Novel therapeutic strategies targeting innate immune responses and early inflammation after stroke

Authors

  • Takashi Shichita,

    1. Department of Microbiology and Immunology, School of Medicine, Keio University, Tokyo, Japan
    2. Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Tokyo, Japan
    3. Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
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  • Tetsuro Ago,

    1. Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
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  • Masahiro Kamouchi,

    1. Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
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  • Takanari Kitazono,

    1. Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
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  • Akihiko Yoshimura,

    1. Department of Microbiology and Immunology, School of Medicine, Keio University, Tokyo, Japan
    2. Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Tokyo, Japan
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  • Hiroaki Ooboshi

    Corresponding author
    1. Department of Internal Medicine, Fukuoka Dental College Medical and Dental Hospital, Fukuoka, Japan
    • Department of Microbiology and Immunology, School of Medicine, Keio University, Tokyo, Japan
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Address correspondence and reprint requests to Hiroaki Ooboshi, Department of Internal Medicine, Fukuoka Dental College Medical and Dental Hospital, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan. E-mail: ooboshi@college.fdcnet.ac.jp

Abstract

Post-ischemic inflammation is an essential step in the progression of ischemic stroke. This review focuses on the function of infiltrating immune cells, macrophages, and T cells, in ischemic brain injury. The brain is a sterile organ; however, the activation of Toll-like receptor (TLR) 2 and TLR4 is pivotal in the beginning of post-ischemic inflammation. Some endogenous TLR ligands are released from injured brain cells, including high mobility group box 1 and peroxiredoxin family proteins, and activate the infiltrating macrophages and induce the expression of inflammatory cytokines. Following this step, T cells also infiltrate into the ischemic brain and mediate post-ischemic inflammation in the delayed phase. Various cytokines from helper T cells and γδT cells function as neurotoxic (IL-23/IL-17, IFN-γ) or neuroprotective (IL-10, IL-4) mediators. Novel neuroprotective strategies should therefore be developed through more detailed understanding of this process and the regulation of post-ischemic inflammation.

Abbreviations used
BBB

blood-brain barrier

BM

bone marrow

DAMPs

danger-associated molecular patterns

HMGB1

high mobility group box 1

ICAM-1

intercellular adhesion molecule-1

MMPs

matrix metalloproteinases

NKT

natural killer T

Prx

peroxiredoxin

ROS

reactive oxygen species

S1P

Sphingosine-1-phosphate

TCR

T-cell receptor

TLRs

Toll-like receptors

TNF

Tumor necrosis factor

tPA

tissue plasminogen activator

Stroke is a leading cause of death and disability worldwide, and approximately, 70–80% of all cases are ischemic stroke. The only globally approved treatment for ischemic stroke is the intravenous administration of alteplase [tissue plasminogen activator (tPA)], which is a time-dependent therapy that must be provided within 4.5 h after the stroke onset. Thus, the beneficial effect of tPA is limited to a small proportion of stroke patients. There is a need for an efficacious therapy that can be administered beyond this time window (Lo 2010; Moskowitz et al. 2010).

Post-ischemic inflammation is an essential step in ischemic brain injury (Eltzschig and Eckle 2011; Iadecola and Anrather 2011). The primary (acute) and secondary (delayed) progression of the infarct lesion are directly linked to the prognosis of ischemic stroke patients. The deprivation of oxygen, glucose, and other nutrients caused by the insufficient blood supply results in the dysfunction of cerebrovascular units, which consist of glial cells, endothelial cells, pericytes, and neurons (del Zoppo 2006; Iadecola 2004; Kamouchi et al. 2011). Brain cells fail to maintain the neuronal microenvironment, leading to blood-brain barrier (BBB) breakdown. Following the breakdown of the BBB, various blood-borne immune cells infiltrate into the ischemic brain from disrupted vessels. These infiltrating immune cells and the injured brain cells produce inflammatory mediators, which exaggerate brain edema or directly promote the death of brain cells in the penumbra, resulting in the secondary progression of the infarct lesion.

Effectors of innate immunity, especially macrophages and T cells, appear to play a pivotal role in post-ischemic inflammation (Barone and Feuerstein 1999; Macrez et al. 2011). To prevent the primary and secondary progression of an infarct lesion, it is indispensable to clarify the detailed mechanism of post-ischemic inflammation evoked by these immune cells. Despite intensive studies, the complexity of the brain inflammation mechanisms has thus far remained insufficiently clarified.

It is known that macrophages and neutrophils are key players in the various processes of post-ischemic inflammation, but the mechanism responsible for their activation is still unknown. In addition, T cells have been also reported to participate in the delayed cerebral inflammation. This article focuses on the potential mechanisms underlying the post-ischemic inflammation induced by infiltrating immune cells, and explores potential approaches for developing neuroprotective therapies based on regulating post-ischemic inflammation.

DAMPs in ischemic brain tissue

Ischemia induces the production of reactive oxygen species (ROS) from injured brain cells, and ROS activate platelets and endothelial cells, leading to microvascular occlusion. Oxidative stress and the inflammatory cascade alter the permeability of the BBB and exacerbate leukocyte extravasation. Recently, ROS and the regulators of their production have been implicated in the activation of infiltrating leukocytes (Chen and Nuñez 2010), and several molecules have been regarded as activators of infiltrating immune cells in the ischemic brain (Iadecola and Anrather 2011). Because the brain is a sterile organ, pathogens derived from bacteria or viruses are completely lacking in both the normal and ischemic brain. Certain endogenous molecules must be released from the necrotic brain to activate infiltrating immune cells. Such endogenous molecules are called danger-associated molecular patterns (DAMPs) and are considered danger signals or alarm molecules that alert immune cells to the presence of brain tissue injury.

So far, heat shock proteins, β-amyloid (Aβ), hyaluronan, heparin sulfate, DNA or RNA immune complexes, oxidized low-density lipoproteins, and several other molecules, have been considered as possible DAMPs in the ischemic brain (Marsh et al. 2009; Rivest 2009; Yanai et al. 2009; Stewart et al. 2010; Zhang et al. 2010). Interestingly, many reports have demonstrated that heat shock proteins exert neuroprotective roles in post-ischemic injury (Whitesell et al. 2003; Zheng et al. 2008; Stetler et al. 2012).

Despite numerous studies, it has not been known which molecule is the most important for triggering inflammation and activating leukocytes in the ischemic brain. Among these DAMPs, high mobility group box 1 (HMGB1) is a well-investigated DAMP shown to be involved in ischemic brain injury (Kim et al. 2006; Liu et al. 2007; Hayakawa et al. 2010). HMGB1, which is localized in cell nuclei in the normal brain, translocates into the cytosol, and is released into the extracellular compartment under ischemic conditions. Such an extracellular release of HMGB1 is observed within 2–4 h after ischemia-reperfusion, and increases the vascular permeability and promotes BBB breakdown (Zhang et al. 2011). The neutralization of extracellular HMGB1 improves the BBB permeability and reduces the infarct volume in mice and rats. Therefore, HMGB1 is an essential DAMP in the hyperacute phase of ischemic brain injury.

For the development of a novel neuroprotective agent with a wide therapeutic time window, it is important to elucidate the time course of DAMPs release. It has already been demonstrated that the extracellular release of HMGB1 is diminished within 12 h after ischemia-reperfusion (Qiu et al. 2008). Because the infiltration of immune cells and the production of inflammatory mediators become evident thereafter, the direct activation of infiltrating immune cells is not achieved by HMGB1, suggesting that it may not be an ideal therapeutic target in the delayed phase.

By investigating other potential DAMPs in brain homogenate lysates, peroxiredoxin (Prx) family proteins have been identified as strong inducers of inflammatory cytokines in infiltrating macrophages (Shichita et al. 2012). Originally, the Prx family proteins are ubiquitous antioxidant enzymes and are more abundant in brain than in other tissues. Prx proteins contain one [1-cysteine (1-Cys): Prx6] or two (2-Cys: Prx1–5) cysteine residues that allow them to scavenge ROS in cooperation with thioredoxin, another important redox modulator without cytokine-inducing activity in macrophages (Wood et al. 2003; Ago et al. 2008). In various brain tissue injuries, the increased expression of Prx proteins within brain cells has been observed and considered to diminish ROS (Patenaude et al. 2005). Consistent with this hypothesis, such an intracellular expression of Prx proteins has been reported to play a neuroprotective role (Rashidian et al. 2009). These results indicate that Prx family members are strongly induced inside the injured ischemic cells to improve their survival. However, they are released into the extracellular compartment once the cells are about to die, and such an extracellular release of Prx proteins functions as DAMPs. Therefore, Prx proteins can have two opposing functions, one inside and one outside the brain cells.

Compared with HMGB1, it may take more time for Prxs to function in the ischemic brain. In ischemic brain tissue, the Prx expression is evident on day 1, but disappears by day 4 after ischemia-reperfusion in mice. The highly expressed Prx is released into the extracellular compartment around injured brain cells (Fig. 1) and co-localizes with the membranes of infiltrating macrophages. Neutralization of extracellular Prx proteins decreases the expression of inflammatory cytokines by infiltrating inflammatory cells, and thereby attenuates ischemic damage in mice. Because Prx family proteins have a common β4 sheet and α3 helix region that is involved in their cytokine-inducing activity, a specific anti-inflammatory therapy may be developed by targeting this region of Prx proteins. Interestingly, an increased amount of Prx proteins in the extracellular fluid of the ischemic brain has been also reported in rats and stroke patients (Chou et al. 2011; Dayon et al. 2011). Thus, the extracellularly released Prx from injured brain cells appears to activate infiltrating macrophages and promotes post-ischemic inflammation (Fig. 2).

Figure 1.

Double immunohistochemical staining for Prx6 and TUNEL in the peri-infarct area of the mouse ischemic brain on day 1. The yellow arrow shows ischemic brain cells, which strongly expressed peroxiredoxin (Prx)6. The yellow arrowhead shows a dying brain cell, which released Prx6 into the extracellular compartment (bar: 20 μm).

Figure 2.

Post-ischemic inflammation is triggered by danger-associated molecular patterns (DAMPs). During the hyperacute phase of brain ischemia (within 6 h after stroke onset), high mobility group box 1 (HMGB1) is released from necrotic brain cells and induces blood-brain barrier (BBB) breakdown. Following this, macrophages begin to infiltrate into the ischemic brain tissue via disrupted vessels during the acute phase of ischemia (12 h to 24 h after stroke onset). In this phase, the expression of peroxiredoxin (Prx) is induced in the wounded brain cells by ischemic stress to catalyze the destruction of reactive oxygen species (ROS) to improve their survival. However, when the ischemic phenomena finally result in the death of brain cells, Prx is released into the extracellular compartment from the cell body. The extracellular release of Prx activates infiltrating macrophages through the Toll-like receptor (TLR)2 and TLR4 signaling pathways, leading to the production of various inflammatory cytokines. IL-1β and TNF-α directly act on neuronal cells in the penumbral region, which manage to survive because of the neuroprotective effects of intracellular Prx. The IL-23 and IL-12 produced from infiltrating macrophages act on T cells, which infiltrate during the delayed phase (more than 24 h after stroke onset) and induce IL-17 and IFN-γ production, respectively. These inflammatory cytokines promote post-ischemic inflammation and neuronal damage. On the other hand, regulatory T cells (Tregs) produce IL-10 and exert an anti-inflammatory effect by suppressing the expression of TNF-α and IFN-γ.

TLRs as DAMPs receptors

Toll-like receptors (TLRs) are essential receptors involved in the innate immune response to general pathogens such as bacteria and viruses. Recently, TLR2 and TLR4 have been reported to contribute to non-infectious immune-mediated injury, including ischemic brain injury (Chen et al. 2007; Tang et al. 2007). Indeed, HMGB1, Prx proteins, and other DAMPs can stimulate TLR2 and TLR4. The activation of macrophages and T cells through TLR pathway induces strong inflammatory responses. Post-ischemic inflammation and subsequent ischemic damage depend on TLR2 and TLR4, but not TLR9 (Hyakkoku et al. 2010), because TLR2- or TLR4-deficiency significantly attenuates ischemic brain damage and suppresses inflammatory cytokine expression in infiltrating immune cells on day 1 after ischemia-reperfusion in mice (Shichita et al. 2012). The clinical relevance of TLR2 and TLR4 in stroke patients has been also demonstrated (Brea et al. 2011). Despite these findings about the importance of TLR, the mechanisms responsible for activating them in infiltrating immune cells, such as macrophages and T cells, are not yet fully understood. CD36, RAGE, SRA, Mincle, and other pattern recognition receptors are also possible receptors for DAMPs (Akashi-Takamura and Miyake 2008).

In addition, the fact that TLRs are expressed on both leukocytes and brain cells makes it difficult to fully understand the complex inflammatory mechanisms of TLR activation in the ischemic brain. It remains to be clarified whether the effect of TLRs on brain cells is neurotoxic or neuroprotective. Brain cells, including astrocytes, oligodendrocytes, endothelial cells, and pericytes, constitute a neurovascular unit and maintain the neuronal microenvironment by increasing their antioxidant activities to scavenge ROS, and support the essential metabolic needs of neurons (Fraser 2011; Arimura et al. 2012). On the other hand, these brain cells, which are activated by ischemic injury, also contribute to triggering post-ischemic inflammation by producing inflammatory mediators. Tumor necrosis factor (TNF)-α, IL-1β, nitric oxide, and matrix metalloproteinases (MMPs) produced from brain cells regulate the cerebrovascular permeability and exaggerate brain edema (Takano et al. 2009; Morancho et al. 2010). Microglia, resident macrophages in the brain, are also activated through the TLR signaling pathway, and not only act as an inflammatory mediator by producing neurotoxic cytokines but also produce neurotrophic factors for tissue repair (Lai and Todd 2006).

Using bone marrow (BM) chimeric mice, the function of TLRs in brain cells has been investigated. Microglia, which are radiation-resistant, are a well-known rapid effector in the ischemic brain (microglia are derived from recipient brain cells in BM chimeric mice). However, in an experiment using TLR2- or TLR4-deficient mice with wild-type BM, no improvement was observed in ischemic brain injury (Yang et al. 2011; Shichita et al. 2012). Moreover, mice lacking MyD88, the adaptor protein required for almost all TLR signaling cascades (other than TLR3), showed either no improvement or exaggeration of ischemic brain injury (Famakin et al. 2011). These results indicate that the function of TLRs is dependent of the cell types in the brain and the timing of their activation. Recently, the administration of an anti-TLR blocking antibody has emerged as a neuroprotective therapy for ischemic myocardial or renal injury (Arslan et al. 2010; Farrar et al. 2012). To explore the therapeutic potential of this strategy, more detailed knowledge about the functions of TLRs in the ischemic brain is needed.

Inflammatory cytokines and mediators

Activated infiltrating immune cells and injured brain cells produce various inflammatory cytokines and mediators. IL-1β is expressed in the ischemic brain within 30 min after ischemia-reperfusion (Lakhan et al. 2009). Recently, the previously unknown mechanism of IL-1β production and caspase-1 activation mediated by the inflammasome has attracted particular attention. IL-1β is produced in an inactivate form, pro-IL-1β, whose mRNA expression is regulated by TLRs or other pattern recognition receptor signaling pathways (signal 1). Pro-IL-1β is cleaved to become an active 17-kDa form by caspase-1 inside multiprotein complexes called the inflammasome, which is activated by hypoxia, ATP, or endogenous molecules from damaged or dying cells (signal 2) (Martinon et al. 2002; Rathinum et al. 2012). The inflammasome is present in brain cells (neurons, astrocytes, and microglia) and macrophages (Abulafia et al. 2009; Chakraborty et al. 2010). Several types of inflammasomes have been discovered, and are denoted NALP1, NALP3, AIM, and so on, but the specific type most important in ischemic brain injury remains unknown. IL-1β is considered to be a neurotoxic mediator, given that the loss of IL-1β function is reported to reduce infarct size (Boutin et al. 2001). IL-1β directly induces neuronal cell death and enhances the expression of chemokines in microglia and astrocytes (Allan et al. 2005). Inhibiting IL-1β or its induction may represent a possible therapeutic approach.

TNF-α is another essential cytokine involved in ischemic brain injury. TNF-α is expressed in ischemic brain tissue within 1 h after ischemia-reperfusion (Liu et al. 1994). TNF-α exerts neurotoxic effects by promoting neuronal cell death and the expression of MHC class II and intercellular adhesion molecule-1 (ICAM-1) in astrocytes, resulting in leukocyte infiltration and BBB breakdown. The neuroprotective effect of TNF-α gene deficiency or the administration of an anti-TNF-α neutralizing antibody has been demonstrated. On the other hand, TNFR KO mice, which lack both p75 and p50 genes, exhibit enlargement of the infarct volume, indicating that TNF-α can be considered to function as both a neurotoxic and a neuroprotective mediator (Hallenbeck 2002). TNF-α promotes post-ischemic inflammation but also participates in a negative feedback loop to suppress inflammatory signal cascades, and it controls the duration of post-ischemic inflammation by regulating these two functions. It appears that which of the opposing functions is active, toxic, or protective depends on the type of involved brain cells. The application of anti-TNF-α neutralizing therapy for ischemic stroke may need to wait for a better elucidation of the various functions of TNF-α.

IL-6 is an important cytokine in various types of inflammation, but ischemic brain damage is not attenuated by IL-6 deficiency or administration of an anti-IL-6R antagonistic antibody (Yamashita et al. 2005). However, it has recently been reported that IL-6 produced from brain cells contributes to neoangiogenesis and neuronal survival through STAT3 activation (Jung et al. 2011; Gertz et al. 2012). Consistent with this observation, the inhibition of the JAK/STAT pathway or the enhancement of SOCS3 (a negative regulator of the JAK/STAT pathway) has been reported to promote neuronal cell death (Yadav et al. 2005). Thus, it is possible that IL-6 contributes to the process of tissue repair after ischemic brain injury.

Chemokines are also important enhancers of post-ischemic inflammation. RANTES, monocyte chemotactic protein-1, and IL-8 have been reported to promote leukocyte infiltration into the ischemic brain and to increase infarct growth (Terao et al. 2008, 2009; Strecker et al. 2011). The overexpression of dominant negative monocyte chemotactic protein-1 has been demonstrated to attenuate macrophage infiltration and reduce the infarct size (Kumai et al. 2004). Although the chemokines for T-cell infiltration remain unknown, CCL12, CCL20, and their receptor, CCR6, have been reported to be essential for the induction of IL-17-producing helper T cells in experimental autoimmune encephalomyelitis (Martin et al. 2009; Reboldi et al. 2009). Nevertheless, the mechanism of T-cell infiltration in the ischemic brain tissue remains to be elucidated.

ICAM-1 is essential for chemotaxis and the infiltration of leukocytes. Increased expression of ICAM-1 in cerebrovascular endothelial cells is observed in the ischemic brain, and ICAM-1 deficiency or neutralization of ICAM-1 by an antibody leads to the improvement of ischemic brain damage (Connolly et al. 1996; Liesz et al. 2011b). MMPs are also important mediators that promote post-ischemic inflammation by enhancing the breakdown of the BBB. The neurotoxic function of MMP-9 has been established, and MMP-9-deficient mice exhibit a smaller infarct size compared with control mice (Asahi et al. 2000).

Sphingosine-1-phosphate (S1P) is a bioactive phospholipid. At sites of tissue injury, S1P is mainly released from platelets and mediates its effect via the activation of cell-surface S1P receptors, which are ubiquitously expressed in neurons, astrocytes, and microglial cells (Dev et al. 2008). S1P directly acts on brain cells and induces astrocyte proliferation and migration, oligodendrocyte differentiation and survival, and neurite outgrowth and neurogenesis. S1P receptors are also present on the surface of T cells; therefore, S1P is considered to be essential for the infiltration of T cells. The neuroprotective effect of FTY720 (fingolimod), a functional S1P receptor antagonist, has recently been examined, and the intravenous administration of FTY720 was found to decrease the number of infiltrating T cells in the ischemic brain and reduce infarct size (Shichita et al. 2009; Hasegawa et al. 2010). Therefore, S1P is considered to be an important inflammatory mediator in ischemic brain injury. Because a clinical trial of FTY720 for multiple sclerosis showed favorable results (Cohen et al. 2010), FTY720 may be a safe and promising new drug for the treatment of ischemic stroke.

Infiltrating immune cells in the ischemic brain

Macrophages are the main inflammatory effector among the various infiltrating immune cells from blood. The infiltration of macrophages becomes evident from 12 h to 24 h after ischemia-reperfusion and reaches a peak on day 3 (Clausen et al. 2008). Infiltrating macrophages produce various inflammatory cytokines, such as IL-1β, TNF-α, IL-23, and promote post-ischemic inflammation. Mice deficient in CD11b, a specific marker of macrophages, exhibited attenuated ischemic damage (Soriano et al. 1999).

T cells are also important effectors in the delayed phase of brain ischemia (Yilmaz et al. 2006). The number of infiltrating T cells in the ischemic brain increases over 24 h after ischemia-reperfusion and reaches a peak in the delayed phase (around day 3) (Schroeter et al. 1994; Jander et al. 1995). T cells appear to be localized to the infarct boundary zones, typically close to blood vessels. T cells consist of 30~40% CD4+ helper T cells, 20~30% γδT cells, and 20~30% CD8+ cytotoxic T cells. Although the function of T cells in the ischemic brain has not been fully clarified, on the whole, T cells are considered to exert neurotoxic effects. This hypothesis is suggested by the following observations: First, recombination activating gene-deficient mice and severe combined immunodeficient mice, both of which lack T and B cells, show a significant reduction of infarct volume compared with wild-type mice (Yilmaz et al. 2006; Hurn et al. 2007). Second, the depletion of CD4+ helper T cells or CD8+ cytotoxic T cells, but not of B cells, led to a significant reduction of the infarct volume. Thus, T cells are a promising target for the development of a neuroprotective treatment with a prolonged therapeutic time window.

B cells, NK cells, and natural killer T (NKT) cells are also observed in ischemic brain tissue. However, their function in the ischemic brain remains to be clarified. There was one report that regulatory B cells protect the brain from ischemic damages (Ren et al. 2011). Another report revealed a protective effect for hepatic invariant NKT cells against stroke-associated infections (Wong et al. 2011). It is important to elucidate the roles of these immune cells in ischemic brain injury.

T cells in ischemic brain injury

Because post-ischemic inflammation occurs in the acute phase, T cells are considered to act mainly as an effector of an antigen-independent innate inflammatory response. However, it is possible that acquired immunity by T cells also plays a role in ischemic brain injury, even though it has not been clarified whether a specific antigen in the ischemic brain is involved in the activation of T cells. Some reports suggest the importance of antigen recognition by T cells in ischemic brain injury. Treatment with a T-cell receptor (TCR) ligand, which consisted of major histocompatibility complex class II molecules bound to myelin peptides, was protective against ischemic brain injury (Subramanian et al. 2009). In myelin basic protein-tolerized animals, a reduction of the infarct size has been demonstrated (Becker et al. 2003). Therefore, the possibility exists that some T-cell subsets specifically tolerized to neuronal proteins can be neuroprotective (Becker 2009). This idea is supported by the recent finding that regulatory T cells are protective against ischemic brain injury (Liesz et al. 2009).

Various cytokines produced from T cells are strong regulators of post-ischemic inflammation (Fig. 3). These cytokines seem to function as innate immune mediators, given that the needs of antigen-specific TCR stimulation have not been demonstrated for the production of these cytokines. IFN-γ and IL-4 are well-known classical cytokines released from T cells. IFN-γ is mainly produced from CD4+ helper T cells and is thought to be neurotoxic, as it directly acts on neurons and induces neuronal cell death (Lambertsen et al. 2004). However, the neuroprotective effect of IFN-γ deficiency is controversial (Lambertsen et al. 2004; Yilmaz et al. 2006). IL-12 is produced from myeloid cells such as macrophages, dendritic cells, neutrophils, and is important for the differentiation of IFN-γ-producing helper T cells. In the ischemic brain, IL-12 is produced from infiltrating macrophages, but its function has not been fully elucidated. IL-4 may have a neuroprotective function to promote tissue repair, given that IL-4 deficiency exacerbates ischemic brain damage and neurological deficits in mice (Xiong et al. 2011). Although previously unknown functions of IL-4 in tissue repair have been receiving increasing attention (Chen et al. 2012), the involvement of IL-4 in tissue repair after brain ischemia remains to be clarified.

Figure 3.

Regulation of T-cell function in ischemic brain injury. For the development of a neuroprotective strategy targeting T cells, it is important to inhibit the inflammatory T cell (γδT-IL-17, Th1) function and promote the anti-inflammatory T cell (Treg) function.

IL-10 is an immunosuppressive cytokine and is thought to have a neuroprotective effect against ischemic brain injury (Ooboshi et al. 2006). Although the IL-10-producing T-cell population is small in the ischemic brain, a neuroprotective therapy could be developed by enhancing IL-10 production or promoting the differentiation of cells into the IL-10-producing T cells. Regulatory T cells are reported to be important IL-10-producing cells in ischemic brain injury and may exert a neuroprotective effect by suppressing the neurotoxic functions of TNF-α and IFN-γ (Liesz et al. 2009). Recently, the transcription factor E4BP4 and the expression of lymphocyte activation gene 3 have been discovered to be essential for the regulation of IL-10 production from T cells (Okamura et al. 2009; Motomura et al. 2011). Because TGF-β, an important cytokine for IL-10 production from T cells, is highly expressed in the brain tissue, infiltrating T cells may easily differentiate into IL-10-producing T cells in the brain. Although the antigen specificity of the helper T cell response remains unknown, the elucidation of the detailed mechanism(s) responsible for IL-10 production in the ischemic brain has the potential to lead to the development of a neuroprotective therapy.

IL-17 is an emerging therapeutic target for various organ injuries. IL-23 has been reported to be essential for IL-17 induction from CD4+ helper T cells, γδT cells, and NKT cells, and to play a critical role in experimental autoimmune encephalomyelitis (Cua et al. 2003). In ischemic brain injury, IL-23 is produced by infiltrating macrophages on day 1 after ischemia-reperfusion (Shichita et al. 2009), and IL-23 induces IL-17 production from γδT cells in the delayed phase (Fig. 2). Either IL-23 or IL-17 deficiency prevents infarct growth in the delayed phase (from days 1 to 4) in mice, although IL-23 exerts its primary effects earlier in the pathological course, given that IL-23 deficiency or the neutralization of IL-23 attenuate ischemic damage on day 1 (Shichita et al. 2009; Konoeda et al. 2010). The IL-17 receptor is ubiquitously expressed in brain cells and modifies various inflammatory responses in the central nervous system. IL-17 has been reported to promote the expression of inflammatory cytokines and chemokines from macrophages (Fossiez et al. 1996). IL-17 also modulates the epithelial barrier function by promoting the expression of MMPs and ICAM-1 (Kebir et al. 2007; Ifergan et al. 2008). Although it remains unknown whether IL-17 directly affects neurons, it appears to be a pivotal inflammatory cytokine in the delayed phase of brain ischemia. The main source of IL-17 in the ischemic brain is γδT cell (Shichita et al. 2009). This can be reasonably explained by the fact that the IL-17 production from γδT cells requires only IL-1β and IL-23 stimulation, not specific TCR stimulation, and IL-6 and TGF-β stimulation are indispensable for the IL-17-producing CD4+ helper T cell differentiation (Sutton et al. 2009). Therefore, compared with helper T cells, IL-23-induced IL-17-producing γδT cells are rapid inflammatory effectors involved in the delayed phase (days 1 to 4) of brain ischemia. Clinical trials of IL-23 or IL-17 neutralizing antibodies are now ongoing for the treatment of various inflammatory diseases. Targeting IL-23- or IL-17-producing γδT cells may be a promising therapeutic strategy for ischemic stroke.

The possibility of medical intervention for post-ischemic inflammation

As previously described, FTY720 is one of the important therapeutic candidates that can modulate the post-ischemic inflammation to treat ischemic stroke (Shichita et al. 2009; Wei et al. 2011). FTY720 decreases the number of infiltrating T cells, including γδT cells, although one report demonstrated no improvements in ischemic brain injury in mice (Liesz et al. 2011a). The most troublesome side effect of FTY720 administration may be an increased incidence of bacterial pneumonia after ischemic stroke (Meisel and Meisel 2011). It is possible that FTY720 interferes with peripheral T cell distribution in the body, leading to the inhibition of the defensive function of peripheral T cells against bacterial infections. In fact, it has been suggested that the dysfunction of T-cell-mediated immune responses after ischemic stroke is a possible cause of the increased incidence of bacterial pneumonia (Offner et al. 2006; Dirnagl et al. 2007; Vogelgesang et al. 2008). Although it has been reported that FTY720 does not promote spontaneous bacterial infection after experimental stroke in mice, future clinical studies will be needed to minimize the detrimental effects of FTY720 by adjusting the dose and timing of administration (Pfeilschifter et al. 2011).

Another important point is that post-ischemic inflammation is also associated with the repair of injured brain tissue. It is therefore important to regulate the balance between the neurotoxic and neuroprotective effects of post-ischemic inflammation. This problem can be solved by targeting specific inflammatory mediators. For example, a specific therapy that suppresses the inflammatory subset of T cells (e.g., IL-17-producing γδT cells) but promotes the neuroprotective function of regulatory T cells may be desirable (Fig. 3).

Finally, one of the most advantageous points of the T-cell targeting therapy is its prolonged therapeutic time window. It is possible that treatment against infiltrating T cells may work in the later phase (Fig. 2) because the administration of a γδTCR-depleting antibody delivered 1 day after the initiation of brain ischemia reduces the infarct size (Shichita et al. 2009). As a result, a neuroprotective therapy targeting inflammatory T cells possesses great promise.

In conclusion, recent research has gradually shed light on the mechanisms of cerebral post-ischemic inflammation mediated by immune cells. A deeper understanding of the intricacies of these processes should enable us to develop a more effective treatment with a prolonged therapeutic time window (Fig. 4).

Figure 4.

The time course of post-ischemic inflammation mediated by immune cells. After the onset of ischemic stroke, danger-associated molecular patterns (DAMPs) are released from injured brain cells. Thereafter, the infiltrating macrophages activated by DAMPs promote inflammation. During the delayed phase (around day 3), T cells also play a pivotal role in the inflammatory regulation. Targeting these toxic T cells may be a promising approach to develop a neuroprotective therapy with a prolonged therapeutic time window.

Acknowledgements

We have no financial conflict of interest.

This work was supported by the grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (S0801084, 23591262), PRESTO and CREST from Japan Science and Technology Agency.

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