Microglial toll-like receptors and Alzheimer’s disease

Brain, Behavior, and Immunity xxx (2015) xxx–xxx

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Microglial toll-like receptors and Alzheimer’s disease Fan Su, Feng Bai ⇑, Hong Zhou, Zhijun Zhang Department of Neurology, Affiliated ZhongDa Hospital, School of Medicine, Southeast University, Nanjing, Jiangsu 210009, China

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Article history: Received 16 August 2015 Received in revised form 9 October 2015 Accepted 15 October 2015 Available online xxxx Keywords: Alzheimer’s disease Toll-like receptors Microglia Neuroinflammation

a b s t r a c t Microglial activation represents an important pathological hallmark of Alzheimer’s disease (AD), and emerging data highlight the involvement of microglial toll-like receptors (TLRs) in the course of AD. TLRs have been observed to exert both beneficial and detrimental effects on AD-related pathologies, and transgenic animal models have provided direct and credible evidence for an association between TLRs and AD. Moreover, analyses of genetic polymorphisms have suggested interactions between genetic polymorphisms in TLRs and AD risk, further supporting the hypothesis that TLRs are involved in AD. In this review, we summarize the key evidence in this field. Future studies should focus on exploring the mechanisms underlying the potential roles of TLRs in AD. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction Alzheimer’s disease (AD), a neurodegenerative disease that is characterized by a progressive decline in cognitive and functional abilities, is one of the leading causes of disability among the elderly, and the number of people affected by AD is expected to increase in

Abbreviations: AD, Alzheimer’s disease; TLRs, toll-like receptors; AbPP, amyloidb protein precursor; NSAIDs, non-steroidal anti-inflammatory drugs; CNS, central nervous system; TNF-a, tumor necrosis factor-a; IL-1b, interleukin 1-b; IL-6, interleukin-6; NO, nitric oxide; ROS, reactive oxygen-nitrogen species; DAMPs, danger-associated molecular patterns; PAMPs, pathogen-associated molecular patterns; PRRs, pattern recognition receptors; NF-jB, nuclear factor jB; MAPK, mitogen-activated protein kinase; Ab, amyloid-b; PET, positron emission tomography; PS1, presenilin 1; iNOS, inducible nitric oxide synthase; PGN, peptidoglycan; PI3K, phosphatidylinositide 3-kinases; IKK, IjB kinase; MyD88, myeloid differentiation factor; FPRL1, formyl peptide receptor-like 1; MMP-9, matrix metalloproteinase-9; SIGIRR, single-Ig-interleukin-1 related receptor; PPARc, peroxisome proliferator-activated receptor c; LPS, lipopolysaccharide; MPL, Monophosphoryl lipid A; IFN, interferon; TRIF, Toll/interleukin-1 receptor domain-containing adaptor inducing IFNb; ISGs, IFN-stimulated genes; TRAF6, TNF receptor associated factor 6; TIRAP, Toll-IL-1R domain-containing adapter protein; IRAK, IL-1R-associated kinase family; TRAM, TRIF-related adaptor molecule; FoxO1, forkhead box O1; IRF3, interferon regulatory factor 3; ICV, intracerebroventricular; CpG, cytosine–guanosine-containing oligodeoxynucleotides; NFT, neurofibrillary tangles; HO-1, heme oxygenase-1; CCR1, C–C chemokine receptor type 1; CX3CR1, CX3C chemokine receptor 1; mFPR2, mouse homologue formyl peptide receptor 2; HSP70, heat shock protein 70; AIF, apoptosis-inducing factor; GT repeat, Guanine–thymine repeat; LOAD, late-onset Alzheimer’s disease; SNPs, single nucleotide polymorphisms; JNK, c-Jun N-terminal kinas. ⇑ Corresponding author at: Department of Neurology, Affiliated ZhongDa Hospital, School of Medicine, Southeast University, Nanjing, Jiangsu 210009, China. E-mail addresses: [email protected] (F. Su), [email protected] (F. Bai), [email protected] (H. Zhou), [email protected] (Z. Zhang).

the coming years unless effective methods are found to cure the disease or to halt the associated declines in cognitive functions (Brookmeyer et al., 2007). Substantial effort has been made to explore the exact mechanisms underlying AD, and emerging evidence has suggested that inflammation may exacerbate or even cause AD. This concept is not new; approximately two decades ago, the demonstration of the activation of both the complement system and the innate immune system in AD patients implied an association between AD and neuroinflammation (Retz et al., 1998; Yasojima et al., 1999). Moreover, increased levels of inflammatory cytokines and chemokines have been detected in the postmortem brains of both AD patients and amyloid-b protein precursor (AbPP) transgenic animals (Cacabelos et al., 1991; Grammas and Ovase, 2001; Perry et al., 2001; Xia and Hyman, 1999; Xia et al., 1998). Although with some controversies (Arvanitakis et al., 2008; Breitner et al., 2009), epidemiological investigations have provided further support for this concept by showing that the long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) can reduce the risk of developing AD, especially in individuals who are treated in middle ages (Landi et al., 2003; Vlad et al., 2008). In some experiments, the explanation for why NSAIDs failed to show protective effects may be that the drugs were provided for elderly individuals, whose AD pathology had already developed prior to treatment with anti-inflammatory medications (Arvanitakis et al., 2008; Breitner et al., 2009). Dysfunctions in microglia, which are brain-specific macrophages and the most important immune cells in the central nervous system (CNS), are receiving an increasing amount of attention in the context of the neuroinflammatory process in AD. In healthy brains, microglia are in a ‘resting’ state, and they

http://dx.doi.org/10.1016/j.bbi.2015.10.010 0889-1591/Ó 2015 Elsevier Inc. All rights reserved.

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perform immune surveillance (Nimmerjahn et al., 2005). Under pathological conditions, microglia can be activated by various stimuli, and different types of stimulation induce microglial activation toward a ‘classical (M1)’ or ‘alternative (M2)’ state (Colton and Wilcock, 2010). The M1 activated microglia secrete various pro-inflammatory cytokines [such as tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), and interleukin 1-b (IL-1b)] and cytotoxic factors [such as nitric oxide (NO) and reactive oxygen species (ROS)] (Aloisi, 2001) to promote the destruction of pathogens (Kettenmann et al., 2011). However, the non-specific immune responses can simultaneously induce neurotoxicity in the healthy tissue (Colton and Wilcock, 2010). Hence, M2-activated microglia play key roles in maintaining homeostasis by secreting anti-inflammatory cytokines [for example, IL-10 and transforming growth factor-b] to down-regulate the pro-inflammatory processes and promote tissue reconstruction (Colton and Wilcock, 2010). The activation of microglia has been demonstrated in AD tissue, especially in surrounding amyloid plaques (Cagnin et al., 2001; Higuchi, 2009), and the proinflammatory factors released from activated microglia appear to contribute to detrimental effects (Galasko and Montine, 2010; Zaheer et al., 2008). However, the beneficial effects of microglial activation also deserve substantial attention. In particular, activated microglia clear amyloid plaques (Hickman et al., 2008; Maier et al., 2008; Tahara et al., 2006), which are believed to be not only the core hallmark of AD but also the initiator of downstream responses that exacerbate neurodegeneration. The complexity of microglial activation makes it difficult to completely understand the role of microglia in AD, but it has been suggested that microglial dysfunction augments their neurotoxic effects and suppresses their neuroprotective functions, thus contributing to the process of the disease. If this hypothesis is true, the modulation of microglial activation may be a promising target for AD therapy. Toll-like receptors (TLRs) are important members of the family of pattern recognition receptors (PRRs). In mammals 12 types of TLRs have been detected in various cell types and human microglia express TLRs 1–9. TLRs are transmembrane proteins that are composed of highly conserved structural domains, containing the binding sites for both their ligands and their coreceptors. They recognize specific ligands to initiate the inflammatory process, activating signaling molecules such as the transcription factor nuclear factor jB (NF-jB) and the mitogen-activated protein kinase (MAPK) to promote microglial phagocytosis, cytokine release and the expression of the co-stimulatory molecules needed for adaptive immune responses (Hanke and Kielian, 2011). Thus, TLRs serve as the first line of defense against pathogens, and their activations result in the death or disposal of the invading pathogen. Moreover, in recent decades, increasing evidence has suggested the involvement of TLRs in AD. In this review, the key evidence supporting the important roles of microglia in AD is summarized. Additionally, we elucidate the influences of manipulating microglial TLRs on AD pathology, including the operations of TLRs gene in AD animal models. Finally, the genetic polymorphisms that suggest the disruption of TLRs in the brains of patients with AD are illustrated. 2. Microglial activation in AD As early as the 1990s, various postmortem experiments demonstrated microglial activations in the brains of AD patients, especially in the vicinity of amyloid-b (Ab) peptide-containing plaques (McGeer et al., 1987; Styren et al., 1990; Wisniewski et al., 1992). Ultrastructural analyses demonstrated individual microglia extending their finger-like processes into the core of plaques (Wisniewski et al., 1992), suggesting a specific microglia-Ab

association and indicating that Ab may be the major driving force for microglial activation. Furthermore, the development of in vivo positron emission tomography (PET) imaging has greatly expanded our understanding of microglial activation in AD. Several studies reported an increased activated microglial load in AD brains, and this increase was directly correlated with the degree of cognitive deficits (Edison et al., 2008; Okello et al., 2009; Versijpt et al., 2003; Yasuno et al., 2012). Additionally, activated microglia have been detected in various animal models. For instance, in the AbPP, P301S and tripletransgenic (3
Tg-AD) mice, an increased number of activated microglia labeled by immunostaining were reported (Bellucci et al., 2004; Bornemann et al., 2001; Janelsins et al., 2005), and ultrastructural analysis showed microglia extending finger-like processes into Ab fibrils (Stalder et al., 2001), similar to findings reported in the human brain (Wisniewski et al., 1992). Lines of in vivo PET imaging studies have further demonstrated the activation and both Ab-driven and age-dependent activation occur (Meyer-Luehmann et al., 2008; Rapic et al., 2013; Venneti et al., 2009). Microglial activation in AD is suggested to be heterogeneous, and Ab plaques are considered to be the major driving forces. Numerous in vitro experiments have demonstrated the Abinduced microglial expression of inflammatory factors, including pro-inflammatory cytokines, chemokines, ROS, reactive nitrogen species and acute phase proteins (Mandrekar-Colucci and Landreth, 2010). Most of these factors were found to be neurotoxic in AD process as they disrupted synaptic plasticity (Tong et al., 2012), suppressed microglial clearance of Ab (Yamamoto et al., 2008) and promoted tauopathies (Li et al., 2003). Moreover, microglia taken from AD patients (Lue et al., 2001) showed increased reactivity to Ab stimulation. Similarly, microglia from various AD animal models, such as AbPP/presenilin 1 (PS1), P301S and triple-transgenic (3
Tg-AD) mice (Bellucci et al., 2004; Hickman et al., 2008; Janelsins et al., 2005) released increased levels of a set of pro-inflammatory factors but expressed decreased levels of Ab-degrading enzymes and Ab-binding receptors, such as scavenging receptor A, CD36 and the receptor for advanced glycosylation end products (Hickman et al., 2008). These findings imply that activated microglia overexert their pro-inflammatory activity but down-regulate their Ab-clearing capabilities in AD. Thus, Abinduced activated microglia directly exert deleterious effects on neurons and promote the generation of Ab plaques, which further promotes microglial activation, forming in a vicious cycle. Studies have been performed to clarify other forces that drive microglial activation in addition to Ab plaques. For instance, AbPP and soluble Ab fragments are thought to trigger neuroinflammation (Barger and Harmon, 1997; Szczepanik et al., 2001). Tauopathies have also been demonstrated to activate microglia (Bellucci et al., 2004). Moreover, injuries to neurons and synapses represent key pathological markers of AD, and the substances released by impaired neurons can initiate microglial activation (Lehnardt et al., 2008). Furthermore, impaired brain metabolism, which may contribute to AD pathology and precede the clinical symptoms, was very recently shown to promote microglial activation (Turano et al., 2015; Yokokura et al., 2011). Moreover, microglial activation may partially arise from genetic factors, as recent genome wide association studies have identified several microgliarelated genetic polymorphisms that are associated with AD, such as triggering receptor expressed on myeloid cells 2 (TREM2), CD33 and CD2-associated protein (Karch and Goate, 2015). Therefore, multiple factors result in microglial activation in AD, and activated microglia exhibit complex functional alterations. The exact mechanisms by which microglia become activated and alter their function remain unclear. Higher levels of TLR mRNAs were detected in the brains of AD patients (Walter et al., 2007),

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suggesting the involvement of TLRs in AD. In addition, TLRs recognize a variety of danger- and pathogen-associated molecular patterns (DAMPs and PAMPs), including Ab, and TLR activation can lead to neuroinflammation and the microglial clearance of Ab. Thus, studying the roles of TLRs could enhance our understanding of the involvement of microglial dysfunctions in AD. In the next part of this review, we will discuss the implications of microglial TLRs in AD.

3. Toll-like receptors implicated in AD 3.1. TLR2 Increased expression levels of TLR2 have been found in the brains in both AD patients and AD animal models (Letiembre et al., 2009), and several agents have been identified to drive the activation of TLR2. Axonal injury has been observed in AD processes (Zhan et al., 2015), and damage to axons activated microglial TLR2 (Babcock et al., 2006). Further, Lewy bodies, consisting of alpha-synuclein, have been detected in AD brains (Hamilton, 2000), and alpha-synuclein caused pro-inflammatory responses by triggering microglial TLR2 activation (Daniele et al., 2015). Oxidized low density lipoprotein (oxLDL) also initiated microglial TLR2 activation (Bai et al., 2014), and emerging evidence has suggested that oxLDL is involved in AD (Dias et al., 2014; Kankaanpaa et al., 2009; Murr et al., 2014). Moreover, oligomeric and fibrillar Ab peptide-induced TLR2-dependent microglial activation has been frequently reported. First, Ab42 failed to induce the expression of inducible nitric oxide synthase (iNOS) or CD11b in the cortex of TLR2-deficient mice (Jana et al., 2008). Second, an antibody-induced blockade of TLR2 function was reported to neutralize Ab-induced pro-inflammatory cytokine release and neural cell injury, and similar results were obtained in microglia isolated from TLR2-deficient mice (Lin et al., 2013; Udan et al., 2008). Additionally, Liu and colleagues provided further evidence showing that inhibiting TLR2 could be beneficial in AD, as they demonstrated that genetic deficiency in TLR2 resulted in a conversion of microglial from the M1 to the M2 state in the brains of AbPP/ PS1-transgenic mice, accompanied by improvements in the expression of BDNF and in neuronal functions (Liu et al., 2012). More recently, TLR2 deficiency was proposed to mediate its beneficial effects via alternative mechanisms and to inhibit Abtriggered neuroinflammation. First, TLR2 deficiency increased the clearance of Ab, as it was observed to attenuate Ab burden in the brains of AbPP mice. This effect may be due to the TLR2 deficiency-promoted microglial phagocytosis of Ab, as has been observed in cultured macrophages (Liu et al., 2012). Second, TLR2 deficiency protected neurons and synapses against Ab-induced neurotoxicity. In Ab-treated organotypic hippocampal slice cultures (OHSCs), decreased synaptophysin, increased propidium iodide uptake (a measure of cell death) and changes in neuronal ultrastructure. Alterations including nuclear shrinkage, swollen mitochondria and degraded organelles were observed. However, these changes were not detected in TLR2-deficient OHSCs (Suh et al., 2013). Third, TLR2 deficiency relieved tauopathies, as it was observed that TLR2-deficient OHSCs showed resistance to the Ab-induced expression of phosphorylated tau (Suh et al., 2013). All of these data strongly suggest that TLR2 activation contributes to AD deterioration and that inhibiting TLR2 function may inhibit disease progression. However, conflicting results have been reported and TLR2-mediated Ab clearance has also been demonstrated in various experiments. In brains collected from AbPP/PS1–TLR2-/- mice, elevated Ab loads were detected compared with those from TLR2-wild type AbPP/PS1 mice (Richard et al., 2008). This concept has been further supported by the

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finding that activating TLR2 with peptidoglycan (PGN) can promote Ab uptake by microglia in various experimental systems (Chen et al., 2006; Tahara et al., 2006). More importantly, genetic deficiency in TLR2 contributes to more serious spatial and contextual memory impairments in AbPP/PS1 mice, and the reintroduction of TLR2 gene reversed this cognitive damage (Richard et al., 2008). Thus, apparently contradictory influences have been reported for the effects of TLR2 activation on AD pathology, especially in relation to Ab clearance by microglia, and the reasons for these discrepancies remain unclear. Great efforts have been made to explore the mechanisms by which TLR2 activation influences AD pathology, and parts of the TLR2-mediated signaling pathways have been identified. (A) TLR2 activation promotes M1 microglial activation. This effect is mediated by the MyD88/TNF receptor-associated factor 6 (TRAF6)/ MAPK/IjB kinases (IKKs)/NF-jB pathway and the MyD88/phosphatidylinositide 3 (PI3)-kinase (PI3K)/IKKs/NF-jB pathway (Jana et al., 2008; Lin et al., 2011; Lin et al., 2010; Lin et al., 2013; Qi et al., 2013). (B) TLR2 activation influences other functional components that have been implicated in AD. First, TLR2 activation was reported to promote the activity of formyl peptide receptor-like 1 (FPRL1) and matrix metalloproteinase-9 (MMP-9) (Bai et al., 2014; Chen et al., 2008), both of which are involved in Ab clearance and neuroinflammation in AD (Chen et al., 2006; Hu et al., 2014). Second, the anti-inflammatory and neuroprotective effects of TLR2 inhibition result, in part, from the consequent up-regulation of single-Ig-interleukin-1 related receptor (SIGIRR) and peroxisome proliferator-activated receptor c (PPARc) in microglia by the PI3/AKT pathway (Costello et al., 2015). The mechanisms underlying the involvement of TLR2 in AD are illustrated in Fig. 1, and they strongly suggest that TLR2 is implicated in the complex networks controlling Ab clearance and pro-inflammation. 3.2. TLR4 Similar to the effects of activating TLR2, the activation of TLR4 in AD has been shown to produce both beneficial and detrimental effects, as TLR4 is involved in both Ab clearance and release of neurotoxic factors during neuroinflammatory processes in AD. Primary microglia isolated from TLR4 mutant mice (C3H/HeJ mice) show a deficiency in mediating Ab-induced neuronal death (Walter et al., 2007). The mechanisms by which TLR4-mediated signaling pathways contribute to neurotoxicity are not completely understood, although Ab-induced TLR4-mediated nitrite and cytokine release has been demonstrated. First, pharmacological inhibition of TLR4 activation reduced Ab-induced microglial activation and the release of pro-inflammatory cytokines (Capiralla et al., 2012). Second, the antibody-induced blockade of TLR4 function was found to reduce the Ab-triggered release of nitrite and cytokines in vitro (Udan et al., 2008). Third, significantly increased levels of cytokines were detected in the brains of APP/PS1 mice, but not in TLR4mutant APP/PS1 mice compared with their non-transgenic littermates (Jin et al., 2008), further supporting the idea that Abinduced TLR4 activation promotes neuroinflammation in AD, and inhibiting TLR4 activation may be beneficial in remitting neuroinflammatory processes. In addition to Ab, high-mobility group box1 (HMGB1) and heat shock proteins (HSPs) have been identified as DAMPs in AD and they are suggested to influence the disease process by activating microglial TLR4. HMGB1 is a very important nonhistone chromosomal protein, with the cytokine-type functions upon its nuclear-to-cytoplasmatic translocation and subsequent celluar release. Elevated levels of HMGB1 were detected in AD brains, and they were thought to originate from dead neurons during the progression of AD (Scaffidi et al., 2002; Takata et al., 2003). The over-expression of HMGB1 could promote the neuroinflammation that exacerbates AD through microglial TLR4. This idea was

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Fig. 1. TLR2 implicated in AD. TLR2, located on the surface of microglia, activates downstream signaling pathways following ligand binding. This leads to the activation of NFkB and the subsequent induction of microglial M1 activation. In addition, TLR2 activation also promotes MMP9 and mFPR expressions, both of which are involved in Ab clearance and neuroinflammation. Moreover, TLR2 blockage facilitates microglial M2 activation, partly as a result of the increased expression of the anti-inflammatory factors, PPARc and SIGIRR. Straight arrows, activation; single-headed arrows, inhibition; dotted arrows, unknown mechanism; blue arrows: TLR2 inhibition-mediated signaling pathway; red arrows: PGN-activated signaling pathway. (For interpretation of the references to colour in this figure caption, the reader is referred to the web version of this article.)

supported by the observation that intracerebroventricular (ICV) administration of HMGB1 to mice resulted in memory deficits, and these deficits were not detected in TLR4 deficient mice (Mazarati et al., 2011). In the CNS, apoptotic and necrotic cells also produce HSPs, which in turn induce CNS damage. Both in vivo and in vitro, administration of HSP60 triggered neurotoxicity, including extensive axonal damage, neuronal death and the loss of oligodendrocytes (Lehnardt et al., 2008; Rosenberger et al., 2015). Moreover, in vitro studies demonstrated that HSP32, HSP70 and HSP90 induced microglial production of proinflammatory cytokines (Kakimura et al., 2002). These effects were demonstrated to be mediated by the microglial TLR4. Taking into considerations the fact that HSPs may be overexpressed in AD brains (Anthony et al., 2003; Calabrese et al., 2006; Kakimura et al., 2002; Perez et al., 1991), it is reasonable to postulate that endogenous HSPs could act as DMAPs in AD process by activating microglial TLR4. However, TLR4-mediated Ab clearance and neuroprotection have also frequently been reported. Microglia activated by lipopolysaccharide (LPS), a typical TLR4 ligand, showed an increased ability to uptake Ab in vitro (Tahara et al., 2006). Additionally, the LPS-derived TLR 4 agonist, monophosphoryl lipid A (MPL) was shown to significantly reduce the Ab load in the brain and to enhance cognitive function in AD animal models, by inducing a potent phagocytic response in microglia while also triggering a moderate inflammatory reaction (Michaud et al., 2013). In vitro, HSP32-, HSP70- and HSP90-treated microglia phagocytosed

increased Ab, and the facilitation of phagocytosis was not detected in TLR4-mutant microglia (Kakimura et al., 2002). Furthermore, a series of experiments in which the TLR4 gene was manipulated in animal models provided more direct and credible evidence. According to Fukuchi and colleagues, a destructive mutation of the TLR4 gene led to an increased in the Ab load compared to TLR4 wild-type AD mice (Tahara et al., 2006). Therefore, TLR4 has been suggested to be involved in both Ab clearance and neuroinflammation in AD animal models. A subsequent study further supported this model by demonstrating that mutated TLR4 caused a reduction in microglial activation and an increase in Ab deposition in AbPP/PS1 AD mice, and these effects were accompanied by further cognitive impairment (Song et al., 2011). Taken together, these results indicate that TLR4 activation may hamper AD progression via promoting microglia-mediated Ab clearance. Therefore, regulating TLR4 activation to promote Ab clearance without inducing neuroinflammation should be a promising treatment option for AD. An exploration of the TLR4-mediated signaling pathways would provide support for this proposal. Upon stimulation, TLR4 activation initiates the production of pro-inflammatory cytokines via both the MyD88/MAPKs/NF-jB and the TIR (Toll/interleukin-1 receptor) domain-containing adaptor inducing IFNb (TRIF)/PI3K pathways (Regen et al., 2011; Suh et al., 2009). In AD scenario, Ab was shown to activate microglia via receptor complexes containing TLR4, such as the myeloid differentiation protein 2/TLR4/

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CD14, TLR2/TLR4/CD14 and TLR4/TLR6/CD36 complexes (ReedGeaghan et al., 2009; Stewart et al., 2010; Walter et al., 2007). In addition, the Ab-induced activation of MyD88, p38 MAPK, IKK and NF-jB has been demonstrated in various studies (Bachstetter et al., 2011; Capiralla et al., 2012; Yu et al., 2013), and STAT activation has indirectly indicated the involvement of the TLR4-mediated MyD88-independent pathway in AD neuroinflammation (Capiralla et al., 2012). Furthermore, microglial production of Ab could be augmented by inflammatory stimuli or Ab (Bitting et al., 1996; Spitzer et al., 2010) and could thereby contribute to neurotoxicity in AD, and this effect has been suggested to be mediated by TLR4/ NF-jB activation (Paris et al., 2007). For other DAMPs, signaling molecules that are known to be associated with exogenous stimulator-induced activation of TLR4 also participate in pathways that are activated by HMGB1 and HSPs, such as MyD88, TRAF6, p38, IKKs and NF-jB (Park et al., 2004). However, differences exist between the endogenous and exogenous stimulator-induced TLR4 activations. Firstly, compared to LPS, HMGB1 activates PI3K/Akt and ERK to a lesser extent (Park et al., 2003). Secondly, LPS primarily activates IKKb while HMGB1 activates both IKKa and IKKb (Park et al., 2004). Thirdly, the observations that HSP60 does not induce massive glial activation and inflammatory reactions in vivo, as LPS dose (Rosenberger et al., 2015), strongly suggests that HSP60 and LPS activate distinct pathways downstream of TLR4/MyD88. Furthermore, because HMGB1 also activates TLR2 (Park et al., 2006), the possibility that HMGB1-induced memory deficits are also mediated by TLR2 cannot be excluded (Mazarati et al., 2011). Meanwhile, studies have been performed to clarify the mechanisms of TLR4-mediated phagocytosis, and it has been suggested that the up-regulation of receptors involved in phagocytosisrelated process and the induction of actin reorganizations both contribute to the Ab uptake. (A) TLR4 activation up-regulated the phagocytosis-related receptors. First, TLR4/MyD88/TRAK4/p38 activation promoted the expression of scavenger receptor A (SRA) (Doyle et al., 2004), which plays important roles in microglial adherence and phagocytosis of Ab (Paresce et al., 1996). Second, TLR4 activation-induced phagocytosis was also shown to be mediated by CD14 and mFPR2, because blocking these receptors partially neutralized the LPS-stimulated microglial uptake of Ab (Tahara et al., 2006). (B) TLR4 activation also promoted actin cytoskeletal assembly, which is required for microglial phagocytosis, and this process was mediated via activating the parallel signaling cascades of Src-Vav-Rac and the p38 MAPK (Chen et al., 2012b; Kong and Ge, 2008). (C) Conflicting results have also been reported. TLR4 activation reduced CD36 expression in a prostaglandin E2 receptor-dependent manner, thereby inhibiting the CD36-mediated phagocytosis of Ab (Li et al., 2015). Moreover, the involvement of the purinergic P2X7 receptor in both microglia-mediated release of pro-inflammatory cytokines and phagocytosis of injured cells following TLR4 activation has been demonstrated (Clark et al., 2010). In addition, PPARc, which is overexpressed in the brain in AD patients, has been shown to inhibit microglial TLR4, CD14 and MyD88 expression and to downregulate the release of pro-inflammatory cytokine (Saravanan et al., 2015). We illustrate the mechanisms underlying the roles of TLR4 in AD in Fig. 2. This complex network consists of TLR4, other microglial receptors and signaling molecules, which allows for greater signaling diversity and for the regulation of the release of TLR4-mediated pro-inflammatory factors and microglial phagocytosis. 3.3. TLR9 Unlike the activations of TLR2 and TLR4, microglial TLR9 activation, which is induced by cytosine–guanosine-containing

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oligodeoxynucleotides (CpGs), the TLR9-specific agonists, has been shown to have only beneficial effects on AD-related pathologies. It should be noted that at least three types of CpGs have so far been reported: classes A, B and C. They are structurally distinct and differentially trigger TLR9-mediated signaling pathways (Kaisho and Akira, 2006; Klinman, 2004). Because CpGs-B are strong stimulators of microglial activation (Kaisho and Akira, 2006), they were applied in studies examining the significance of microglial TLR9 in AD. ICV administration of Ab to cognitively normal mice resulted in impaired recognition memory, and this impairment was ameliorated by ICV injection of CpGs-B (Doi et al., 2009). Further, the administration of CpGs-B to Tg2576 mice resulted in significantly improved cognitive function, accompanied by a reduced Ab burden in animal brains (Doi et al., 2009; Scholtzova et al., 2009), suggesting that TLR9 activation protects against Ab-mediated neurotoxicity by promoting Ab clearance. This concept was supported by the finding that CpGs-B-stimulated microglia show enhanced Ab phagocytosis and produce increased levels of the Abdegrading enzyme MMP-9 (Doi et al., 2009; Iribarren et al., 2005; Tahara et al., 2006). Alternatively, CpGs-B-induced TLR9 activation was shown to reduce the level of neurofibrillary tangles (NFTs) in 3xTg AD mice (Scholtzova et al., 2014). More importantly, microglia alleviated the amount of Ab and NFTs present in the brain without producing neurotoxic molecules, and TLR9 activation also protected against neuroinflammation and apoptosis. In a microglia-neuron coculture system, Ab-induced neuronal death was significantly suppressed by stimulation with CpGs-B, and this effect was accompanied by increased microglial expression of heme oxygenase-1 (HO-1), an antioxidant enzyme (Doi et al., 2009). Therefore, TLR9 activation benefited AD pathology not only by promoting Ab clearance but also by reducing the levels of NFTs and oxidative stress, all of which are closely associated with cognitive benefits. Moreover, Doi et al. compared the influences of all the three classes of CpGs on microglial function. They demonstrated that microglia activated by CpGs-C showed similar neuroprotective effects to the CpGs-B treated microglia, while CpGs-A neither activated microglia nor induced neuro-protective effects against Ab (Doi et al., 2009). The different intracellular localizations of these CpGs may be the cause of these distinct outcomes, but the mechanisms that contribute to these differences are not fully understood (Honda et al., 2005). TLR9 is expressed intracellularly, within endosomal compartments and it functions by activating downstream signaling molecules including ERK, PI3K and p38 in a MyD88-dependent manner (Butchi et al., 2010; Ravindran et al., 2010). The mechanism by which TLR9 activation promotes Ab clearance has been partially defined and we summarize these findings in Fig. 3. Firstly, upon TLR9 stimulation, microglia up-regulate the expression of chemotactic factors, such as C–C chemokine receptor type 1 (CCR1) and CX3C chemokine receptor 1 (CX3CR1), which supports microglial chemotaxis to Ab (Ravindran et al., 2010; Wu et al., 2014). Additionally, TLR9 activation was found to increase levels of the mouse homologue formyl peptide receptor 2 (mFPR2) in microglia via activating p38, which promotes microglial chemotaxis and Ab uptake (Iribarren et al., 2005). Furthermore, TLR9/ ERK activation led to increased microglial expression of TREM2 (Wu et al., 2014), which plays a neuroprotective role not only by promoting Ab phagocytosis but also by inhibiting neuroinflammation (Doens and Fernandez, 2014). As mentioned above, TLR9 activation protected against neuroinflammation and apoptosis. TLR9/ PI3K/AKT activation resulted in increased levels of Bcl-2, an important anti-apoptotic factor, and reduced levels of pro-apoptotic factors, such as Bax and caspase-3/7 (Lu et al., 2014). Moreover, TLR9induced HSP70 activity may also mediate this effect via the MyD88/PI3K/AKT pathway (Kuo et al., 2006). The overexpression of HSP70 protects cells from apoptotic death induced by various

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Fig. 2. TLR4 implicated in AD. TLR4, located on the surface of microglia, activates downstream signaling pathways following ligand binding. This activates NF-kB and INFb and inhibits FoxO1, leading to the induction of microglial M1 activation. Additionally, TLR4 activation also promotes microglial phagocytosis of Ab by initiating actin remodeling and the up-regulation of phagocytosis-related receptors, such as mFPR, CD14 and SR-A. However, TLR4 activation also reduces CD36 expression, thus the CD36-mediated phagocytosis of Ab was inhibited. P2X7R is involved in the TLR4-mediated production of pro-inflammatory cytokines, and it also mediates Ab phagocytosis by microglia. Straight arrows, activation; single-headed arrows, inhibition; dotted arrows, unknown mechanism; red arrows: LPS-activated signaling pathway.

factors, and HSP70 contributes to microglial anti-apoptotic activity by promoting Bcl-x expression and inhibiting the nuclear translocation of apoptosis-inducing factor (AIF) (Kuo et al., 2006). Furthermore, TLR9 activation promoted the secretion of IL-9, IL10 and granulocyte-colony stimulating factor from microglia (Butchi et al., 2010), all of which may suppress inflammation and apoptosis. Further evidence relating to the influence of TLR9 inhibition on AD-related pathology will contribute to a better understanding of the relationships between TLR9 and AD. However, no studies have been performed so far. In other CNS disease, including multiple sclerosis (Marta et al., 2008; Prinz et al., 2006), spinal cord injury (David et al., 2013) and cerebral infarction (Hyakkoku et al., 2010), TLR9 deficiency has been reported to make beneficial, detrimental or no influences on the disease process (David et al., 2013; Hyakkoku et al., 2010; Marta et al., 2008; Prinz et al., 2006). Additionally, the influence of TLR9 deficiency on cognition has also studied, and spatial learning and memory were not significantly different between C57BL/6 mice that were TLR9-deficient or strain-matched TLR9 wild-type mice (Khariv et al., 2013). More importantly, TLR9-deficient macrophage exerted protective effects against chronic stress-induced apoptosis, and this process was mediated via the up-regulation of Bcl-2 and the down-regulation of Bax and caspase-3 (Xiang et al., 2015). These observations strongly imply that TLR9 reacts differently to various stimulations, and that the influences of TLR9 inhibition on AD should be investigated.

All of the data mentioned above suggest that the microglial TLR system exerts complicated effects on AD. On one hand, TLR activation enhances the clearance of neurotoxic Ab by microglia; on the other hand, TLR activation triggers the release of proinflammatory factors. TLR-based treatments might serve as promising therapeutic strategies, but studies should be performed to clarify the roles of TLRs in AD. Furthermore, a strong genetic predisposition has also been demonstrated for AD, and advanced gene analysis technology has provided additional insights into the pathways and proteins, including TLRs, related to AD onset and progression. In the next section, selected genetic polymorphisms that have been implicated in the dysfunction of TLRs in AD are discussed. 4. Genetic polymorphisms that imply the involvement of TLR dysfunctions in AD 4.1. TLR2 In recent years, two genetic variations in TLR2 have been suggested to influence AD risk in the Han Chinese population. Wang et al. explored the interaction between the guanine–thymine (GT) repeat microsatellite polymorphisms in intron II of the TLR2 gene and AD risk, and the S allele (which contains fewer than 16 GT repeats) was associated with an increased risk of late-onset AD (LOAD) (Wang et al., 2011a). Additionally, this genetic polymorphism has been associated with other inflammatory diseases,

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Fig. 3. TLR9 implicated in AD. TLR9, located on the endosome of microglia, activates downstream signaling pathways following CpGs-B binding. This activates Akt, p38 and ERK to mediate the subsequent induction of anti-inflammation and anti-apoptosis. TLR9 activation also promotes Ab clearance, by increasing microglial expression of the chemotactic factors, TREM2 and mFPR2. Straight arrows, activation; single-headed arrows, inhibition.

such as rheumatoid arthritis, nontuberculous mycobacterial lung disease, tuberculosis and leprosy (Lee et al., 2006; Suryadevara et al., 2013; Yim et al., 2008; Yim et al., 2006), and its functional effects have been studied. The S allele was identified to increase the promoter activity of the TLR2 gene in response to stimulation with IFNc (Yim et al., 2004), and peripheral blood mononuclear cells collected from individuals harboring the S allele displayed increased mRNA levels of TLR2 and decreased IL-10 release when stimulated with M. leprae soluble antigen (Suryadevara et al., 2013). These phenomena may partially account for the associations between the S alleles and AD risk, because enhanced TLR2mediated neuroinflammation may occur in these individuals, and the production of neuroprotective IL-10 in response to Ab may be inhibited. However, as reported by Yim et al., increased TLR2 promoter activity was related to a greater GT repeat length under both untreated conditions and during stimulation by either LPS or M. tuberculosis (Yim et al., 2008). Although these results appear to be inconsistent, it should be noted that during the AD process, microglia are maintained in an activated rather than a resting state. Moreover, this polymorphism may differentially modify TLR2 gene promoter activity under specific conditions. The 196 to 174 del polymorphism (a 22-bp nucleotide deletion from position 196 to 174) of TLR2 has been reported to influence AD risk in the Han Chinese population, and the 196 to 174 del/del genotype has been suggested to increase AD risk (Yu et al., 2011). This genetic polymorphism has also been found to increase the risk of developing various cancers (Kutikhin, 2011) and infectious diseases, such as pulmonary tuberculosis

(Velez et al., 2010), suggesting that this polymorphism inhibits immune functions. Functional analysis via luciferase reporter assay supported the concept, as this polymorphism was demonstrated to reduce the transcriptional activity of the TLR2 gene (Junpee et al., 2010; Noguchi et al., 2004). Additionally, monocytes collected from individuals harboring the del allele displayed reduced TLR2 expression upon stimulation with PGN (Nischalke et al., 2012). Thus, it could be assumed that this polymorphism decreases TLR2 expression, which likely leads to the inhibition of TLR2-mediated Ab clearance, thereby promoting AD. However, it should be noted that this polymorphism is also related to the decreased release of IL-8, a pro-inflammatory cytokine, by monocytes upon stimulation (Nischalke et al., 2012). Additionally, in AD patients TLR2 appears to be overexpressed (Letiembre et al., 2009). Therefore, the functional effects of these polymorphisms should be further investigated, and the associations between TLR2 polymorphisms and AD risk should be verified in other ethnic groups. 4.2. TLR4 The gene variations in TLR4 appear to be the most studied among variations in all TLRs, and several single nucleotide polymorphisms (SNPs) in TLR4 have been reported to influence the susceptibility to AD. Minoretti et al. first demonstrated a relationship between a TLR4 gene variation and AD risk in an Italian cohort, as they observed that a minor allele (G) of rs4986790 might protect against AD (Minoretti et al., 2006). Balistreri et al. replicated that result, also in an Italian population (Balistreri et al., 2008).

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Rs4986790 A/G is a common missense mutation that causes the replacement of an aspartic acid residue with a glycine at amino acid 299. This mutation was frequently reported to be coincident with rs4986791 (T399I), and these SNPs have been found to reduce the responsiveness of the TLR4 signaling pathway, for instance, by inhibiting the capacity of this pathway to induce inflammation in human macrophages (Arbour et al., 2000). The mechanisms underlying these effects have been only partially defined. On one hand, these polymorphisms inhibited the expression of functional TLR4 (Prohinar et al., 2010); on the other hand, they caused the inefficient recruitment of MyD88 and TRIF to TLR4 (Figueroa et al., 2012), which leads to inadequate mediation of neuroinflammation by the TLR4 signaling pathway. Moreover, whole blood cells collected from individuals carrying this mutation produced higher levels of an anti-inflammatory cytokine in response to stimulation with LPS (Balistreri et al., 2011). These data suggest that microglia in individuals carrying this mutation may exhibit weakened neuroinflammatory reactions to stimulation with Ab, and this deficiency may partially account for the protective effects of this SNP against AD. Additionally, TLR4 11367 G/C, a common polymorphism that was identified in the 30 -untranslated region of the TLR4 gene in a Han Chinese population, has been associated with an increased risk of AD (Wang et al., 2011b). The functional significance and the clinical relevance of this polymorphism have also been investigated. Hypo-responsiveness of the TLR4 signaling pathway was reported in healthy individuals carrying the minor allele: their peripheral leukocytes expressed reduced TLR4 levels in response to LPS stimulation. Furthermore, this hypo-responsiveness was suggested to result from the attenuation of the post-transcriptional expression of the TLR4 30 -untranslated region and gene (Duan et al., 2009). Therefore, it was hypothesized that the TLR4/11367 polymorphism increases AD risk by suppressing TLR4 expression and TLR4mediated Ab clearance (Wang et al., 2011b). However, this polymorphism was also reported to influence inflammatory processes. For example, patients with trauma who carry the C allele are less likely to experience sepsis and multiple organ dysfunctions, and this effect may be due to the suppression of the TLR4-mediated release of pro-inflammatory cytokines, such as IL-6 and TNF-a. Additionally, reduced levels of TNF-a were detected in healthy individuals carrying the C allele (Duan et al., 2009; Duan et al., 2007). Thus, this polymorphism may influence both TLR4mediated Ab clearance and neuroinflammation in AD. Further studies investigating the influence of the TLR4 11367 G/C polymorphism on microglial functions may help to reveal its significance in AD, and its associations with AD risk must be explored in other ethnic populations. Moreover, several other SNPs in TLR4 have been shown to exert either detrimental (e.g., rs1927907) or protective (e.g., rs10759930, rs1927914, rs1927911, rs12377632, rs2149356, rs7037117, and rs7045953) effects on the risk of developing AD in the Han Chinese population (Chen et al., 2012a; Yu et al., 2012). These results have not been replicated in other ethnic groups, and the functional significance of these SNPs has not been determined. 4.3. TLR9 For TLR9, rs187084 appears to be the only polymorphism that has been observed to modify AD risk. The GG genotype of rs187084 was associated with significantly decreased AD risk in a Han Chinese population (Wang et al., 2013). Additionally, higher levels of TLR9 were detected in the peripheral blood of individuals carrying the GG genotype (Wang et al., 2013). Thus, it could be assumed that the GG genotype protects against AD by promoting TLR9-mediated neuroprotection. However, conflicting results have been reported. A luciferase reporter assay indicated that the GC

haplotype, which consists of rs352139 and rs187084, has been shown to down-regulate TLR9 expression (Tao et al., 2007). Moreover, the TTA haplotype consisting of rs187084, rs5743836 and rs352139 enhanced the promoter activity of the TLR9 gene (Omar et al., 2012). All of these data suggest that the G allele of rs187084, in combination with other genetic polymorphisms of TLR9, reduces TLR9 expression. Meanwhile, it should be noted that these polymorphisms also influence TLR9 signaling pathways by creating potential binding sites for transcription factors (Hamann et al., 2006), and the G allele is associated with increased TNF-a production by peripheral blood leukocytes in response to TLR9 stimulation in vitro (Chen et al., 2011). Therefore, it could be assumed that this polymorphism promotes TLR9-mediated inflammation, which leads to exacerbated chronic inflammation and increased susceptibility to inflammation-related diseases, such as cervical cancer (Wan et al., 2014), Graves’ ophthalmopathy (Liao et al., 2010), rheumatoid arthritis (Lee et al., 2013) and inflammatory bowel disease (Bank et al., 2014). Thus, to further clarify the associations between TLR9 polymorphisms and AD risk, the combination of rs187084, other polymorphisms and their influences on neuroinflammation should be explored in future studies. Briefly, these genetic analyses further support the association of TLRs with AD development and progression [listed in Table 1]. However, much additional investigation must be conducted in this field, as the interactions mentioned above must be confirmed in additional independent populations and across different ethnic groups. Moreover, the associations between genetic polymorphisms of other TLRs and AD remain poorly understood, and this may represent a significant field of research that can be explored to further define the etiology of AD. 4.3.1. Conclusions and future perspectives In the current review, the evidence supporting the multiple roles of microglial activation in AD has been briefly summarized. We have also illustrated the key findings related to the involvement of microglial TLRs in AD progression. In summary, modifying receptor activation and gene expression has been shown to ameliorate or worsen AD-related pathologies, and the associations between genetic polymorphisms in TLR and AD have been identified in certain ethnic groups. These data strongly imply that TLRs are a promising target for better understanding the etiology of AD and for exploring effective AD treatments. Several issues should be explored in further studies. Firstly, it must be emphasized that exogenous TLRs ligands are frequently used to explore the influence of TLR activations on AD. While in the AD scenarios, it is the endogenous substances that affect microglial function. As TLRs could discriminate among different ligands and specific responses may be generated depending on the stimulus, there is a pressing need to find out what affect TLR activations in the AD brains and how these effects take place. Particularly, little is known about how endogenous TLRs ligands influence microglial Ab clearance. It has been observed that HSP90 promoted microglial Ab phagocytosis, and this effect may be mediated via activating TLR4/NF- jB and TLR4/p38. In contrast, HMGB1 could inhibit the microglial uptake and degradation of Ab (Takata et al., 2003; Takata et al., 2012). Since TLRs are receptors for HMGB1 and they are also involved in microglial Ab phagocytosis (Tahara et al., 2006), there is a possibility that the interactions of HMGB1 with TLRS may be related to the inhibitory clearance of Ab. Future works should expand our understanding in this field. Moreover, in addition to HSPs and HMGB1, other endogenous TLRs ligands have been identified and some of them may be related to AD, including b-defensin (Williams et al., 2013), hyaluronan (Jenkins and Bachelard, 1988), 4-hydroxynonenal (Tang et al., 2008) and mitochondria DNA (Podlesniy et al., 2013). Additional works should be performed to make it clear whether they influence AD process

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F. Su et al. / Brain, Behavior, and Immunity xxx (2015) xxx–xxx Table 1 Genetic polymorphisms of TLRs associated with AD. Gene

Polymorphism

Association with AD

Ethnic line

Functional activity

References

TLR2

S allele in intron II

Increased risk

Han Chinese

TLR2 TLR4

196 to 174 del rs4986790

Increased risk Decreased risk

Han Chinese Italian

Increased promoter activity of the TLR2 gene, decreased IL10 production Reduced transcriptional activity of the TLR2 gene Reduced TLR4-mediated inflammation, increased IL- 10 production

TLR4

11367 G/C

Increased risk

Han Chinese

TLR4 TLR4

rs1927907 rs10759930 rs1927914 rs1927911 rs12377632 rs2149356 rs7037117 rs7045953 rs187084

Increased risk Decreased risk

Han Chinese Han Chinese

Decreased TLR4 expression, reduced LPS- induced inflammation Unknown Unknown

(Suryadevara et al., 2013; Wang et al., 2011a; Yim et al., 2004) (Nischalke et al., 2012; Yu et al., 2011) (Arbour et al., 2000; Balistreri et al., 2011; Balistreri et al., 2008; Figueroa et al., 2012; Minoretti et al., 2006; Prohinar et al., 2010) (Duan et al., 2009; Duan et al., 2007; Wang et al., 2011b) (Chen et al., 2012a) (Yu et al., 2012)

Decreased risk

Han Chinese

Higher TLR9 expression

(Wang et al., 2013)

TLR9

S allele, less than 16 guanine–thymine repeats in intron II of the TLR2 gene; 196 to 174 del, a 22-bp nucleotide deletion at position 196 to 174 of the TLR2 gene; AD, Alzheimer’s disease; TLRs, Toll-like receptors; IL-10, interleukin- 10.

via activating microglial TLRs. Secondly, emerging evidence has demonstrated the detrimental effects of anti-inflammatory factors, such as TREM2 and IL-10, on AD pathology (Chakrabarty et al., 2015; Jay et al., 2015), which implies that neuroinflammation is more complex than was previously appreciated. These evidences strongly indicates that it is inappropriate to regard certain inflammatory mediators as exclusively detrimental or beneficial, and imbalance in the immune system, which consists of various cytokines, chemokines, receptors and signaling molecules, contribute to the progress of the disease. With regard to TLRs, networks between TLRs and other coreceptors play vital roles in the CNS. It is unreasonable to consider the manipulation of certain TLRs to be a therapeutic option for AD without accounting for their involvement in these complex regulatory networks. Lastly, TLRs are among the first lines of defense in the innate immune system. When exploring TLR-related treatments for AD, the influences of manipulating of these receptors on the immune system should be taken into consideration. Modulating the activity of TLRs to strengthen their beneficial effects and decrease their adverse influences may be the best goal of further investigations. This strategy will require a profound and comprehensive understanding of TLR signaling pathways. Competing interests The authors declare that they have no competing interests. Authors’ contributions Feng Bai contributed to the concept of this review and revised the manuscript. Fan Su performed the literature review and drafted the manuscript. Zhijun Zhang and Hong Zhou helped to improve the English language and revised the manuscript. All authors read and approved the final manuscript. Acknowledgements This review was partly supported by the National Natural Science Foundation of China (No. 91332104; 81201080; 81271218); Key Program for Clinical Medicine and Science and Technology: Jiangsu Provence Clinical Medical Research Center (No.BL2013025); Natural Science Foundation of Jiangsu Province (No. BK2012337; BK2012746) and Doctoral Fund of Ministry of Education of China (No. 20120092120068).

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Please cite this article in press as: Su, F., et al. Microglial toll-like receptors and Alzheimer’s disease. Brain Behav. Immun. (2015), http://dx.doi.org/10.1016/ j.bbi.2015.10.010