Pathogen-specific TLR signaling in mucosa: mutual contribution of microbial TLR agonists and virulence factors

Detection of microorganisms through microbe-associated molecular patterns (MAMP) by Toll-like receptors (TLR) is crucial to trigger protective immunity. In the mucosa, sentinel cells are exposed to MAMP from both pathogens and commensals; however, the TLR response is tightly controlled to avoid inflammation in response to commensals. Uropathogenic Escherichia coli (UPEC) trigger innate responses during urinary tract infection in a TLR4-dependent and CD14-independent manner. UPEC express virulence factors, such as type 1 fimbriae and/or P fimbriae, allowing bacterial attachment to the epithelium. In this issue of the European Journal of Immunology, Fisher et al. show that fimbriae are required to induce a TLR4-specific epithelial response. Depending on the fimbriae expressed by UPEC, different adaptor molecules are involved in TLR4 signaling. These data add to the recent body of evidence suggesting that TLR responses are regulated by co-receptors, such as receptors for virulence factors. In conclusion, the "pathogenic" TLR stimulation provides a novel way for the host to ignore commensal bacteria.     

Molecular analysis of a novel Toll/interleukin-1 receptor (TIR)-domain containing virulence protein of Y. pseudotuberculosis among Far East scarlet-like fever serotype I strains

Pathogenicity of Yersinia pseudotuberculosis is determined by an arsenal of virulence factors. Particularly, the Yersinia outer proteins (Yops) and the Type III secretion system (T3SS) encoded on the pYV virulence plasmid are required for Yersinia pathogenicity. A specific group of Y. pseudotuberculosis, responsible for the clinical syndrome described as Far East scarlet-like fever (FESLF), is known to have an altered virulence gene cluster. Far East strains cause unique clinical symptoms for which the pYV virulence plasmid plays apparently a rather secondary role. Here, we characterize a previously unknown protein of Y. pseudotuberculosis serotype I strains (TcpYI) which can be found particularly among the FESLF strain group. The TcpYI protein shares considerable sequence homology to members of the Toll/IL-1 receptor family. Bacterial TIR domain containing proteins (Tcps) interact with the innate immune system by TIR-TIR interactions and subvert host defenses via individual, multifaceted mechanisms. In terms of virulence, it appears that the TcpYI protein of Y. pseudotuberculosis displays its own virulence phenotype compared to the previously characterized bacterial Tcps. Our results clearly demonstrate that TcpYI increases the intracellular survival of the respective strains in vitro. Furthermore, we show here that the intracellular survival benefit of the wild-type strain correlates with an increase in tcpYI gene expression inside murine macrophages. In support of this, we found that TcpYI enhances the survival inside the spleens of mice in a mouse model of peritonitis. Our results may point toward involvement of the TcpYI protein in inhibition of phagocytosis, particularly in distinct Y. pseudotuberculosis strains of the FESLF strain group where the pYV virulence plasmid is absent.     

TNF receptor-associated factor-1 (TRAF1) negatively regulates Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF)-mediated signaling

Toll-like receptor 3 (TLR3) plays an important role in antiviral responses through recognizing viral double-stranded RNA produced during viral infection and mediating induction of type I IFN. TRIF is a Toll/IL-1 receptor (TIR) domain-containing adaptor protein that is associated with TLR3 and critically involved in TLR3-mediated signaling. In yeast two-hybrid screens, we identified TNF receptor-associated factor (TRAF)1 as a TRIF-interacting protein. The TRAF-C domain of TRAF1 and the TIR domain of TRIF were responsible for their interaction. Overexpression of TRAF1 inhibited TRIF- and TLR3-mediated activation of NF-kappaB, IFN-stimulated response element and the IFN-beta promoter. Overexpression of TRIF caused caspase-dependent cleavage of TRAF1. The cleaved N-terminal but not C-terminal fragment of TRAF1 was responsible for inhibiting TRIF signaling. Mutation of the caspase cleavage site of TRAF1 or addition of the caspase inhibitor crmA inhibited TRAF1 cleavage and abolished the ability of TRAF1 to inhibit TRIF signaling, suggesting that TRIF-induced cleavage of TRAF1 is required for its inhibition of TRIF signaling. Our findings provide a novel mechanism for negative regulation of TRIF-mediated signaling.     

Pseudomonas aeruginosa lipopolysaccharide: A major virulence factor, initiator of inflammation and target for effective immunity

Pseudomonas aeruginosa is one of the most important bacterial pathogens encountered by immunocompromised hosts and patients with cystic fibrosis (CF), and the lipopolysaccharide (LPS) elaborated by this organism is a key factor in virulence as well as both innate and acquired host responses to infection. The molecule has a fair degree of heterogeneity in its lipid A and O-antigen structure, and elaborates two different outer-core glycoforms, of which only one is ligated to the O-antigen. A close relatedness between the chemical structures and genes encoding biosynthetic enzymes has been established, with 11 major O-antigen groups identified. The lipid A can be variably penta-, hexa- or hepta-acylated, and these isoforms have differing potencies when activating host innate immunity via binding to Toll-like receptor 4 (TLR4). The O-antigen is a major target for protective immunity as evidenced by numerous animal studies, but attempts, to date, to produce a human vaccine targeting these epitopes have not been successful. Newer strategies employing live attenuated P. aeruginosa, or heterologous attenuated bacteria expressing P. aeruginosa O-antigens are potential means to solve some of the existing problems related to making a P. aeruginosa LPS-specific vaccine. Overall, there is now a large amount of information available about the genes and enzymes needed to produce the P. aeruginosa LPS, detailed chemical structures have been determined for the major O-antigens, and significant biologic and immunologic studies have been conducted to define the role of this molecule in virulence and immunity to P. aeruginosa infection.     

P物质/神经激肽1受体系统在肿瘤治疗中的研究进展

P物质（SP）是第1个被发现的速激肽家族成员,广泛分布于哺乳动物的中枢神经系统和外周神经系统,参与多种生理和病理过程。神经激肽受体（NKRs）包括NK1R、NK2R和NK3R,它们是G-蛋白耦联受体家族成员。SP是NK1R内源性的高亲和性和高选择性配体。越来越多的实验结果表明,SP/NK1R系统参与了肿瘤的发生过程。因此,NK1R有可能成为肿瘤治疗的新靶点。我们就SP/NK1R系统在肿瘤中的表达水平,以及SP/NK1R系统作为新靶点在肿瘤治疗领域的研究进展做一简要综述。     

Association between interleukin 1 receptor antagonist gene 86-bp VNTR polymorphism and sepsis: A meta-analysis

ObjectiveMany studies have focused on the relationship between interleukin 1 receptor antagonist (IL1RN) gene 86-bp VNTR polymorphism and sepsis, but the results remain inconsistent. Thus, a meta-analysis was carried out to derive a more precise estimation of the association between IL1RN 86-bp VNTR polymorphism and risk of sepsis and sepsis-related mortality.MethodsRelevant publications were searched in several widely used databases and six eligible studies were included in the meta-analysis. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to evaluate the strength of the association between IL1RN 86-bp VNTR polymorphism and risk of sepsis and sepsis-related mortality.ResultsSignificant associations between IL1RN 86-bp VNTR polymorphism and sepsis risk were observed in both overall meta-analysis for L2 versus 22 (OR = 0.75, 95% CI = 0.59–0.94) and severe sepsis subgroup for LL + L2 versus 22 (OR = 0.67, 95% CI = 0.47–0.93). L stands for long alleles containing three to six repeats; 2 stands for short allele containing two repeats. However, no significant sepsis mortality variation was detected for all genetic models.ConclusionsAccording to the results of our meta-analysis, the IL1RN 86-bp VNTR polymorphism probably associates with sepsis risk but not with sepsis-related mortality.     

HIV-1 proteins in infected cells determine the presentation of viral peptides by HLA class I and class II molecules and the nature of the cellular and humoral antiviral immune responses—A review

The goals of molecular virology and immunology during the second half of the 20th century have been to provide the conceptual approaches and the tools for the development of safe and efficient virus vaccines for the human population. The success of the vaccination approach to prevent virus epidemics was attributed to the ability of inactivated and live virus vaccines to induce a humoral immune response and to produce antiviral neutralizing antibodies in the vaccinees. The successful development of antiviral vaccines and their application to most of the human population led to a marked decrease in virus epidemics around the globe. Despite this remarkable achievement, the developing epidemics of HIV-caused AIDS (accompanied by activation of latent herpesviruses in AIDS patients), epidemics of Dengue fever, and infections with respiratory syncytial virus may indicate that conventional approaches to the development of virus vaccines that induce antiviral humoral responses may not suffice. This may indicate that virus vaccines that induce a cellular immune response, leading to the destruction of virus-infected cells by CD8⁺ cytotoxic T cells (CTLs), may be needed. Antiviral CD8⁺ CTLs are induced by viral peptides presented within the peptide binding grooves of HLA class I molecules present on the surface of infected cells. Studies in the last decade provided an insight into the presentation of viral peptides by HLA class I molecules to CD8⁺ T cells. These studies are here reviewed, together with a review of the molecular events of virus replication, to obtain an overview of how viral peptides associate with the HLA class I molecules. A similar review is provided on the molecular pathway by which viral proteins, used as subunit vaccines or inactivated virus particles, are taken up by endosomes in the endosome pathway and are processed by proteolytic enzymes into peptides that interact with HLA class II molecules during their transport to the plasma membrane of antigen-presenting cells. Such peptides are identified by T-cell receptors present on the plasma membrane of CD4⁺ T helper cells. The need to develop viral synthetic peptides that will have the correct amino acid motifs for binding to HLA class I A, B, and C haplotypes is reviewed.The development of HIV vaccines that will stimulate, in an uninfected individual, the humoral (antibody) and cellular (CTL) immune defenses against HIV and HIV-infected cells, respectively, and may lead to protection from primary HIV infection are discussed. The need to eliminate the release of HIV virions from infected cells introduced by an infected donor to an uninfected recipient may require both the humoral and cellular immune responses. However, such CTLs may fail to identify HIV-infected cells with integrated HIV proviral DNA that do not express viral genes and proteins. Based on reported results on the immunization of monkeys with uninfected cells, which prevented infection with SIV grown in the same type of cells, it may be possible to consider immunization of specific human populations against HLA haplotypes prevalent in HIV-infected donors. Since HIV virions may carry the HLA class I molecules present in the infected donors' cells, synthesis of CTLs to the mutated amino acid sequence in peptide binding grooves of the foreign HLA haplotypes may induce anti-HLA CTLs in the immunized individual, which may destroy HIV-infected, virus synthesizing donor cells, as well as donor cells containing latent proviral DNA. Such anti-foreign HLA CTLs may prevent the release of virions from the infecting donor's cells. The importance of HLA haplotypes for protection against HIV will be discussed.Dedicated to the memory of Dr. Albert Sabin (1906–1993).     

Comparative genomic analysis for the presence of potential enterococcal virulence factors in the probiotic Enterococcus faecalis strain Symbioflor 1

Enterococci are members of the natural microbiota of animal and human intestinal tracts and are capable of causing opportunistic infections. They are also used as starter cultures in the food industry as well as in health supplements and probiotics by the pharmaceutical industry. This Janus-faced status requires a careful evaluation on the basis of pathogenic traits to ensure the safety of the strain used to produce food and pharmaceuticals. We performed gapped-genome sequencing of a probiotic strain Enterococcus faecalis Symbioflor 1 and present initial results deriving from comparative genome analysis with that of the previously sequenced pathogenic clinical isolate E. faecalis V583. There was strong overall conservation of synteny between both strains and a detailed analysis revealed the absence of large genomic regions from the chromosome of the probiotic strain, indicating gene loss. Genes absent from the Symbioflor 1 strain included those encoding the enterococcal cytolysin, enterococcal surface protein, and gelatinase (coccolysin) as well as hyaluronidase and the peptide antibiotic AS-48. This data was confirmed using PCR primers specific for the respective genes. However, other enterococcal determinants such as aggregation substance, collagen adhesion protein, the ability to resist oxygen anions as well as capsule formation were detected. The presence of these traits may be advantageous for the strain Symbioflor 1 since they potentially enable colonization and proliferation of the bacterium on mucosal surfaces thereby conferring on it probiotic traits.     

G蛋白参与C1q／C1qR系统介导的信号转导

ａｇｇ－Ｃ１ｑ可抑制人Ｔ细胞系Ｊｕｒｋａｔ和Ｍψ系Ｕ９３７细胞受霍乱毒素刺激的（ｃＡＭＰ）ｉ增高，而百日咳毒素能遏止人Ｂ细胞系Ｒａｊｉ细胞ａｇｇ－Ｃ１ｑ诱导的（ｃＡＭＰ）ｉ升高和Ｃ１ｑＲ交联所促成的磷脂酰肌醇水解。表明：Ｇ蛋白介入了Ｃ１ｑ／Ｃ１ｑＲ系统介导的信号转导途径。     

Role of endogenous RGS proteins on endothelial ERK 1/2 activation

Endothelial cells are maintaining atherosclerotic signaling mediated by Extracellular Regulated Kinases 1 and 2 (ERK). Signaling gets activated upon stimulation of G protein-coupled receptors mediated by G_q and G_i/o proteins subjected to regulation by RGS proteins. The goal of the study was to delineate the specificity of RGS proteins modulating induced ERK phosphorylation. We used stimulated HUVEC, silenced specifically RGS proteins and compared assessed ERK 1/2 activation with immunohistochemical stainings on atherosclerotic plaques.Increased ERK phosphorylation was detected upon stimulation with Phenylephrine (2.6 ± 0.1 times over basal), Endothelin-1 (1.8 ± 0.2), Dopamine (5.1 ± 0.2), TNF (9.8 ± 0.7) or IL-4 (3.1 ± 0.3). RGS silencing increased activation of ERK 1/2: Phen (RGS3, 5), ET-1 (RGS3, 4), Dopa (RGS3), TNF (RGS2, 3, 4) or IL-4 (RGS2, 3, 4). Immunohistochemically, increased ERK activation was detected on atherosclerotic plaques.This data supports the role of RGS proteins on ERK activation in human atherosclerosis which identifies RGS proteins as new therapeutical targets.     

Inhibition of sigma-1 receptor reduces N-methyl-d-aspartate induced neuronal injury in methamphetamine-exposed and -naive hippocampi

Acute and prolonged methamphetamine (METH) exposure has been reported to moderate the function of N-methyl-d-aspartate type glutamate receptors (NMDAr) in the hippocampus. These effects have been found to be associated with enhanced NMDAr-dependent release of Ca²⁺ from IP₃-sensitive intracellular stores. The present studies were designed to extend these findings and examine the role of the endoplasmic membrane (ER) bound orphan receptor, the sigma-1 receptor, in NMDA-induced neuronal injury and METH withdrawal-potentiated NMDA-induced neuronal injury. Organotypic hippocampal slice cultures were exposed to METH (0 or 100 μM) for 6 days and withdrawn for 7 days, then exposed to NMDA (0 or 5 μM) for 24 h. Additional cultures were also exposed to this regimen and were co-incubated with BD1047 (100 μM), a specific inhibitor of ER-bound sigma-1 receptors, for the 24 h NMDA exposure. Cytotoxicity was assessed by analysis of propidium iodide uptake. These studies demonstrated that protracted METH exposure and withdrawal significantly potentiated the neuronal injury produced by NMDA exposure. Further, co-exposure to BD1047 with NMDA markedly attenuated neuronal injury in METH-naïve and METH-withdrawn organotypic cultures. As a whole, these data demonstrate that prolonged METH exposure, even at non-toxic concentrations, significantly alters glutamate receptor signaling. Inhibition of sigma-1 receptor-dependent Ca²⁺ release from the ER entirely prevented NMDA-induced toxicity in METH-naïve cultures and markedly reduced METH-potentiated toxicity. These findings demonstrate the importance of Ca²⁺-induced intracellular Ca²⁺ release in excitotoxic insult and suggest that blockade of glutamatergic overactivity may represent a therapeutic target in the treatment of METH withdrawal.     

A transgenic mouse model for studying the clearance of blood-borne pathogens via human complement receptor 1 (CR1)

Complement receptor 1 (CR1) on the surface of human erythrocytes facilitates intravascular clearance of complement-opsonized pathogens. The need for complement activation can be circumvented by directly coupling the organism to CR1 using a bispecific monoclonal antibody heteropolymer (HP). Lack of a functional homologue to CR1 on mouse erythrocytes has made it difficult to study HP-dependent clearance of pathogens in small animals. We have developed a transgenic mouse that expresses human CR1 on erythrocytes. CR1 antigen is of appropriate size and in a clustered distribution as confirmed by immunoblotting and fluorescence microscopy, respectively. HP that immobilized bacteriophage PhiX174 prototype pathogen to erythrocyte CR1 of the transgenic mice increased the rate of clearance of the virus compared with HP that bound bacteriophage, but not CR1. This transgenic mouse model will allow evaluation of different HPs for their in vivo efficacy and potential as human therapeutics.     

甘氨酸受体α_1亚基在乳鼠心肌细胞的表达及损害性因素对其表达的影响

目的:研究甘氨酸受体α1亚基(GlyRα1)在乳鼠心肌细胞中的表达以及脂多糖(LPS)、缺氧/复氧(H/R)、异丙肾上腺素(ISO)和高糖(HG)对其表达的影响。方法:体外培养乳鼠心肌细胞,Western blotting方法检测心肌细胞上GlyRα1的表达;心肌细胞分别用LPS、H/R、ISO以及HG处理24 h,采用CCK-8试剂检测细胞活力,Western blotting方法检测心肌细胞上GlyRα1的表达。结果:Western blotting方法检测到乳鼠心肌细胞上GlyRα1的表达;LPS(20 mg/L)、ISO(100μmol/L)以及HG(25mmol/L)处理心肌细胞24 h与心肌细胞H/R 3 h对心肌细胞存活率无明显影响;LPS组、H/R 3 h组以及ISO组心肌细胞上GlyRα1表达均高于对照组(P0.01),而HG组心肌细胞上GlyRα1表达低于对照组(P0.01)。结论:乳鼠心肌细胞上存在GlyRα1,并且一定浓度的LPS、ISO与一定时间的H/R均可上调乳鼠心肌细胞GlyRα1的表达,而HG可下调乳鼠心肌细胞GlyRα1的表达。     

1-磷酸鞘氨醇受体2介导PI3K/AKT/eNOS通路抑制甲型流感病毒诱导的病毒性肺炎

目的:探讨1-磷酸鞘氨醇受体2(S1PR2)对甲型流感病毒诱导的病毒性肺炎的作用及机制。方法:采用甲型流感病毒鼠肺适应株FM1滴鼻感染野生C57BL/6小鼠和S1pr2~(-/-)小鼠,建立甲型流感病毒性肺炎动物模型。病毒感染4和6 d时观察比较对照组(模型组的野生小鼠)、JTE-013(S1PR2高效拮抗剂)处理的小鼠及S1pr2~(-/-)小鼠肺组织的病理改变,检测支气管肺泡灌洗液(BALF)中的蛋白浓度、细胞总数及细胞因子[白细胞介素(IL)-1β、IL-6和肿瘤坏死因子α(TNF-α)]的表达,Western blot法检测小鼠肺组织的AKT和e NOS的磷酸化水平。结果:与模型对照组的野生鼠比较,JTE处理组和S1pr2~(-/-)组甲型流感病毒性肺炎更加严重;BALF中的蛋白浓度,总细胞数及炎性细胞因子表达显著增加;且PI3K下游靶点AKT和e NOS磷酸化显著增高(P0.01)。结论:S1PR2通过介导PI3K/AKT/e NOS信号转导通路,调节NO生成,抑制血管通透性和炎性细胞因子释放,从而减轻甲型流感病毒诱导的病毒性肺炎。