Ischaemic tolerance in the brain is a powerful adaptive defence that involves an endogenous programme of neuroprotection culminating in marked protection against brain injury from ischaemia. A range of preconditioning stimuli exist that differ in ligand and target characteristics but share the common feature of causing mild stress or insult without inducing overt injury. The protective phenotype that emerges confers tolerance to subsequent exposure to injurious insults. Tolerance to injury is the result of genomic reprogramming, an adaptation comprising regulatory processes that countermand injurious effectors and invoke novel neuroprotective pathways. TLRs (Toll-like receptors) play important roles in sensing potential danger/insult in the form of pathogens as well as endogenous stress molecules that occur in response to mild injury (e.g. heat-shock proteins). Recent studies suggest that TLRs are novel and potent preconditioning targets that offer substantial promise to protect the brain from ischaemic injury.
Ischaemic tolerance in the brain (in which brief ischaemia increases resistance to subsequent injurious ischaemia) is a powerful adaptive defence that involves an endogenous programme of neuroprotection (reviewed in ). This neuroprotective programme sets into motion a complex cascade of signalling events leading to synthesis of new proteins that ultimately re-programmes the transcriptional response to subsequent injury. The sequence of events that leads to ischaemic tolerance is only partially known, although evidence is emerging that diverse stimuli that trigger preconditioning may share a common process that confers neuroprotection [2–4]. Thus identification of the central mediators involved in preconditioning may define essential pathways responsible for this adaptive cellular response programme and thereby provide novel candidates that offer promise as potential stroke therapeutics.
Tolerance to ischaemic brain injury can be induced by several distinct preconditioning stimuli including non-injurious ischaemia, cortical spreading depression, brief episodes of seizure, exposure to anaesthetic inhalants and low doses of endotoxin [LPS (lipopolysaccharide)] [5–8]. The precise mechanisms that underlie these neuroprotective processes are not completely defined; however, they appear to share a common link that small doses of an otherwise harmful stimulus induce protection against subsequent injurious challenge .
Endotoxin-induced tolerance to ischaemic injury in the brain
Endotoxin (LPS) confers ischaemic tolerance in several systems (e.g. heart, liver and brain) [9–11]. LPS preconditioning in the brain shares several hallmark characteristics with ischaemic preconditioning in the brain , such as the delayed induction of tolerance following preconditioning and a dependency on de novo protein synthesis [12,13]. Importantly, similar to ischaemic preconditioning wherein preconditioning by exposure to brief ischaemia does not induce brain damage, a preconditioning dose of LPS does not cause brain injury. Ischaemic tolerance conferred by LPS appears to occur through stimulation of TLR4 (Toll-like receptor 4), one of a class of molecules involved in sensing the presence of pathogens.
The TLR system
TLRs are pattern recognition receptors thought to be involved in the recognition and response to foreign pathogens. At least ten TLRs have been reported in humans  and nine in the mouse . A common pathway in all the TLRs is the ability to induce the transcription factor NF-κB (nuclear factor κB), which subsequently leads to the transcription of various cytokines, chemokines and cell-surface molecules. As this field is quite extensive, TLR4 and TLR9 will be discussed due to their relevance in neuroprotection. TLR4 is thought to be specific for molecules characteristic of extracellular pathogens, such as LPS. In addition to activation by LPS, TLR4 can be activated by molecules that are considered endogenous ligands such as Hsp60 (heat-shock protein 60), Hsp70, fibrinogen and certain fragments of fibronectin . The TLRs have remarkably similar signalling cascades; thus it seems likely that the phenomenon of tolerance induction may be a general feature of TLR activation. In support of this is the finding that activation of TLR2 by its agonist, lipoteichoic acid, has been shown to induce tolerance in both a rat heart and renal model of ischaemia/reperfusion injury [17,18].
TLR9 as a new target for induction of tolerance to ischaemic injury in the brain
TLR9 is required for responses to unmethylated CpG motifs found in bacterial DNA  and, more recently, has been shown to recognize self-DNA complexes . CpG ODNs (oligodeoxynucleotides) have been studied extensively for activation of the innate immune response resulting in protection of mice against subsequent challenge from a variety of bacteria, viruses, parasites and prions . The protection typically begins within 2 days and lasts for several weeks. In addition, the protection against these pathogens can be extended by repeated administration of CpG ODNs (reviewed in ). The delayed initiation of protection is similar to what has been seen for LPS-induced ischaemic tolerance [10,23]. We have found recently that a preconditioning dose of CpG ODNs administered in advance of ischaemia confers marked neuroprotection (S.L. Stevens and M.P. Stenzel-Poore, unpublished work). As CpG ODNs have been approved for human trials in allergy and cancer therapy , this TLR9 agonist offers substantial promise as a target for use as stroke therapy. While slight differences in the intracellular signalling cascades downstream of TLR4 and TLR9 may account for the different incidence of toxic side effects caused by LPS compared with CpG, similarities in the signalling cascades led to the elucidation of known neuroprotective molecules by each ligand. Moreover, differences between species with regard to TLR9 localization may explain the presence of fewer TLR9-induced unwanted side effects in primates compared with rodents .
LPS and CpG ODNs induce critical mediators of ischaemic tolerance
The activation of inflammatory pathways plays an important role in LPS-induced ischaemic tolerance. TNFα (tumour necrosis factor α) and its downstream signalling mediator, ceramide, are critical effectors of LPS-induced neuroprotection . An essential role for TNFα in LPS preconditioning has been shown in studies where LPS treatment was accompanied by concurrent neutralization of TNFα in the circulation [with a soluble TNFR (TNF receptor)]. Blockade of TNFα reversed the protective effect of LPS preconditioning . Thus proximal members of the TNFα pathway, namely TNFα and its receptors TNFR1 (p55) and TNFR2 (p75), as well as sphingomyelin-based second messengers such as ceramide, are likely mediators of the protective effects of TNFα in LPS preconditioning. TNFα activation of NF-κB may also be involved, as inflammatory molecules regulated by NF-κB, such as superoxide dismutase, are known to be important in LPS preconditioning . It is important to emphasize that studies have shown that TNFα on its own induces tolerance to ischaemia/hypoxia and that this protective effect depends, in part, on ceramide [26,27]. Importantly, like LPS, CpG ODNs are also known to induce TNFα expression in murine microglia and monocytes and in human neutrophils and plasmacytoid dendritic cells [28,29].
Type I IFNs (interferons) are a family of cytokines comprising type I (IFNα and IFNβ) and type II IFNs (IFNγ). First characterized based on antiviral properties, type I IFNs have many immunomodulatory functions, some of which link innate and adaptive immunity . Generally, IFNα/β are associated with anti-inflammatory cytokines. IFNα/β production is induced by many viral infections and LPS of Gram-negative micro-organisms. LPS preconditioning in the setting of ischaemia depends, in part, on IFNα/β expression . Importantly, CpG ODNs induce IFNα/β, which may provide one possible explanation for its ability to induce tolerance to ischaemic injury [28,32].
The idea that IFNβ may be neuroprotective in stroke is supported by the finding that systemic administration of IFNβ improves stroke outcome in rodents  and rabbits . The mitigating role of IFNβ in stroke is primarily ascribed to its anti-inflammatory properties that reduce cell infiltration into the injured brain via regulation of matrix metalloproteinase-9. In addition, IFNβ has been shown to decrease reactive oxygen species, suppress inflammatory cytokines  and promote cell survival . These functions could contribute to improved outcomes in stroke injury. The anti-inflammatory effects of IFNβ have long been valued therapeutically in the setting of multiple sclerosis  and as such have been approved for clinical use.
Evidence is emerging that in addition to antiviral effects, type I IFNs inhibit and/or modulate activation of cells in the immune system [38,39]. The basis for some of IFNα/β's regulatory functions lies in its action as a facilitator of expression of other IFN-inducible proteins known as IRFs (IFN regulatory factors), which in turn transactivate additional IFN-inducible genes. IRFs constitute a family of transcription factors whose functions in some instances are distinct and independent of one another, whereas in others, appear to be critically interdependent . IRF3 and IRF7 activation occurs following LPS stimulation (reviewed in ), which has important implications for LPS preconditioning, as LPS induction of IFNβ requires IRF3 . Thus IFR3 may be an important link between LPS preconditioning and neuroprotection. In addition, IRF3 binding to its ISRE (IFN-stimulated response elements) induces additional genes  that may be involved in the acquisition of neuroprotection. It is possible that a parallel story exists with CpG ODN activation of TLR9.
Reprogramming the genomic response to injury: a proposed mechanism of TLR-induced ischaemic tolerance
We have proposed previously that ischaemic tolerance reflects a fundamental change in the genomic response to injury that shifts the outcome from cell death to cell survival . We refer to this transcriptional change as genomic reprogramming. This concept was first advanced to explain the novel gene expression profiles observed in animals made tolerant to ischaemic injury by prior preconditioning with a brief period of ischaemia . Ischaemic preconditioning caused pronounced suppression of gene expression in response to ischaemia that is ordinarily injurious. Such suppression contrasted sharply with the up-regulation of RNA transcripts seen in animals subjected to ischaemic injury without prior preconditioning. This change was not simply the lack of a response, but rather a reprogramming of the genetic response to ischaemia, whereby down-regulation of genes that control metabolism, cell-cycle regulation and ion-channel activity was found to be the dominant response. These features mimic specific adaptive neuroprotective strategies seen in hibernation and other hypoxia-tolerant states. Thus, as in hibernation, preconditioning elicits endogenous genetic adaptations that confer tolerance to the injurious effects of oxygen deprivation.
The finding that exposure to a brief ischaemic event (preconditioning) causes a complex reprogramming of the cellular responses to ischaemia may reflect a fundamental regulatory mechanism for governing host responses to harmful stimuli. Our genomic studies with LPS preconditioning provide another compelling demonstration of a complex reprogramming event that alters the transcriptional response to brain ischaemia [45,46]. A similar process occurs with exposure of macrophages to a small dose of endotoxin before a large dose, evinced by the finding that macrophages not so primed respond entirely differently. This reprogramming capacity is viewed as protective to the host because the outcome is a finely controlled shift in the balance of pro-inflammatory and anti-inflammatory cytokines . Mechanisms known to mediate endotoxin tolerance in macrophages that lead to suppression of specific cytokines and inflammatory molecules involve attenuation of NF-κB and AP-1 (activator protein 1) and enhanced expression of the signalling mediators IRAK-M (interleukin-1-receptor-associated kinase M) and SOCS-1 (suppressor of cytokine signalling 1) [48,49]. A case for gene reprogramming can also be made where preconditioning with LPS induces cardiac resistance to ischaemia . In this scenario, exposure to LPS may induce non-specific myocardial adaptations through gene changes (e.g. Hsp70, c-Jun and c-Fos) that allow the heart greater resistance to subsequent injurious stimuli, such as ischaemia.
Preconditioning activates endogenous signalling pathways that culminate in marked protection against brain injury from ischaemia. Although a range of preconditioning stimuli exist that differ in ligand and target characteristics, they share the feature of acting as a mild insult without causing overt injury. Thus preconditioning may serve as a warning of impending danger or injury. The protective phenotype that emerges confers tolerance to subsequent exposure to injurious insults. Tolerance to injury is the result of genomic reprogramming, an adaptation that yields counter-regulatory processes that mitigate injurious effectors and induction of novel neuroprotective pathways. These powerful adaptive processes occur following priming by mild danger that may well herald impending injury. That such adaptations have evolved emphasizes the importance of protective physiological responses that are shaped by previous signals and thereby governed by the context in which they are generated. TLRs sense danger in the form of pathogens and endogenous molecules made in response to injury or stress (e.g. Hsps); as such, TLRs are novel and potent preconditioning targets that offer substantial promise to protect the brain from ischaemic injury.
International Symposium on Neurodegeneration and Neuroprotection: Independent Meeting held at University of Münster, Germany, 23–27 July 2006. Organized and Edited by S. Klumpp and J. Krieglstein (Münster, Germany).
This work was supported by the National Institutes of Health (Bethesda, MD, U.S.A.) grants NS50567 and NS35965.