R in mice . This protective mechanism may be due to alteration of the intestinal symbiotic microflora structure, resulting in the production of Tregs in intestinal lymph nodes derived from the small intestine. Treg homing in the intestine inhibits the differentiation of IL-17+ γδT cells via IL-10 secretion. After stroke, effector T cells migrate from the intestine to the meninges, because the decrease in IL-17+ γδT cells reduces CXCL1 and CXCL2 expression in ischaemic brain tissue, thereby reducing the migration and infiltration of leukocytes into the ischaemic brain tissue and the resulting brain tissue damage .
The role of the spleen in stroke
Splenectomy has been shown to play a protective role in various brain injury models, including permanent/temporary middle cerebral artery occlusion (p/t MCAO), ICH and traumatic brain injury (TBI) [113,114,115,116,117,118]. Splenectomy before pMCAO significantly reduces the infarct size, numbers of neutrophils and activated microglia in the damaged brain tissue , IFN-γ level and number of infiltrating immune cells . Splenectomy before tMCAO results in a significantly lower cerebral infarction volume and IFN-γ level after ischaemia and does not increase the risk of post-stroke infection . Splenectomy immediately after different TBI injury models can also reduce nerve injury. For instance, vascular injury and brain oedema in the cerebral ischaemic region were significantly reduced in the splenectomy group [116,117,118]. Similar protective effects were observed in aged rats either before tMCAO or immediately after reperfusion with splenectomy . However, splenectomy fails to provide long-term protection against I/R. In one study, splenectomy was performed 3 days after reperfusion, and the infarct volume, nerve function and peripheral blood immune cell count were assessed 28 days after stroke. The results showed that delayed splenectomy neither reduced brain tissue loss nor alleviated sensorimotor and cognitive impairment . Although splenectomy immediately upon reperfusion significantly reduced the infarct size and immune cell infiltration 3 days after MCAO, the procedure failed to promote long-term recovery . This finding indicates that the acute neuroprotective effect achieved by immediate splenectomy after stroke does not provide long-term protection and that immune regulation by the spleen may play different roles in different pathological stages of stroke. As an alternative to splenectomy, exposure of the spleen to radiation 4 h after tMCAO has similar protective effects. Exposure of the spleen to radiation causes a temporary decrease in splenic cells and does not cause extensive immunosuppression .
The changes in the spleen after stroke are mainly reflected in three aspects: first, the splenic morphology; second, the numbers of immune cells derived from the spleen; and third, inflammatory cytokine production by the spleen.
In different stroke animal models, splenic atrophy similar to that of the thymus appears after brain injury [102, 114]. The splenic morphology decreases gradually 24 to 72 h after pMCAO. The increase in catecholamines in the insular cortex after I/R may be an important cause of splenic atrophy after injury. Activation of alpha 1 adrenergic receptor (α1-AR) in the splenic smooth muscle sac causes contraction of the splenic envelope, which leads to a reduction of the splenic volume. Prazosin, which is an α1-AR antagonist, can effectively alleviate splenic atrophy after CI . Clinical studies have also assessed changes in the shape of the spleen in stroke patients. One study showed that loss of splenic volume in ischaemic stroke patients began less than 6 h after stroke and that the process of splenic atrophy continued until approximately the third day after stroke, gradually increased from the fourth day to the eighth day and then basically returned to the pre-stroke state. At the same time, the size of the spleen after tMCAO is negatively correlated with the infarct volume, and more severe atrophy of the spleen is associated with a larger infarct volume . The spleen of patients with ischaemic stroke may initially contract after onset and then re-expand, which contributes to ischaemic brain damage via splenic cell components . Atrophy of the spleen is also accompanied by apoptosis of splenic cells and loss of B cells in the germinal centre. Moreover, this study also showed that the only subset of immune cells that decreased after tMCAO was B cells .
The spleen is the largest natural reservoir of immune cells, many of which also change in the spleen after stroke, including lymphocytes, monocytes, neutrophils and NKs. These cells are mobilised from the spleen to the brain after CI and play an important role in the pathological process after stroke . Removal of blood neutrophils with an anti-neutrophil antibody has been shown to alleviate nerve damage and splenic atrophy in hypoxic ischaemic neonatal rats . Neutrophils are the first immune cells to respond to ischaemic injury and infiltrate into the damaged brain within hours of stroke. Animal models show that neutrophil infiltration reaches a peak during days 1–3 after CI. In acute ischaemic stroke patients, neutrophils are recruited within 24 h after symptom onset . Studies have shown that neutrophil infiltration plays an important role in the pathological process of stroke [113, 127]. Clinical data show that the splenic volume is negatively correlated with the percentage of blood lymphocytes and positively correlated with the percentage of neutrophils after acute stroke . In addition, neutrophils may increase BBB permeability by releasing matrix metalloproteinase (MMP)-9, resulting in more leukocyte infiltration and increased neuroinflammation [129, 130]. Animal data also show that arginase I released by activated neutrophils after CI can induce peripheral immunosuppression . As expected, the protective effect of splenectomy 2 weeks before pMCAO is also reflected in a reduction of neutrophils at the injury site .
Peripheral blood monocytes/macrophages have also been shown to migrate to and infiltrate into ischaemic brain tissue under the action of C-C chemokine ligand 2 (CCL-2) and to promote inflammation and tissue damage in the brain after stroke . Researchers have promoted tissue repair and remodelling after brain ischaemia by injecting clodronate liposomes into mice to deplete macrophages and especially by reducing the numbers of macrophages in the spleen . Interestingly, both pro-inflammatory Ly-6Chi and anti-inflammatory Ly-6Clow were mobilised to migrate from the spleen to ischaemic brain tissue. However, another study reported that complete removal of splenic-derived monocytes/macrophages by splenectomy did not provide any protection against ischaemic brain tissue . Therefore, only selective clearance of pro-inflammatory Ly-6Chi and anti-inflammatory Ly-6Clow monocytes/macrophages can determine the different effects of different groups of cells on the stroke prognosis. However, systemic injection of low-dose lipopolysaccharide (LPS) induces a Ly6Chi monocyte response that protects the brain after tMCAO in mice . Remarkably, adoptive transfer of monocytes isolated from LPS-preconditioned mice into naïve mice 7 h after tMCAO reduces brain injury, although the protective effect still depends on an intact spleen .
Many studies have confirmed that T lymphocytes play a harmful role in I/R damage [135,136,137]. T cells contribute to the lymphopaenia induced by CI and are the most crucial lymphocytes for immunodepression after stroke . In young mice, RTL1000 (a type of recombinant T cell receptor ligand) therapy inhibited the splenocyte efflux while reducing the frequency of T cells and macrophages in the spleen. Older mice treated with RTL1000 exhibited a significant reduction in inflammatory cells in the brain and inhibition of splenic atrophy . The protective effect of splenectomy on the brain is also accompanied by a decrease in the number of T cells at the brain injury site . However, some studies have also shown that certain T cell subsets, such as Tregs, may play a protective role in the pathological process of stroke [140, 141]. Animal data showed that the therapeutic effect of adoptive injection of Tregs could be maintained for at least 12 days . The increase in the number of Tregs in the spleen after stroke is known and may reflect an endogenous protective mechanism . Intraperitoneal injection of CD28SA (a CD28 superagonistic monoclonal antibody) after MCAO in mice increased the Treg levels in the brain and spleen, thereby attenuating the inflammatory response and improving the outcome after experimental stroke . Gu et al. studied the effects of the absence of T cell subsets on brain infarction after in vivo stroke and then used an in vitro coculture system of splenocytes and neurons to further identify the roles of T cell subsets in neuronal death. The data displayed the detrimental versus beneficial effects of Th1 and Th2 cells both in vivo and in vitro .
B cells are the main type of splenic lymphocyte, but few studies have focused on the role of B cells in stroke injury. The lack of B cells does not improve brain injury in mice after I/R, suggesting that endogenous B cells do not have harmful effects after acute CI . However, the animal data showed a genetic deficiency, and pharmacologic ablation of B lymphocytes using an anti-CD20 antibody prevented the appearance of delayed cognitive deficits . Similar to Tregs, Bregs that secrete IL-10 also protect against I/R injury , but further studies are needed to confirm whether these Bregs are released by the spleen after stroke. However, exogenous transplantation of spleen-derived Bregs via intravenous injection has been shown to protect against CI [42, 146]. In addition, adoptive injection of Bregs into tMCAO rats can increase the level of CD8+CD122+ Tregs in the spleen and CNS after CI .
NKs, which also travel from the spleen into the ischaemic brain, are a type of cytotoxic cell that forms part of the innate immune system. Studies have shown that chemokines produced by ischaemic neurons cause NKs to migrate and infiltrate into the brain, where they promote further brain damage [148, 149]. In addition, splenic T lymphocytes are known to be activated by antigen-presenting cells (APCs), especially DCs. An increase in the number of DCs has been observed in both pMCAO and tMCAO . Immature DCs patrol the blood and invade injured tissues, where they pick up antigens and acquire the ability to stimulate T cells in lymphoid tissues, such as the lymph nodes and spleen [151, 152]. Therefore, DCs can present antigens to T lymphocytes in the spleen and activate adaptive immunity after stroke. However, the exact role of splenic DCs in the prognosis of stroke is unknown.
From these studies, we can deduce that brain-spleen cell cycling after stroke can affect systemic inflammation and the brain inflammatory milieu, which may be a target for a novel therapeutic strategy.
Immune cells in the spleen contribute to the rise of cytokines in the blood and in turn in the brain after stroke. For example, spleen cells collected from stroke model mice show a stronger ability to secrete inflammatory cytokines, including TNF-α, IL-6, monocyte chemoattractant protein-1 (MCP-1) and IFN-γ , than those collected from normal mice. Many of these inflammatory cytokines and chemokines, such as IFN-γ and IFN-induced protein 10 (IP-10), have been shown to be key factors in stroke-induced neurodegenerative diseases [119, 153, 154]. Offner et al. also confirmed that the IL-2 and IL-10 levels in the spleen of experimental animals increased to varying degrees after CI . In a clinical trial involving 158 healthy volunteers and 158 stroke patients, the levels of various inflammatory factors were elevated in patients with splenic contraction, with significant differences in IFN-γ, IL-6, IL-10, IL-12 and IL-13 .
Effects of splenic cells on stroke
These responses in the spleen after CI may provide new opportunities for the development of novel stroke therapies. What effect does adoptive reinfusion of splenic cells have on stroke? Syngeneic transplantation of newborn splenocytes in a murine model of neonatal I/R achieved long-term survival of the grafts, exerted an influence on the microenvironment in the injured brain and showed improvement in behavioural tests 2 weeks after onset, but these parameters were not significantly different from those of the control groups after four weeks of brain injury . The effect of adoptive transfer of splenic immune subsets on CI outcomes still depends on the splenic integrity of the mice . From the above research, we can deduce that different subsets of splenic cells may play distinct roles in different pathological stages of stroke through the release of various cytokines.
Brain-spleen cross-talk after stroke
The mechanism underlying the splenic responses after stroke and methods to improve the prognosis of stroke by intervening with the splenic reactions have become urgent issues in need of clarification. Although the exact mechanisms underlying the initiation of splenocyte responses to stroke have not been identified, several events, including ANS activation, release of CNS antigens and chemokine/chemokine receptor interactions, have been documented to be essential for efficient brain-spleen cross-talk after stroke.
Clinical and experimental studies have shown that the ANS is also involved in the regulation of immune-related pathological processes and neuroprotective effects after stroke [159, 160]. Symptoms of ANS dysfunction often occur after stroke, and in many cases, they show a special association with the location and exacerbation of the brain damage. Because many pathological risk factors of stroke are closely related to changes in ANS function, we can speculate that an interdependence exists between the ANS and stroke. The ANS may affect the immune system through many pathways. Many PIOs, including the spleen, are controlled by the ANS (mainly the SNS). At the same time, immune cells and organs also express various receptors for sympathetic neurotransmitters [159, 160]. SNS markers are increased and parasympathetic nervous system (PNS) markers are decreased after stroke . Increasing SNS activity or decreasing PNS activity is associated with a worsening prognosis in patients with acute stroke [162,163,164,165]. Blocking SNS by chemical methods can effectively relieve the immunosuppression and the reduction in the splenic volume, which improve the outcomes of experimental animals after CI [166,167,168].
The CNS regulates immune system activity mainly through complex humoural and neural pathways, including the hypothalamic-pituitary-adrenal (HPA) axis, vagus nerve (VN) and SNS . Elevated cortisone, corticosterone and metanephrine levels and associated lymphocytopaenia are often observed after extended brain infarction. The differential effects and complex interplay between the SNS and the HPA axis on systemic immune cells have to be considered when targeting the neurohormonal systems in the acute phase of severe stroke . The hypothalamus is associated with the central function of the ANS by synchronising the neuroendocrine (glucocorticoid) response and cholinergic pathways, which together inhibit the release of inflammatory cytokines from peripheral T cells, monocytes and macrophages and promote the release of anti-inflammatory cytokines, such as IL-10. Similarly, norepinephrine (NE) released from dense neural networks throughout the brain and from peripheral organs, including the spleen, induces significant anti-inflammatory phenotypes in lymphocytes, monocytes and macrophages. In addition, the release of catecholamine from nerve endings can induce the release of acetylcholine (ACh) from splenic T memory cells, which can inhibit inflammation and increase the risk of infection after stroke . A recent work showed that transcutaneous auricular VN stimulation (ta-VNS) reduced the infarct volume and induced angiogenesis in focal cerebral I/R rats. Ma suggested that the neurobehavioural recovery induced by ta-VNS might involve spleen-brain communication triggered by redistribution of growth differentiation factor (GDF)-11 .
The brain and viscera interact through the ANS, and the VN, which contains 80% afferent fibres and 20% efferent fibres, plays multiple key roles in regulation of visceral homeostasis and anti-inflammatory processes . These VN functions are mediated by many pathways, some of which are controversial. In the splenic sympathetic nerve-mediated anti-inflammatory pathway, VN fibres stimulate the splenic sympathetic nerve, causing NE release from the distal splenic nerve to act directly on the β2-AR of splenic lymphocytes, thereby inducing the release of ACh. Finally, ACh inhibits the release of TNF-α from spleen macrophages through the α-7-nicotinic ACh receptor (α7nAChR) . At the same time, activation of the β2-AR receptor on some splenic lymphocytes may trigger activation of the cAMP-PKA pathway , which is related to inhibition of the NF-κB pathway and IL-10 production in the spleen [174, 175] (Fig. 5).
Generally, the immune response to stroke can be divided into two stages. The immune response at the early stage of acute stroke is pro-inflammatory and is driven by an increase in SNS activity. Later, immunosuppression starts when the spleen has depleted its immune cell reserve, and the risk of infection increases after stroke during this window . Rasouli et al. also proposed a similar viewpoint of “brain-spleen inflammatory coupling” by which autonomic control of splenic macrophages could modulate systemic inflammation after injury. Stimulation of α/β-AR located on splenic macrophages leads to the release of TNF-α and IL-1β, which enhance and exacerbate inflammation. Conversely, parasympathetic stimulation of α7nAChR inhibits the release of these cytokines, thereby attenuating the inflammatory response to injury .
In response to stroke, the ischaemic brain may secrete a variety of antigens that activate adaptive immune responses and recruit immune cells from the spleen. During acute stroke, neo-antigens, such as microtubule-associated protein 2 (MAP2), N-methyl-d-aspartic acid receptor subunit 2 (NR-2A), myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG), can all be released into the periphery and captured by APCs, especially DCs and macrophages. This response is thought to eventually trigger the activation of T cell-dependent adaptive immune responses in the T cell zone [177, 178].
Although the immune system does not directly initiate the pathological process of stroke, stroke-induced immune activation has been increasingly recognised to enhance the neuropathological outcomes or nerve repair. Elucidating novel neo-antigens that are targets for immune cells offers unique insights into potential cellular and systemic consequences of autoimmunity during post-stroke neuronal plasticity. Mice with small infarct volumes exhibited high autoreactivity to MAP2 and MBP. This autoimmunity was maintained through splenic CD4+ and CD8+ T cells as well as CD19+ B cells during the first 10 days post-stroke . An early splenic CD4+ T cell autoimmune response to neuronal and myelin antigens is associated with better recovery . Ren et al. applied adoptive transfer of MOG-reactive splenocytes capable of secreting toxic Th1 cytokines (such as IFN-γ and TNF-α) to mice with severe combined immunodeficiency. This manoeuver resulted in an increased infarct size, increased neurological deficits and a higher accumulation of immune cells in the ischaemic brain in the treated mice relative to those of the control animals . The MBP-specific phenotype of the splenocytes was obtained from donor animals 1 month after stroke and adoptively transferred to naïve recipient animals at the time of CI. Animals that received either MBP-specific TH1 or TH17 cells suffered worse neurological outcomes, and the degree of impairment correlated with the robustness of the MBP-specific TH1 and TH17 responses . Hurn et al. put forward a hypothesis of “brain-spleen cell cycling” that stated that adaptive immune cells could be triggered through an encounter with CNS antigens in either soluble form or presented by macrophages or DCs .
Chemokines (CCL2, CCL3, CCL5, CX3CL1, CXCL8, CXCL12, etc.) are secreted by damaged central cells to recruit inflammatory cells into the damaged brain. The corresponding chemokine receptors are also increased in splenocytes after I/R .
CCL2 can effectively mediate monocyte/macrophage and neutrophil infiltration during CI . Inadequate CCR2 expression results in decreased monocyte and neutrophil infiltration into the ischaemic brain, followed by decreased inflammation and cerebral infarction. Bao also showed that the CCL2-CCR2 interaction might play an important role in the distribution and migration of monocytes from the spleen to the injured brain . Moxifloxacin treatment can effectively inhibit CCR2 expression in monocytes, thereby significantly reducing the infarct size after CI .
The CXCR4-CXCL12 axis is closely related to the pathology of ischaemic stroke. CI leads to a rapid and long-lasting increase in CXCL12 in the ischaemic penumbra. Transplanted GFP-labelled bone marrow cells are recruited in proximity to these CXCL12+ vessels and display characteristics of activated microglial cells. Therefore, we can speculate that CXCL12 plays an important role in homing of bone marrow-derived monocytes, which transform into microglia at the site of ischaemic injury . The CXCR4 antagonist AMD3100 blocks the interaction between CXCR4 and CXCL12, which not only alleviates cerebral inflammation and cerebral infarction but also prevents splenic atrophy after tMCAO. Therefore, CXCR4-CXCL12 may play a regulatory role in the splenic response after stroke .
Many other cytokines have also been shown to play important roles in the recruitment of immune cells into the ischaemic brain. For example, CCL3 is closely related to the accumulation of monocytes and neutrophils in damaged brain tissue [186, 187]. CCL5 is involved in leukocyte infiltration after I/R . CX3CR1-knockout mice show reduced neuroinflammation after focal CI, suggesting that CX3CL1 promotes post-stroke inflammation, which may be related to chemotaxis of monocytes, T cells and NKs [148, 189, 190]. CXCL8 has been considered as an important chemotactic factor for neutrophil recruitment after ischaemic stroke . IP-10 expands the NK-induced damage of the BBB through CXCR3 . The increased CXCL-1 and CXCL-2 levels in the brain tissue after stroke lead to accelerated leukocytes and particularly granulocyte accumulation and aggravate ischaemic tissue damage . CCL20 is upregulated in the thymus and spleen 24 h after TBI and in the cortex and hippocampus 48 h after TBI . The roles of these cytokines in splenic cell mobilisation warrant further study.
Stem cell therapy targeting the spleen
In various experimental models, hUCBs [32,33,34,35,36], HSCs , BMSCs [36, 97, 193], hAECs  and NSCs  have been shown to reduce the neurological damage caused by stroke. Compared with that of intracerebral administration, all stem cells show better therapeutic effects when administered systemically. Stem cells migrate to the injured brain and spleen and in some cases have been shown to modulate the immune response to stroke [32, 35,36,37, 48], which may be one reason that this injection route is more efficacious. Ninety-five percent of BMSCs are found in the spleen following systemic administration after MCAO  (Fig. 6).
Intravenous hBMSCs preferentially migrate to the spleen and alleviate chronic inflammation in rats with CI. hBMSC treatment reduces the TNF-α level in the spleen after CI by 40%. Correlation analysis revealed negative correlations between hBMSC migration in the spleen and the infarct areas, peri-infarct areas, volume of MHCII− activated cells in the striatum and TNF-α level in the spleen . Even NSCs migrate to the spleen following ICH, but the therapeutic effect disappears after splenectomy. NSCs have been found to be in direct contact with CD11b+ cells in the spleen , which partly demonstrates that the neuroprotective effect of NSCs during stroke involves their interaction with the spleen.
hUCBs are another cell type that has been shown to affect the spleen during the pathological process of stroke. hUCBs alter cell populations in the peripheral circulation and spleen after pMCAO due to the interactions of all subpopulations together  (Fig. 7). Systemic administration of hUCBs 24 h after MCAO significantly alters splenic T cell responses to concanavalin A, decreases the proliferation activity of splenic T cells, decreases production of the inflammatory factors TNF-a and IFN-γ and increases production of the anti-inflammatory cytokine IL-10 [32, 196]. hUCBs also inhibit splenic atrophy in rats 48 h after MCAO. This effect is thought to be achieved by regulation of immune cells in the spleen by hUCBs after MCAO and inhibition of their release into the systemic circulation . Kadam et al. used intravenous injection of CD34+-enriched hUCBs to treat CI mice; the experimental results showed that neurogenic niche proliferation and glial brain responses to CD34+-enriched hUCBs after neonatal stroke might involve interactions with the spleen and were sex-dependent .
hAECs are derived from the epithelial layer of the amnion, which is the sac that encloses the developing foetus and is attached to the placenta. Evans recently tested the efficacy of systemically delivered hAECs to improve a number of outcome measures in four animal models of ischaemic stroke . Based on their experimental results, they put forward the hypothesis that administration during the acute phase (within 1.5 h) after ischaemic attack allowed the hAECs to migrate preferentially to the spleen and damaged brain; subsequently, cell apoptosis and inflammation were inhibited. Early brain infiltration of immune cells, aggravation of infarction and systemic immunosuppression were also alleviated .
HSCs injected intravenously 24 h after reperfusion were first detected at 24 h after injection in the spleen and later in the ischaemic brain parenchyma . In addition, compared with that of the sham-operated control group, the immune environment after CI increased HSC migration to the spleen 72 h after reperfusion. In the absence of induction of an injury, the cells did not preferentially accumulate in the spleen . HSC treatment reduced the infarct volumes, apoptotic neuronal cell death in the peri-infarct areas and immune cell (T cells and macrophages) infiltration into the ischaemic hemispheres. Moreover, HSC therapy decreased the TNF-α, IL-1β, CCR2 and CX3CR1 levels in the spleen after CI .
These experiments suggest that stem cell therapy works to some extent by regulating the immune response after stroke, especially at the spleen level, which may be crucial and is an important potential therapeutic target.
Important therapeutic targets
Many factors and regulatory pathways change significantly during the entire pathophysiological process of stroke. They may play different roles in different pathological stages after CI. Fully understanding their effects on stroke may identify new targets for development of novel therapeutic strategies.
IL-10 is a multicellular, multifunctional cytokine that regulates cell growth and differentiation and participates in inflammatory and immune responses. It is mainly produced by cells such as Tregs, Th2 cells and Bregs and currently is recognised as an anti-inflammatory and immunosuppressive factor. IL-10 is an important component of the endogenous repair mechanism after stroke. The IL-10 levels in the brain and spleen increase after stroke [90, 155, 156]. The use of exogenous IL-10 for anti-inflammatory therapeutic approaches has been shown to provide neuroprotection during ischaemic stroke . However, an excessive IL-10 response can contribute to immunosuppression after CI, which worsens outcomes. Additionally, sex differences may exist in the role of IL-10 in stroke recovery .
Adoptive transfer of IL-10-secreting cells is widely used for the treatment of experimental stroke. However, after the earlier use of Tregs , Bregs are attracting increasing attention. Adoptive reinfusion of spleen-derived Bregs can improve neurological injury and motor dysfunction after CI in experimental rats [34, 140]. Elevated Breg and Treg levels in the spleen at different time points after CI have been found in some studies investigating the preconditioning protection of I/R [208, 209]. In addition to their anti-inflammatory effects, Bregs can also promote the activation and proliferation of other anti-inflammatory immune cells and have a cascade amplification effects on the repair mechanism after CI [147, 210] (Fig. 8). Therefore, we have proposed that Bregs may represent an important potential strategy to rapidly start the endogenous protective mechanism during the early stage of stroke and increase the Breg levels in the body, especially in the spleen.
IFN-γ is the only member of the type II IFNs and is produced only by activated T cells, NKs and natural killer T cells (NKTs). IFN-γ is a marker cytokine of Th1 cells that can activate APCs and promote the differentiation of Th1 cells by upregulating related transcription factors. Deletion of the IFN-γ gene has been shown to reduce brain damage after CI . In the first 3 days after I/R, intraventricular administration of an IFN-γ neutralising antibody can protect the brain from CI injury . IFN-γ participates in the Th1 inflammatory response by activating monocytes, microglia and macrophages. Since activation of microglia/macrophages is part of the cause of delayed cell injury following ischaemic injury, IFN-γ may play a role in the splenic response by aggravating the inflammation associated with ischaemic injury. Seifert et al. previously proposed that the spleen contributed to stroke-induced neurodegeneration through IFN-γ signalling . IFN-γ is increased early in the spleen but later in the brain following I/R. Splenectomy reduces the IFN-γ level in the infarct after MCAO. The protective effect of splenectomy was eliminated by systemic recombinant IFN-γ accompanied by an increase in IFN-γ expression in the brain post-pMCAO . Moreover, IP-10 has been shown to be a key factor in stroke-induced neurodegenerative diseases . NKs participate in CI and promote neuronal necrosis through IFN-γ. IP-10 expands the damage of the BBB induced by NKs through CXCR3 . Furthermore, hUCB therapy inhibits the proliferation of splenic T cells after MCAO by increasing IL-10 and IFN-γ production .
IFN-β is a type I IFN that binds to the IFN-α/β receptor. IFN-β exerts anti-inflammatory effects, and systemic administration of recombinant IFN-β has been used for the treatment of multiple sclerosis, which is a neuroinflammatory condition of the CNS . Therefore, IFN-β has been considered to be able to decrease neuronal death and promote functional recovery after CI by limiting inflammation. In agreement with this view, IFN-β has been put forward as a candidate drug for the treatment of stroke. Several animal experiments have shown that systemic administration of recombinant IFN-β at different time points before and after MCAO results in neuroprotection [212,213,214]. This outcome may be related to endogenous IFN-β signalling not only to reduce CNS inflammation but also to reduce autoreactive T cell proliferation via inhibiting the antigen presenting capacity of astrocytes and microglia. Inácio’s research also showed that endogenous IFN-β signalling could alleviate local inflammation and regulate peripheral immune cells, thereby contributing positively to stroke outcomes .
Cholinergic anti-inflammatory pathway
As mentioned above, VNS causes prominent attenuation of the systemic inflammatory response evoked by CI in experimental animals. This effect is mediated by ACh stimulation of acetylcholine receptors on splenic macrophages. Therefore, the circuit is known as the “cholinergic anti-inflammatory pathway”, which casts the spleen as the major effector . α7nAChR is considered to be an important target for alleviating the release of pro-inflammatory cytokines from macrophages and DCs.
Noradrenergic neurons provide innervation to all primary and secondary lymphoid tissues, including the bone marrow, spleen and lymph nodes [217, 218]. Most immune cells express one or more ARs, but β2-AR is the most widely distributed and mediates most of the effects of sympathetic nerves on immune function [219,220,221,222]. Sympathetic nerves affect the innate and adaptive immune responses through stimulating β2-AR. Most evidence shows that β2-AR activation has immunosuppressive effects on monocytes and macrophages [221, 222]. Stimulation of β2-AR-naïve CD4+ T cells (Th0) results in their differentiation into Th1 cells, which enhance cellular immunity, or Th2 cells, which decrease cellular immunity [221, 222]. Norepinephrine (NE) can also produce β2-mediated anti-inflammatory effects by releasing ACh from cholinergic spleen cells [221, 222]. Blocking adrenocortical receptors has been shown to inhibit the splenic response after pMCAO and reduce injury . The spleen receives noradrenergic innervation from the postganglionic sympathetic neurons . Splenic T cells are the source of ACh and may be a link between NE and splenic macrophage suppression . β2-AR on T cells is essential for the anti-inflammatory effect of VNS [225, 226]. Splenectomy eliminates the role of VNS in increasing plasma NE, which supports the conclusion that plasma NE is released from the spleen into circulation during VNS . The spleen plays a central role in the pathophysiology of the hyperinflammatory state triggered by threatening conditions, such as stroke, and splenic macrophages are the dominant source of pro-inflammatory cytokines .
Targeted therapy for α7nAChR on microglia and macrophages after stroke has long been considered an important potential strategy. α7nAChR expression on activated microglia and infiltrated macrophages after CI plays an important role in the pathological process of stroke. Nicotine therapy has been shown to significantly attenuate the increase in microglia and inflammatory cytokines (i.e., TNF-α and IL-1β) induced by CI through mediation of α7nAChR . In addition, α7 agonists can reduce the infarct volume and functional deficits in different animal models of stroke [229, 230]. In contrast, blockade of α7nAChRs with a selective antagonist increases the infarct volume, which suggests some degree of α7nAChR stimulation by the endogenous agonist . The endogenous choline released from damaged brain tissue may fulfil this role. The use of an allosteric modulator of α7nAChRs 6 h after tMCAO can reduce the infarct volume and improve neurological performance [231, 232]. In addition, regulation of α7nAChRs has also been shown to be associated with changes in M1 and M2 microglia/macrophages after CI .
Multipotent adult progenitor cells
MAPCs are a unique type of adult adhesion cells (MSC-like) that extend understanding of how intravenous cell therapy participates in and regulates the peripheral immune system after stroke. MAPCs can be isolated from bone marrow and other tissues , are well characterised and can be easily distinguished from bone marrow mononuclear cells (BMMNCs) and MSCs based on their size , transcriptome , microRNA profile , differentiation capability  and secretome .
Intravenous injection of MAPCs has been shown to have beneficial effects on other CNS injuries, such as TBI . MAPC therapy attenuates activated microglial/macrophage responses, preserves the BBB, reduces cerebral oedema and improves spatial learning after TBI [118, 239, 240], and MSPC treatment within 24 h after injury can achieve better outcomes . After TBI, intravenous MAPCs also tend to migrate to the spleen .
MAPCs have become a new hotspot of cell therapy for stroke after BMSCs, hUCBs and HSCs. MAPCs can significantly inhibit the inflammatory reaction of injured brain tissue [242, 243] and improve the motor function and neurological outcomes of experimental stroke animals [242, 243]. Moreover, MAPCs exhibit more robust tissue sparing and mitigation of glial activation than MSCs regardless of whether intravenous or intraparenchymal administration is used [244, 245]. Similarly, recent animal data support a role for intravenous MAPCs in regulation of the peripheral immune system through specific interactions between MAPCs and splenocytes, thereby promoting stroke recovery and inhibiting splenic atrophy in the 24 h after CI. Similar to results obtained from testing hUCBs, stroke can also accelerate the migration of splenocyte MAPCs to the spleen and inhibit splenocyte apoptosis. In addition, MAPCs decreased the levels of CD3+, CD4+ and CD8+ cells in the spleen and increase Tregs after tMCAO compared with the levels in the vehicle-treated group . In terms of inflammatory factors, IL-1β and TNF-α were significantly lower in the splenic cells of rats with I/R after MAPC treatment than those of the saline-treated animals, whereas the IL-10 level was higher .
MAPCs not only have a good protective effect on acute CI but also maintain a stable and sustained therapeutic effect on chronic stroke. Compared with those of the saline-treated group, the MAPC treatment group exhibited advantages in locomotor and neurological outcomes that persisted for more than 28 days. In addition, the results of a comparative trial of MAPCs for stroke in splenectomized and sham splenectomized mice also demonstrated that MAPCs could inhibit expansion of the infarct volume through a non-spleen-mediated mechanism, but the MAPC-induced IL-10 production after tMCAO was still dependent on an intact spleen. Moreover, no significant difference was found in the improvement of neurological outcomes between the splenectomy group treated with MAPCs 24 h after tMCAO and the splenectomy group treated with saline .
Preclinical animal data support the benefits of intravenous MAPC therapy for stroke. A phase I/II clinical trial is under way to test the safety and efficacy of MultiStem (the MAPC clinical-grade product) for treatment of patients with acute ischaemic stroke. Previous in vitro studies have shown that MACPs inhibit CD8+ T cells, which are harmful to stroke . The MASTERS trial (MultiStem in Acute Stroke Treatment to Enhance Recovery Study) was conducted in 33 clinical centres in the USA and UK from October 2011 through December 2015. First, MultiStem treatment was proven to be safe. No infusion-related allergic reaction was observed in the MultiStem and placebo-treated groups, and no cases showed neurological worsening. MultiStem treatment can reduce the risk of life-threatening adverse events or death and secondary infections in stroke patients. Furthermore, MultiStem treatment also greatly shortened the time in the intensive care unit and the overall hospitalisation time compared with those of the placebo-treated patients. Compared with those of the placebo, MultiStem treatment significantly reduced the biomarkers of post-stroke inflammation (circulating CD3+ T cells and inflammatory factors). Importantly, MultiStem treatment significantly improved the chance for an excellent outcome at 1 year of onset [248, 250].
Currently, the phase III MASTERS-2 trial aims to expand our knowledge and understanding of treatment of ischaemic stroke patients with MultiStem and plans to start recruiting patients in 2018. Another MultiStem clinical study named TREASURE (Treatment evaluation of acute stroke for using in regenerative cell elements) was officially launched in 31 medical centres in Japan in 2017. The research project recruited 220 patients with acute ischaemic stroke, including speech or motor deficits, as defined by a National Institution of Health Stroke Scale (NIHSS) score of 8–20 at baseline. TREASURE is a randomised, double-blind, placebo-controlled, multicentre phase 2/3 trial to evaluate the efficacy and safety of intravenous administration of MultiStem® compared with those of a placebo in patients with ischaemic stroke .
Intravenous cell therapy can modulate the acute and adverse contributions of the peripheral immune system after ischaemic stroke . A single intravenous administration of cells 24 to 36 h after stroke onset that can mitigate and rebalance the immune response to the initial focal ischaemic injury is sufficient for nerve repair and improvement of long-term outcomes .
As mentioned earlier, the activation, migration and participation of peripheral immune cells, and perhaps most importantly immune cells from the spleen, are critical steps in the pathophysiological progression after stroke. Therefore, we believe that targeted inhibition of peripheral blood immune cell (especially splenic cell) activation, reduction of inflammatory cytokine production and inhibition of their entry into the brain parenchyma through the BBB are key steps in attenuating the expansion of pro-inflammatory microglial activation, neuronal die-back and tissue loss.
Simply inhibiting the participation of splenic components of the peripheral immune system in post-stroke pathophysiological processes allows tissue sparing but is not sufficient to enable neurological and locomotor benefits . As mentioned above, splenectomy fails to provide long-term protection against ischaemic stroke, and delayed splenectomy neither reduces brain tissue loss nor alleviates sensorimotor and cognitive impairment . These results suggest that the spleen may be a double-edged sword that plays completely opposite roles during different pathological stages of stroke. In the early stage of stroke, the spleen is mainly characterised by inflammation and harmfulness but then gradually transforms into an anti-inflammatory and protective phenotype. Therefore, inhibiting the inflammatory immune response of the spleen in the early stage of stroke and accelerating the initiation of its anti-inflammatory mechanism have become the keys for the use of stem cells in the treatment of acute stroke. We also summarise that many stem cells, including MAPCs, can simultaneously inhibit potentially harmful aspects of the innate immune system response to stroke while speeding up beneficial aspects or reparative responses, which confirms our viewpoint (Fig. 9).
Through a number of preclinical and clinical studies, we have obtained a certain understanding of the biological distribution of stem cells after injection for the treatment of stroke and their effects on many immune cell subsets and cytokines in the CNS and PIOs. However, little is known about the effects of stem cell therapy on the immune regulation mediated by the ANS and SNS. Many trials will provide an opportunity to better evaluate and examine hypothetical mechanisms, and we believe that stem cell therapy provides benefits for stroke; however, further preclinical and clinical studies are needed to advance a more comprehensive understanding. Evaluating the safety and efficacy of various stem cell therapies at multiple doses and at different time points may also yield new information.
In summary, we can draw the following conclusions. First, the splenic response after stroke is critical for pathological damage and tissue repair. The key of immunomodulatory therapy for stroke may be to inhibit splenic inflammation at the early stage of pathology and accelerate the initiation of its anti-inflammatory/repair mechanism. Second, during the course of stem cell therapy for stroke, allowing more stem cells to migrate to the spleen may inhibit the splenic inflammatory response and accelerate the initiation of its anti-inflammatory/repair mechanism, which will have a better therapeutic effect than increasing the number of stem cells delivered to the injured brain tissue. Finally, inflammatory factors, such as TNF-α and IL-1β, produced after stroke can activate inflammatory pathways, such as NF-κB, in stem cells, thereby enabling stem cells to acquire stronger immune regulation potential. Therefore, stroke-like stimuli can be applied to stem cells for treatment before injection, which may lead to a better therapeutic effect. In the future, we believe that the spleen will become a potential target of various stem cell therapies for stroke represented by MAPC treatment. The research results will move closer to clinical application and ultimately benefit the majority of stroke patients.
Autonomic nervous system
Bone marrow mononuclear cells
Bone marrow stem cells
Regulatory B cells
C-C chemokine ligand
Central nervous system
C-X-C chemokine ligand
C-X-C chemokine receptor
Damage-associated molecular patterns
Growth differentiation factor
Human amnion epithelial cells
Haematopoietic progenitor cells
Haematopoietic stem cells
Human umbilical cord blood cells
Insulin-like growth factor
Invariable natural killer T cells
Multipotent adult progenitor cells
Myelin basic protein
Monocyte chemoattractant protein
Myelin oligodendrocyte glycoprotein
Mesenchymal stem cells
The MAPC clinical product
Nerve growth factor
National Institution of Health Stroke Scale
Natural killer cells
Natural killer T cells
Neural progenitor cells
N-methyl-d-aspartic acid receptor subunit 2
Neural stem cells
- p/t MCAO:
Permanent/temporary middle cerebral artery occlusion
Pathogen-associated molecular patterns
Peripheral immune organs
Parasympathetic nervous system
Reactive oxygen species
Sympathetic nervous system
Transcutaneous auricular VN stimulation
Traumatic brain injury
Transforming growth factor
Helper T cell
Tumour necrosis factor
T regulatory 1 cells
Regulatory T cells
Vascular endothelial growth factor
α-7-nicotinic ACh receptors
Benjamin EJ, Blaha MJ, Chiuve SE, Cushman M, Das SR, Deo R, de Ferranti SD, Floyd J, Fornage M, Gillespie C, Isasi CR, Jiménez MC, Jordan LC, Judd SE, Lackland D, Lichtman JH, Lisabeth L, Liu S, Longenecker CT, Mackey RH, Matsushita K, Mozaffarian D, Mussolino ME, Nasir K, Neumar RW, Palaniappan L, Pandey DK, Thiagarajan RR, Reeves MJ, Ritchey M, Rodriguez CJ, Roth GA, Rosamond WD, Sasson C, Towfighi A, Tsao CW, Turner MB, Virani SS, Voeks JH, Willey JZ, Wilkins JT, Wu JH, Alger HM, Wong SS, Muntner P, American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Heart Disease and Stroke Statistics-2017 Update: A Report From the American Heart Association. Circulation. 2017;135(10):e146–603.
Wei L, Wei ZZ, Jiang MQ, Mohamad O, Yu SP. Stem cell transplantation therapy for multifaceted therapeutic benefits after stroke. Prog Neurobiol. 2017;157:49–78.
Anrather J, Iadecola C. Inflammation and stroke: an overview. Neurotherapeutics. 2016;13(4):661–70.
Liu ZJ, Chen C, Li FW, Shen JM, Yang YY, Leak RK, et al. Splenic responses in ischemic stroke: new insights into stroke pathology. CNS Neurosci Ther. 2015;21(4):320–6.
Maya-Espinosa G, Collazo-Navarrete O, Millán-Aldaco D, Palomero-Rivero M, Guerrero-Flores G, Drucker-Colín R, et al. Mouse embryonic stem cell-derived cells reveal niches that support neuronal differentiation in the adult rat brain. Stem Cells. 2015;33(2):491–502.
Rosenblum S, Smith TN, Wang N, Chua JY, Westbroek E, Wang K, et al. BDNF pretreatment of human embryonic-derived neural stem cells improves cell survival and functional recovery after transplantation in hypoxic-ischemic stroke. Cell Transplant. 2015;24(12):2449–61.
Huang L, Wong S, Snyder EY, Hamblin MH, Lee JP. Human neural stem cells rapidly ameliorate symptomatic inflammation in early-stage ischemic-reperfusion cerebral injury. Stem Cell Res Ther. 2014;5(6):129.
Chen L, Qiu R, Li L, He D, Lv H, Wu X, et al. The role of exogenous neural stem cells transplantation in cerebral ischemic stroke. J Biomed Nanotechnol. 2014;10(11):3219–30.
Cheng Y, Zhang J, Deng L, Johnson NR, Yu X, Zhang N, et al. Intravenously delivered neural stem cells migrate into ischemic brain, differentiate and improve functional recovery after transient ischemic stroke in adult rats. Int J Clin Exp Pathol. 2015;8(3):2928–36.
Wakao S, Kuroda Y, Ogura F, Shigemoto T, Dezawa M. Regenerative effects of mesenchymal stem cells: contribution of muse cells, a novel pluripotent stem cell type that resides in mesenchymal cells. Cell. 2012 Nov 8;1(4):1045–60.
Chen J, Li Y, Katakowski M, Chen X, Wang L, Lu D, et al. Intravenous bone marrow stromal cell therapy reduces apoptosis and promotes endogenous cell proliferation after stroke in female rat. J Neurosci Res. 2003;73(6):778–86.
Kondziolka D, Steinberg GK, Wechsler L, Meltzer CC, Elder E, Gebel J, et al. Neurotransplantation for patients with subcortical motor stroke: a phase 2 randomized trial. J Neurosurg. 2005;103(1):38–45.
Li Y, Chopp M, Chen J, Wang L, Gautam SC, Xu YX, et al. Intrastriatal transplantation of bone marrow nonhematopoietic cells improves functional recovery after stroke in adult mice. J Cereb Blood Flow Metab. 2000;20(9):1311–9.
Steinberg GK, Kondziolka D, Wechsler LR, Lunsford LD, Coburn ML, Billigen JB, et al. Clinical outcomes of transplanted modified bone marrow-derived mesenchymal stem cells in stroke: a phase 1/2a study. Stroke. 2016;47(7):1817–24.
Otero L, Zurita M, Bonilla C, Aguayo C, Vela A, Rico MA, et al. Late transplantation of allogeneic bone marrow stromal cells improves neurologic deficits subsequent to intracerebral hemorrhage. Cytotherapy. 2011;13(5):562–71.
Wang L, Ji H, Li M, Zhou J, Bai W, Zhong Z, et al. Intrathecal administration of autologous CD34 positive cells in patients with past cerebral infarction: a safety study. ISRN Neurol. 2013;2013:128591.
Danielyan L, Schäfer R, von Ameln-Mayerhofer A, Buadze M, Geisler J, Klopfer T, et al. Intranasal delivery of cells to the brain. Eur J Cell Biol. 2009;88(6):315–24.
Janowski M, Wagner D-C, Boltze J. Stem cell-based tissue replacement after stroke: factual necessity or notorious fiction? Stroke. 2015;46(8):2354–63.
Eckert A, Huang L, Gonzalez R, Kim HS, Hamblin MH, Lee JP. Bystander effect fuels human induced pluripotent stem cell-derived neural stem cells to quickly attenuate early stage neurological deficits after stroke. Stem Cells Transl Med. 2015;4(7):841–51.
Pendharkar AV, Chua JY, Andres RH, Wang N, Gaeta X, Wang H, et al. Biodistribution of neural stem cells after intravascular therapy for hypoxic-ischemia. Stroke. 2010;41(9):2064–70.
Detante O, Valable S, de Fraipont F, Grillon E, Barbier EL, Moisan A, et al. Magnetic resonance imaging and fluorescence labeling of clinical-grade mesenchymal stem cells without impacting their phenotype: study in a rat model of stroke. Stem Cells Transl Med. 2012;1(4):333–41.
Rosado-de-Castro PH, Schmidt Fda R, Battistella V, Lopes de Souza SA, Gutfilen B, Goldenberg RC, Kasai-Brunswick TH, Vairo L, Silva RM, Wajnberg E, Alvarenga Americano do Brasil PE, Gasparetto EL, Maiolino A, Alves-Leon SV, Andre C, Mendez-Otero R, Rodriguez de Freitas G, Barbosa da Fonseca LM. Biodistribution of bone marrow mononuclear cells after intra-arterial or intravenous transplantation in subacute stroke patients. RegenMed. 2013;8(2):145–55.
Watanabe M, Yavagal D. Intra-arterial delivery of mesenchymal stem cells. Brain Circ. 2016;2:114–7.
Gutiérrez-Fernández M, Rodríguez-Frutos B, Alvarez-Grech J, Vallejo-Cremades MT, Expósito-Alcaide M, Merino J, et al. Functional recovery after hematic administration of allogenic mesenchymal stem cells in acute ischemic stroke in rats. Neuroscience. 2011;175:394–405.
Li Y, Chen J, Wang L, Lu M, Chopp M. Treatment of stroke in rat with intracarotid administration of marrow stromal cells. Neurology. 2001;56(12):1666–72.
Banerjee S, Bentley P, Hamady M, Marley S, Davis J, Shlebak A, et al. Intra-arterial Immunoselected CD34+ stem cells for acute ischemic stroke. Stem Cells Transl Med. 2014;3(11):1322–30.
Ohshima M, Taguchi A, Tsuda H, Sato Y, Yamahara K, Harada-Shiba M, et al. Intraperitoneal and intravenous deliveries are not comparable in terms of drug efficacy and cell distribution in neonatal mice with hypoxia-ischemia. Brain Dev. 2015;37(4):376–86.
Vahidy FS, Rahbar MH, Zhu H, Rowan PJ, Bambhroliya AB, Savitz SI. Systematic review and meta-analysis of bone marrow-derived mononuclear cells in animal models of ischemic stroke. Stroke. 2016;47:1632–9.
Hess DC, Wechsler LR, Clark WM, Savitz SI, Ford GA, Chiu D, et al. Safety and efficacy of multipotent adult progenitor cells in acute ischaemic stroke (MASTERS): a randomised, double-blind, placebo-controlled, phase 2 trial. Lancet Neurol. 2017;16:360–8.
Bacigaluppi M, Pluchino S, Peruzzotti-Jametti L, Kilic E, Kilic U, Salani G, et al. Delayed post-ischaemic neuroprotection following systemic neural stem cell transplantation involves multiple mechanisms. Brain. 2009;132:2239–51.
Acosta SA, Tajiri N, Hoover J, Kaneko Y, Borlongan CV. Intravenous bone marrow stem cell grafts preferentially migrate to spleen and abrogate chronic inflammation in stroke. Stroke. 2015;46(9):2616–27.
Vendrame M, Gemma C, Pennypacker KR, Bickford PC, Davis Sanberg C, Sanberg PR, et al. Cord blood rescues stroke-induced changes in splenocyte phenotype and function. Exp Neurol. 2006;199(1):191–200.
Vendrame M, Cassady J, Newcomb J, Butler T, Pennypacker KR, Zigova T, et al. Infusion of human umbilical cord blood cells in a rat model of stroke dosedependently rescues behavioral deficits and reduces infarct volume. Stroke. 2004;35(10):2390–5.
Yan T, Venkat P, Ye X, Chopp M, Zacharek A, Ning R, et al. HUCBCs increase angiopoietin 1 and induce neurorestorative effects after stroke in T1DM rats. CNS Neurosci Ther. 2014;20(10):935–44.
Schwarting S, Litwak S, Hao W, Bahr M, Weise J, Neumann H. Hematopoietic stem cells reduce postischemic inflammation and ameliorate ischemic brain injury. Stroke. 2008;39(10):2867–75.
Keimpema E, Fokkens MR, Nagy Z, Agoston V, Luiten PG, Nyakas C, et al. Early transient presence of implanted bone marrow stem cells reduces lesion size after cerebral ischaemia in adult rats. Neuropathol Appl Neurobiol. 2009;35(1):89–102.
Lee ST, Chu K, Jung KH, Kim SJ, Kim DH, Kang KM, et al. Anti-inflammatory mechanism of intravascular neural stem cell transplantation in haemorrhagic stroke. Brain. 2008;131(Pt 3):616–29.
Barone FC, Feuerstein GZ. Inflammatory mediators and stroke: new opportunities for novel therapeutics. J Cereb Blood Flow Metab. 1999;19(8):819–34.
del Zoppo G, Ginis I, Hallenbeck JM, Iadecola C, Wang X, Feuerstein GZ. Inflammation and stroke: putative role for cytokines, adhesion molecules and iNOS in brain response to ischemia. Brain Pathol. 2000;10(1):95–112.
Iadecola C, Alexander M. Cerebral ischemia and inflammation. Curr Opin Neurol. 2001;14(1):89–94.
Li P, Mao L, Zhou G, Leak RK, Sun BL, Chen J, et al. Adoptive regulatory T cell therapy preserves systemic immune homeostasis following cerebral ischemia. Stroke. 2013;44(12):3509–15.
Ren X, Akiyoshi K, Dziennis S, Vandenbark AA, Herson PS, Hurn PD, et al. Regulatory B-cells limit CNS inflammation and neurologic deficits in murine experimental stroke. J Neurosci. 2011;31(23):8556–63.
Bodhankar S, Chen Y, Vandenbark AA, Murphy SJ, Offner H. Treatment of experimental stroke with IL-10-producing B-cells reduces infarct size and peripheral and CNS inflammation in wild-type B-cell-sufficient mice. Metab Brain Dis. 2014;29(1):59–73.
Luo Y, Zhou Y, Xiao W, Liang Z, Dai J, Weng X, et al. Interleukin-33 ameliorates ischemic brain injury in experimental stroke through promoting Th2 response and suppressing Th17 response. Brain Res. 2015;1597:86–94.
Kim E, Yang J, Beltran CD, Cho S. Role of spleen-derived monocytes/macrophages in acute ischemic brain injury. J Cereb Blood Flow Metab. 2014;34(8):1411–9.
Yang B, Strong R, Sharma S, Brenneman M, Mallikarjunarao K, Xi X, et al. Therapeutic time window and dose response of autologous bone marrow mononuclear cells for ischemic stroke. J Neurosci Res. 2011;89(6):833–9.
Becerra-Calixto A, Cardona-Gómez GP. The role of astrocytes in neuroprotection after brain Stroke_ potential in cell therapy. Front Mol Neurosci. 2017;10:88.
Evans MA, Broughton BRS, Drummond GR, Ma H, Phan TG, Wallace EM, et al. Amnion epithelial cells - a novel therapy for ischemic stroke? Neural Regen Res. 2018;13(8):1346–9.
Otero-Ortega L, Gómez de Frutos MC, Laso-García F, Rodríguez-Frutos B, Medina-Gutiérrez E, López JA, Vázquez J, Díez-Tejedor E, Gutiérrez-Fernández M. Exosomes promote restoration after an experimental animal model of intracerebral hemorrhage. J Cereb Blood Flow Metab. 2018;38(5):767–79.
Han Y, Seyfried D, Meng Y, Yang D, Schultz L, Chopp M, et al. Multipotent mesenchymal stromal cell-derived exosomes improve functional recovery after experimental intracerebral hemorrhage in the rat. J Neurosurg. 2018 Jul;20:1–11.
Venkat P, Chen J, Chopp M. Exosome-mediated amplification of endogenous brain repair mechanisms and brain and systemic organ interaction in modulating neurological outcome after stroke. J Cereb Blood Flow Metab. 2018;11. https://doi.org/10.1177/0271678X18782789.
Woodruff TM, Thundyil J, Tang SC, Sobey CG, Taylor SM, Arumugam TV. Pathophysiology, treatment, and animal and cellular models of human ischemic stroke. Mol Neurodegener. 2011;6:11.
Meisel C, Schwab JM, Prass K, Meisel A, Dirnagl U. Central nervous system injury-induced immune deficiency syndrome. Nat Rev Neurosci. 2005;6:775–86.
Famakin BM. The immune response to acute focal cerebral ischemia and associated post-stroke immunodepression: a focused review. Aging Dis. 2014;5(5):307–26.
Wattananit S, Tornero D, Graubardt N, Memanishvili T, Monni E, Tatarishvili J, et al. Monocyte-derived macrophages contribute to spontaneous long-term functional recovery after stroke in mice. J Neurosci. 2016;36:4182–95.
Carden DL, Granger DN. Pathophysiology of ischaemia-reperfusion injury. J Pathol. 2000;190:255–66.
Peerschke EI, Yin W, Ghebrehiwet B. Complement activation on platelets: implications for vascular inflammation and thrombosis. Mol Immunol. 2010;47:2170–5.
Hurley SD, Olschowka JA, O’Banion MK. Cyclooxygenase inhibition as a strategy to ameliorate brain injury. J Neurotrauma. 2002;19:1–15.
Minghetti L. Role of COX-2 in inflammatory and degenerative brain diseases. Subcell Biochem. 2007;42:127–41.
Iadecola C, Abe T, Kunz A, Hallembeck J. Cerebral ischemia and inflammation. In: Mohr JP, Wolf PA, Grotta JC, Moskowitz MA, Mayberg M, von Kummer R, editors. Stroke: pathophysiology, diagnosis and management. 5th ed. Philadelphia: Elsevier Health Sciences; 2011. p. 138–53.
Zhang Q, Raoof M, Chen Y, Sumi Y, Sursal T, Junger W, et al. Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature. 2010;464:104–7.
Matzinger P. The evolution of the danger theory. Interview by Lauren constable, commissioning editor. Expert Rev Clin Immunol. 2012;8:311–7.
Caso JR, Pradillo JM, Hurtado O, Lorenzo P, Moro MA, Lizasoain I. Toll-like receptor 4 is involved in brain damage and inflammation after experimental stroke. Circulation. 2007;115:1599–608.
Savage CD, Lopez-Castejon G, Denes A, Brough D. NLRP3-Inflammasome activating DAMPs stimulate an inflammatory response in glia in the absence of priming which contributes to brain inflammation after injury. Front Immunol. 2012;3:288.
Fann DY, Lee SY, Manzanero S, Chunduri P, Sobey CG, Arumugam TV. Pathogenesis of acute stroke and the role of inflammasomes. Ageing Res Rev. 2013;12:941–66.
Strbian D, Karjalainen-Lindsberg ML, Tatlisumak T, Lindsberg PJ. Cerebral mast cells regulate early ischemic brain swelling and neutrophil accumulation. J Cereb Blood Flow Metab. 2006;26:605–12.
Iadecola C, Anrather J. The immunology of stroke: from mechanisms to translation. Nat Med. 2011;17:796–808.
Huang J, Choudhri TF, Winfree CJ, McTaggart RA, Kiss S, Mocco J, et al. Postischemic cerebrovascular E-selectin expression mediates tissue injury in murine stroke. Stroke. 2000;31(12):3047–53.
Allan SM, Rothwell NJ. Cytokines and acute neurodegeneration. Nat Rev Neurosci. 2001;2(10):734–44 Review.
Burnstock G. Purinergic signalling and disorders of the central nervous system. Nat Rev Drug Discov. 2008;7:575–90.
Shichita T, Sugiyama Y, Ooboshi H, Sugimori H, Nakagawa R, Takada I, et al. Pivotal role of cerebral interleukin-17-producing gammadeltaT cells in the delayed phase of ischemic brain injury. Nat Med. 2009;15:946–50.
Meisel A, Meisel C, Harms H, Hartmann O, Ulm L. Predicting poststroke infections and outcome with blood-based immune and stress markers. Cerebrovasc Dis. 2012;33(6):580–8.
Hannawi Y, Hannawi B, Rao CP, Suarez JI, Bershad EM. Stroke-associated pneumonia: major advances and obstacles. Cerebrovasc Dis. 2013;35(5):430–43.
Kawanokuchi J, Shimizu K, Nitta A, Yamada K, Mizuno T, Takeuchi H, et al. Production and functions of IL-17 in microglia. J Neuroimmunol. 2008;194:54–61.
Morrison HW, Filosa JA. A quantitative spatiotemporal analysis of microglia morphology during ischemic stroke and reperfusion. J Neuroinflammation. 2013;10:4.
Denes A, Vidyasagar R, Feng J, Narvainen J, McColl BW, Kauppinen RA, et al. Proliferating resident microglia after focal cerebral ischaemia in mice. J Cereb Blood Flow Metab. 2007;27:1941–53.
Villarreal A, Rosciszewski G, Murta V, Cadena V, Usach V, Dodes-Traian MM, et al. Isolation and characterization of ischemia-derived astrocytes (IDAs) with ability to transactivate quiescent astrocytes. Front Cell Neurosci. 2016;10:139.
Liu Z, Chopp M. Astrocytes, therapeutic targets for neuroprotection and neurorestoration in ischemic stroke. Prog Neurobiol. 2016;144:103–20.
Fathali N, Ostrowski RP, Hasegawa Y, Lekic T, Tang J, Zhang JH. Splenic immune cells in experimental neonatal hypoxia-ischemia. Transl Stroke Res. 2013;4(2):208–19.
Li M, Li Z, Yao Y, Jin WN, Wood K, Liu Q, et al. Astrocyte-derived interleukin-15 exacerbates ischemic brain injury via propagation of cellular immunity. Proc Natl Acad Sci U S A. 2017;114(3):E396–405.
Cuadrado E, Jansen MH, Anink J, De Filippis L, Vescovi AL, Watts C, et al. Chronic exposure of astrocytes to interferon-α reveals molecular changes related to Aicardi-Goutieres syndrome. Brain. 2013;136(Pt 1):245–58.
Sims NR, Yew WP. Reactive astrogliosis in stroke: contributions of astrocytes to recovery of neurological function. Neurochem Int. 2017;107:88–103.
Hayakawa K, Esposito E, Wang X, Terasaki Y, Liu Y, Xing C, et al. Transfer of mitochondria from astrocytes to neurons after stroke. Nature. 2016;535(7613):551–5.
Mattila OS, Strbian D, Saksi J, Pikkarainen TO, Rantanen V, Tatlisumak T, et al. Cerebral mast cells mediate blood-brain barrier disruption in acute experimental ischemic stroke through perivascular gelatinase activation. Stroke. 2011;42:3600–5.
Strbian D, Kovanen PT, Karjalainen-Lindsberg M-L, Tatlisumak T, Lindsberg PJ. An emerging role of mast cells in cerebral ischemia and hemorrhage. Ann Med. 2009;41:438–50.
Strbian D, Karjalainen-Lindsberg ML, Kovanen PT, Tatlisumak T, Lindsberg PJ. Mast cell stabilization reduces hemorrhage formation and mortality after administration of thrombolytics in experimental ischemic stroke. Circulation. 2007;116(4):411–8.
McKittrick CM, Lawrence CE, Carswell HV. Mast cells promote blood brain barrier breakdown and neutrophil infiltration in a mouse model of focal cerebral ischemia. J Cereb Blood Flow Metab. 2015;35(4):638–47.
Fink SL, Cookson BT. Caspase-1-dependent pore formation during pyroptosis leads to osmotic lysis of infected host macrophages. Cell Microbiol. 2006;8:1812–25.
Schilling M, Besselmann M, Müller M, Strecker JK, Ringelstein EB, Kiefer R. Predominant phagocytic activity of resident microglia over hematogenous macrophages following transient focal cerebral ischemia: an investigation using green fluorescent protein transgenic bone marrow chimeric mice. Exp Neurol. 2005;196:290–7.
Nathan C, Ding A. Nonresolving inflammation. Cell. 2010;140:871882.
Li S, Overman JJ, Katsman D, Kozlov SV, Donnelly CJ, Twiss JL, et al. An age-related sprouting transcriptome provides molecular control of axonal sprouting after stroke. Nat Neurosci. 2010;13:1496–504.
Hayakawa K, Nakano T, Irie K, Higuchi S, Fujioka M, Orito K, et al. Inhibition of reactive astrocytes with fluorocitrate retards neurovascular remodeling and recovery after focal cerebral ischemia in mice. J Cereb Blood Flow Metab. 2010;30:871–82.
Hao Q, Chen Y, Zhu Y, Fan Y, Palmer D, Su H, et al. Neutrophil depletion decreases VEGF-induced focal angiogenesis in the mature mouse brain. J Cereb Blood Flow Metab. 2007;27:1853–60.
Vidale S, Consoli A, Arnaboldi M, Consoli D. Postischemic inflammation in acute stroke. J Clin Neurol. 2017 Jan;13(1):1–9.
Wang J, Yu L, Jiang C, Fu X, Liu X, Wang M, et al. Cerebral ischemia increases bone marrow CD4+CD25+FoxP3+ regulatory T cells in mice via signals from sympathetic nervous system. Brain Behav Immun. 2015;43:172–83.
Hori E, Hayakawa Y, Hayashi T, Hori S, Okamoto S, Shibata T, et al. Mobilization of pluripotent multilineage-differentiating stress-enduring cells in ischemic stroke. J Stroke Cerebrovasc Dis. 2016;25(6):1473–81.
Wang LL, Chen D, Lee J, Gu X, Alaaeddine G, Li J, et al. Mobilization of endogenous bone marrow derived endothelial progenitor cells and therapeutic potential of parathyroid hormone after ischemic stroke in mice. PLoS One. 2014;9(2):e87284.
Shyu WC, Lin SZ, Yen PS, Su CY, Chen DC, Wang HJ, et al. Stromal cell-derived factor-1α promotes neuroprotection, angiogenesis, and mobilization/homing of bone marrow derived cells in stroke rats. J Pharmacol Exp Ther. 2008;324(2):834–49.
Gendron A, Teitelbaum J, Cossette C, Nuara S, Dumont M, Geadah D, et al. Temporal effects of left versus right middle cerebral artery occlusion on spleen lymphocyte subsets and mitogenic response in Wistar rats. Brain Res. 2002;955(1–2):85–97.
Prass K, Meisel C, Höflich C, Braun J, Halle E, Wolf T, et al. Stroke-induced immunodeficiency promotes spontaneous bacterial infections and is mediated by sympathetic activation reversal by poststroke T helper cell type 1-like immunostimulation. J Exp Med. 2003;198(5):725–36.
Prass K, Ruscher K, Karsch M, Isaev N, Megow D, Priller J, et al. Desferrioxamine induces delayed tolerance against cerebral ischemia in vivo and in vitro. J Cereb Blood Flow Metab. 2002;22(5):520–5.
Offner H, Subramanian S, Parker SM, Wang C, Afentoulis ME, Lewis A, et al. Splenic atrophy in experimental stroke is accompanied by increased regulatory T cells and circlating macrophages. J Immunol. 2006;176(11):6523–31.
Braun JS, Prass K, Dirnagl U, Meisel A, Meisel C. Protection from brain damage and bacterial infection in murine stroke by the novel caspase-inhibitor Q-VD-OPH. Exp Neurol. 2007;206(2):183–91.
Ishibashi S, Maric D, Mou Y, Ohtani R, Ruetzler C, Hallenbeck JM. Mucosal tolerance to E-selectin promotes the survival of newly generated neuroblasts via regulatory T-cell induction after stroke in spontaneously hypertensive rats. J Cereb Blood Flow Metab. 2009;29(3):606–20.
Laman JD, Weller RO. Drainage of cells and soluble antigen from the CNS to regional lymph nodes. J NeuroImmune Pharmacol. 2013;8(4):840–56.
Urra X, Miró F, Chamorro A, Planas AM. Antigen-specific immune reactions to ischemic stroke. Front Cell Neurosci. 2014;8:278.
van Zwam M, Huizinga R, Melief MJ, Wierenga-Wolf AF, van Meurs M, Voerman JS, Biber KP, Boddeke HW, Höpken UE, Meisel C, Meisel A, Bechmann I, Hintzen RQ, ‘t Hart BA, Amor S, Laman JD, Boven LA. Brain antigens in functionally distinct antigen-presenting cell populations in cervical lymph nodes in MS and EAE. J Mol Med (Berl). 2009;87(3):273–86.
Weller RO, Massey A, Kuo YM, Roher AE. Lymphatic drainage of the brain and the pathophysiology of neurological. Ann N Y Acad Sci. 2000;903:110–7.
Dirnagl U, Klehmet J, Braun JS, Harms H, Meisel C, Ziemssen T, et al. Stroke-induced immunodepression: experimental evidence and clinical relevance. Stroke. 2007;38(2 Suppl):770–3.
Schulte-Herbrüggen O, Quarcoo D, Meisel A, Meisel C. Differential affection of intestinal immune cell populations after cerebral Ischemiia in mice. Neuroimmunomodulation. 2009;16(3):213–8.
Mazmanian SK, Liu CH, Tzianabos AO, Kasper DL. An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell. 2005;122:107–18.
Benakis C, Brea D, Caballero S, Faraco G, Moore J, Murphy M, et al. Commensal microbiota affects ischemic stroke outcome by regulating intestinal γδT cells. Nat Med. 2016;22:516–23.
Ajmo CT Jr, Vernon DO, Collier L, Hall AA, Garbuzova-Davis S, Willing A, et al. The spleen contributes to stroke-induced neurodegeneration. J Neurosci Res. 2008;86(10):2227–34.
Jin R, Zhu X, Liu L, Nanda A, Granger DN, Li G. Simvastatin attenuates stroke-induced splenic atrophy and lung susceptibility to spontaneous bacterial infection in mice. Stroke. 2013;44(4):1135–43.
Lin JN, Lin CL, Lin MC, Lai CH, Lin HH, Yang CH, et al. Increased risk of hemorrhagic and ischemic strokes in patients with splenic injury and splenectomy: a Nationwide cohort study. Medicine (Baltimore). 2015;94(35):e1458.
Das M, Leonardo CC, Rangooni S, Mohapatra SS, Mohapatra S, Pennypacker KR. Lateral fluid percussion injury of the brain induces CCL20 inflammatory chemokine expression in rats. J Neuroinflammation. 2011;8:148.
Li M, Li F, Luo C, Shan Y, Zhang L, Qian Z, et al. Immediate splenectomy decreases mortality and improves cognitive function of rats after severe traumatic brain injury. J Trauma. 2011;71(1):141–7.
Walker PA, Shah SK, Jimenez F, Gerber MH, Xue H, Cutrone R, et al. Intravenous multipotent adult progenitor cell therapy for traumatic brain injury: preserving the blood brain barrier via an interaction with splenocytes. Exp Neurol. 2010;225(2):341–52.
Seifert HA, Leonardo CC, Hall AA, Rowe DD, Collier LA, Benkovic SA, et al. The spleen contributes to stroke induced neurodegeneration through interferon gamma signaling. Metab Brain Dis. 2012;27(2):131–41.
Chauhan A, Al Mamun A, Spiegel G, Harris N, Zhu L, McCullough LD. Splenectomy protects aged mice from injury after experimental stroke. Neurobiol Aging. 2018;61:102–11.
Ran Y, Liu Z, Huang S, Shen J, Li F, Zhang W, et al. Splenectomy fails to provide long-term protection against ischemic stroke. Aging Dis. 2018;9(3):467–79.
Ostrowski RP, Schulte RW, Nie Y, Ling T, Lee T, Manaenko A, et al. Acute splenic irradiation reduces brain injury in the rat focal ischemic stroke model. Transl Stroke Res. 2012;3:473–81.
Ajmo CT Jr, Collier LA, Leonardo CC, Hall AA, Green SM, Womble TA, et al. Blockade of Adrenoreceptors inhibits the splenic response to stroke. Exp Neurol. 2009;218(1):47–55.
Sahota P, Vahidy F, Nguyen C, Bui TT, Yang B, Parsha K, et al. Changes in spleen size in patients with acute ischemic stroke: a pilot observational study. Int J Stroke. 2013;8(2):60–7.
Jin R, Yang G, Li G. Inflammatory mechanisms in ischemic stroke: role of inflammatory cells. J Leukoc Biol. 2010;87:779–89.
Doycheva DM, Hadley T, Li L, Applegate RL, Zhang JH, Tang J. Anti-neutrophil antibody enhances the neuroprotective effects of G-CSF by decreasing number of neutrophils in hypoxic ischemic neonatal rat model. Neurobiol Dis. 2014;69:192–9.
Price CJ, Menon DK, Peters AM. Cerebral neutrophil recruitment, histology, and outcome in acute ischemic stroke: an imaging-based study. Stroke. 2004;35:1659–64.
Chiu NL, Kaiser B, Nguyen YV, Welbourne S, Lall C, Cramer SC. The volume of the spleen and its correlates after acute stroke. J Stroke Cerebrovasc Dis. 2016;25(12):2958–61.
Amantea D, Nappi G, Bernardi G, Bagetta G, Corasaniti MT. Post-ischemic brain damage: pathophysiology and role of inflammatory mediators. FEBS J. 2009;276:13–26.
Justicia C, Panés J, Solé S, Cervera A, Deulofeu R, Chamorro A, et al. Neutrophil infiltration increases matrix metalloproteinase-9 in the ischemic brain after occlusion/reperfusion of the middle cerebral artery in rats. J Cereb Blood Flow Metab. 2003;23:1430–40.
Sippel TR, Shimizu T, Strnad F, Traystman RJ, Herson PS, Waziri A. Arginase I release from activated neutrophils induces peripheral immunosuppression in a murine model of stroke. J Cereb Blood Flow Metab. 2015;35(10):1657–63.
Bao Y, Kim E, Bhosle S, Mehta H, Cho S. A role for spleen monocytes in post-ischemic brain inflammation and injury. J Neuroinflammation. 2010;7:92.
Ma Y, Li Y, Jiang L, Wang L, Jiang Z, Wang Y, et al. Macrophage depletion reduced brain injury following middle cerebral artery occlusion in mice. J Neuroinflammation. 2016;13:38.
Garcia-Bonilla L, Brea D, Benakis C, Lane DA, Murphy M, Moore J, et al. Endogenous protection from ischemic brain injury by preconditioned monocytes. J Neurosci. 2018;38(30):6722–36.
Gu L, Xiong X, Wei D, Gao X, Krams S, Zhao H. T cells contribute to stroke-induced lymphopenia in rats. PLoS One. 2013;8(3):e59602.
Hurn PD, Subramanian S, Parker SM, Afentoulis ME, Kaler LJ, Vandenbark AA, et al. T-and B-cell-deficient mice with experimental stroke have reduced lesion size and inflammation. J Cereb Blood Flow Metab. 2007;27:1798–805.
Yilmaz G, Arumugam TV, Stokes KY, Granger DN. Role of T lymphocytes and interferon-gamma in ischemic stroke. Circulation. 2006;113:2105–12.
Dotson AL, Zhu W, Libal N, Alkayed NJ, Offner H. Different immunological mechanisms govern protection from experimental stroke in young and older mice with recombinant TCR ligand therapy. Front Cell Neurosci. 2014;8:284.
Zhang BJ, Men XJ, Lu ZQ, Li HY, Qiu W, Hu XQ. Splenectomy protects experimental rats from cerebral damage after stroke due to anti-inflammatory effects. Chin Med J. 2013;126(12):2354–60.
Liesz A, Suri-Payer E, Veltkamp C, Doerr H, Sommer C, Rivest S, et al. Regulatory T cells are key cerebroprotective immunomodulators in acute experimental stroke. Nat Med. 2009;15:192–9.
Li P, Gan Y, Sun BL, Zhang F, Lu B, Gao Y, et al. Adoptive regulatory T-cell therapy protects against cerebral ischemia. Ann Neurol. 2013;74:458–71.
Na SY, Mracsko E, Liesz A, Hünig T, Veltkamp R. Amplification of regulatory T cells using a CD28 superagonist reduces brain damage after ischemic stroke in mice. Stroke. 2015;46(1):212–20.
Gu L, Xiong X, Zhang H, Xu B, Steinberg GK, Zhao H. Distinctive effects of T cell subsets in neuronal injury induced by cocultured splenocytes in vitro and by in vivo stroke in mice. Stroke. 2012;43(7):1941–6.
Doyle KP, Quach LN, Solé M, Axtell RC, Nguyen TV, Soler-Llavina GJ, et al. B-lymphocyte-mediated delayed cognitive impairment following stroke. J Neurosci. 2015;35(5):2133–45.
Bodhankar S, Chen Y, Vandenbark AA, Murphy SJ, Offner H. IL-10-producing B-cells limit CNS inflammation and infarct volume in experimental stroke. Metab Brain Dis. 2013;28:375–86.
Chen Y, Bodhankar S, Murphy SJ, Vandenbark AA, Alkayed NJ, Offner H. Intrastriatal B-cell administration limits infarct size after stroke in B-cell deficient mice. Metab Brain Dis. 2012;27(4):487–93.
Bodhankar S, Chen Y, Lapato A, Vandenbark AA, Murphy SJ, Saugstad JA, et al. Regulatory CD8(+) CD122 (+) T-cells predominate in CNS after treatment of experimental stroke in male mice with IL-10-secreting B-cells. Metab Brain Dis. 2015;30(4):911–24.
Gan Y, Liu Q, Wu W, Yin JX, Bai XF, Shen R, et al. Ischemic neurons recruit natural killer cells that accelerate brain infarction. Proc Natl Acad Sci U S A. 2014;111:2704–9.
Zhang Y, Gao Z, Wang D, Zhang T, Sun B, Mu L, Li H, Wang G, et al. Accumulation of natural killer cells in ischemic brain tissues and the chemotactic effect of IP-10. J Neuroinflammation. 2014;11:79.
Gelderblom M, Leypoldt F, Steinbach K, Behrens D, Choe CU, Siler DA, et al. Temporal and spatial dynamics of cerebral immune cell accumulation in stroke. Stroke. 2009;40:1849–57.
Alvarez D, Vollmann EH, von Andrian UH. Mechanisms and consequences of dendritic cell migration. Immunity. 2008;29:325–42.
Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, et al. Immunobiology of dendritic cells. Annu Rev Immunol. 2000;18:767–811.
Offner H, Subramanian S, Parker SM. Experimental stroke induces massive, rapid activation of the peripheral immune system. J Cereb Blood Flow Metab. 2006;26:654–65.
Seifert HA, Collier LA, Chapman CB, Benkovic SA, Willing AE, Pennypacker KR. Pro-inflammatory interferon gamma signaling is directly associated with stroke induced neurodegeneration. J NeuroImmune Pharmacol. 2014;9:679–89.
Offner H, Vandenbark AA, Hurn PD. Effect of experimental stroke on peripheral immunity: CNS ischemia induces profound immunosuppression. Neuroscience. 2009;158(3):1098–111.
Vahidy FS, Parha KN, Rahbar MH, Lee M, Bui TT, Nguyen C, Barreto AD, Bambhroliya AB, Sahota P, Yang B, Aronowski J, Savitz SI. Acute splenic responses in patients with ischemic stroke and intracerebral hemorrhage. J Cereb Blood Flow Metab. 2016;36(6):1012–21.
Wang F, Shen Y, Tsuru E, Yamashita T, Baba N, Tsuda M, et al. Author information syngeneic transplantation of newborn splenocytes in a murine model of neonatal ischemia-reperfusion brain injury. J Matern Fetal Neonatal Med. 2015;28(7):842–7.
Wang J, Dotson AL, Murphy SJ, Offner H, Saugstad JA. Adoptive transfer of immune subsets prior to MCAO does not exacerbate stroke outcome in splenectomized mice. J Syst Integr Neurosci. 2015;1(1):20–8.
Mignini F, Streccioni V, Amenta F. Autonomic innervation of immune organs and neuroimmune modulation. Auton Autacoid Pharmacol. 2003;23(1):1–25.
Kenney MJ, Ganta CK. Autonomic nervous system and immune system interactions. Compr Physiol. 2014;4(3):1177–200.
Sörös P, Hachinski V. Cardiovascular and neurological causes of sudden death after ischaemic stroke. Lancet Neurol. 2012;11(2):179–88.
Bassi A, Colivicchi F, Santini M, Caltagirone C. Cardiac autonomic dysfunction and functional outcome after ischaemic stroke. Eur J Neurol. 2007;14(8):917–22.
Colivicchi F, Bassi A, Santini M, Caltagirone C. Prognostic implications of right-sided insular damage, cardiac autonomic derangement, and arrhythmias after acute ischemic stroke. Stroke. 2005;36(8):1710–5.
Korpelainen JT, Sotaniemi KA, Huikuri HV, Myllyä VV. Abnormal heart rate variability as a manifestation of autonomic dysfunction in hemispheric brain infarction. Stroke. 1996;27(11):2059–63.
Sander D, Winbeck K, Klingelhöfer J, Etgen T, Conrad B. Prognostic relevance of pathological sympathetic activation after acute thromboembolic stroke. Neurology. 2001;57(5):833–8.
Deng QW, Yang H, Yan FL, Wang H, Xing FL, Zuo L, et al. Blocking sympathetic nervous system reverses partially stroke-induced immunosuppression but does not aggravate functional outcome after experimental stroke in rats. Neurochem Res. 2016;41(8):1877–86.
Zierath D, Olmstead T, Stults A, Shen A, Kunze A, Becker KJ. Chemical Sympathectomy, but not adrenergic blockade, improves stroke outcome. J Stroke Cerebrovasc Dis. 2018;S1052-3057(18):30384–7.
Yan FL, Zhang JH. Role of the sympathetic nervous system and spleen in experimental stroke-induced Immunodepression. Med Sci Monit. 2014;20:2489–96.
Chamorro Á, Meisel A, Planas AM, Urra X, van de Beek D, Veltkamp R. The immunology of acute stroke. Nat Rev Neurol. 2012;8(7):401–10.
Mracsko E, Liesz A, Karcher S, Zorn M, Bari F, Veltkamp R. Differential effects of sympathetic nervous system and hypothalamic-pituitary-adrenal axis on systemic immune cells after severe experimental stroke. Brain Behav Immun. 2014;41:200–9.
Ma J, Zhang L, He G, Tan X, Jin X, Li C. Transcutaneous auricular vagus nerve stimulation regulates expression of growth differentiation factor 11 and activin-like kinase 5 in cerebral ischemia/reperfusion rats. J Neurol Sci. 2016 Oct;369:27–35.
Bonaz B, Sinniger V, Pellissier S. Anti-inflammatory properties of the vagus nerve: potential therapeutic implications of vagus nerve stimulation. J Physiol. 2016;594(20):5781–90.
Zuo L, Shi L, Yan F. The reciprocal interaction of sympathetic nervous system and cAMP-PKA-NF-kB pathway in immune suppression after experimental stroke. Neurosci Lett. 2016;627:205–10.
Pongratz G, Melzer M, Straub RH. The sympathetic nervous system stimulates anti-inflammatory B cells in collagen-type II-induced arthritis. Ann Rheum Dis. 2012;71(3):432–9.
Pongratz G, Straub RH. The B cell, arthritis, and the sympathetic nervous system. Brain Behav Immun. 2010;24(2):186–92.
Rasouli J, Lekhraj R, Ozbalik M, Lalezari P, Casper D. Brain-spleen inflammatory coupling: a literature review. Einstein J Biol Med. 2011;27(2):74–7.
Planas AM, Gómez-Choco M, Urra X, Gorina R, Caballero M, Chamorro Á. Brain-derived antigens in lymphoid tissue of patients with acute stroke. J Immunol. 2012;188:2156–63.
Tsuchida T, Parker KC, Turner RV, McFarland HF, Coligan JE, Biddison WE. Autoreactive CD8+ T-cell responses to human myelin protein-derived peptides. Proc Natl Acad Sci U S A. 1994;91:10859–63.
Ortega SB, Noorbhai I, Poinsatte K, Kong X, Anderson A, Monson NL, et al. Stroke induces a rapid adaptive autoimmune response to novel neuronal antigens. Discov Med. 2015;19(106):381–92.
Ren X, Akiyoshi K, Grafe MR, Vandenbark AA, Hurn PD, Herson PS, et al. Myelin specific cells infiltrate MCAO lesions and exacerbate stroke severity. Metab Brain Dis. 2012;27(1):7–15.
Zierath D, Schulze J, Kunze A, Drogomiretskiy O, Nhan D, Jaspers B, et al. The immunologic profile of adoptively transferred lymphocytes influences stroke outcome of recipients. J Neuroimmunol. 2013;263(1–2):28–34.
Hurn PD. 2014 Thomas Willis award lecture: sex, stroke, and innovation. Stroke. 2014;45(12):3725–9.
Dimitrijevic OB, Stamatovic SM, Keep RF, Andjelkovic AV. Absence of the chemokine receptor CCR2 protects against cerebral ischemia/reperfusion injury in mice. Stroke. 2007;38:1345–53.
Hill WD, Hess DC, Martin-Studdard A, Carothers JJ, Zheng J, Hale D, et al. SDF-1 (CXCL12) is upregulated in the ischemic penumbra following stroke: association with bone marrow cell homing to injury. J Neuropathol Exp Neurol. 2004;63(1):84–96.
Ruscher K, Kuric E, Liu Y, Walter HL, Issazadeh-Navikas S, Englund E, et al. Inhibition of CXCL12 signaling attenuates the postischemic immune response and improves functional recovery after stroke. J Cereb Blood Flow Metab. 2013;33(8):1225–34.
Kim JS, Gautam SC, Chopp M, Zaloga C, Jones ML, Ward PA, et al. Expression of monocyte chemoattractant protein-1 and macrophage inflammatory protein-1 after focal cerebral ischemia in the rat. J Neuroimmunol. 1995;56(2):127–34.
Reichel CA, Rehberg M, Lerchenberger M, Berberich N, Bihari P, Khandoga AG, et al. Ccl2 and Ccl3 mediate neutrophil recruitment via induction of protein synthesis and generation of lipid mediators. Arterioscler Thromb Vasc Biol. 2009;29(11):1787–93.
Terao S, Yilmaz G, Stokes KY, Russell J, Ishikawa M, Kawase T, et al. Blood cell-derived RANTES mediates cerebral microvascular dysfunction, inflammation, and tissue injury after focal ischemia-reperfusion. Stroke. 2008;39(9):2560–70.
Bazan JF, Bacon KB, Hardiman G, Wang W, Soo K, Rossi D, et al. A new class of membrane-bound chemokine with a CX3C motif. Nature. 1997;385(6617):640–4.
Dénes A, Ferenczi S, Halász J, Környei Z, Kovács KJ. Role of CX3CR1 (fractalkine receptor) in brain damage and inflammation induced by focal cerebral ischemia in mouse. J Cereb Blood Flow Metab. 2008;28(10):1707–21.
Domac FM, Misirli H. The role of neutrophils and interleukin-8 in acute ischemic stroke. Neurosciences (Riyadh). 2008;13(2):136–41.
Herz J, Hagen SI, Bergmüller E, Sabellek P, Göthert JR, Buer J, et al. Exacerbation of ischemic brain injury in hypercholesterolemic mice is associated with pronounced changes in peripheral and cerebral immune responses. Neurobiol Dis. 2014;62:456–68.
Goldmacher GV, Nasser R, Lee DY, Yigit S, Rosenwasser R, Iacovitti L. Tracking transplanted bone marrow stem cells and their effects in the rat MCAO stroke model. PLoS One. 2013;8(3):e60049.
Carrero R, Cerrada I, Lledó E, Dopazo J, García-García F, Rubio MP, et al. IL1β induces mesenchymal stem cells migration and leucocyte chemotaxis through NF-κB. Stem Cell Rev. 2012;8(3):905–16.
Bai X, Xi J, Bi Y, Zhao X, Bing W, Meng X, et al. TNF-α promotes survival and migration of MSCs under oxidative stress via NF-κB pathway to attenuate intimal hyperplasia in vein grafts. J Cell Mol Med. 2017;21(9):2077–91.
Golden JE, Shahaduzzaman M, Wabnitz A, Green S, Womble TA, Sanberg PR, et al. Human umbilical cord blood cells alter blood and spleen cell populations after stroke. Transl Stroke Res. 2012;3(4):491–9.
Kadam SD, Chen H, Markowitz GJ, Raja S, George S, Verina T, et al. Systemic injection of CD34(+)-enriched human cord blood cells modulates poststroke neural and glial response in a sex-dependent manner in CD1 mice. Stem Cells Dev. 2015;24(1):51–66.
Peroni JF, Borjesson DL. Anti-inflammatory and immunomodulatory activities of stem cells. Vet Clin North Am Equine Pract. 2011;27(2):351–62.
Ulrich H, do Nascimento IC, Bocsi J, Tárnok A. Immunomodulation in stem cell differentiation into neurons and brain repair. Stem Cell Rev. 2015;11(3):474–86.
Racz GZ, Kadar K, Foldes A, Kallo K, Perczel-Kovach K, Keremi B, et al. Immunomodulatory and potential therapeutic role of mesenchymal stem cells in periodontitis. J Physiol Pharmacol. 2014;65(3):327–39.
Zhang R, Liu Y, Yan K, Chen L, Chen XR, Li P, et al. Anti-inflammatory and immunomodulatory mechanisms of mesenchymal stem cell transplantation in experimental traumatic brain injury. J Neuroinflammation. 2013;10:106.
Dorronsoro A, Ferrin I, Salcedo JM, Jakobsson E, Fernández-Rueda J, Lang V, et al. Human mesenchymal stromal cells modulate T-cell responses through TNF-α-mediated activation of NF-κB. Eur J Immunol. 2014;44(2):480–8.
Prockop DJ, Oh JY. Mesenchymal stem/stromal cells (MSCs): role as guardians of inflammation. Mol Ther. 2012;20(1):14–20.
Gao S, Mao F, Zhang B, Zhang L, Zhang X, Wang M, et al. Mouse bone marrow-derived mesenchymal stem cells induce macrophage M2 polarization through the nuclear factor-κB and signal transducer and activator of transcription 3 pathways. Exp Biol Med (Maywood). 2014;239(3):366–75.
Evans MA, Lim R, Kim HA, Chu HX, Gardiner-Mann CV, Taylor KWE, Kwan W, Teo L, Bourne JA, Neumann S, Young S, Gowing EK, Drummond GR, Clarkson AN, Wallace EM, Sobey CG, Broughton BRS, et al. Acute or delayed systemic administration of human amnion epithelial cells improves outcomes in experimental stroke. Stroke. 2018;49(3):700–9.
Spera PA, Ellison JA, Feuerstein GZ, Barone FC. IL-10 reduces rat brain injury following focal stroke. Neurosci Lett. 1998;251(3):189–92.
Conway SE, Roy-O'Reilly M, Friedler B, Staff I, Fortunato G, McCullough LD. Sex differences and the role of IL-10 in ischemic stroke recovery. Biol Sex Differ. 2015;6:17.
Wang Z, Zhou Y, Yu Y, He K, Cheng LM. Lipopolysaccharide Preconditioning Increased the Level of Regulatory B cells in the Spleen after Acute Ischaemia/Reperfusion in Mice. Brain Res. 2018;(18):30293–2.
Yang H, Zhang A, Zhang Y, Ma S, Wang C. Resveratrol pretreatment protected against cerebral ischemia/reperfusion injury in rats via expansion of T regulatory cells. J Stroke Cerebrovasc Dis. 2016;25(8):1914–21.
Mauri C, Bosma A. Immune regulatory function of B cells. Annu Rev Immunol. 2012;30:221–41.
Dhib-Jalbut S, Marks S. Interferon-beta mechanisms of action in multiple sclerosis. Neurology. 2010;74(Suppl 1):S17–24.
Liu H, Xin L, Chan BP, Teoh R, Tang BL, Tan YH. Interferon-beta administration confers a beneficial outcome in a rabbit model of thromboembolic cerebral ischemia. Neurosci Lett. 2002;327:146–8.
Veldhuis WB, Derksen JW, Floris S, Van Der Meide PH, De Vries HE, Schepers J, et al. Interferon-beta blocks infiltration of inflammatory cells and reduces infarct volume after ischemic stroke in the rat. J Cereb Blood Flow Metab. 2003;23:1029–39.
Marsh B, Stevens SL, Packard AE, Gopalan B, Hunter B, Leung PY, et al. Systemic lipopolysaccharide protects the brain from ischemic injury by reprogramming the response of the brain to stroke: a critical role for IRF3. J Neurosci. 2009;29:9839–49.
Inácio AR, Liu Y, Clausen BH, Svensson M, Kucharz K, Yang Y, et al. Endogenous IFN-β signaling exerts anti-inflammatory actions in experimentally induced focal cerebral ischemia. J Neuroinflammation. 2015;12:211.
Hoover DB. Cholinergic modulation of the immune system presents new approaches for treating inflammation. Pharmacol Ther. 2017;179:1–16.
Jung WC, Levesque JP, Ruitenberg MJ. It takes nerve to fight back: the significance of neural innervation of the bone marrow and spleen for immune function. Semin Cell Dev Biol. 2017;61:60–70.
Nance DM, Sanders VM. Autonomic innervation and regulation of the immune system (1987-2007). Brain Behav Immun. 2007;21(6):736–45.
Bellinger DL, Lorton D. Autonomic regulation of cellular immune function. Auton Neurosci. 2014;182:15–41.
Elenkov IJ, Wilder RL, Chrousos GP, Vizi ES. The sympathetic nerve--an integrative interface between two supersystems: the brain and the immune system. Pharmacol Rev. 2000;52(4):595–638.
Lorton D, Bellinger DL. Molecular mechanisms underlying β-adrenergic receptor-mediated cross-talk between sympathetic neurons and immune cells. Int J Mol Sci. 2015;16(3):5635–65.
Padro CJ, Sanders VM. Neuroendocrine regulation of inflammation. Semin Immunol. 2014;26(5):357–68.
Bellinger DL, Felten SY, Lorton D, Felten DL. Origin of noradrenergic innervation of the spleen in rats. Brain Behav Immun. 1989;3(4):291–311.
Rosas-Ballina M, Olofsson PS, Ochani M, Valdés-Ferrer SI, Levine YA, Reardon C, et al. Acetylcholine-synthesizing T cells relay neural signals in a vagus nerve circuit. Science. 2011;334(6052):98–101.
Peña G, Cai B, Ramos L, Vida G, Deitch EA, Ulloa L. Cholinergic regulatory lymphocytes re-establish neuromodulation of innate immune responses in sepsis. J Immunol. 2011;187(2):718–25.
Vida G, Peña G, Kanashiro A, Thompson-Bonilla Mdel R, Palange D, Deitch EA, et al. β2-Adrenoreceptors of regulatory lymphocytes are essential for vagal neuromodulation of the innate immune system. FASEB J. 2011;25(12):4476–85.
Vida G, Peña G, Deitch EA, Ulloa L. α7-cholinergic receptor mediates vagal induction of splenic norepinephrine. J Immunol. 2011;186(7):4340–6.
Guan YZ, Jin XD, Guan LX, Yan HC, Wang P, Gong Z, et al. Ncotine inhibits microglial proliferation and is neuroprotective in global ischemia rats. Mol Neurobiol. 2015;51(3):1480–8.
Han Z, Shen F, He Y, Degos V, Camus M, Maze M, et al. Activation of α-7 nicotinic acetylcholine receptor reduces ischemic stroke injury through reduction of pro-inflammatory macrophages and oxidative stress. PLoS One. 2014;9(8):e105711.
Parada E, Egea J, Buendia I, Negredo P, Cunha AC, Cardoso S, et al. The microglial α7-acetylcholine nicotinic receptor is a key element in promoting neuroprotection by inducing heme oxygenase-1 via nuclear factor erythroid-2-related factor 2. Antioxid Redox Signal. 2013;19(11):1135–48.
Sun F, Jin K, Uteshev VV. A type-II positive allosteric modulator of α7 nAChRs reduces brain injury and improves neurological function after focal cerebral ischemia in rats. PLoS One. 2013;8(8):e73581.
Sun F, Johnson SR, Jin K, Uteshev VV. Boosting endogenous resistance of brain to ischemia. Mol Neurobiol. 2017;54(3):2045–59.
Mays RW. Clinical development of MultiStem® for treatment of injuries and diseases of the central nervous system. In: Hess DC, editor. Cell therapy for brain injury. Switzerland: Springer International; 2015. p. 47–64.
Fischer UM, Harting MT, Jimenez F, Monzon-Posadas WO, Xue H, Savitz SI, et al. Pulmonary passage is a major obstacle for intravenous stem cell delivery: the pulmonary first-pass effect. Stem Cells Dev. 2009;18(5):683–92.
Roobrouck VD, Clavel C, Jacobs SA, Ulloa-Montoya F, Crippa S, Sohni A, et al. Differentiation potential of human postnatal mesenchymal stem cells, mesoangioblasts, and multipotent adult progenitor cells reflected in their transcriptome and partially influenced by the culture conditions. Stem Cells. 2011;29(5):871–82.
Boozer S, Lehman N, Lakshmipathy U, Love B, Raber A, Maitra A, et al. Global characterization and genomic stability of human MultiStem, a multipotent adult progenitor cell. J Stem Cells. 2009;4(1):17–28.
Aranda P, Agirre X, Ballestar E, Andreu EJ, Román-Gómez J, Prieto I, et al. Epigenetic signatures associated with different levels of differentiation potential in human stem cells. PLoS One. 2009;4(11):e7809.
Burrows GG, Van’t Hof W, Newell LF, Reddy A, Wilmarth PA, David LL, et al. Dissection of the human multipotent adult progenitor cell secretome by proteomic analysis. Stem Cells Transl Med. 2013;2(10):745–57.
Bedi SS, Hetz R, Thomas C, Smith P, Olsen AB, Williams S, et al. Intravenous multipotent adult progenitor cell therapy attenuates activated microglial/macrophage response and improves spatial learning after traumatic brain injury. Stem Cells Transl Med. 2013;2(12):953–60.
Walker PA, Bedi SS, Shah SK, Jimenez F, Xue H, Hamilton JA, et al. Intravenous multipotent adult progenitor cell therapy after traumatic brain injury: modulation of the resident microglia population. J Neuroinflammation. 2012;9:228.
Rahimzadeh A, Mirakabad FS, Movassaghpour A, Shamsasenjan K, Kariminekoo S, Talebi M, et al. Biotechnological and biomedical applications of mesenchymal stem cells as a therapeutic system. Artif Cells Nanomed Biotechnol. 2016;44(2):559–70.
Brenneman M, Sharma S, Harting M, Strong R, Cox CS Jr, Aronowski J, et al. Autologous bone marrow mononuclear cells enhance recovery after acute ischemic stroke in young and middle-aged rats. J Cereb Blood Flow Metab. 2010;30(1):140–9.
Liu N, Chen R, Du H, Wang J, Zhang Y, Wen J. Expression of IL-10 and TNF-alpha in rats with cerebral infarction after transplantation with mesenchymal stem cells. Cell Mol Immunol. 2009;6(3):207–13.
Mora-Lee S, Sirerol-Piquer MS, Gutiérrez-Pérez M, Gomez-Pinedo U, Roobrouck VD, López T, et al. Therapeutic effects of hMAPC and hMSC transplantation after stroke in mice. PLoS One. 2012;7(8):e43683.
Mays R, Borlongan C, Yasuhara T, Hara K, Maki M, Carroll J, et al. Development of an allogeneic adherent stem cell therapy for treatment of ischemic stroke. J Exp Stroke Transl Med. 2010;3:34–46.
Yang B, Hamilton JA, Valenzuela KS, Bogaerts A, Xi X, Aronowski J, et al. Multipotent adult progenitor cells enhance recovery after stroke by modulating the immune response from the spleen. Stem Cells. 2017;35(5):1290–302.
Plessers J, Dekimpe E, Van Woensel M, Roobrouck VD, Bullens DM, Pinxteren J, et al. Clinical-grade human multipotent adult progenitor cells block CD8+ cytotoxic T lymphocytes. Stem Cells Transl Med. 2016;5(12):1607–19.
Mays R, Deans R. Adult adherent cell therapy for ischemic stroke: clinical results and development experience using MultiStem. Transfusion. 2016;56(4):6S–8S.
Osanai T, Houkin K, Uchiyama S, Minematsu K, Taguchi A, Terasaka S. Treatment evaluation of acute stroke for using in regenerative cell elements (TREASURE) trial: rationale and design. Int J Stroke. 2018;13(4):444–8.
Mays RW, Savitz SI. Intravenous cellular therapies for acute ischemic stroke. Stroke. 2018;49(5):1058–65.
Sincere thanks to La-Mei Cheng, Professor of Reproductive and Stem Cell Research Institute of Central South University, for guidance on this subject.
Funding was obtained through the National High Technology Research and Development Program of China (863 project) (No. 2011AA020113), Hunan Province Key Scientific and Technological Project (No. 2013SK5070), the National Natural Science Fund of China (No. 81460244) and Scientific Research Plan of Hunan Provincial Health Department (No. B2010-107).
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