The Chaperone UNC93B1 Regulates Toll-like Receptor Stability Independently of Endosomal TLR Transport

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UNC93B1影响TLRs信号通路及其与疾发病机制关系的研究进展

UNC93B1存在于内质网上,具多次跨膜区域,它能与TLR(Toll-like receptors,TLRs)-3,-7,-9发生特异性结合并将它们从内质网上运输(trafficking)至溶酶体中。UNC93B1与TLRs相互作用并通过MyD88/TRIF途径进行着信号传递。TLRs通过识别相应的高度保守的病原相关分子模式(PAMPs),在介导防御外来微生物入侵的先天性免疫反应并桥连或触发获得性免疫反应中起重要作用。在3d(N-乙基-N-亚硝基脲诱导的染色体基因的隐性突变)小鼠中,UNC93B1上H412R位点的突变阻断了其和TLRs的结合,进而阻碍了TLRs信号的传递,这使得该小鼠易被各种病原体感染。另外,UNC93B1的突变阻止了小鼠体内抗原的交叉呈递途径,也降低了MHC-II类分子呈递途径。UNC93B1缺陷使得人体易受单纯疱疹病毒I型(HSV-1)病毒感染而引起单纯疱疹病毒1型脑炎(HSE)。此外,通过调节UNC93B1和TLR7/9的接触可控制或避免系统性红斑狼疮、关节炎等自身性免疫疾病的发生。     

Nucleic acid-sensing TLRs and autoimmunity: novel insights from structural and cell biology

Invasion of pathogenic microorganisms or tissue damage activates innate immune signaling receptors that sample subcellular locations for foreign molecular structures, altered host molecules, or signs of compartment breaches. Upon engagement of innate immune receptors an acute but transient inflammatory response is initiated, aimed at the clearance of pathogens and cellular debris. Among the molecules that are sensed are nucleic acids, which activate several members of the transmembrane Toll-like receptor (TLR) family. Inappropriate recognition of nucleic acids by TLRs can cause inflammatory pathologies and autoimmunity. Here, we review the mechanisms involved in triggering nucleic acid-sensing TLRs and indicate checkpoints that restrict their activation to endolysosomal compartments. These mechanisms are crucial to sample the content of endosomes for nucleic acids in the context of infection or tissue damage, yet prevent accidental activation by host nucleic acids under physiological conditions. Decoding the molecular mechanisms that regulate nucleic acid recognition by TLRs is central to understand pathologies linked to unrestricted nucleic acid sensing and to develop novel therapeutic strategies.     

Oxidized mitochondrial DNA sensing by STING signaling promotes the antitumor effect of an irradiated immunogenic cancer cell vaccine

Exposure to ionizing radiation, a physical treatment that inactivates live tumor cells, has been extensively applied to enhance the antitumor responses induced by cancer cell vaccines in both animal research and human clinical trials. However, the mechanisms by which irradiated cells function as immunogenic tumor vaccines and induce effective antitumor responses have not been fully explored. Here, we demonstrate that oxidized mitochondrial DNA (mtDNA) and stimulator of interferon genes (STING) signaling play a key roles in the enhanced antitumor effect achieved with an irradiated tumor cell vaccine. Elevations in ROS and oxidized mtDNA 8-OHG content could be induced in irradiated tumor cells. Oxidized mtDNA derived from irradiated tumor cells gained access to the cytosol of dendritic cells (DCs). Oxidized mtDNA, as a DAMP or adjuvant, activated the STING-TBK1-IRF3-IFN-β pathway in DCs, which subsequently cross-presented irradiated tumor cell-derived antigens to CD8⁺ T cells and elicited antitumor immunity. The results of our study provide insight into the mechanism by which an irradiated cell vaccine mediates antitumor immunity, which may have implications for new strategies to improve the efficacy of irradiated vaccines.     

Regulatory molecules required for nucleotide-sensing Toll-like receptors

Summary:  Toll-like receptors (TLRs) play an important role in innate immune responses against bacteria and viruses. TLRs localize either on the cell surface or in intracellular vesicular compartments. The cell-surface TLRs, including TLR1, TLR2, TLR4, and TLR6, recognize microbial membrane lipids, whereas TLR3, TLR7, TLR8, and TLR9 recognize pathogen-derived nucleotides in intracellular compartments. TLR7 and TLR9 respond to host-derived nucleotides as well, and they have been implicated in a variety of autoimmune diseases. Safety mechanisms are required to avoid detrimental autoimmune responses. TLR7 and TLR9 are sequestered in the endoplasmic reticulum (ER) in a resting state and traffic to endolysosomes upon ligand-induced stimulation. Sequestration in the ER is a mechanism controlling TLR7/9 responses. A chaperone, gp96, in the ER is reported to regulate TLR7/9 maturation. gp96 is associated with TLR9 and is required for ligand-induced activation of TLR7/9. Two molecules in the ER are reported to regulate TLR7/9 trafficking to endolysosomes. PRAT4A (a protein associated with TLR4 A) is associated with TLR9 and is required for ligand-induced trafficking of TLR9 to endolysosomes. UNC93B1 is specifically associated with TLR3, TLR7, TLR9, and TLR13 and regulates ligand-induced trafficking of TLR7 and TLR9 from the ER to endolysosomes. These molecules are potential therapeutic targets for controlling dysregulated TLR7/9 responses in autoimmune diseases.     

Human plasmacytoid dendritic cells elicit a Type I Interferon response by sensing DNA via the cGAS‐STING signaling pathway

Plasmacytoid dendritic cells (pDCs) are a major source of type I interferon (IFN) and are important for host defense by sensing microbial DNA via TLR9. pDCs also play a critical role in the pathogenesis of IFN‐driven autoimmune diseases. Yet, this autoimmune reaction is caused by the recognition of self‐DNA and has been linked to TLR9‐independent pathways. Increasing evidence suggests that the cytosolic DNA receptor cyclic GMP‐AMP (cGAMP) synthase (cGAS) is a critical component in the detection of pathogens and contributes to autoimmune diseases. It has been shown that binding of DNA to cGAS results in the synthesis of cGAMP and the subsequent activation of the stimulator of interferon genes (STING) adaptor to induce IFNs. Our results show that the cGAS‐STING pathway is expressed and activated in human pDCs by cytosolic DNA leading to a robust type I IFN response. Direct activation of STING by cyclic dinucleotides including cGAMP also activated pDCs and knockdown of STING abolished this IFN response. These results suggest that pDCs sense cytosolic DNA and cyclic dinucleotides via the cGAS‐STING pathway and that targeting this pathway could be of therapeutic interest.     

Endocytosis‐free DNA sensing by cell surface TLR9 in neutrophils: Rapid defense with autoimmune risks

TLR9 senses microbial DNA, but may also respond to self‐DNA. To prevent the initiation of innate immune responses to self‐DNA, TLR9 is thought to sense microbial DNA in endolysosomes, and not at the cell surface. A report by Lindau et al. in this issue of the European Journal of Immunology [Eur. J. Immunol. 2013. 43: 2101–2113] shows that TLR9 is expressed on the surface of human and mouse neutrophils and, furthermore, shows that cell surface TLR9, instead of endosomal TLR9, senses DNA in neutrophils. These findings demonstrate that DNA sensing by TLR9 in neutrophils is quite distinct from that in DCs or macrophages. The unique DNA sensing by cell surface TLR9 in neutrophils may reflect their role in inducing rapid inflammation by degranulation with a minimal role in engulfing microbial products for antigen presentation.     

Blockade of the Phagocytic Receptor MerTK on Tumor-Associated Macrophages Enhances P2X7R-Dependent STING Activation by Tumor-Derived cGAMP

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Blockade of endogenous B7-H1 suppresses antibacterial protection after primary Listeria monocytogenes infection

B7-H1 (also known as CD274 and PD-L1) is a cosignalling molecule regulating T-cell immunity positively or negatively in vivo. However, little is known about the role of endogenous B7-H1 in bacterial infection. We found that B7-H1 expression was up-regulated in various cell populations including CD4+ and CD8+ T cells, natural killer (NK) cells and macrophages following Listeria monocytogenes infection. Administration of the antagonistic B7-H1 monoclonal antibody resulted in a significant increase in mortality in mice infected with a lethal dose of L. monocytogenes compared with mice given the control immunoglobulin. In vivo blockade of B7-H1 greatly inhibited the production of tumour necrosis factor (TNF)-alpha and nitric oxide, key effector molecules responsible for intracellular killing by macrophages. B7-H1 blockade also suppressed the expression of granzyme B and interferon (IFN)-gamma by NK cells. Interestingly, blocking of endogenous B7-H1 selectively inhibited CD8+ T cells rather than CD4+ T cells in response to L. monocytogenes infection, as evidenced by the reduction of IFN-gamma production and the expression of effector surface markers including CD62L(int/low) and CD44(high) in CD8+ T cells from mice given anti-B7-H1 monoclonal antibody. In addition, we found that the proliferation of listeriolysin-O (LLO)-specific and IFN-gamma-producing L. monocytogenes-reactive CD8+ T cells was significantly decreased not only in the effector phase but also in the memory phase in the presence of anti-B7-H1 antibody. Our findings thus suggest that endogenous B7-H1 can provide positive costimulatory signals for innate and adaptive immunity leading to protection against intracellular bacterial infection.     

Polyreactive antigen-binding B (PAB-) cells are widely distributed and the PAB population consists of both B-1+ and B-1- phenotypes

B cells that make polyreactive antibodies (PAB+ cells) express polyreactive Ig receptors on their surface and can bind a variety of different antigens. The present study shows that PAB+ cells are widely distributed, are present in varying numbers in different lymphoid organs and that their phenotype varies depending on the organs from which they are isolated. Up to 10 times more cells in PAB+ enriched populations bind antigens as compared to PAB- populations. Comparison of PAB+ with B-1+ cells showed that a high percentage of PAB+ cells are B-1+, but that many PAB+ cells do not express B-1 cell surface markers and, in fact, are B-1-. It is concluded that the B cell population consists of PAB+/B-1+, PAB+/B-1-, PAB-/B-1+, and PAB-/B-1- cells. The presence of PAB+ cells in the thymus points to the possibility that PAB+ cells may carry endogenous host antigens from peripheral tissues to the thymus where they may contribute to immunological tolerance.     

日本血吸虫组织蛋白酶B DNA疫苗的免疫保护效果研究

目的 :观察已构建日本血吸虫大陆株 31kDa组织蛋白酶BDNA疫苗VR1 0 1 2 Sj31及VR1 0 1 2 SjGST Sj31在BALB c小鼠体内的免疫保护效果。方法 :用纯化的空白质粒载体VR1 0 1 2、重组质粒VR1 0 1 2 Sj31及VR1 0 1 2 SjGST Sj31于股四头肌注射免疫BALB c鼠 :将 36只 6～ 8周龄BALB c鼠随机分为 3组 ,每组 1 2只 ,对照组 (A组 )注射空白质粒载体VR1 0 1 2 ,B组注射重组质粒VR1 0 1 2 Sj31 ,C组注射重组质粒VR1 0 1 2 SjGST Sj31。质粒剂量均为 1 0 0 μg ,每隔 2周免疫 1次 ,共免疫 3次 ,第 3次免疫后第 2 1天 ,每只鼠经腹部感染 1 0条尾蚴 ,4 2天后剖杀计数各小鼠成虫数和每克肝卵数 ,观察诱导产生的减虫和减卵效果 ,并用ELISA分析小鼠血清中的抗体。结果 :ELISA分析表明 ,第 3次免疫后小鼠出现特异性IgG抗体。与空白质粒对照组比较 ,2个实验组的减虫率分别为 2 5 0 %及 30 0 % (P <0 0 5 ) ,肝组织减卵率分别为 5 4 90 %及 6 9 74 % (P <0 0 0 1 ) ,每雌肝减卵率分别为 4 7 96 %、5 4 6 2 % (P <0 0 0 1 )。与B组相比 ,C组的肝组织减卵率为 32 92 % (P <0 0 0 1 ) ,每雌肝减卵率为 1 2 79% (P <0 0 5 )。结论 :DNA疫苗VR1 0 1 2 Sj31和VR1 0 1 2 SjGST Sj31能诱导小鼠产生一定水平的抗     

RETRACTED: MiR-93 blocks cell cycle progression and promotes apoptosis in uterine leiomyoma cells by targeting CCND1

Uterine leiomyoma (UL) is the most common type of benign tumor in the women's reproductive system. A number of genes has been found to play an important role in the initiation and progression of UL, including miRNAs. In this study, our results exhibited that miR-93, a member of mir-106b-25 cluster, significantly reduced the cell viability, promoted cell cycle arrest, caused apoptosis, and inhibited migration in UL cells (p < .01). Moreover, our results have provided experimental evidence that miR-93 regulated the biological functions of UL cells by targeting CCND1.     

RAGE-independent autoreactive B cell activation in response to chromatin and HMGB1/DNA immune complexes

Increasing evidence suggests that the excessive accumulation of apoptotic or necrotic cellular debris may contribute to the pathology of systemic autoimmune disease. HMGB1 is a nuclear DNA-associated protein, which can be released from dying cells thereby triggering inflammatory processes. We have previously shown that IgG2a-reactive B cell receptor (BCR) transgenic AM14 B cells proliferate in response to endogenous chromatin immune complexes (ICs), in the form of the anti-nucleosome antibody PL2-3 and cell debris, in a TLR9-dependent manner, and that these ICs contain HMGB1. Activation of AM14 B cells by these chromatin ICs was inhibited by a soluble form of the HMGB1 receptor, RAGE-Fc, suggesting HMGB1–RAGE interaction was important for this response. To further explore the role of HMGB1 in autoreactive B cell activation, we assessed the capacity of purified calf thymus HMGB1 to bind dsDNA fragments and found that HMGB1 bound both CG-rich and CG-poor DNA. However, HMGB1–DNA complexes could not activate AM14 B cells unless HMGB1 was bound by IgG2a and thereby able to engage the BCR. To ascertain the role of RAGE in autoreactive B cell responses to chromatin ICs, we intercrossed AM14 and RAGE-deficient mice. We found that spontaneous and defined DNA ICs activated RAGE⁺ and RAGE^? AM14 B cells to a comparable extent. These results suggest that HMGB1 promotes B cell responses to endogenous TLR9 ligands through a RAGE-independent mechanism.     

Hepatitis regulation by the inflammasome signaling pathway

Hepatitis is damage and inflammation of the liver. It is triggered by both environmental and endogenous insults and is a platform for developing liver cirrhosis and cancer. Both innate and adaptive immune activation contribute to hepatic inflammation and disease. Viral hepatitis is the most common form of hepatitis and is typically associated with chronic viral infection. Alcohol-induced and non-alcoholic steatohepatitis are two rising hepatic problems. The innate immune inflammasome signaling cascade mediates the production of essential proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. These cytokines regulate hepatic cell interaction and crosstalk of the various inflammatory pathways and influence disease outcome.     

Regulation of B lymphocytes and plasma cells by innate immune mechanisms and stromal cells in rheumatoid arthritis

B cells mediate multiple functions that influence immune and inflammatory responses in rheumatoid arthritis. Production of a diverse array of autoantibodies can happen at different stages of the disease, and are important markers of disease outcome. In turn, the magnitude and quality of acquired humoral immune responses is strongly dependent on signals delivered by innate immune cells. Additionally, the milieu of cells and chemokines that constitute a niche for plasma cells rely strongly on signals provided by stromal cells at different anatomical locations and times. The chronic inflammatory state therefore importantly impacts the developing humoral immune response and its intensity and specificity. We focus this review on B cell biology and the role of the innate immune system in the development of autoimmunity in patients with rheumatoid arthritis.     

IL—12和B7—1协同抗肿瘤作用和疫苗效应的研究

研究了IL-12和B7-1基因导入小鼠EL-4胸腺瘤细胞后在小鼠体内诱导的协同抗肿瘤免疫作用。方法将逆转录病毒载体重组的小鼠IL-12,B7-1基因表达质粒分别导入小鼠EL-4胸腺瘤细胞,利用转基因细胞治疗小鼠观察其抗肿瘤免疫效果。结果转基因细胞的肿瘤原性较EL-4/Wt和EL-4/Neo组明显降低(P<0.001)。同时免疫原性明显增强,以     

Cytosolic d-type CpG-oligonucleotides induce a type I interferon response by activating the cGAS-STING signaling pathway

Cytosolic DNA receptor cyclic GMP-AMP (cGAMP) synthase (cGAS) has been shown to be critically involved in the detection of cytosolic, self- and non-self-DNA, initiating a type I IFN response through the adaptor protein Stimulator of Interferon Genes (STING) and interferon regulatory factor 3 (IRF3). Current studies propose that canonical binding of dsDNA by cGAS depends on DNA length, but not on base sequence. In contrast, activation of TLR9 is sequence dependent. It requires unmethylated CpG dinucleotides in microbial DNA, which is mimicked by synthetic oligodeoxynucleotides (ODN). Here, we provide evidence that d -type ODN (D-ODN), but not K-type ODN (K-ODN), bind to human cGAS and activate downstream signaling. Transfection of D-ODN into a TLR9-deficient, human monocytic cell line (THP-1) induced phosphorylation of IRF3 and secretion of IFN. This response was absent in cells with CRISPR/Cas9-mediated cGAS- or STING-deficiency. Utilizing a protein pulldown approach, we further demonstrate direct binding of D-ODN to cGAS. Induction of a type I IFN response by D-ODN was confirmed in human primary monocytes and monocyte-derived macrophages. These results are relevant to our understanding of self–nonself-discrimination by cGAS and to the pharmacologic effects of ODN, which currently are investigated in clinical studies.     

Impact of class A, B and C CpG-oligodeoxynucleotides on in vitro activation of innate immune cells in human immunodeficiency virus-1 infected individuals

Oligodeoxynucleotides (ODN) with unmethylated deoxycytidyl-deoxyguanosine dinucleotides (CpG-ODNs) stimulate Toll-like receptor 9 (TLR9) in plasmacytoid dendritic cells (pDC) and B cells and activate innate and adaptive immunity. Three classes of synthetic CpG-ODNs, class A, B and C, activate cells through TLR9; our goal was to evaluate their effect on cells from human immunodeficiency virus (HIV)-1(+) individuals. We compared the frequencies and the unstimulated activation status of immune effector cells in HIV-1(+) and HIV-1(-) individuals. Fewer pDC, myeloid dendritic cells (mDC), B cells, natural killer (NK) cells and invariant natural killer T cells (iNKT) were present in HIV-1(+) peripheral blood mononuclear cells (PBMC) and their baseline activation status was higher than HIV-1(-) PBMC. Exposure of HIV-1(+) PBMC to all classes of CpG-ODNs led to activation and maturation of pDC based on CD86, CD80, and CD83 expression similar to that of cells from HIV-1(-) individuals. The percentage of CpG-ODN stimulated pDC that express CD40 was dramatically higher when cells were obtained from HIV-1(+) than from HIV-1(-) individuals. B-lymphocytes were activated similarly in HIV-1(+) and HIV-1(-) individuals. mDC, NK and iNKT cell, which lack TLR9, were indirectly activated. Interferon-alpha (IFN-alpha) and interferon inducible protein 10 (IP-10) secretion was induced by class A or C but not class B CpG-ODN, but the concentrations were less than those produced by HIV-1(-) PBMC. HIV-1 infected individuals have fewer innate effector cells that are chronically activated, but these cells can be further activated by CpG-ODN, which suggests that synthetic CpG-ODNs could be used to enhance the immune system in HIV-1 infected individuals.