Biological role of Toll

Biological role of Toll
Biological role of Toll

iologiBcal role of Toll-like receptor-4 in the brain

The 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. Introduction

Mammalian 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 functional

in 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 (Schr?der 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 signaling

In 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 brain

TLRs 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 polymorphism

It 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 (Schr?der 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 neurodegeneration

Microglia, 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-κ

B pathways as well as the production of inflammatory mediators by astrocytes (Alfonso-Loeches et al., 2010).

6. Role of TLR4 in traumatic brain injury

Several 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 TLR4

AD 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 sclerosis

MS 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 na?ve 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 encephalomyelitis

Experimental 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). Na?ve 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

al., 2005). Both Th17 and Th1 cells are involved in promoting autoimmune inflammation, especially during MS and EAE (Park et al., 2005, Yang et al., 2008 and Lees et al., 2008), however, Th17 cells may be the major inflammatory subset involved in promoting EAE (Dong, 2008), and it was shown that the Th17 subset can regulate Th1 cells during EAE pathogenesis (Yamazaki et al., 2008).

In the EAE model, the loss of TLR4 solely in CD4 + T cells was reported to abrogate disease symptoms almost completely, mainly through blunted Th17 and, to a lesser degree, Th1 responses, thus suggesting an important role of this innate receptor in the direct regulation of T-cell activation and survival during autoimmune inflammation (Reynolds et al., 2012).

10. Parkinson's disease

PD is the second most common neurodegenerative disease after AD (Tansey and Goldberg, 2010).

PD is an age-related neurodegenerative disorder of the extrapyramidal motor neurons, characterized by loss of dopaminergic neurons from the substantia nigra pars compacta (SNpc) to the striatum (caudate and putamen) of the basal ganglia (Duttaa et al., 2008). The presence of dystrophic projections to the striatum, intracellular proteinaceous inclusions containing large amounts of alpha-synuclein (AS) (Lewy bodies) and activated microglia has been observed (Béraud and Maguire-Zeiss, 2012). The clinical signs include tremor, rigidity, bradykinesia and postural instability. Although the pathogenesis of PD and the chronic disease progression mechanisms are still unknown, recent studies on postmortem PD patients and animal PD models suggest that neuroinflammation and microglia activation play important roles in PD pathogenesis (Tufekci et al., 2011), as demonstrated by several authors (McGeer et al., 1988, Banati et al., 1998 and Imamura et al., 2003). Several studies have confirmed the pres ence of inflammation related enzymes iNOS and COX2 in SNpc.; TNFα, β2-microglobulin, EGF, TGFα, TGFβ1, and IL 1β, 6, and 2 levels were found to be increased in the striatum of PD brain at the molecular level, while IL-2, TNFα, IL-6, and RANTES levels were found to be increased in the cerebrospinal fluid and serum of PD patients (Tufekci et al., 2011).

It is well known that LPS is a potent stimulator of both peripheral immune cells (macrophages and monocytes) and CNS glia (microglia and astrocytes), although it does not seem to have a direct effect on neurons, probably because they lack a functional expression of TLR4 (Duttaa et al., 2008).

In this respect, damaged neurons can cause the microglia activation defined as reactive microgliosis, through the release of injury signals (such as neuromelanin and AS). The interaction between microglia and neurons may lead to a self-amplification of neuronal injury and microglial activation, which may finally result in the neurodegenerative disease (Liu and Bing, 2011).

AS cytoplasmic inclusions are the pathological hallmark of PD, dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) commonly called alpha-synucleinopathies (ASPs) (Yasuda et al., 2013).

PD is characterized by AS positive intracytoplasmic inclusions in neuronal and glial cells. Genetic (Al-Chalabi et al., 2009, Gasser, 2009 and Scholz et al., 2009) and experimental studies (Shults et al., 2005,Zhang et al., 2005 and Xilouri et al., 2009) suggested that AS play a major role in the pathogenesis of ASP. Recent studies have found an up-regulation of TLRs in ASP (Stefanova et al., 2007 and Letiembre et al., 2009) and suggested that just TLR4 may be involved in the pathogenesis of ASP. Up-regulation of TLR4 was shown in ASP postmortem tissue as well as in a transgenic mouse model (Stefanova et al., 2007 and Letiembre et al., 2009).

Fellner et al. (2013) demonstrated that TLR4 is essential for the AS-dependent activation of microglial cells, including phagocytic activity, release of pro-inflammatory cytokines and ROS. TLR4 plays a modulatory role on glial pro-inflammatory responses and ROS production triggered by AS. In contrast to microglia, the uptake of AS by astroglia is not dependent on TLR4 (Fellner et al., 2013). Functional blocking or knockdown of TLR4 in microglia resulted in suppression of AS phagocytosis in vitro, and also TLR4 ablation in vivo led to reduced phagocytosis by microglia associated with accumulation of AS in the mouse brain. These results suggest the role of TLR4-mediated clearance of extracellular AS by microglia, although the exact mechanisms need further elucidation. Therefore, an impaired TLR4-regulated AS clearance appears to exacerbate neurodegeneration by increasing AS accumulation, which is especially toxic to nigral dopaminergic neurons, thus contributing to the pathogenesis of ASPs (Zhang et al., 2005 and Cookson, 2009). However, it is true that TLR4 agonist treatment could also lead to dangerous adverse effects associated with the induction of high levels of proinflammatory mediators, as previously described.

Experimental TLR4 deficiency led to decreased AS clearance by murine microglia (Stefanova et al., 2011). These authors showed, in a transgenic mouse model of MSA with an over-expression of oligodendroglial AS, that TLR4 ablation increased motor disability and augmented loss of dopaminergic neurons. These observations were associated with increased brain levels of AS due to impaired phagocytosis of AS by TLR4-mediated microglia. These results demonstrated that TLR4 may have a neuroprotective role through clearance of AS (Stefanova et al., 2011).

Beraud et al. (2011) demonstrated that misfolded AS composed of monomers, dimers and oligomers of ~ 250 kDa is responsible for microglial activation by upregulation of cytokines, increased expression of antioxidant response enzymes and alteration of TLR gene expression (Beraud et al., 2011).

Recent studies have focused on the effects of LPS challenge in toxin-based and genetic models of PD. Increased mRNA and protein expression of both CD14 and TLR4 in the SN, but not in the caudate putamen nuclei of mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), suggests that the endotoxin receptors are

over-expressed in specific areas of the CNS during experimental PD (Panaro et al., 2008). Thus, the neurotoxin challenge may also induce a predisposition to the exacerbation of chronic neuroinflammation (Tufekci et al., 2011).

In mice lacking a functional TLR4, it has been demonstrated that TLR4 and ROS work in concert to mediate microglia activation. Both TLR4?/? and TLR4+/+ microglia showed an increased production of extracellular superoxide when treated with LPS, indicating a TLR4-independent pathway in microglia. ROS derived from the production of extracellular superoxide in microglia mediates the LPS-induced TNF-α response of both the TLR4-dependent and independent pathways (Qin et al., 2005).

11. Amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating chronic neurodegenerative disease, characterized by loss of motoneurons and extensive astrogliosis and microglial activation in the motor cortex and spinal cord (Casula et al., 2011). ALS manifests as muscle weakness and paralysis, and most patients die within 5 years of symptom onset (Zhang et al., 2011). Although the etiology and pathogenesis of ALS are still elusive, almost 90% of ALS patients have been shown to have sporadic ALS (sALS), 10% of ALS cases are familial (fALS) with 20–25% of these cases resulting from various mutations in the SOD1 gene (Gurney et al., 1998 and Zhang et al., 2011).

Several studies have demonstrated the involvement of activated microglia in both the familial and sporadic forms of ALS disease (Zhang et al., 2011). Beers et al. (2006) have demonstrated that microglia with mutant superoxide dismutase 1 (mSOD1) release more superoxide, nitrate and nitrite and induce more neuronal death, and that treatment of mSOD1 mice with wild-type microglia improved the pathogenesis (Beers et al., 2006).

Interestingly, increased levels of TLR have been observed in mSOD1 mice, as compared to controls (Letiembre et al., 2009) and mutant SOD1 expression in ALS has been suggested to facilitate microglial neurotoxic inflammatory responses via TLR2 (Liu et al., 2009).

Human mutations in (Cu/Zn) SOD1 G93A and G85R, responsible for non-cell-autonomous motor neuron death, interact with CD14 in microglia, which induces the production of inflammatory mediators, including TNF-α, IL-1β, and NO via TLR2 and TLR4 (Zhao et al., 2010). mSOD1 activates microglia through the MyD88-dependent pathway (Kang and Rivest, 2007).

Recently, Zhao et al. (2010) have shown that mutant SOD1 binds to CD14, which has a role in the activation and toxicity of microglia treated with extracellular mSOD1, and that this microglial activation can be attenuated using TLR2, TLR4 and CD14 blocking antibodies (Zhao et al., 2010).

In addition to LPS and misfolded proteins like mutant SOD1, various endogenous molecules such as HMGB1, FAβ, as well as fibro nectin, have been shown to bind to TLR4 and CD14 (Parajuli et al., 2012). Surface expression of TLR4 and CD14 in microglia has been shown to be elevated in ALS. TLR4 expression in microglia is reportedly increased in the spinal cord of ALS patients. Nguyen et al. (2004) showed in a mouse model of ALS that chronic activation of TLR4 and CD14 by LPS exacerbated disease. Simila rly, Aβ has been shown to bind both TLR4 and CD14 and to induce inflammation.Parajuli et al. (2012) showed that the GM-CSF receptor complex, GM-CSFRα and GM-CSFRβ, is strongly expressed in microglia. GM-CSF augmented mRNA and surface expression of both TLR4 and CD14 in microglia, with an increase of LPS-induced NF-κB nuclear translocation and cytokines production such as IL-6, TNF-α, and NO medi ated by LPS. GM-CSF induced expression of TLR4 and CD14 via ERK1/2 and p38, respectively. Thus, GM-CSF may act to exacerbate ALS and AD by increasing TLR4 and CD14 expression in microglia (Parajuli et al., 2012).

sALS is characterized by elevated levels of abnormally activated monocyte/macrophages in peripheral blood (Zhang et al., 2005) and by large numbers of activated microglia/macrophages in the regions of neurodegenerative spinal cord (Graves et al., 2004 and Henkel et al., 2004). Furthermore, recent studies have demonstrated an important role for these cells in ALS disease progression in vivo (Beers et al., 2006 and Boillée et al., 2006). Zhang et al. (2011) found upregulation of genes associated to LPS/TLR4 signal transduction pathways in the PMBCs from patients with sALS after short-term cultivation, and elevated levels of gene expression correlated with the degree of peripheral blood monocyte activation and plasma LPS levels in sALS.

These data suggest that chronic activation of monocyte/macrophages via LPS/TLR4 signaling pathways would have a deleterious effect on disease progression in ALS (Zhang et al., 2011).

Casula et al. (2011) have investigated the expression patterns of TLR2, TLR4, RAGE and HMGB1 in normal and ALS spinal cord from patients with sporadic ALS and different disease duration, to define the possible involvement of TLR/RAGE signaling in the pathophysiology of ALS.

In ALS spinal cord, the HMGB1 signal was increased in the cytoplasm of reactive glia, indicating a possible release of this molecule from glial cells. An up-regulation of TLR2, TLR4 and HMGB1 expression was demonstrated in specimens from patients with sALS; in particular, it was observed in ALS spinal cord, with a different expression pattern of TLR4 from that of TLR2. TLR2 was predominantly detected in cells of the microglia/macrophage lineage, whereas TLR4 showed a prominent expression in both glial and neuronal cells (Casula et al., 2011). Although reactive astrocytes present within gray (ventral horn) and white matter of ALS spinal cord express TLR4, its expression in cultured astrocytes appears controversial (Kielian, 2006 and Crack and Bray, 2007), because some studies were unable to detect TLR4 expression (Farina et al., 2005 and Kielian, 2006), whereas others showed a constitutive expression of TLR4 in astrocytes and an up-regulation following activation (Bsibsi et al., 2002, Bowman et al., 2003 and Carpentier et al., 2005). TLR4 expression was also observed in motor neurons (Crack and Bray, 2007 and Mallard et al., 2009) and its activation could play

a role in the progressive degeneration of motor neurons in ALS. The activation of the TLR/RAGE signaling pathways may contribute to the progression of inflammation, resulting in motor neuron injury. Neuronal TLR4 expression has recently been shown in both experimental and human epileptic tissue (Maroso et al., 2010).

12. Therapeutic approaches

It is hoped that a more complete understanding of TLRs and their signaling pathways in the CNS will lead to the development of more effective treatments for a number of CNS diseases such as stroke, AD, MS and infectious diseases. However, in order for this to occur better tools to study human TLRs are needed (Carty and Bowie, 2011).

A great number of studies indicate that inhibiting inflammation is one of the most important mechanisms in the prevention of oxidative and brain damage during the neurodegenerative process (Kinsner et al., 2005 and Wang et al., 2006). Since TLR4 is closely associated to a number of neurodegenerative disorders including AD, MS and ischemic stroke, targeted TLR4-based therapies have been proposed. Although there is an increasing body of evidence that TLR signaling mediates beneficial effects in the CNS, it has become clear that TLR-induced activation of microglia and the release of proinflammatory molecules are responsible for neurotoxic processes in the course of various CNS diseases. This is primarily attributable to the fact that increased TLR4 activation by either microbial or endogenous ligands is often responsible for an increased secretion of proinflammatory cytokines, exacerbating the neurodegeneration.

The proinflammatory signaling induced by TLR4 agonists may be counteracted by co-application of specific antagonists able to neutralize the involved proinflammatory cytokines, without altering other TLR4 functions, such as the phagocytic activity related to an improved clearance of the tox ic protein species (AS, Aβ, for example). In this respect, it was recently reporte d that a novel analog of thalidomine, 3,6′-dithiothalidomide (DT), an agent with anti-TNF-α activity, resulted able to restore neuronal function and significantly reverse hippocampus-dependent cognitive deficits induced by LPS chronic neuroinflammation in a rat model (Belarbi et al., 2012). TNF-α decrease led to an attenuated expression of genes involved within the TLR mediated signaling pathway associated with classical microglia activation, thus providing a potential therapeutic approach to several human neurodegenerative diseases, as suggested by Belarbi et al. (2012).

Of note, although interrogating the functional role of TLRs using KO mice is useful, it should be acknowledged that the absence of particular TLRs during development may have an impact on later responses in the adult and/or elicit compensatory reactions that are normally not operative. In this case, it would be useful to examine cell-type-specific and/or inducible KO systems to better fine-tune the system and allow a kinetic assessment of receptor activity to be assessed (Hanke and Kielian, 2011). TLR4 knockout mice, for example, are prone to infection (Wu et al., 2010).

Hyakkoku et al. (2010) found that TLR4 but not TRL3 or TRL9 KO mice have smaller cerebral infarctions because these inflammation-associated cytokines via TLR4 were not expressed, suggesting that the TLR4 signaling pathway is involved in I/R brain injury, demonstrating that the attenuation of TRL4 could have a neuroprotective role in ischemic brain injury.

siRNA is a more promising and advantageous strategy, as in chronic neuropathic pain the suppression of TLR4 with intrathecal siRNA delivery was able to alleviate pain responses in a rat chronic constriction injury (CCI) model, suggesting that siRNA targeting TLR4 could be another strategy to treat CNS diseases (Wu et al., 2010). However, it is also true that TLR4 exhibits a broad range of functions in the brain, including neuroplasticity and AS clearance.

13. Conclusion

In this scenario, targeting the TLR4 might be promising as an effective regimen to fight neuroinflammation and to assure a better life quality in patients with neurodegenerative diseases. Thus, an understanding of the function and regulation of the TLR signaling pathways and further study of the effect of TLR activation in the brain Cookson essential for the control of inflammatory-based neurodegenerative diseases.

How plausible is it to target TLR4 responses in neurological disorders to modify disease progression without affecting the TLR4-mediated physiological functions of the brain? The engagement of TLR4 with endogenous or exogenous ligands may be related to beneficial effects, such as AS or Aβ clearance, but TLR4 agonist treatment could also lead to dangerous adverse effects associated with the induction of high levels of proinflammatory mediators, deleterious for the cerebral tissue. These considerations have significant implications on the development of TLR4-based novel therapies aimed at implementing a controlled manipulation of this receptor in relation to the disease stage.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We gratefully thank Mary Victoria Pragnell, B.A., for English language assistance.

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Toll样受体信号通路的研究进展

Toll样受体信号通路的研究进展 摘要Toll样受体(Toll-like receptor,TLR)是近年来发现的一类模式识别受体,通过识别病原相关分子模式(pathogen-associated molecular pattern,PAMP)激活天然免疫。而髓样分化因子(myeloid differentiation factor 88,MyD88)是TLR信号通路中的一个关键接头分子,在传递上游信息和疾病发生发展中具有重要的作用。本文对Toll样受体、髓样分化因子88的分子结构和基本功能,及Toll样受体的信号传导通路进行了综述。 关键词Toll样受体;髓样分化因子88;信号通路;负调控机制 免疫系统识别“非我”和“自我”的过程是依赖于不同的受体来完成的,作为先天性免疫系统的重要组成部分及连接获得性免疫与先天性免疫的“桥梁”, TLRs 是生物的一种模式识别受体(pattern recognition receptor, PRR),它主要通过识别病原相关分子模式PAMPs来启动免疫反应。而MyD88是Toll受体信号通路中的一个关键接头分子,是第一个被鉴定的含TIR结构域的接头蛋白分子,在传递上游信息和疾病发生发展中具有重要的作用。 1TLR的结构与基本功能 Toll样受体一词来自对果蝇的研究,是决定果蝇背腹分化的基因所编码的一种跨膜受体蛋白,同时还参与果蝇的免疫反应,具有介导抗真菌感染信号转导的功能[1]。后来在哺乳动物也发现有与Toll受体同源的受体分子,统称为称为Toll 样受体TLRs。 TLRs是广泛分布在免疫细胞尤其非特异免疫细胞以及某些体细胞表面的一类模式识别受体,它们可以直接识别结合某些病原体或其产物所共有的高度保守的特定分子结构,即病原相关分子模式。迄今为止,已经发现哺乳动物至少有13种toll样受体,其中人的toll样受体鉴定出11种(TLR1-TLR11) [2]。TLRs识别的配基各不相同,其中TLR1-TLR5的结构已被确定,但只有TLR2与TLR4的功能被部分揭示。TLR4主要介导G-菌感染后LPS的信号转导,而TLR2主要介导G+感染后脂蛋白、脂多肽等的信号转导。它们都最终导致该转录因子的转位与相应免疫基因的活化而转录,释放前炎症因子及辅助刺激分子起到调节炎症反应的作用,从而提示TLRs可能在先天性免疫系统中起重要作用[3-4]。 TLRs家族成员具有相似的结构特征。它们均为Ⅰ型跨膜受体,由胞外区、跨膜区和胞内区3个功能区组成。胞外区序列差异大,是与配体结合的特异部位,主要包括十几至二十几个串联的富亮氨酸重复基序(leucine-rich repeats, LRRs),LRR

Toll样受体信号通路图

Toll样受体信号通路图 TLR家族成员(TLR3除外)诱导的炎症反应都经过一条经典的信号通路(图1),该通路起始于TLRs的一段胞内保守序列—Toll/IL-1受体同源区(Toll/IL-1receptorhomologousregion,TIR).TIR可激活胞内的信号介质—白介素1受体相关蛋白激酶(IL-1Rassociatedkinase,IRAK)IRAK-1和IRAK-4、肿瘤坏死因子受体相关因子6(TNFR-associatedfactor6,TRAF-6)、促分裂原活化蛋白激酶(mitogenactivatedproteinkinase,MAPK)和IκB激酶(IκBkinase,IκK),进而激活核因子κB(nuclearfactorκB,NF-κB),诱导炎症因子的表达。 Toll-liker Receptor Signaling 本信号转导涉及的信号分子主要包括: CD14,MD-2,TRAM,TRIF,TIRAP,MyD88,TLR1,TLR2,TLR3,TLR4,TLR5,TLR6,TLR7,TLR8,TLR9,IRAK-1,IRAK-2,IRAK-4,IRAK-M,TRAF6,TRIAD3A,ST2L,SOCS1,RIG-I,FADD,TOLLIP,RIP1,A20,UEV1A,Ubc13,ECSIT,MEKK-1,TAK1,

TBK1,MKK3/6,p38,TAB1/2,MKK4/7,JNK,IKKα,IKKβ,IKKγ,IKKε,NEMO,IκBα,NF-κB,p65/RelA,Casp-8,IRF-3,IRF-7,MA VS等

细胞受体及重要的细胞信号转导途径

细胞受体类型、特点 及重要的细胞信号转导途径 学院:动物科学技术学院 专业:动物遗传育种与繁殖 姓名:李波

学号:2015050509

目录 1、细胞受体类型及特点 (4) 1.1离子通道型受体 (4) 1.2 G蛋白耦联型受体 (4) 1.3 酶耦联型受体 (5) 2、重要的细胞信号转导途径 (5) 2.1细胞内受体介导的信号传递 (5) 2.2 G蛋白偶联受体介导的信号转导 (6) 2.2.1激活离子通道的G蛋白偶联受体所介导的信号通路 (7) 2.2.2激活或抑制腺苷酸环化酶的G蛋白偶联受体 (7) 2.2.3 激活磷脂酶C、以lP3和DAG作为双信使 G蛋白偶联受体介导的信号通 路 (8) 2.2 酶联受体介导的信号转导 (9) 2.2.1 受体酪氨酸激酶及RTK-Ras蛋白信号通路 (10) 2.2.2 P13K-PKB(Akt)信号通路 (10) 2.2.3 TGF-p—Smad信号通 (11) 2.2.4 JAK—STAT信号通路 (12)

1、细胞受体类型及特点 受体(receptor)是一种能够识别和选择性结合某种配体(信号分子)的大分子物质,多为糖蛋白,一般至少包括两个功能区域,与配体结合的区域和产生效应的区域,当受体与配体结合后,构象改变而产生活性,启动一系列过程,最终表现为生物学效应。受体与配体问的作用具有3个主要特征:①特异性;②饱和性;③高度的亲和力。 根据靶细胞上受体存在的部位,可将受体分为细胞内受体(intracellular receptor)和细胞表面受体(cell surface receptor)。细胞内受体介导亲脂性信号分子的信息传递,如胞内的甾体类激素受体。细胞表面受体介导亲水性信号分子的信息传递,膜表面受体主要有三类:①离子通道型受体(ion—channel—linked receptor);②G蛋白耦联型受体(G—protein —linked receptor);③酶耦联的受体(enzyme—linked recep—tor)。第一类存在于可兴奋细胞。后两类存在于大多数细胞,在信号转导的早期表现为激酶级联事件,即为一系列蛋白质的逐级磷酸化,借此使信号逐级传送和放大。 1.1离子通道型受体 离子通道型受体是一类自身为离子通道的受体,即配体门通道(1igand—gated channel),主要存在于神经、肌肉等可兴奋细胞,其信号分子为神经递质。神经递质通过与受体的结合而改变通道蛋白的构象,导致离子通道的开启或关闭,改变质膜的离子通透性,在瞬间将胞外化学信号转换为电信号,继而改变突触后细胞的兴奋性。如:乙酰胆碱受体以三种构象存在,两分子乙酰胆碱的结合可以使之处于通道开放构象,但该受体处于通道开放构象状态的时限仍十分短暂,在几十毫微秒内又回到关闭状态。然后乙酰胆碱与之解离,受体则恢复到初始状态,做好重新接受配体的准备。离子通道型受体分为阳离子通道,如乙酰胆碱、谷氨酸和五羟色胺的受体,和阴离子通道。 1.2 G蛋白耦联型受体 三聚体GTP结合调节蛋白(trimeric GTP—binding regulatory protein)简称G蛋白,位于质膜胞质侧,由a、p、-/三个亚基组成,a和7亚基通过共价结合的脂肪酸链尾结合在膜上,G蛋白在信号转导过程中起着分子开关的作用,当a亚基与GDP结合时处于关闭状态,与GTP结合时处于开启状态,“亚基具有GTP酶活性,能催化所结合的ATP 水解,恢复无活性的三聚体状态,其GTP酶的活性能被RGS(regulator of G protein signaling)增强。RGS也属于GAP(GTPase activating protein)。 G蛋白耦联型受体为7次跨膜蛋白(图10—6),受体胞外结构域识别胞外信号分子并与之结合,胞内结构域与G蛋白耦联。通过与G蛋白耦联,调节相关酶活性,在细胞内

TOLL样受体7(TLR7)增殖分化信号通路论文

TOLL样受体7(TLR7)增殖分化信号通路论文 【提示】本文仅提供摘要、关键词、篇名、目录等题录内容。为中国学术资源库知识代理,不涉版权。作者如有疑义,请联系版权单位或学校。 【摘要】目的探讨TLR7的激活对HaCaT细胞增殖与分化的影响及其可能的机制。方法培养HaCaT细胞,以不同剂量的TLR7配体Gardiquimod经不同的时间体外刺激HaCaT细胞,MTT及流式细胞术分析TLR7的激活对HaCaT细胞增殖的影响。以不同剂量的TLR7配体Gardiquimod经不同的时间体外刺激HaCaT细胞,加入氯化钙诱导HaCaT细胞分化,Western-Blot分析HaCaT细胞的分化Markers(颗粒层:Keratin1,基底层:Keratin5和棘层:Involucrin)并以此分析TLR7的激活对氯化钙诱导HaCaT细胞分化的影响。 Western-blotting分析TLR7在HaCaT细胞中激活的信号通路 PI3K-AKT和RAS-MAPK等。在TLR7配体Gardiquimod处理HaCaT细胞前1h,分别加入特异性阻断剂(PD98059及LY2940002)阻断TLR7配体Gardiquimod激活的相关信号通路,然后分析阻断剂对TLR7配体Gardiquimod调控HaCaT细胞增殖及分化影响,从而探讨PI3K-AKT 和RAS-MAPK信号通路在TLR7配体Gardiquimod对HaCaT细胞增殖及分化调控中的作用。结果MTT及流式细胞分析结果显示:TLR7配体Gardiquimod促进HaCaT细胞增殖,且具有时间及剂量依赖性;TLR7配体Gardiquimod能够抑制氯化钙诱导的HaCaT细胞分化markers (Keratin1及Involucrin)的表达,存在时间效应及剂量效应;信号通路分析揭示TLR7配体Gardiquimod能够增加ERK1/2和MAPK的水平;阻断剂的研究发现TLR7配体Gardiquimod部分依赖PI3K-AKT

toll样受体信号通路

Toll 样受体(TLRs)是一个模式识别受体家族,它们在进化上高度保守,从线虫到哺乳 动物都存在TLRs,目前在哺乳动物中已发现 12 个成员[1].TLRs 主要表达于抗原递 呈细胞及一些上皮细胞,为玉型跨膜蛋白,胞外区具有富含亮氨酸的重复序列,能够 特异识别病原微生物进化中保守的抗原分子———病原相关分子模式 (pathogen-associatedmolecular patterns, PAMPs)[2].为了有效地抵抗入侵的病原体,机体需要对多种 PAMPs 产生适当的免疫应答,TLRs 可以通过识别 PAMPs 诱发抵抗病原体的免疫反应.而且 TLRs 也参与识别有害的内源性物质.TLRs 的激活可诱导很强的免疫反应,有利于机体抵抗病原体感染或组织损伤,但是过度的免疫反应也会带来不利影响,如产生内毒素休克、自身免疫性疾病等.为了保证 TLRs 介导正确的免疫应答,机体 存在精密的负调控机制,及时抑制 TLRs 信号,维持机体的免疫平衡[3]TLR 家族成员(TLR3 除外)诱导的炎症反应都经过一条经典的信号通路(图 1),该通路起始于TLRs 的一段胞内保守序列———Toll/IL-1 受体同源区(Toll/IL-1 receptor homologous region,TIR).TIR可激活胞内的信号介质———白介素 1 受体相关蛋白激酶 (IL-1R associated kinase, IRAK) IRAK-1 和IRAK-4、肿瘤坏死因子受体相关因子 6(TNFR-associated factor 6, TRAF-6)、促分裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)和 I资B激酶 (I资B kinase, I资K ),进而激活核因子资 B(nuclear factor 资B,NF-资B),诱导炎症因子的表达.TLRs 信号通路上的许多接头蛋白都具有 TIR结构域:髓系分化因子 88(myeloid differentiationfactor 88, MyD88)、MyD88- 接头蛋白相似物(MyD88-adaptor like,Mal)、含有 TIR 结构能诱导干扰 素茁的接头分子 (TIR domain-containingadaptor inducing interferon 茁,TRIF)、TRIF 相关接头分子(TRIF-related adaptor molecule,TRAM)和SARM (sterile 琢 and armadillo motif-containingprotein)[4].它们参与 TLRs 所介导的信号转导,其中 MyD88 最重要,参与了除 TLR3 外所有 TLRs介导的信号转导.MyD88 首先通过 TIR 与 TLRs 相结合,接着募集下游信号分子 IRAK-4,IRAK-4 磷酸化激活IRAK-1,随后 活化 TRAF6.活化的 TRAF6 具有泛素连接酶(E3)的活性,能够结合泛素结合酶(E2),进而泛素化降解 IKK-酌.这种泛素化降解可以活化TGF-茁激酶(TGF-茁 activated kinase 1, TAK1) 和TAK1 结合蛋白 (TAK1 binding protein, TAB1、TAB2、 TAB3).活化的 TAK1 会催化 IKK-茁磷酸化,最终激活 NF-资B,促使炎症因子的表达.除了共同的 NF-资B 激活通路,不同的 TLRs 还存在着其特有的信号通路,一些TLRs 具有募集 Mal、TRAM 和 TRIF 的作用.不同的接头分子在信号传导中发挥的作 用不同[5],TRIF 在脂多糖(LPS)激活的 TLR4 途径和 Poly(I∶C)激活的 TLR3 途径中都起到了重要的作用,而 TRAM 仅在 TLR4 的途径中发挥作用.TLRs 的激活是一把双刃剑,它可以通过刺激先天性免疫应答和提高获得性免疫反应来保护机体,但是它所引 起的持续性炎症反应也会对机体产生损伤,自身免疫、慢性炎症和感染性疾病都与它 有一定关系.例如LPS 持续刺激TLR4 就可以引起严重的败血病和感染性休克,此外,类风湿性关节炎、慢性阻塞性肺心病、结肠炎、哮喘、心肌病、狼疮和动脉粥样硬化

toll样受体及其研究进展

Toll样受体、信号通路及其免疫的研究 Toll样受体最早是在研究果蝇胚胎发育过程中发现的,它不仅是果蝇胚胎发育过程中的必需蛋白,而且在免疫应答过程中具有重要作用[1]。Toll 样受体(TLRs)是一个模式识别受体家族,它们在进化上高度保守,从线虫到哺乳动物都存在TLRs,它能识别病原微生物进化中保守分子,如脂多糖(LPs)、肽聚糖、酵母多糖以及病原微生物的核酸等等.脂多糖受体TLR4是发现的第一个TLRs,至今在动物中已经发现15种TLRs(在人体已经发现11个成员,即TLRl~TLRl0和TLRl4,小鼠不表达TLR10,但发现了TLR11—13[2],在鸡中发现了TLR15[3]。哺乳动物的TLRs同果蝇的TLRs一样,同属于I型跨膜蛋白,主要由3个功能区构成:胞外区、跨膜区和胞内区。胞外区具有富含亮氨酸的重复序列,能够特异识别病原微生物进化中保守的抗原分子——病原相关分子模式(pathogen-associated molecular patterns, PAMPs)[4]。为了有效地抵抗入侵的病原体,机体需要对多种PAMPs产生适当的免疫应答,TLRs可以通过识别PAMPs诱发抵抗病原体的免疫反应。而且TLRs也参与识别有害的内源性物质. 1. Toll样受体 1.1 Toll样受体的发现Toll是在昆虫中发现的一个受体蛋白,参与昆虫胚胎发育时背腹肌极性的建立。进一步研究发现,Toll胞内区与哺乳动物中自介素-1受体(IL-1R)的胞内区具有很高的同源性,下游的信号转导通路通过NF—kB样因子发挥作用。IL-1R是免疫相关分子,而且昆虫中抗微生物的多肽基因上游大多有NF—kB样因子结合位点,是否Toll蛋白也参与昆虫的天然免疫反应调控?研究证实Toll参与昆虫的抗真菌免疫.真菌感染时果蝇Toll 通路被激活,诱导大量的抗真菌肽Drosomycin,Toll的突变导致果蝇极易受到真菌的感染[1]。.哺乳动物存在Toll的同源分子,即TLRs。TLRs是一个受体家族。 1.2 TLRs分子特征TLRs为一类Ⅰ型跨膜蛋白,其细胞外区域存在由18~31个氨基酸组成的富含亮氨酸的重复单位(LRR motif)XLXXLXLXXL(X代表任何氨基酸,L为亮氨酸)每个LRR由24~29个氨基酸组成,为8折叠一环一a螺旋的结构。整个LRR结构域形成一个马蹄型的结构,参与识别各种病原体。它们的细胞外区域较长,在550~980氨基酸之间,而且同源性较差,如TLR2与TLR4细胞外区域的同源性只有24%。提示TLRs各个分子之间所结合的配体具有不同的结构、性质;但各个分子种属间的差异较小,如人和小鼠的TLR4胞外区有53%相同,而胞质区则高达83%,提示着它们是一组非常保守的分子,执行着相似的功能。TLRs的胞内区含有Toll/IL-1受体同源(Toll/IL-1 receptor homologous region, TIR), 其中包括3个保守盒(conserved boxes),参与信号转导。TIR是一个保守结构,其中的23个氨基酸的位置是固定的,所形成的三个结构域分别为这些分子的标志区域和信号介导区域。具有TIR结构域[5]分子现在发现的共有31种,如MyD88、IL-1相关蛋白激酶(IRAK)、肿瘤坏死因子受体相关因子6(TRAF6)等。 1.3 TLRs的配体(PAMP)及其特异性TLRs配体按来源可分为外源性和内源性配体。外源性配体主要来自病原微生物,是微生物进化过程中的保守成分,如细菌的脂多糖、胞壁酸、肽聚糖以及细菌和病毒的核酸等。内源性配体来自宿主细胞,如热休克蛋白、细胞外基质降解成分等等,内源性配体在机体应激或是组织损伤时释放[6,7]。TLR4识别G-菌的LPS;TLR2可识别G+菌、分枝杆菌及真菌的PAMP。TLR9识别细菌特殊序列胞嘧啶磷酸鸟(CpG-DNA);TLR5 识别细菌鞭毛蛋白。 目前对TLR生物学作用研究的焦点集中在介导对LPS的反应,而LPS的生物活性成分是脂质A。3种天然对大剂量LPS耐受的小鼠C3H/HeJ、C57BL/10ScCr、C57BL/10ScN,

第二节_膜表面受体介导的信号转导

第二节膜表面受体介导的信号转导亲水性化学信号分子: * 有神经递质、蛋白激素、生长因子等 * 它们不能直接进入细胞 只能通过膜表面的特异受体,传递信号 使靶细胞产生效应 膜表面受体主要有三类(图8-7): ①离子通道型受体(ion-channel-linked receptor) 存在于可兴奋细胞 ②G蛋白耦联型受体(G-protein-linked receptor) ③酶耦联的受体(enzyme-linked receptor) 后2类存在于大多数细胞 在信号转导的早期 表现为一系列蛋白质的逐级磷酸化 使信号逐级传送和放大。

图8-7 膜表面受体主要有3类 一、离子通道型受体 离子通道型受体(图8-8): * 离子通道的受体 即,配体门通道(ligand-gated channel) * 主要存在于神经、肌肉等,可兴奋细胞其信号分子为神经递质 * 神经递质+受体,而改变通道蛋白的构象

离子通道,开启or关闭 改变质膜的离子通透性 瞬间(1/1000秒),胞外化学信号→电信号 继而改变突触后细胞的兴奋性 * 位于细胞膜上的受体,一般4次跨膜 位于质网上的受体,一般6次跨膜 * 离子通道型受体分为 阳离子通道,如乙酰胆碱、谷氨酸、五羟色胺的受体阴离子通道,如甘氨酸&γ-氨基丁酸的受体 * 如:乙酰胆碱受体(图8-9、10)以三种构象存在2分子乙酰胆碱的结合 使通道处于开放构象 但受体处于通道开放构象状态,时限十分短暂 在几十毫微秒,又回到关闭状态 然后,乙酰胆碱与受体解离 受体恢复到初始状态 做好重新接受配体的准备

图8-8 离子通道型受体 synaptic cleft:突触间隙 图8-9 乙酰胆碱受体结构模型

肿瘤免疫及其免疫检验

肿瘤免疫及其免疫检验 一、单项选择题 1、机体抗肿瘤免疫的主要机制是 A. NK 细胞的非特异性杀瘤作用 B. 体液中非特异性免疫成分抗肿瘤作用 C.细胞免疫 D.体液免疫 E. TIL、LAK 细胞杀伤肿瘤细胞作用 2、可特异杀伤肿瘤细胞的是 A. NK 细胞 B. 巨噬细胞 C. CTL D. LAK E. γδT 细胞 3、用于主动免疫特异治疗肿瘤的物质是 A. 酵母多糖 B. 卡介苗 C. 抗独特型抗体疫苗 D. 生物导弹 E. IL-2 4、抗体抗肿瘤的机制不包括 A. CDC B. ADCC C. 调理作用 D. 抗体封闭肿瘤细胞上的某些受体 E. 封闭因子(增强抗体)的作用 5、不用于肿瘤被动免疫治疗的是 A. DNA 疫苗 B. TNF C. TIL D. 外源的免疫效应物 E. 生物导弹 6、高水平血清甲胎蛋白(AFP)见于 A. 孕妇 B. 重度嗜烟者 C. 酒精性肝硬化 D. 结肠癌切除术后病人 E. 原发性肝细胞性肝癌 7、大多数人类肿瘤抗原属于 A、胚胎抗原 B、病毒诱发的肿瘤抗原 C. 化学或物理因素诱发的肿瘤抗原 D. 细菌诱发的肿瘤抗原 E. 自发肿瘤抗原 8、可致机体对肿瘤免疫耐受的因素有 A.肿瘤特异抗原(TSA)及相关抗原(TAA)密度低 B.肿瘤细胞MHC 分子表达下调或丢失 C. 患者APC 的B7、CD40 下调 D. 上列A 和B 两项 E. 上列A、B 和C 三项 9、打破机体对肿瘤免疫耐受可用的方法是 A. 可用抗TGF-β抗体治疗 B. 对瘤细胞转染MHC 基因及B7 或CD40 基因 C.制备TSA/TAA 基因克隆重组蛋白作肿瘤多肽疫苗 D.上列A 和B 两项 E. 上列A、B 和C 三项 10、与HTLV-1 有关的疾病是 A. 成人T 细胞白血病 B. 鼻咽癌 C. 原发性肝癌 D. B 细胞淋巴瘤 E. 胰腺癌 11、关于肿瘤逃避免疫监视的机制,错误的是 A. 瘤细胞表面的转铁蛋白被封闭 B. 增强抗体 C. 瘤细胞的“漏逸” D. 宿主抗原提呈细胞功能低下 E. 某些细胞因子对机体免疫应答的抑制 12、介导补体溶解肿瘤的主要抗体是 A. IgA B. IgD C. IgE D. IgG E. IgM 13、介导ADCC 杀伤肿瘤细胞的抗体主要是 A. IgG B. IgM C. IgE D. IgA E. IgD 14、抗体抗肿瘤的机制不包括 A. 增强抗体 B. ADCC C. 调理作用 D. CDC E. 封闭肿瘤细胞上的转铁蛋白受体 15、NK 杀伤瘤细胞的机制不包括 A. ADCC B. CDC C. 诱导瘤细胞凋亡 D. 释放穿孔素 E. 释放IL-2、IFN-γ

第二十七章肿瘤免疫及免疫学检验TumorimmunityandI

第二十七章肿瘤免疫及免疫学检验Tumor immunity and Immunological assay 第一部分目的要求和教学内容 一、目的要求:掌握肿瘤抗原的类型、肿瘤相关抗原的检测及其临床意义,熟悉 肿瘤与宿主的免疫相关性,了解肿瘤的免疫学治疗手段。 二、教学内容 1、肿瘤抗原:肿瘤抗原的类型(肿瘤特异性抗原、肿瘤相关抗原),肿瘤抗原的诱发因素。 2、肿瘤与宿主的免疫相关性:机体对肿瘤的免疫应答、肿瘤的免疫逃避机制。 3、肿瘤的免疫学检测及免疫疗法:肿瘤相关抗原的检测、宿主免疫状态的检测、肿瘤的免 疫治疗(肿瘤的主动免疫治疗,过继性免疫治疗,抗体导向治疗,基因治疗等)。 第二部分测试题 一、选择题 (一)单项选择题(A型题) 1.甲胎蛋白(AFP)是:() A.隐蔽的自身抗原 B.同种异型抗原 C.肿瘤特异性抗原 D.肿瘤相关抗原 A.交叉反应抗原 2.关于肿瘤相关抗原,下列哪一项不对:( ) A.存在于肿瘤细胞表面的糖蛋白或糖脂 B.在正常细胞上表达量很低 C.在肿瘤细胞上高表达 D.只表达于特定的肿瘤细胞 E.肿瘤胚胎抗原是最常见的肿瘤相关抗原 3.肿瘤患者体内的癌胚抗原对该患者来说是:() A.自身抗原 B.变应原 C.肿瘤特异性抗原 D.肿瘤相关抗原 E.半抗原 4.巨噬细胞活化的条件是: A.有T细胞产生的MAF B.有肿瘤特异性抗体的介导 C.Ⅰ类MHC分子介导 D.Ⅱ类MHC分子介导 E.肿瘤表面表达有B7分子 5.机体对抗原性极弱的肿瘤细胞发挥免疫效应的机制是:

A.通过巨噬细胞释放TNF发挥杀瘤作用 B.通过巨噬细胞的直接吞噬作用发挥杀瘤作用 C.通过NK 细胞释放一系列细胞毒因子发挥杀瘤作用 D.通过树突细胞的抗原提呈作用,促进肿瘤细胞特异性的细胞免疫 E.通过Ⅰ类MHC分子介导CTL发挥杀瘤作用 6.AFP升高可见于下列哪种疾病? A.原发性肝癌 B.重症肝炎 C.转移性肝癌 D.肝硬化 E.以上都是 7.对消化道肿瘤有较大诊断价值的CEA的诊断标准是: A.>2.5μg/L B. >5μg/L C. >10μg/L D. >20μg/L E. >100μg/L 8. 目前.常用来作为直肠癌、结肠癌治疗后的随访项目,检测肿瘤扩散和复发的非创伤性指标是: A.AFP B.CEA C.CA125 D.CA19-9 E.PSA 9. 分化抗原是指: A. 不同肿瘤的特异性抗原 B. 是特定组织正常分化到一定阶段所特有的标志 C. 同一肿瘤不同分化阶段的特征性抗原 D. 同一组织良、恶性肿瘤的标志性抗原 E. 区分正常组织和癌变组织的特异性抗原 10. 分化抗原的主要作用是: A. 刺激机体产生抗肿瘤抗体 B. 可直接作用于CD8+ T细胞介导机体抗肿瘤免疫应答 C. 可作为肿瘤分化程度的标志 D. 可协助判断预后 E. 可作为肿瘤起源的诊断性标志 11.与肿瘤发生有关的因素有: A.机体细胞免疫功能的强弱 B.化学因素,化学诱变剂 C.X 线,紫外线照射、辐射 D.某些病毒感染 E.以上都包括 12. 肿瘤细胞表达的能介导CTL凋亡的是: A.FasL

Toll样受体4信号转导研究进展(1)

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万方数据

万方数据

Toll样受体4信号转导研究进展 作者:李立斌, 朱红, 方强 作者单位:李立斌,朱红(浙江大学医学院附属第二医院ICU,浙江,杭州,310009), 方强(浙江大学医学院附属第一医院ICU,浙江,杭州,310003) 刊名: 国外医学(生理、病理科学与临床分册) 英文刊名:FOREIGN MEDICAL SCIENCES(SECTION OF PATHOPHYSIOLOGY AND CLINICAL MEDICINE) 年,卷(期):2005,25(2) 被引用次数:3次 参考文献(20条) 1.Poltorak A;He X;Smirnova I Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice:mutations in the Tlr gene[外文期刊] 1998(5396) 2.Muroi M;Ohnishi T;Tanamoto K Regions of the mouse CD14 molecule required for toll-like receptor 2-and 4-mediated activation of NF-kappa B[外文期刊] 2002(44) 3.VISINTIN A;Latz E;Monks BG Lysines 128 and 132 enable lipopolysaccharide binding to MD-2, leading to Toll-like receptor-4aggregation and signal transduction[外文期刊] 2003(48) 4.Akashi S;Saitoh S;Wakabayashi Y Lipopolysaccharide interaction with cell surface Toll-like receptor 4-MD-2: higher affinity than that with MD-2 or CD14[外文期刊] 2003(07) https://www.360docs.net/doc/0516507347.html,ea MG;van Deuren M;Kullberg B J Does the shape of lipid A determine the interaction of LPS with Tool-like receptors?[外文期刊] 2002(3) 6.Triantafilou M;Brandenburg K;Kusumoto S Combinational clustering of receptors following stimulation by bacterial products determines LPS responses 2004 7.Wesche H;Henzel WJ;Shillinglaw W MyD88:an adaptor that recruits IRAK to the IL-1 receptor complex [外文期刊] 1997(06) 8.Horng T;Barton GM;Flavell RA The adaptor molecule TIRAP provides signaling specificity for Toll-like receptors[外文期刊] 2002(6913) 9.Horng T;Barton GM;Medzhitov R TIRAP: an adaptor molecule in the Toll signaling pathway[外文期刊] 2001(09) 10.Yamamoto M;Sato S;Hemmi H Essential role for TIRAP in activation of the signaling cascade shared by TLR2 and TLR4[外文期刊] 2002(6913) 11.Burns K;Clatworthy J;Martin L Tollip,a new component of the IL-1RI pathway,links IRAK to the IL-1 receptor 2000(04) 12.Jefferies CA;Doyle S;Brunner C Bruton's tyrosine kinase is a Toll/interleukin-1 receptor domain-binding protein that participates in nuclear factor kappa B activation by Toll-like receptor 4[外文期刊] 2003(28) 13.Yamamoto M;Sato S;Hemmi H Role of adaptor TRIF in the MyD88-independent Toll-like receptor signaling pathway[外文期刊] 2003(5633) 14.Oshiumi H;Sasai M;Shida K TICACM-2:a bridging adaptor recruiting to Toll-like receptor 4 TICAM-1 that induces interferon-beta[外文期刊] 2003(50) 15.Wietek C;Miggin SM;Jefferies CA Interferon regulatory factor-3-mediated activation of the

Toll样受体信号传导与炎症相关肿瘤的关系

《中国癌症杂志》2011年第21卷第6期 CHINA ONCOLOGY 2011 Vol.21 No.6 489 Toll样受体信号传导与炎症相关肿瘤的 关系 曾治民 何静 刘安文 南昌大学第二附属医院肿瘤科,江西 南昌 330006 [摘要] Toll样受体(toll-like receptors,TLRs)属先天性免疫的病原相关分子模式(pathogen-associated molecular patterns,PAMPs)识别受体,主要表达于天然免疫细胞,在机体抵抗外来病原微生物入侵中起关键作用。TLRs在多种恶性肿瘤细胞及组织中均有表达,大量研究认为TLRs对肿瘤的发生、发展有重要影响,特别是与炎症相关肿瘤,如肝癌、结肠癌、胃癌和宫颈癌等。TLRs可能通过促进肿瘤细胞增殖、抑制肿瘤细胞凋亡及免疫逃逸等机制参与炎症相关性肿瘤的发生、发展。 [关键词] Toll样受体; 信号传导; 炎症; 肿瘤 DOI:10.3969/j.issn.1007-3969.2011.06.014 中图分类号:R730.231 文献标志码:A 文章编号:1007-3639(2011)06-0489-06 The relationship between TLRs signaling and inflammation-related-cancers ZENG Zhi-min, HE Jing, LIU An-wen (The Oncology Department, the Second Affiliated Hospital, Nanchang University, Nanchang Jiangxi 330006, China) Correspondence to:LIU An-wen E-mail:awliu666@https://www.360docs.net/doc/0516507347.html, [Abstract ] Toll-like receptors (TLRs) play a critical role in the innate immune system, acting as pathogen-recognition receptors against microorganisms. TLRs also express on a wide variety of cancer cells and tissues. Many evidences showed that TLRs have an effect on the tumorigenesis and progress, especially in ? ammation related cancers such as liver cancer, colorectal cancer, gastric cancer and cervical cancer and so on. This review focused on the relationship between the TLRs signaling and the developing of in ? ammation related cancers. [Key words ] Toll-like receptors; Signal conduct; In ? ammation; Neoplasm 通信作者:刘安文 E-mail:awliu666@https://www.360docs.net/doc/0516507347.html, Toll样受体(toll-like receptors,TLRs)属于病原相关分子模式识别受体,在天然免疫及其继发的获得性免疫中起重要作用。TLRs通过激活核转录因子(nuclear factor-kappa B, NF-κB)进而调控多种重要的细胞因子、黏附分子和趋化因子的表达,在机体的免疫应答、炎症反应及组织修复等方面发挥重要的作用,其活化也参与细胞增殖和凋亡的过程。近来大量研究表明,TLRs与肿瘤密切相关,特别是与炎症相关肿瘤。本文就TLRs及其诱导的信号途径与炎症相关肿瘤的关系作一综述。1 TLRs概述 人类天然性免疫系统具有高度特异性,能正确地区分自我与异己。这种能力是通过发达的高度保守的识别受体家族来实现,其中TLRs 在宿主防卫病原微生物入侵中起到极为重要的 作用,并且其激活也参与了机体获得性免疫反应。目前在哺乳动物中共发现11种:TLR1~TLR11[1]。TLRs与白介素-1受体(interleukin-1 receptors,IL-1Rs)同属一个超家族成员,主要的区别是细胞外区域:TLRs胞外富含亮氨酸重复序列,辅助识别病原微生物及其产物。而IL-1Rs含有3个免疫球蛋白样功能区域。TLRs是Ⅰ型跨膜糖蛋白,胞质区与IL-1Rs胞质区结构相似,称TIR结构域。TLRs胞内结构域含3个高度保守区,在TLRs和信号转导衔接蛋白的启动中发挥作用。TLRs胞外富含亮氨酸区域是受体接受区域,不同的受体识别不同的配体。TLR2与TLR1、TLR6的二聚体识别细菌脂肽/蛋白或脂膜酸,不同的是TLR1/TLR2二聚体识别三酰基脂肽,而TLR2~TLR6识别二酰基脂肽;TLR4与MD2及共刺激分子CD14结合识别革兰氏阴性菌特有的内毒素成分脂多糖(LPS);TLR3识别病

Toll样受体与细胞自噬

· 902 · 《生命的化学》2010年30卷6期CHEMISTRY OF LIFE 2010,30(6) ● Mini Review 文章编号: 1000-1336(2010)06-0902-03 Toll样受体与细胞自噬 熊励晶 童 煜 毛 萌 四川大学华西第二医院儿科,成都 610041 摘要:Toll样受体(Toll-like receptor, TLR)是存在于一线防御细胞上的一种识别病原体相关分子模式(pathogen-asso-ciated molecular pattern, PAMP)的受体,在免疫反应中发挥重要作用。自噬是一种进化过程中保留的细胞反应机制,不仅是细胞适应各种代谢压力的生存机制,还被认为参与了天然免疫和获得性免疫过程。本文将TLR与自噬在免疫反应中的研究进展进行综述。关键词:Toll样受体;自噬;天然免疫中图分类号:R392.12 收稿日期:2010-06-25 作者简介:熊励晶(1985-),女,硕士生,E-mail:ljxiong@hotmail.com;童煜(1978-),女,博士,助理研究员,E-mail:zisu_yu@163.com;毛萌(1956-),女,博士,教授,博士生导师,主任医师,通讯作者,E-mail:dffmmao@126.com 病原微生物在和宿主相互作用的过程中保留了一些相对保守的结构,即病原体相关分子模式(pathogen-associated molecular pattern, PAMP),人体内的模式识别受体(pattern recognition receptor, PRR)通过对PAMP的识别发现病原体的存在,触发不同的信号级联反应从而清除病原体、诱发获得性免疫。Toll样受体(Toll-like receptor, TLR)家族即是体内一种重要的PRR。自噬是维持细胞内容物质和量平衡的重要机制,是细胞面对生存压力的抵抗机制。近来发现自噬反应能够清除入侵细胞的病原微生物,在免疫反应中发挥作用。现将Toll样受体与细胞自噬在免疫反应中相关性研究进展进行综述。1. Toll样受体1.1 TLR结构  TLR是一种跨膜受体,由富含亮氨酸重复序列(leucine-rich repeat, LRR)的胞外区、富含半胱氨酸的跨膜区以及具有核心元件的胞内区组成。胞内区与白介素-1型受体胞内结构域具有高度同源性,故称Toll/白介素-1受体域(Toll/IL-1 receptor domain, TIR),确定了TLR在细胞内的定位以及激活下游信号分子。TIR结构域氨基酸序列和空间结构高度保守,使 其保持稳定的空间结构,并直接与下游的信号分子形成复合体参与信号转导[1]。1.2 TLR的信号传递 TLR通过结构各异的胞外结合域识别不同的PAMP后激活下游信号反应。根据募集配体分子的不同,总体分为MyD88依赖性和MyD88非依赖性两大途径依。 MyD88在TLR信号传导中起重要作用,不仅可通过一系列信号转导激活核因子-κB (nuclear factorkappa B, NF-κB )使其转位进入核内调节相关基因的表达(图1),还可通过p38、JNK 等丝裂原活化蛋白 图1 Toll样受体MyD88依赖途径[2]

免疫学及其免疫学检验学汇总题库规范标准答案

一、名词解释 第1章概论 1.免疫学: 2.免疫分子: 3.补体: 4.临床免疫学: 第2章抗原抗体反应 5.抗原抗体反应: 6.抗原抗体反应特异性 7.可逆性 8.比例性 9.抗原抗体反应的等价带(zoneofequivalence) 10.最适比(optimalratio) 11.带现象(zonephenomenon) 第3章免疫原和抗血清的制备 12.免疫原(immunogen) 13.半抗原 14.免疫佐剂 15.多克隆抗体(polyclonal antibody, pcAb) 第5章凝集反应 16.凝集反应 17.直接凝集反应 18.间接凝集反应 19.明胶凝集试验 第6章沉淀反应

20.沉淀反应 21.絮状沉淀试验 22.免疫浊度测定 23.凝胶内沉淀试验 24.单项扩散试验 25.双向扩散试验 26.免疫电泳技术 27.对流免疫电泳 28.火箭免疫电泳 29.免疫电泳 30.免疫固定电泳 第19章补体检测及应用 31.补体 32.免疫溶血法 33.补体结合试验 第22章感染性疾病与感染免疫检测34.感染 第23章超敏反应性疾病及其免疫检测 35.超敏反应 36.Ⅰ型超敏反应 37.Ⅱ型超敏反应 38.Ⅲ型超敏反应 39.Ⅳ型超敏反应 第24章自身免疫性疾病及其免疫检测 40.自身耐受 41.自身免疫

42.自身免疫病 43.自身抗体 44.抗核抗体 第25章免疫增殖性疾病及其免疫检测 45.免疫增殖性疾病 46.免疫球蛋白增殖病 47.本周蛋白 48.血清区带电泳 49.免疫电泳 50.免疫固定电泳 第26章免疫缺陷性疾病及其免疫检验 51.免疫缺陷病 52.获得性免疫缺陷综合征 第27章肿瘤免疫与免疫学检验 53.肿瘤免疫学 54.肿瘤抗原 55.肿瘤标志物 第28章移植免疫及其免疫检测 56.移植 57.主要组织相容性复合体 58.移植排斥反应 59.移植物抗宿主反应(GVHR) 60.血清学分型法 二、填空题。

Toll样受体及其信号通路研究进展

Toll样受体及其信号通路研究进展 摘要:Toll样受体(TLRs)是一类模式识别受体,可以识别微生物并对其作出反应。TLRs家族成员在免疫系统中起着重要作用,既是参与先天免疫的重要分子,也是连接先天免疫和特异性免疫的桥梁。该受体可以特异性地识别微生物,并启动免疫应答。本文对TLRs结构、功能和信号通路等方面进行综述。 关键词:Toll样受体免疫系统信号通路 在天然免疫系统的研究中,Toll样受体的发现是最重要的进展之一。TLRs 最早是1980年在果蝇胚胎中发现的,此基因决定了果蝇背腹侧的分化[1]。1991年Gay等发现,TLRs蛋白的结构与哺乳动物中IL-1具有同源性[2]。随后,TLRs 被发现能够激活获得性免疫[3]。至今,已经发现21种TLRs,其中人13种(TLR1-13),小鼠12种(TLR1-9及TLR11-13),斑马鱼18种(TLR1-9、TLR11-14和TLR18-22)。 1、TLRs的结构 TLRs结构由三部分组成,胞外区、跨膜区和胞浆区。胞外区是亮氨酸富集的重复序列,识别病原体细胞表面的分子;跨膜区富含半胱氨酸;胞浆区与哺乳动物IL-1受体高度同源,称为TIR[5]。TIR的构型与病原识别相关,不同种类TLRs,识别不同种类的微生物。 2、TLRs的功能 TLRs是抵御感染性疾病的第一道屏障,在免疫系统中起识别微生物的作用。TLRs通过TIR识别相应的配体来激活免疫反应。TLR1可识别细菌的三酰脂肽;TLR2可识别革兰氏阳性细菌的脂蛋白、肽聚糖等;TLR3主要识别dsDNA;TLR4能识别革兰氏阴性菌的脂多糖;TLR5特异识别细菌的鞭毛蛋白;TLR6主要识别细菌的肽聚糖;TLR7、TLR8可识别单链RNA病毒;TLR9可识别CpGDNA。 另外树突细胞可表达TLRs。TLRs在识别脂多糖、肽聚糖、脂蛋白及病毒后,树突细胞被活化并成熟,提供获得性免疫的共刺激信号。TLRs是微生物成分引起树突细胞活化的桥梁。 3、TLRs信号通路 TLRs信号通路由下游信号分子构成,包括髓样分化因子(MyD88)、IL-1R相关蛋白激酶(IRAK)、TRAF6、TAK1和TAB1、TAB2。研究表明,信号转导过程中,存在两个信号通路,包括MyD88依赖途径和MyD88非依赖途径。

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