Biological role of Toll

iologiBcal role of Toll-like receptor-4 in the brainThe Toll-like receptors (TLRs) are a family of microbe-sensing receptors that play a central role in the regulation of the host immune system. TLR4 has been described in the brain and seems to regulate some physiological processes, such as neurogenesis. TLR4 has also been reported to play a role during neurodegenerative disorders, including Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis and Parkinson's disease. This review is focused on reports concerning recent insights into the role and activation mechanisms of TLR4 in the brain, in pathological and physiological conditions, as well as the therapeutic benefit that could derive from TLR4 modulation.TLR4 在大脑中的生物角色TLRs 是细菌传感器家族的一员，在宿主免疫系统中发挥着核心作用。

TLE4 据称在大脑中似乎调节一些生理过程，如神经再生。

TLR4 也被报道在神经退行性疾病中发挥作用，包括1. IntroductionMammalian Toll-like receptors (TLRs) were initially discovered because of their sequence similarities to Toll involved in Drosophila dorsoventral embryonic development and antifungal immunity ( Nüsslein-Volhard and Wieschaus, 1980, Steward et al., 1984 and Lemaitre et al., 1996). In 1997, Medzhitov et al. cloned a human homolog of the Drosophila Toll protein, now known as TLR4, and showed that Toll signaling was able to stimulate adaptive immune responses ( Medzhitov et al., 1997). Shortly after, the Toll gene was discovered to be an important component for the detection of microbes in Drosophila melanogaster, as well as demonstrations that TLR4 mediates the inflammatory response to lipopolysaccharide (LPS) in mice (Poltorak et al., 1998 and Poltorak et al., 2000). This led to the identification of the target molecule of LPS on the cellular surface of macrophages. These discoveries substantially extended the knowledge of pathogen-mediated intra-cellular signal transduction, and were crucial for understanding the mechanisms that govern innate immunity ( Bode et al., 2012).In general, mammalian cells recognize the presence of pathogens through a group of receptor complexes, also termed pattern recognition receptors (PRR), that are specialized in detecting conserved molecular structures that are essential to the life cycle of a pathogen. These pathogen-borne molecular structures are also termed pathogen-associated molecular patterns (PAMP) (Takeda and Akira, 2005 and Akira et al., 2006).Thus, the term PRR encompasses a heterogeneous group of soluble, membrane-bound or cytoplasmic receptor structures involved in the detection of PAMPs.These molecular sensors are crucial to the initiation of innate immunity, constituting the first line defense against microorganisms (Bode et al., 2012).Specifically, TLRs are a family of PRR that enable the recognition of conserved structural motifs in a wide array of pathogens. They are homologs of Toll, a receptor found in insects, involved both in establishing dorsoventral polarity during embryogenesis and in immune response against fungal infections (Hashimoto et al., 1988 and Lemaitre et al., 1996). These receptors are type I integral membrane glycoproteins characterized by three major domains: (1) a leucine-rich extracellular domain; (2) a transmembrane domain; and (3) a cytoplasmic TIR domain homologous to that of the interleukin 1 (IL-1) receptor, termed the Toll/IL-1 receptor (TIR) domain (O'Neill and Dinarello, 2000). Ligand recognition by TLRs is mediated by the extracellular domain that harbors a leucine-rich repeat (LRR) composed of 19–25 tandem copies of the “xLxxLxLxx” motif (Jin and Lee, 2008).To date, 10 members of the human receptors and about 13 mammalian TLRs have been described (Akira et al., 2006 and McGuire et al., 2006) and more recently,10 bovine TLRs have been mapped (McGuire et al., 2006). Among these, TLR1–TLR10 are conserved between humans and mice, although TLR10 is not functionalin mice because of a retroviral insertion, whereas TLR11–13 are not present in humans (Akira et al., 2006, Beutler et al., 2007 and Medzhitov, 2007).The TLR family can be divided into extracellular and intracellular members. TLR1, TLR2, TLR4, TLR5, TLR6, and TLR11 are localized on the cell surface to recognize PAMPs. Conversely, TLR3, TLR7, TLR8, and TLR9 are intracellularly expressed in endosomal or lysosomal compartments and the endoplasmic reticulum (ER) (Bode et al., 2012). TLRs are described not only in the immune cells, such as macrophages, dendritic cells, B and T cells, but also in non-immune cells, including fibroblasts and epithelial cells (Wang et al., 2006).In 1998, TLR-4 was identified as the signaling receptor for LPS or endotoxin from the outer membrane of Gram-negative bacteria (Poltorak et al., 1998).The TLR4 specificity for LPS from Gram negative bacteria has also been demonstrated with TLR4 knock-out mice (Hoshino et al., 1999). Furthermore, a mouse strain possessing a point mutation in the TLR4 gene was shown to be unresponsive to LPS (Poltorak et al., 1998).There is accumulating evidence that not only does TLR4 activation affect the immune response against invading Gram-negative bacteria but it is also involved in the development and progression of a number of neurodegenerative diseases (Schröder and Schumann, 2005). In the CNS, constitutive expression of TLR4 transcripts has been described in distinct anatomical areas of the brain (Lacroix et al., 1998 and Laflamme and Rivest, 2001). In this respect,it was reported that microglia but not astrocytes or oligodendrocytes express TLR4, and that TLR4 is required for LPS-induced oligodendrocyte death in vitro (Lehnardt et al., 2002). Recently, TLR2 has also been reported to be involved in neurodegeneration (Koedel et al., 2007, Okun et al., 2009 and Ziegler et al., 2011). Among TLRs, TLR2 seems to be the most promiscuous TLR receptor capable of recognizing the widest set of different pathogens. TLR2 complexes with TLR1 or TLR6 are involved in the recognition of bacterial lipoproteins (Akira and Takeda, 2004 and Gay and Gangloff, 2007). TLR2 can also interact with other molecules such as CD36 (Triantafilou et al., 2006) or CD14 (Yang et al., 1999 and Flo et al., 2002) and can induce multimerization in response to different microbial ligands (Triantafilou et al., 2006). Interestingly, LPS is able to cause a robust transcriptional induction of TLR2 in the brain, but this receptor does not modulate the immune response to LPS in the brain (Naert et al., 2009).By contrast, in the central nervous system (CNS), TLR4 has been reported to be constitutively expressed in microglia (Lehnardt et al., 2003) and its ligand, LPS, induces the production of inflammatory mediators including tumor necrosis factor alpha (TNF-α), IL-6, and nitric oxide (NO) (Akira and Takeda, 2004) via the nuclear factor κ B (NF-κB) signaling pathway.Considering the critical role of TLR4 in neuroinflammation and brain injury, the aim of this review is to focus on reports concerning recent insights into the role and activation mechanisms of TLR4 in the brain, not only in pathological events but also in physiological conditions, as well as the therapeutic benefit that could derive from TLR4 modulation.2. TLR-4 signalingIn the host system, LPS capture is facilitated by the LPS binding protein (LBP) which transfers it to the receptor complex composed of CD14, MD-2 (or LY96) and TLR4. Upon LPS binding, TLR4 recruits, through its short intracellular TIR domain, adaptor molecules and kinases, thus initiating a downstream signaling cascade that culminates in the secretion of pro-inflammatory cytokines and chemokines (Takeuchi and Akira, 2002 and Takeda and Akira, 2005). Activation of TLR4 by LPS induces two signaling pathways known as the myeloid differentiation primary response gene 88 (MyD88) dependent and independent pathways (Akira et al., 2006).The MyD88 dependent pathway in TLR4 signaling requires the adaptor proteins TIRAP (TIR domain containing adaptor protein) and MyD88 to initiate a downstream cascade leading to nuclear translocation of the nuclear factor (NF)-κ B and mitogen associated protein (MAP) kinase signaling pathways (such as the ERK-CREB pathway, the JNK-AP1 pathway, and the p38 pathways), resulting in the production of pro-inflammatory cytokines (Kagan and Medzhitovd, 2006). This leads to the rapid expression of inducible nitric oxide synthase (iNOS) and a wide variety of proinflammatory cytokines, chemokines, and their receptors, including tumor necrosis factor alpha (TNF-α), IL-1α, IL-1β, IL-1ra, IL-6, IL-8, IL-10, IL-12p40, IL-23, and macrophage inflammatory protein (MIP)-1α, and MIP-1β (Lee and Kim, 2007). These factors initiate the inflammatory response, increase vascular permeability, direct dendritic cells (DC) and macrophage migration from the periphery to the central lymphoid organs, and regulate various aspects of adaptive immunity development. On the other hand, the independent signaling pathway is controlled by the adaptors TICAM (Toll-like receptor adaptor molecule) 1 or TRIF (TIR-domain-containing adaptor inducing interferon-β) and TICAM 2 or TRAM (TRIF-related adaptor molecule), which activate the transcription factor IRF3 (IFN regulatory factor 3) and the production of IFN-βand chemokine RANTES (Regulated on Activation Normal T cell Expressed and Secreted) (Yamamoto et al., 2003a and Yamamoto et al., 2003b).TLR4 engagement leads to the production of neurotoxic molecules such as proinflammatory cytokines, NO,reactive oxygen species (ROS), and peroxynitrite (Xie et al., 2002). Moreover, LPS-activated microglia produce a large amount of glutamate, an important neurotransmitter which in some circumstances acts as a potent neurotoxin (Takeuchi et al., 2006). LPS challenge may also activate TLR4 on the microglia surface, leading to oligodendrocyte injury (Lehnardt et al., 2002). Recently, CNS-relevant in vitro and in vivo studies have highlighted the function of suppressor of cytokine signaling (SOCS) proteins under various neuroinflammatory or neuropathological conditions. SOCS1 and SOCS3 have been described as having a short half-life (1–2 h) and their expression levels are reported to increase rapidly following macrophage exposure to inflammatory cytokines and TLR ligands. Expression of SOCS1 and SOCS3 is regulated primarily by activation of STAT1 and STAT3, respectively, although their expression can be mediated through other signaling cascades, including the mitogen activated protein kinase (MAPK) and NF-κ B pathways (Wang and Campbell, 2002 and Blach-Olszewska and Leszek, 2007). Moreover, SOCSs not only influence cytokine and growth hormone signaling, but also the signaling pathway initiated by the reaction of a TLR with PAMP or an endogenous molecule. SOCS-1 negatively regulates TLR signaling by mediating the degradation of the adapter protein Mal. This protein is involved in signaling via TLR2 and TLR4. Because of their obvious biological importance, the SOCS proteins have been the subject of intense investigation, including the development of strategies to utilize these proteins to control cytokine-induced JAK/STAT signal transduction for therapeutic purposes (Blach-Olszewska and Leszek, 2007).inflammatory diseases such as arthritis and atherosclerosis (Erridge, 2010). Paradoxically, TLR activation by endogenous ligands following ischemia worsens stroke damage, therefore these molecules, recen tly termed “alarmins”, have been suggested to serve as mediators of inflammation that may be expressed or released in response to tissue damage and, therefore, have also been described as DAMPs (Bianchi, 2007).In the brain the following molecules have been reported as DAMPs: heat shock proteins (HSPs), β-amyloid (Aβ), hyaluronan, heparin sulfate, DNA or RNA immune complex, oxidized low-density lipoproteins (oxLDL), and others, all able to stimulate TLRs (Marsh et al., 2009, Rivest, 2009, Yanai et al., 2009, Stewart et al., 2010 and Zhang et al., 2010).Among DAMPs, high-mobility group box-1 (HMGB1), a non-histone nuclear protein, has been reported to exert its biological effects through the activation of signaling pathways coupled to TLRs, including TLR4 and RAGE, both of which are involved in inflammatory responses (Park et al., 2004, Yang et al., 2010, Rauvala and Rouhiainen, 2010 and Volz et al., 2010). Recent studies show that elevated brain levels of HMGB1 induce memory abnormalities which may be mediated by either TLR4, or RAGE. This mechanism may contribute to memory deficits under various neurological and psychiatric conditions associated with increased HMGB1 levels, such as epilepsy, Alzheimer's disease and stroke (Mazarati et al., 2011).3. TLR4 in the brainTLRs are described in the brain where, until recently, their expression was believed to be limited to microglia (Olson and Miller, 2004), astrocytes (Bowman et al., 2003), and oligodendrocytes (Bsibsi et al., 2002). In addition, the expression of certain TLRs has recently been documented in mammalian neurons (Prehaud et al., 2005 and Wadachi and Hargreaves, 2006) and appears to be implicated in several non-immune processes, such as bone metabolism (Bar-Shavit, 2008), neurogenesis (Rolls et al., 2007) and brain development (Ma et al., 2007).In the last decade a number of studies demonstrated the localization of TLR4 in the CNS. Laflamme and Rivest (2001) reported that TLR4 is present in the rat CNS structures that can be reached through the bloodstream: circumventricular organs, choroid plexus and leptomeninges. The constitutive expression of TLR4 may explain the innate immune response in the brain, which originates from the structures devoid of the blood–brain barrier in the presence of circulating LPS, thus suggesting a role for TLR4 acting as a sensor for engaging the cerebral innate immune response in the case of invasion during systemic bacterial infections, which may have detrimental consequences for the neuronal structures (Laflamme and Rivest, 2001).In the CNS, previous findings reported that TLR4 is preferentially expressed on microglia as compared to astrocytes, whereas it is present at very low or undetectable levels on neurons (Kim et al., 2000). However, other findings report that cerebral cortical neurons express both TLR2 and -4, whose expression appears to be increased in response to ischemia/reperfusion injury, whereas the entity of brain damage and neurological deficits caused by stroke is significantly smaller in mice deficient in TLR2 or -4 as compared with WT control mice (Tang et al., 2007a). Although the presence of TLR4 appears controversial on neurons, considerable evidence points to a role for this receptor in neurons in physiological as well as pathological conditions (Mishra et al., 2006;Wadachi and Hargreaves, 2006 and Tang et al., 2007b).LPS is a potent stimulator of microglia, whereas resting murine astrocytes in culture have been shown to express no TLR4 (Sola et al., 2002 and Falsig et al., 2004a) or very low levels (Bowman et al., 2003). In spite of this, astrocytes in culture have been reported to respond to LPS (Bowman et al., 2003, Esen et al., 2004 and Carpentier et al., 2005). Published reports draw different conclusions regarding the TLR4 responsiveness of astrocytes, probably due to differences in the preparation of astrocyte cultures (Bsibsi et al., 2002, Falsig et al., 2004b and Carpentier et al., 2005). For example, Holm et al. (2012) reported that astrocytes in the presence of microglia are capable of responding to TLR2, -3, and -4 ligation, whereas in the absence of functional microglia, the astrocytes no longer responded to TLR4 ligation and responded weakly to TLR2 and -3 stimulation. These observations suggest that the response of astrocytes to TLR agonists is in large part due to bystander activation by microglia, or the release of soluble factors that permit autonomous astrocyte responses. It could thus be concluded that the cells of the CNS may become activated in the presence of TLR agonists even though they do not express the TLR receptor themselves.However, microglia are much more responsive than astrocytes to LPS when assayed in a tissue culture environment, whereas neurons are virtually unresponsive (Saijo et al., 2009). Interestingly, LPS challenge of the neurons has little effect on gene expression or survival as compared to the neurotoxicity observed after treatment of neurons with conditioned media from LPS-treated microglia (Saijo et al., 2009).In this respect, in an in vivo model of neurodegeneration it was reported that the administration of LPS is able to stimulate innate immunity, causing extensive neuronal and axonal loss in the cortex. In contrast, animals bearing a loss-of-function mutation in the tlr4 gene are resistant to neuronal injury, thus demonstrating a mechanistic link among innate immunity, TLRs, and neurodegeneration (Lehnardt et al., 2003).Shichita et al. (2012) reported that TLR2 and TLR4 deficiency suppresses inflammatory cytokine expression in infiltrating immune cells on day 1 after brain ischemia, thus demonstrating that both TLR2 and TLR4signaling cascades are essential to trigger post-ischemic inflammation, and activate infiltrating immune cells.However, a molecular model of LPS-induced neuroprotection from ischemic injury has been proposed, whereby systemic LPS preconditioning reprograms TLR4 signaling in response to stroke, directing it towards a neuroprotective pathway, via TRIF to IRF3, up-regulating IFNβ (Marsh et al., 2009).4. TLR4 polymorphismIt has been reported that genetic variations of TLR4 greatly influence immune responses towards pathogenic challenges and disease outcome. The TLR4 gene was sequenced from 348 human samples and 35 mouse strains by Smirnova et al. (2001) and resulted highly polymorphic. Since the frequency of genetic variations is highest in the extracellular domain, responsible for the recognition of PAMPs, it is possible that the evolutionary pressure exerted by pathogens may explain the more frequent polymorphism observed in this domain (Schröder and Schumann, 2005). Thirteen different polymorphisms of the TLR4 gene have been reported in Caucasian populations (Orange and Geha, 2003), whereas another study conducted in a Dutch population reported a further 13 variants of TLR4 (Puel et al., 2004). There is enormous heterogeneity and controversy among the various reports about the association between TLR4 polymorphism and different diseases, such as viral or fungal infection, cancer and autoimmune diseases. Recently, the association between the TLR4 polymorphism gene and Alzheimer's disease (AD) was investigated by Balistreri et al. (2008) showing the involvement of TLR4 in AD pathophysiology. However, most of the studies indicating this association between TLR4 polymorphisms and brain disease susceptibility are preliminary and have yet to be confirmed (Noreen et al., 2012).5. TLR4 in neurodegenerationMicroglia, the resident cells in the CNS, are described as a distinct cell type at close contact with neurons and astrocytes: in normal conditions microglial immune response assures the CNS parenchymal integrity, whereas sustained overaction of microglia is a detrimental event, since it is responsible for irreversible neuronal damage, contributing to chronic neurodegenerative diseases, such as traumatic brain injury (TBI),Parkinson's disease (PD), AD and multiple sclerosis (MS) (Kim and Joh, 2006 and Panaro and Cianciulli, 2012).In fact, it is well recognized that neuroinflammation constitutes an important risk factor for the progression of several neurodegenerative disorders. In this context, TLRs have been shown to be implicated in several CNS diseases, and accumulating evidence demonstrates that TLR4 contributes to neuronal death, blood brain barrier damage, brain edema, and inflammatory responses in the brain injury induced by ischemia (Caso et al., 2007 and Hua et al., 2007).TLR4-mediated NF-κ B signaling plays a vital role in the initiation of cerebral inflammation in CNS diseases (Kerfoot et al., 2004 and Hua et al., 2007), leading to the transcription of many pro-inflammatory genes encoding cytokines, chemokines, and enzymes such as COX-2 and MMP-9, mediators that are involved in the development of secondary brain injury following TBI (Wang et al., 2000 and Lucas et al., 2006). The upregulation of cytokines and chemokines could activate microglia, thus initiating infiltration of inflammatory cells into the brain which may cause neuronal loss (Allan and Rothwell, 2001 and Morganti-Kossmann et al., 2002).Another cause of brain damage is alcohol abuse, responsible for cognitive dysfunctions, and even neurodegeneration in some cases (Crews and Nixon, 2009). In fact, alcohol, by activating TLR4 receptor signaling in glial cells (Blanco et al., 2005), can induce neuroinflammatory brain injury (Blanco and Guerri, 2007).A recent study supports and demonstrates the hypothesis that TLR4 is critical for the ethanol-induced inflammatory signaling in astrocytes, since the knockdown of TLR4 abolished the activation of the MAPK and NF-κB pathways as well as the production of inflammatory mediators by astrocytes (Alfonso-Loeches et al., 2010).6. Role of TLR4 in traumatic brain injurySeveral animal studies have shown that both TLR4 mRNA and protein are upregulated following TBI (Chen et al., 2008, Chen et al., 2009 and Dong et al., 2011). TBI induces a complex series of inflammatory responses that contribute to neuronal damage and behavioral impairment (Chen et al., 2012). TBI is caused by the primary injury, due to the effects of biomechanical injury, and secondary injury which manifests over a period of hours to days and months following the initial impact, and includes complex biochemical and physiological processes. A robust neuroinflammation occurs after TBI and contributes to the development of the secondary injury. Generally, TBI induces rapid activation of glial cells and recruitment of granulocytes,T cells as well as monocytes/macrophages from the blood stream. However, neuroinflammation can have both beneficial and deleterious roles after TBI (Zhang et al., 2012).TLR4-mediated signaling pathways mainly stimulate the activation of NF-κ B. This important nuclear transcription factor regulates many pro-inflammatory genes, e.g., cytokines, chemokines, cyclooxygenase-2 (COX-2), and matrix metalloproteinase-9 (MMP-9), mediators involved in the pathogenesis of TBI (Medzhitov et al., 1997). Ahmad et al. (2013) demonstrate that TLR4 is critical for the TBI-induced inflammatory signaling in astrocytes, since the knockdown of TLR4 abolished the activation of the MAPK and NF-κ B pathways and the production of inflammatory mediators by astrocytes (Ahmad et al., 2013). These studies suggest that pharmacological inhibition of TLR4/NF-κ B signaling may be a useful strategy for protecting the injured brain (Chen et al., 2012). In fact, Chen (Chen et al., 2012) showed that wogonin treatment decreased the number of microglia, macrophages, and neutrophils recruited to the injured areas of the brain, reduced NF-κ B activation and translocation to the nucleus, and interfered with NF-κ B binding activity, expression of inflammatory mediators (IL-1β, IL-6, MIP-2, and COX-2), and MMP-9 activity in the injured brain. Wogonin treatment leads to improved long-term functional and histological outcomes and reduced brain edema in a clinically relevant model of TBI. This improvement was associated with a modulation of the TLR4/NF-κ B signaling pathway(Chen et al., 2012).HMGB1, originally identified as a nonhistone chromatin DNA binding protein, is now recognized as representative of damage-associated molecular patterns.Once released into the extracellular space from necrotic or activated cells, HMGB1 triggers the inflammatory response through the activation of multiple receptors, including TLR4 (Okuma et al., 2012), and then these receptors activate a common signaling pathway that culminates in the activation of NF-κ B transcription factors.Interestingly, the neuroprotective effects of ethyl-pyruvate treatment in a weight-dropping TBI rat model seem to be associated with inhibiting activation of the HMGB1/TLR4/NF-κB pathway (Su et al., 2011).7. Alzheimer's disease and TLR4AD is the most common neurodegenerative disease, characterized by progressive neuronal death and memory loss.Although the etiology of the disease is unknown, accumulation of β amyloid (Aβ) leads to the development of extracellular senile plaques and intracellular neurofibrillary tangles.Several inflammatory markers, such as cytokines and chemokines or proteins of the acute phase and complement are elevated in the AD brain (Capiralla et al., 2012). Microglia are found in an activated state around senile plaques in the brains of AD patients and are considered to be important in the pathogenesis of the disease. Activation of microglia results in the production of NO, oxygen free radicals, proteases, adhesion molecules and proinflammatory cytokin es such as TNFα, IL-1β, LT-a, and IL-6. It is thought that an overproduction of these inflammatory mediators is important in the degenerative process in patients with AD (Carty and Bowie, 2011).Morales et al. found that cytokine-containing conditioned media of activated rat microglia cells in primary cultures promote degeneration, as well as tau hyperphosphorylation, of cultured hippocampal neurons obtained from rat embryos (Morales et al., 2010), confirming the neurotoxic role of microglia (Morales et al., 2010). There is a growing body of evidence that microglial activation occurs by TLRs in AD even if cellular TLRs-mediated responses may have either a positive or negative impact. Animal models of AD and patients with AD exhibit increased expression of CD14 (a co-receptor for TLR4), TLR4 and TLR2 (Fassbender et al., 2004, Liu et al., 2005, Walter et al., 2007 and Letiembre et al., 2009), which are thought to occur independently in response to the presence of Aβ. Interestingly, a polymorphism in TLR4 Asp299Gl y resulted in a 2.7 fold reduction in the risk for late onset AD (Minoretti et al., 2006). The inhibition of TLR4 or TLR2 through function blocking antibodies or siRNA knockdown also prevented fib rillar Aβ (FAβ)-induced nitrite, IL-6, and TNF-α production (Walter et al., 2007, Jana et al., 2008 and Udan et al., 2008).Senile plaque-associated microglia show increased mRNA levels of TLR 2, 4, 5, 7 and 9 (Frank et al., 2009) and a physical interaction between CD14 and FAβ was demonstrated by Reed-Geaghan et al. (2009). The signal transduction cascades triggered by FAβ are identical to those triggered by TLR agonists, and Aβ induction of NF-κB depend ent genes requires TLR2, TLR4 and CD-14 (Reed-Geaghan et al., 2009), thus indicating that TLRs are important in sensing and responding to the presence of Aβ (Carty and Bowie, 2011).Treatment of microglia with AD plaque material induced a strong up-regulation of TLR2, TLR4, TLR5, TLR7 and TLR9 mRNA compared with age-matched plaque-free tissue (Hanke and Kielian, 2011). The activation of microglia by Aβ may be beneficial by pr omoting CD14-, TLR4-, or TLR2-dependent phagocytosis and clearance of Aβ (Iribarren et al., 2005, Liu et al., 2005 and Chen et al., 2006); Tahara et al. (2006) have demonstrated, in an AD mouse model homozygous for a nonfunctional (loss-of-function) mutation of TLR4, an increase in diffuse and FAβ deposits as well as buffer-soluble and insoluble Aβ in the brain in comparison to a TLR4 wild-type AD mouse model (TgAPPswe/PS1dE9 mice) at 14–16 months of age. In this context, Aβ deposits are able to induce the upregulation of certain cytokines and chemokines in the brain of the same model at 13–15 months of age through TLR4 signaling (Jin et al., 2008).Interestingly, a further study conducted in a 5-month-old AD mouse model reports that TLR4 mutation reduces microglial activation, increases Aβ deposits and exacerbates cognitive deficits. It was concluded that TLR4 is not involved in the initiation of Aβ deposition and that, as Aβ deposits st art, microglia are activated via TLR4 signaling to reduce Aβ deposits and preserve cognitive functions from Aβ-mediated neurotoxicity (Song et al., 2011). On the basis of these observations, activation of microglia via TLR4 in early stages of AD pathogenesis is neuroprotective, and TLR4 signaling pathways may offer potential therapeutic targets.Recent evidence showed that resveratrol acts upstream in the activation cascade by interfering with TLR4 oligomerization upon receptor stimulation. Activation of NF-κB in macrophages requires the phosphorylation of IKK by Akt, and several flavonoids have been proposed to inhibit this pathway by interfering with the Akt activating kinase phosphatidylinositol 3-kinase (Lee et al., 2006, Chen et al., 2007 and Capiralla et al., 2012).8. Multiple sclerosisMS is a chronic and debilitating autoimmune-mediated disease of the CNS. It is characterized by inflammation, demyelination and progressive axonal degeneration. The exact cause of MS is still unknown, although it has been hypothesized that genetic influences, environmental factors and infectious agents might be implicated in the pathogenesis (Urcelay et al., 2007).MS is believed to be principally mediated by CD4+ T cells that are activated in the periphery against myelin antigens, extravasate across the blood–brain barrier and invade the CNS where they contribute to the demyelination and progressive axonal pathology (Racke and Drew, 2009).In addition to lymphocytes (T and B cells), dendritic cells and tissue macrophages also play a role in the pathogenesis. These cells express TLRs and following ligand binding to TLRs, innate immune cells produce proinflammatory cytokines and can serve as antigen-presenting cells to prime naïve T cells to recognize antigens.When chronically activated, glia (astrocytes and microglia) are involved in the pathogenesis, in part through PAMP binding to TLRs present on these cells, which can contribute to the reaction of myelin-specific autoreactive T cells in the CNS (Racke and Drew, 2009).Oligodendrocytes are the myelin producing cells of the central nervous system and their destruction is a central feature of MS (Compston and Coles, 2008). In this regard, Yao et al. reported that TLR4 engagement can activate neuronal NOS in cultured primary oligodendrocyte precursor cells (OPC) as well as in an established human oligodendroglial cell line, MO3.13 (Yao et al., 2010a and Yao et al., 2010b). LPS binding to TLR4 causes a decreased cell viability with a loss of mitochondrial membrane potential and reduction of enzymatic activity of complex I and complex IV proteins associated with the release of cytochrome C from mitochondria into the cytosol. Cell death mediated by mitochondrial injury was shown to be due to the activation of both caspase dependent and independent pathways. Although it is not clear whether these pathways are exclusive to TLR4 activation or common to other PAMP receptors, these mechanisms may play a role in diseases characterized by oligodendrocyte loss and demyelination (Yao et al., 2012).9. Experimental autoimmune encephalomyelitisExperimental autoimmune encephalomyelitis (EAE), a well-established animal model of MS, is characterized by inflammation and demyelination of the CNS, exhibiting some of the symptoms and pathologic processes observed in MS patients (Constantinescu et al., 2011).Two reagents commonly used for EAE induction, Mycobacterium tuberculosis and pertussis toxin (PTX), have been shown to signal through the TLR4 pathway, implicating innate immune mechanisms in the development of CNS autoimmune disease ( Heldwein et al., 2003, Kerfoot et al., 2004 and van de Veerdonk et al., 2010). Kerfoot and colleagues reported that EAE was less severe when induced in TLR4-deficent animals ( Kerfoot et al., 2004). Another group, conversely, found that complete TLR4 deficiency resulted in the exacerbation of EAE symptoms ( Marta et al., 2008), thus, the role of TLR4 in EAE remains controversial.Macrophages play indispensable roles in the development of EAE (Mensah-Brown et al., 2011). In fact, blood-derived macrophages are implicated in the development of EAE because of their ability to present antigens, secrete inflammatory cytokines (Benveniste, 1997) and participate in demyelination by phagocyting degraded myelin (Bauer et al., 1994). A number of studies have implicated TLR ligands in the development of EAE (Waldner, 2009). Among these, HMGB1 was reported to be released in the brain after cytokine stimulation and to be involved in inflammatory response after intracerebroventricular administration. RAGE, TLR2 and -4 are the receptors involved in HMGB1 binding (Andersson et al., 2008). In fact, Andersson et al. (2008) showed that macrophages and microglia are major sources of HMGB1 in both MS lesions and rodent EAE lesions, and demonstrated both in tissues and in cerebrospinal fluid cells that an increased HMGB1 expression was accompanied by a high expression of RAGE, TLR2, and TLR4, amplifying inflammatory responses in MS and EAE diseases (Andersson et al., 2008).It was also shown that the p19 subunit of IL-23, a proinflammatory cytokine, was produced by macrophages and microglia in human MS white matter lesions, and that its expression in human microglia is induced via TLR2 and/or TLR4 signaling, further confirming the role of both TLR2 and TLR4 in EAE induction (Li et al., 2007).Ajami et al. reported that, apart from infiltrating blood-derived monocytes involved in EAE progression, CD4 T cells are responsible for disease initiation (Ajami et al., 2011). Naïve CD4 + T cells differentiate into various effector lineages depending on the environment at the time of activation (Dong and Flavell, 2000). Th1 cells are characterized by a requirement for IL-12 as well as the production of IFN-γ. The recently identified Th17 lineage is characterized by IL-17 expression (Park et al., 2005 and Harrington et。