肺炎克雷伯菌生物膜对小鼠巨噬细胞免疫功能的影响

目的研究肺炎克雷伯菌生物膜(BF)对小鼠腹腔巨噬细胞TLRs mRNA和细胞因子表达的影响,探索机体抗BF感染免疫的特点。方法将雄性昆明种小鼠40只随机分成2组,一组腹腔植入体外形成肺炎克雷伯菌BF的硅胶片,建立留置性医疗装置BF感染模型实验组,另一组植入与实验组同等量的浮游菌作为对照组。实时定量PCR分析2组巨噬细胞TLRs mRNA的表达水平,双抗体夹心ELISA法测定细胞因子的含量。结果实验BF组巨噬细胞TLR2、TLR4 mRNA表达量是对照浮游菌组的0.23和0.24倍;而TLR5、TLR9两组表达差异无显著性。实验BF组刺激前后IL-1、IL-2的差值明显低于对照浮游菌组,而IL-4则相反(P0.01)。结论与浮游菌相比,BF能下调小鼠腹腔巨噬细胞TLR2、TLR4的表达,机体的免疫应答朝着Th2型免疫反应发展,这可能是BF相对浮游菌更容易逃脱机体免疫防御系统、引起慢性感染的机制之一。     

MD-2-mediated Ionic Interactions between Lipid A and TLR4 Are Essential for Receptor Activation

Lipopolysaccharide (LPS) activates innate immune responses through TLR4·MD-2. LPS binds to the MD-2 hydrophobic pocket and bridges the dimerization of two TLR4·MD-2 complexes to activate intracellular signaling. However, exactly how lipid A, the endotoxic moiety of LPS, activates myeloid lineage cells remains unknown. Lipid IV_A, a tetra-acylated lipid A precursor, has been used widely as a model for lipid A activation. For unknown reasons, lipid IV_A activates proinflammatory responses in rodent cells but inhibits the activity of LPS in human cells. Using stable TLR4-expressing cell lines and purified monomeric MD-2, as well as MD-2-deficient bone marrow-derived macrophages, we found that both mouse TLR4 and mouse MD-2 are required for lipid IV_A activation. Computational studies suggested that unique ionic interactions exist between lipid IV_A and TLR4 at the dimerization interface in the mouse complex only. The negatively charged 4′-phosphate on lipid IV_A interacts with two positively charged residues on the opposing mouse, but not human, TLR4 (Lys³⁶⁷ and Arg⁴³⁴) at the dimerization interface. When replaced with their negatively charged human counterparts Glu³⁶⁹ and Gln⁴³⁶, mouse TLR4 was no longer responsive to lipid IV_A. In contrast, human TLR4 gained lipid IV_A responsiveness when ionic interactions were enabled by charge reversal at the dimerization interface, defining the basis of lipid IV_A species specificity. Thus, using lipid IV_A as a selective lipid A agonist, we successfully decoupled and coupled two sequential events required for intracellular signaling: receptor engagement and dimerization, underscoring the functional role of ionic interactions in receptor activation.     

��-arrestin Kurtz inhibits MAPK and Toll signalling in Drosophila development

β‐Arrestins have been implicated in the regulation of multiple signalling pathways. However, their role in organism development is not well understood. In this study, we report a new in vivo function of the Drosophila β‐arrestin Kurtz (Krz) in the regulation of two distinct developmental signalling modules: MAPK ERK and NF‐κB, which transmit signals from the activated receptor tyrosine kinases (RTKs) and the Toll receptor, respectively. Analysis of the expression of effectors and target genes of Toll and the RTK Torso in krz maternal mutants reveals that Krz limits the activity of both pathways in the early embryo. Protein interaction studies suggest a previously uncharacterized mechanism for ERK inhibition: Krz can directly bind and sequester an inactive form of ERK, thus preventing its activation by the upstream kinase, MEK. A simultaneous dysregulation of different signalling systems in krz mutants results in an abnormal patterning of the embryo and severe developmental defects. Our findings uncover a new in vivo function of β‐arrestins and present a new mechanism of ERK inhibition by the Drosophila β‐arrestin Krz.     

A Naturally Occurring Variant in Human TLR9, P99L, Is Associated with Loss of CpG Oligonucleotide Responsiveness

The innate immune system employs Toll-like receptors (TLRs) for the detection of invading microorganisms based on distinct molecular patterns. For example, TLR9 is activated by microbial DNA and also by short therapeutic CpG-containing oligonucleotides (CpG-ODN). TLR9 activation leads to the production of interferons and the priming of humoral adaptive immune responses. Unfortunately, the principles of ligand recognition by TLR9 are poorly understood, and genetic variants of TLR9, which may affect its function, have not been characterized systematically on the molecular level. We therefore sought to functionally characterize reported single nucleotide polymorphisms of TLR9 in the HEK293 model system. We discovered that two variants, P99L and M400I, are associated with altered receptor function regarding NF-κB activation and cytokine induction. Our investigations show that for the most functionally impaired variant, P99L, the ability to respond to physiological and therapeutic TLR9 ligands is severely compromised. However, CpG-ODN binding is normal. CpG-ODN recognition by TLR9 thus appears to involve two separate events, CpG-ODN binding and sensing. Our studies highlight Pro-99 as a residue important for the latter process. In genotyping studies, we confirmed that both M400I (rs41308230) and P99L (rs5743844) are relatively rare variants of TLR9. Our data add rs41308230 and rs5743844 to the list of functionally important TLR variants and warrant further research into their relevance for infectious disease susceptibility or responsiveness to CpG-ODN-based therapies.     

A Role for Toll-like Receptor 3 Variants in Host Susceptibility to Enteroviral Myocarditis and Dilated Cardiomyopathy

The innate antiviral response is mediated, at least in part, by Toll-like receptors (TLRs). TLR3 signaling is activated in response to viral infection, and the absence of TLR3 in mice significantly increases mortality after infection with enteroviruses that cause myocarditis and/or dilated cardiomyopathy. We screened TLR3 in patients diagnosed with enteroviral myocarditis/cardiomyopathy and identified a rare variant in one patient as well as a significantly increased occurrence of a common polymorphism compared with controls. Expression of either variant resulted in significantly reduced TLR3-mediated signaling after stimulation with synthetic double-stranded RNA. Furthermore, Coxsackievirus B3 infection of cell lines expressing mutated TLR3 abrogated activation of the type I interferon pathway, leading to increased viral replication. TLR3-mediated type I interferon signaling required cellular autophagy and was suppressed by 3-methyladenine and bafilomycin A1, by inhibitors of lysosomal proteolysis, and by reduced expression of Beclin 1, Atg5, or microtubule-associated protein 1 light chain 3β (MAP1LC3β). However, TLR3-mediated signaling was restored upon exogenous expression of Beclin 1 or a variant MAP1LC3β fusion protein refractory to RNA interference. These data suggest that individuals harboring these variants may have a blunted innate immune response to enteroviral infection, leading to reduced viral clearance and an increased risk of cardiac pathology.     

Extracellular High Mobility Group Box-1 (HMGB1) Inhibits Enterocyte Migration via Activation of Toll-like Receptor-4 and Increased Cell-Matrix Adhesiveness

Toll-like receptor-4 (TLR4) is the receptor for bacterial lipopolysaccharide, yet it may also respond to a variety of endogenous molecules. Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in newborn infants and is characterized by intestinal mucosal destruction and impaired enterocyte migration due to increased TLR4 signaling on enterocytes. The endogenous ligands for TLR4 that lead to impaired enterocyte migration remain unknown. High mobility group box-1 (HMGB1) is a DNA-binding protein that is released from injured cells during inflammation. We thus hypothesize that extracellular HMGB1 inhibits enterocyte migration via activation of TLR4 and sought to define the pathways involved. We now demonstrate that murine and human NEC are associated with increased intestinal HMGB1 expression, that serum HMGB1 is increased in murine NEC, and that HMGB1 inhibits enterocyte migration in vitro and in vivo in a TLR4-dependent manner. This finding was unique to enterocytes as HMGB1 enhanced migration of inflammatory cells in vitro and in vivo. In seeking to understand the mechanisms involved, TLR4-dependent HMGB1 signaling increased RhoA activation in enterocytes, increased phosphorylation of focal adhesion kinase, and increased phosphorylation of cofilin, resulting in increased stress fibers and focal adhesions. Using single cell force traction microscopy, the net effect of HMGB1 signaling was a TLR4-dependent increase in cell force adhesion, accounting for the impaired enterocyte migration. These findings demonstrate a novel pathway by which TLR4 activation by HMGB1 delays mucosal repair and suggest a novel potential therapeutic target in the amelioration of intestinal inflammatory diseases like NEC.     

The biochemical basis of antimicrobial responses in Manduca sexta

Innate immunity is essential for the wellbeing of vertebrates and invertebrates. Key components of this defense system include pattern recognition receptors that bind to infectious agents, extra-and intra-cellular proteins that relay signals, as well as molecules and cells that eliminate pathogens. We have been studying the defense mechanisms in a biochemical model insect, Manduca sexta. In this insect, hemolin, peptidoglycan recognition proteins, β-1,3-glucan recognition proteins and C-type lectins detect microbial surface molecules and induce immune responses such as phagocytosis, nodulation, encapsulation, melanization and production of antimicrobial peptides. Some of these responses are mediated by extracellular serine proteinase pathways. The proteolytic activation of prophenoloxidase （proPO） yields active phenoloxidase （PO） which catalyzes the formation of quinones and melanin for wound healing and microbe killing. M. sexta hemolymph proteinase 14 （HP 14） precursor interacts with peptidoglycan or β-1,3-glucan, autoactivates, and leads to the activation of other HPs including HP21 and proPO-activating proteinases （PAPs）. PAP-1, -2 and -3 cut proPO to generate active PO in the presence of two serine proteinase homologs. Inhibition of the proteinases by serpins and association of the proteinase homologs with bacteria ensure a localized defense reaction. M. sexta HP1, HP6, HP8, HP17 and other proteinases may also participate in proPO activation or processing of spatzle and plasmatocyte spreading peptide.