Toll样受体与动脉粥样硬化

固有免疫应答与动脉粥样硬化斑块形成有关.固有免疫识别中的模式识别受体Toll样受体信号通路与动脉粥样硬化发病机理的关系,解释了针对外来病原体的非特异性防御以及内源性炎症分子导致动脉粥样硬化的可能机制.     

Toll样受体4与动脉粥样硬化

近年来慢性炎性反应与动脉粥样硬化之间的联系逐渐受到重视,而复杂的炎性反应也对动脉粥样硬化定向有效的治疗提出了挑战.目前体内外研究认为,Toll样受体(TLR)途径介导的免疫反应参与动脉粥样硬化的发生和发展,TLR4途径的激动剂和拮抗剂的使用可能成为未来动脉粥样硬化治疗的新靶点.     

Toll-like receptor 7 stimulation by imiquimod induces macrophage autophagy and inflammation in atherosclerotic plaques

Atherosclerotic plaques tend to rupture as a consequence of a weakened fibrous cap, particularly in the shoulder regions where most macrophages reside. Macrophages express Toll-like receptors to recognize pathogens and eliminate intracellular pathogens by inducing autophagy. Because Toll-like receptor 7 (TLR7) is thought to be expressed in macrophages but not in smooth muscle cells (SMCs), we investigated whether induction of macrophage autophagic death by TLR7 ligand imiquimod can affect the composition of atherosclerotic plaques in favor of their stability. Immunohistochemical staining of human carotid plaques as well as Western blotting of cultured macrophages and SMCs confirmed that TLR7 was expressed in macrophages, but not in SMCs. In vitro experiments showed that only TLR7 expressing cells underwent imiquimod-induced cell death, which was characterized by autophagosome formation. Imiquimod-treated macrophages activated nuclear factor-κB (NF-κB) and released pro-inflammatory cytokines and chemokines. This effect was inhibited by the glucocorticoid dexamethasone. Imiquimod-induced cytokine release was significantly decreased in autophagy-deficient macrophages because these cells died by necrosis at an accelerated pace. Local in vivo administration of imiquimod to established atherosclerotic lesions in rabbit carotid arteries induced macrophage autophagy without induction of cell death, and triggered cytokine production, upregulation of vascular adhesion molecule-1, infiltration of T-lymphocytes, accumulation of macrophages and enlargement of plaque area. Treatment with dexamethasone suppressed these pro-inflammatory effects in vivo. SMCs and endothelial cells in imiquimod-treated plaques were not affected. In conclusion, imiquimod induces macrophage autophagy in atherosclerotic plaques, but stimulates plaque progression through cytokine release and enhanced infiltration of inflammatory cells.     

Toll-like receptor signaling and atherosclerosis

PURPOSE OF REVIEW: Chronic inflammation and disordered lipid metabolism represent hallmarks of atherosclerosis. Considerable evidence has accumulated to suggest that innate immune defense mechanisms might interact with proinflammatory pathways and exacerbate or perhaps even initiate development of arterial plaques. Until recently the preponderance of such evidence has been indirectly emerging from clinical and epidemiologic studies, with some support from experimental animal models of atherosclerosis. RECENT FINDINGS: Recent data now directly implicate signaling by toll-like receptor 4 and the common adaptor molecule MyD88 in the pathogenesis of atherosclerosis, establishing a key link between atherosclerosis and defense against both foreign pathogens and endogenously generated inflammatory ligands. SUMMARY: Here we briefly review these and closely related studies, highlighting areas that should provide fertile ground for future studies aimed at a more comprehensive understanding of the interplay between innate immune defense mechanisms, atherosclerosis and related vascular disorders.     

Toll-like receptor 9 signaling protects against murine lupus

OBJECTIVE: Hypomethylated CpG-containing DNA, which is recognized by Toll-like receptor 9 (TLR-9), has been strongly implicated in the pathogenesis of autoantibody-mediated diseases such as systemic lupus erythematosus. This study was undertaken to determine the role of TLR-9 in the MRL/+ and MRL/lpr models of murine lupus. METHODS: TLR-9-deficient MRL mice were generated by backcrossing a TLR-9-deficient allele against the MRL backgrounds by a speed congenic technique. Parameters of murine lupus were examined by routine methods. Regulatory T cell activity was assessed by autologous mixed lymphocyte reaction (AMLR), an in vitro assay for autoreactivity. RESULTS: Surprisingly, TLR-9-deficient animals of both the MRL/+ and the MRL/lpr backgrounds developed more severe lupus, as judged by anti-DNA and rheumatoid factor autoantibodies, total serum Ig isotypes, lymphadenopathy, inflammatory infiltrates in the salivary gland and kidney, proteinuria, and mortality, in comparison with their TLR-9-sufficient littermates. In vitro, regulatory T cells from TLR-9-deficient animals were impaired in their ability to suppress the AMLR. CONCLUSION: In the MRL model of murine lupus, TLR-9 signaling plays a protective role, perhaps by modulating the activity of regulatory T cells. These results contrast with findings of recent studies that implicate TLR-9 in the pathogenesis of anti-DNA responses, based in part on investigations in incompletely backcrossed TLR-9-deficient MRL/lpr mice in vivo or transgenic B cells in vitro. The present results highlight the need for caution in the assessment of disease paradigms based on the study of isolated cell populations in vitro, as well as in vivo studies of knockout animals involving non-ideal genetic models.     

A Toll-like receptor 7 single nucleotide polymorphism protects from advanced inflammation and fibrosis in male patients with chronic HCV-infection

BACKGROUND/AIMS: HCV-infection leads to development of liver fibrosis, causing morbidity and mortality. Multiple factors influence the progression of fibrosis, including genetic factors. Since HCV is an RNA virus, a role for TLR7 in the immune response against HCV is likely. No systematic analysis of TLR7 single nucleotide polymorphisms (SNPs) has been published. METHODS: We sequenced TLR7 in 52 women and investigated SNPs with an allele frequency >5% in 807 patients with chronic HCV-infection by melting curve analysis. We analyzed the effect of TLR7 SNPs on grade of inflammation and stage of fibrosis as determined by liver biopsy. RESULTS: We detected five TLR7 SNPs, three of which showed a frequency >5%. One variant, c.1-120T>G, was more common in patients with no or little inflammation than in patients with grades 2-4 (10.7% vs. 6.1%; P=0.034). The variant was also enriched in patients with no or little fibrosis compared to those with higher stages (12.6% vs. 6.6%; P=0.005). The difference was fully attributable to male patients. CONCLUSIONS: This is the first analysis of TLR7 SNPs in patients with chronic HCV-infection. Our data suggest that the c.1-120G TLR7 allele offers protection from the development of inflammation and fibrosis in male patients with chronic HCV-infection.     

Recognition of single-stranded RNA viruses by Toll-like receptor 7

Viral infection of mammalian host results in the activation of innate immune responses. Toll-like receptors (TLRs) have been shown to mediate the recognition of many types of pathogens, including viruses. The genomes of viruses possess unique characteristics that are not found in mammalian genomes, such as high CpG content and double-stranded RNA. These genomic nucleic acids serve as molecular signatures associated with viral infections. Here we show that TLR7 recognizes the single-stranded RNA viruses, vesicular stomatitis virus and influenza virus. The recognition of these viruses by plasmacytoid dendritic cells and B cells through TLR7 results in their activation of costimulatory molecules and production of cytokines. Moreover, this recognition required intact endocytic pathways. Mice deficient in either the TLR7 or the TLR adaptor protein MyD88 demonstrated reduced responses to in vivo infection with vesicular stomatitis virus. These results demonstrate microbial ligand recognition by TLR7 and provide insights into the pathways used by the innate immune cells in the recognition of viral pathogens.     

Toll样受体4与气道慢性炎症性疾病

气道慢性炎症性疾病如慢性阻塞性肺疾病、支气管哮喘等是在各种内外界刺激因素作用下由气道固有细胞、炎症细胞和炎症因子参与的非特异性炎症性疾病.迄今已发现11种Toll样受体(TLR),均为I型跨膜受体蛋白,广泛表达于支气管上皮细胞、支气管平滑肌细胞、树突状细胞、肺泡巨噬细胞等,因其能感知病原体并直接或间接作出防御反应而在慢性气道炎症性疾病的发生发展中发挥重要作用,其中又以TLR4的作用最为突出而成为研究的热点.故深入认识TLR4与慢性气道炎症性疾病的关系将为临床治疗开辟广阔的前景.     

Platelet Toll-like receptor expression and activation induced by lipopolysaccharide and sepsis

Platelets and Toll-like receptor (TLR) signalling play a role in the immune response during sepsis. Although preclinical knowledge about the role of platelet TLR signalling is increasing, data during human sepsis are less abundant. Moreover, controversy remains about the effect of the TLR4 agonist lipopolysaccharide (LPS) on platelet activation. We therefore assessed platelet TLR expression during human and murine sepsis. Moreover, we investigated the effect of TLR4 signalling on platelet activation and TLR expression. Platelets from healthy controls stimulated with LPS did not show classical platelet activation (P-selectin, CD63 and phosphatidylserine expression), potentiation of subthreshold agonist stimulation nor platelet-leukocyte complex formation. LPS stimulation however did increase maximal mitochondrial respiration in a TLR4-dependent manner. Platelet stimulation with LPS did not alter TLR expression. Platelet stimulation with thrombin receptor activating peptide increased TLR5 and TLR9, but not TLR2 or TLR4 expression. Platelets from patients with sepsis and mice with experimental sepsis showed platelet activation, but unaltered TLR expression. These results indicate that sepsis-induced platelet activation is not associated with altered platelet TLR expression and, although platelets are responsive to LPS, stimulation of platelet TLR4 does not result in classical platelet activation.     

The GTPase-activating protein GIT2 protects against colitis by negatively regulating Toll-like receptor signaling

G protein-coupled receptor kinase-interactor 2 (GIT2) regulates thymocyte positive selection, neutrophil-direction sensing, and cell motility during immune responses by regulating the activity of the small GTPases ADP ribosylation factors (Arfs) and Ras-related C3 botulinum toxin substrate 1 (Rac1). Here, we show that Git2-deficient mice were more susceptible to dextran sodium sulfate (DSS)-induced colitis, Escherichia coli, or endotoxin-shock challenge, and a dramatic increase in proinflammatory cytokines was observed in Git2 knockout mice and macrophages. GIT2 is a previously unidentified negative regulator of Toll-like receptor (TLR)-induced NF-κB signaling. The ubiquitination of TNF receptor associated factor 6 (TRAF6) is critical for the activation of NF-κB. GIT2 terminates TLR-induced NF-κB and MAPK signaling by recruiting the deubiquitinating enzyme Cylindromatosis to inhibit the ubiquitination of TRAF6. Finally, we show that the susceptibility of Git2-deficient mice to DSS-induced colitis depends on TLR signaling. Thus, we show that GIT2 is an essential terminator of TLR signaling and that loss of GIT2 leads to uncontrolled inflammation and severe organ damage.Inflammatory bowel disease (IBD), which includes the two main forms Crohn’s disease (CD) and ulcerative colitis (UC), is generally thought to develop from an abnormal immune response to the gut luminal microbiota in genetically predisposed individuals (1, 2). The innate immune system, which senses the commensal flora, is critical for intestinal homeostasis and tissue repair after injury (3). Toll-like receptor (TLR) signaling is responsible for the development of colitis (4).Tight control of TLR signaling is crucial for the clearance of pathogens and for avoiding nonresolving inflammation (5, 6). Uncontrolled activation of the TLR-triggered signaling pathway promotes chronic inflammation and autoimmune diseases, such as IBD and obesity (7, 8). TLRs mediate host responses to microorganisms and induce inflammatory responses that are linked to the expression of proinflammatory cytokines and activation of the NF-κB pathway (7). Negative regulation of TLR signaling is crucial to maintain immune homeostasis. The tumor suppressor CYLD is a negative regulator of TLR signaling by deubiquitinating TRAF6 and NEMO (9–11). A deficiency in the catalytic domain of CYLD in mice causes elevated NF-κB activity, and those mice die shortly after birth (12).GIT2 belongs to the group of ADP ribosylation factor (Arf)-directed GTPase activating proteins (GAPs). More than 24 proteins with Arf GAP domains have been identified in humans (13). To our knowledge, none of these proteins have been reported to directly regulate TLR signaling. GIT2 negatively regulates the activation of Arf GTPases through its N-terminal, Arf GTPase activating protein (GAP) domain (14) and of Rac GTPase through its interaction with PIX (p21-activated kinase-interacting exchange factor) (15). Git2 deficiency leads to spontaneous splenomegaly, hypersusceptibility to infection, increased oxygen anion production by neutrophils, and impaired positive selection of CD4 single-positive thymocytes in thymus (16, 17). GIT2 is highly expressed in monocytes and macrophages, especially after stimulation by LPS (18, 19). Combining its known relationships with novel transeQTLs extends the connections of GIT2 to a host of inflammatory mediators (19). We identified that GIT2 interacted with the components of the NF-κB signaling pathway by a large-scale yeast two-hybrid screening (20). Cumulatively, these results show the important roles of GIT2 in innate and adaptive immunity. However, whether GIT2 directly regulates the intestinal immune response remains unknown, and the genetic evidence is lacking to support its physiological roles during intestinal immune responses by directly regulating the TLR signaling pathway.Here, we show that knockout of Git2 induced severe acute and chronic colitis after dextran sulfate sodium (DSS) treatment. Git2-deficient mice were more susceptible to infection by Escherichia coli and endotoxin shock. Git2 deficiency resulted in a greater TLR-induced production of proinflammatory cytokines in vitro and in vivo. GIT2 inhibited the TLR-induced signaling pathway and was critical in inhibiting the activation of TRAF6. Moreover, GIT2 terminated the TLR signaling pathway by recruiting the deubiquitinating enzyme CYLD to the TRAF6 ubiquitin ligase. Thus, GIT2 is a critical terminator of TLR-induced inflammatory responses.     

Toll-like receptor 2 mediates apolipoprotein CIII-induced monocyte activation

Apolipoprotein (apo)CIII predicts risk for coronary heart disease. We recently reported that apoCIII directly activates human monocytes. Recent evidence indicates that toll-like receptor (TLR)2 can contribute to atherogenesis through transduction of inflammatory signals. Here, we tested the hypothesis that apoCIII activates human monocytoid THP-1 cells through TLR2. ApoCIII induced the association of TLR2 with myeloid differentiation factor 88, activated nuclear factor (NF)-kappaB in THP-1 cells, and increased their adhesion to human umbilical vein endothelial cells (HUVECs). Anti-TLR2 blocking antibody, but not anti-TLR4 blocking antibody or isotype-matched IgG, inhibited these processes (P<0.05). ApoCIII bound with high affinity to human recombinant TLR2 protein and showed a significantly higher (P<0.05) and saturable binding to 293 cells overexpressing human TLR2 than to parental 293 cells with no endogenous TLR2. Overexpression of TLR2 in 293 cells augmented apoCIII-induced NF-kappaB activation and beta(1) integrin expression, processes inhibited by anti-apoCIII antibody as well as anti-TLR2 antibody. Exposure of peripheral blood monocytes isolated from C57BL/6 (wild-type) mice to apoCIII activated their NF-kappaB and increased their adhesiveness to HUVECs. In contrast, apoCIII did not activate monocytes from TLR2-deficient mice. Finally, intravenous administration to C57BL/6 mice of apoCIII-rich very-low-density lipoprotein (VLDL), but not of apoCIII-deficient VLDL, activated monocytes and increased their adhesiveness to HUVECs, processes attenuated by anti-TLR2 or anti-apoCIII antibody. ApoCIII-rich VLDL did not activate monocytes from TLR2-deficient mice. In conclusion, apoCIII activated monocytes at least partly through a TLR2-dependent pathway. The present study identifies a novel mechanism for proinflammatory and proatherogenic effects of apoCIII and a role for TLR2 in atherosclerosis induced by atherogenic lipoproteins.     

Association between Toll-like receptor 4 and inflammatory bowel disease

BACKGROUND: The human Toll-like receptor 4 (TLR4) participates in the innate response. Recently, the TLR4 variant Asp299Gly has been described to affect the response of this receptor to lipopolysaccharide. As such, there is a potentially important role of TLR4 in the pathogenesis of inflammatory bowel disease (IBD). We studied the involvement of TLR4 in IBD in a large population of Dutch patients with IBD and in family-based controls. METHODS: In 781 IBD cases and 315 controls, genotyping was performed forAsp299Gly and Thr399Ile variants and for 4 microsatellite markers flanking TLR4. Association analysis and the were applied. In addition, interaction of TLR4 with the caspase recruitment domain containing protein 15 gene (CARD15) was studied in patients with Crohn's disease (CD). RESULTS: The haplotype sharing statistic showed association at microsatellite marker D9S1864 with IBD (P = 0.0019), and in particular with CD (P = 0.0025) and at TLR406 with ulcerative colitis (UC; P = 0.027). No association was found for Asp299Gly and Thr399Ile. However, the frequencies of both variant allele carriers were higher among CD cases with a disease onset > or = 40 years than among controls. No evidence for interaction between TLR4 and CARD15 was found. CONCLUSIONS: Haplotype analysis shows that TLR4 is associated with both CD and UC. The Asp299Gly and Thr399Ile variants do not show an association with CD, UC, or IBD as a group, indicating that these polymorphisms are likely not the causal ones. We propose that the 2 polymorphisms are in linkage with (the) disease susceptibility variant(s) located elsewhere on TLR4.     

Mediation of inflammatory activation of renal tubular epithelial cells by high mobility group protein box 1 interacting with Toll-like receptor 4

<正>Objective To observe functional changes of renal tubular epithelial cells stimulated by high mobility group protein box 1(HMGB1)and associated mechanism.Methods Renal tubular epithelial cells(NRK52E)were divided into control group,HMGB1 group and HMGB1+lipopolysaccharide from Rhodobactersphaeroides(LPS RS)group.Toll-like receptor 4(TLR4)expression was     

A critical cysteine is required for HMGB1 binding to Toll-like receptor 4 and activation of macrophage cytokine release

During infection, vertebrates develop “sickness syndrome,” characterized by fever, anorexia, behavioral withdrawal, acute-phase protein responses, and inflammation. These pathophysiological responses are mediated by cytokines, including TNF and IL-1, released during the innate immune response to invasion. Even in the absence of infection, qualitatively similar physiological syndromes occur following sterile injury, ischemia reperfusion, crush injury, and autoimmune-mediated tissue damage. Recent advances implicate high-mobility group box 1 (HMGB1), a nuclear protein with inflammatory cytokine activities, in stimulating cytokine release. HMGB1 is passively released during cell injury and necrosis, or actively secreted during immune cell activation, positioning it at the intersection of sterile and infection-associated inflammation. To date, eight candidate receptors have been implicated in mediating the biological responses to HMGB1, but the mechanism of HMGB1-dependent cytokine release is unknown. Here we show that Toll-like receptor 4 (TLR4), a pivotal receptor for activation of innate immunity and cytokine release, is required for HMGB1-dependent activation of macrophage TNF release. Surface plasmon resonance studies indicate that HMGB1 binds specifically to TLR4, and that this binding requires a cysteine in position 106. A wholly synthetic 20-mer peptide containing cysteine 106 from within the cytokine-stimulating B box mediates TLR4-dependent activation of macrophage TNF release. Inhibition of TLR4 binding with neutralizing anti-HMGB1 mAb or by mutating cysteine 106 prevents HMGB1 activation of cytokine release. These results have implications for rationale, design, and development of experimental therapeutics for use in sterile and infectious inflammation.     

Molecular basis for the immunostimulatory activity of guanine nucleoside analogs: activation of Toll-like receptor 7

Certain C8-substituted and N7, C8-disubstituted guanine ribonucleosides comprise a class of small molecules with immunostimulatory activity. In a variety of animal models, these agents stimulate both humoral and cellular immune responses. The antiviral actions of these guanosine analogs have been attributed to their ability to induce type I IFNs. However, the molecular mechanisms by which the guanosine analogs potentiate immune responses are not known. Here, we report that several guanosine analogs activate Toll-like receptor 7 (TLR7). 7-Thia-8-oxoguanosine, 7-deazaguanosine, and related guanosine analogs activated mouse immune cells in a manner analogous to known TLR ligands, inducing cytokine production in mouse splenocytes (IL-6 and IL-12, type I and II IFNs), bone marrow-derived macrophages (IL-6 and IL-12), and in human peripheral blood leukocytes (type I IFNs, tumor necrosis factor alpha and IL-12). The guanosine congeners also up-regulated costimulatory molecules and MHC I/II in dendritic cells. Genetic complementation studies in human embryonic kidney 293 cells confirmed that the guanosine analogs activate cells exclusively via TLR7. The stimulation of TLR7 by the guanosine analogs in human cells appears to require endosomal maturation because inhibition of this process with chloroquine significantly reduced the downstream activation of NF-kappaB. However, TLR8 activation by R-848 and TLR2 activation by [S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-R-Cys-S-Ser-Lys4-OH, trihydrochloride)] were not inhibited by chloroquine, whereas TLR9 activation by CpG oligodeoxynucleotides was abolished. In summary, we present evidence that guanosine analogs activate immune cells via TLR7 by a pathway that requires endosomal maturation. Thus, the B cell-stimulating and antiviral activities of the guanosine analogs may be explained by their TLR7-activating capacity.