肽聚糖识别蛋白

天然免疫系统通过一系列高度保守的模式识别受体识别病原体相关分子模式.肽聚糖识别蛋白家族是重要的模式识别受体,从昆虫到人类均高度保守,可识别肽聚糖和含肽聚糖的细菌,在天然免疫和获得性免疫应答中发挥重要的识别和调节功能.     

小鼠PGRP-L分子N端基因片段的克隆与原核表达

采用RT-PCR技术,从BALB/c小鼠肝组织总RNA中扩增到长约500bp的小鼠长型肽聚糖识别蛋白(mPGRP-L)分子N端基因片段,将其克隆入pUCm-T载体构建重组质粒pmGN,DNA测序表明该基因片段长530bp,序列与GenBank中的完全一致。应用PCR技术,从重组质粒pmGN中扩增目的基因片段,插入表达质粒pET-28a构建重组表达载体pET-GN,导入大肠杆菌BL21中诱导表达目的蛋白,表达产物以Ni+-NTAagarose层析柱纯化。SDS-PAGE和Westernblot分析发现,表达产物主要以包涵体形式存在,相对分子质量约29000。ELISA表明,重组蛋白能被抗mPGRP-L分子N端单表位多克隆抗体所识别。为mPGRP-L分子的研究奠定了一定基础。     

No associations established between single nucleotide polymorphisms in human Toll‐like receptor 2 and Toll‐interacting protein and Staphylococcus aureus bloodstream infections

Staphylococcus aureus bloodstream infections (SABSI) are associated with high morbidity and mortality. The Toll‐like receptor 2 (TLR2) and Toll‐interacting protein (TOLLIP) are important in recognition and regulation of human innate immunity response to S. aureus. Single nucleotide polymorphisms (SNPs) in the TLR2 and TOLLIP encoding genes have been associated with disease, including BSI. The aim of this study was to examine potential associations between a selection of SNPs in the genes encoding TLR2 and TOLLIP, and predisposition, severity, and outcome of SABSI. All patients ≥18 years of age with at least one S. aureus positive blood culture collected from March 2011 through February 2014 at Akershus University Hospital, Lørenskog, Norway, were considered for inclusion. Patients attending elective orthopaedic surgery (total hip and knee replacements, lumbar surgery) served as a control group. The TLR2 Arg753Gln, TLR2 Pro631His, TOLLIP rs5743942, and rs5743867 polymorphisms were analysed using TaqMan SNP Genotyping Assays. A total of 209 SABSI patients and 295 controls were included. The TLR2 Arg753Gln and TLR2 Pro631His polymorphisms were infrequent with no homozygotes and <10% heterozygotes. The included TLR2 and TOLLIP polymorphisms were not associated with susceptibility to SABSI, severity, 30‐day all‐cause mortality, or SABSI caused by the clonal complex 30 (CC30) genotype.     

The macrophage soluble receptor AIM/Api6/CD5L displays a broad pathogen recognition spectrum and is involved in early response to microbial aggression

Apoptosis inhibitor of macrophages （AIMs）, a homologue of human Spa, is a mouse soluble member of the scavenger receptor cysteine-rich superfamily （SRCR-SF）. This family integrates a group of proteins expressed by innate and adaptive immune cells for which no unifying function has yet been described. Pleiotropic functions have been ascribed to AIM, from viability support in lymphocytes during thymic selection to lipid metabolism and anti-inflammatory effects in autoimmune pathologies. In the present report, the pathogen binding properties of AIM have been explored. By using a recombinant form of AIM （rAIM） expressed in mammalian cells, it is shown that this protein is able to bind and aggregate Gram-positive and Gram-negative bacteria, as well as pathogenic and saprophytic fungal species. Importantly, endogenous AIM from mouse serum also binds to microorganisms and secretion of AIM was rapidly induced in mouse spleen macrophages following exposure to conserved microbial cell wall components. Cytokine release induced by well-known bacterial and fungal Toll-like receptor （TLR） ligands on mouse splenocytes was also inhibited in the presence of rAIM. Furthermore, mouse models of pathogen-associated molecular patterns （PAMPs）-induced septic shock of bacterial and fungal origin showed that serum AIM levels changed in a time-dependent manner. Altogether, these data suggest that AIM plays a general homeostatic role by supporting innate humoral defense during pathogen aggression.     

Staphylococcus lugdunensis endocarditis following vasectomy – report of a case history and review of the literature

Staphylococcus lugdunensis is a coagulase‐negative Staphylococcus (CoNS), and part of the normal skin flora. The bacterium is an emerging pathogen that, unlike other CoNS, resembles coagulase‐positive Staphylococcus aureus infections in virulence, tissue destruction, and clinical course. We report a fatal case following minor surgery. The frequency of S. lugdunensis infections has probably been underestimated and under‐reported in the past as few clinical laboratories routinely identify coagulase‐negative Staphylococci.     

H‐ficolin binds Aspergillus fumigatus leading to activation of the lectin complement pathway and modulation of lung epithelial immune responses

Aspergillus fumigatus is an opportunistic fungal pathogen that typically infects the lungs of immunocompromised patients leading to a high mortality. H‐Ficolin, an innate immune opsonin, is produced by type II alveolar epithelial cells and could participate in lung defences against infections. Here, we used the human type II alveolar epithelial cell line, A549, to determine the involvement of H‐ficolin in fungal defence. Additionally, we investigated the presence of H‐ficolin in bronchoalveolar lavage fluid from transplant patients during pneumonia. H‐Ficolin exhibited demonstrable binding to A. fumigatus conidia via l ‐fucose, d ‐mannose and N‐acetylglucosamine residues in a calcium‐ and pH‐dependent manner. Moreover, recognition led to lectin complement pathway activation and enhanced fungal association with A549 cells. Following recognition, H‐ficolin opsonization manifested an increase in interleukin‐8 production from A549 cells, which involved activation of the intracellular signalling pathways mitogen‐activated protein kinase MAPK kinase 1/2, p38 MAPK and c‐Jun N‐terminal kinase. Finally, H‐ficolin concentrations were significantly higher in bronchoalveolar lavage fluid of patients with lung infections compared with control subjects (n = 16; P = 0·00726). Receiver operating characteristics curve analysis further highlighted the potential of H‐ficolin as a diagnostic marker for lung infection (area under the curve = 0·77; P < 0·0001). Hence, H‐ficolin participates in A. fumigatus defence through the activation of the lectin complement pathway, enhanced fungus–host interactions and modulated immune responses.     

The xanthine oxidase–NFAT5 pathway regulates macrophage activation and TLR‐induced inflammatory arthritis

NFAT5 (nuclear factor of activated T cells), a well‐known osmoprotective factor, can be activated by isotonic stimuli such as Toll‐like receptor (TLR) triggering. However, it is unclear how NFAT5 discriminates between isotonic and hypertonic stimuli to produce different functional and molecular outcomes. Here, we identified a novel XO–ROS–p38 MAPK–NFAT5 pathway (XO is xanthine oxidase, ROS is reactive oxygen species) that is activated in RAW 264.7 macrophages upon isotonic TLR stimulation. Unlike what is seen under hypertonic conditions, XO‐derived ROS were selectively required for the TLR‐induced NFAT5 activation and NFAT5 binding to the IL‐6 promoter in RAW 264.7 macrophages under isotonic conditions. In mouse peritoneal macrophages and human macrophages, TLR ligation also induced NFAT5 activation, which was dependent on XO and p38 kinase. The involvement of XO in NFAT5 activation by TLR was confirmed in RAW 264.7 macrophages implanted in BALB/c mice. Moreover, allopurinol, an XO inhibitor, suppressed arthritis severity and decreased the expression of NFAT5 and IL‐6 in splenic macrophages in C57BL/6 mice. Collectively, these data support a novel function of the XO–NFAT5 axis in macrophage activation and TLR‐induced arthritis, and suggest that XO inhibitor(s) could serve as a therapeutic agent for chronic inflammatory arthritis.     

Polymorphisms in pattern‐recognition genes in the innate immunity system and risk of non‐Hodgkin lymphoma

The pattern‐recognition pathway plays an important role in infection recognition and immune responses, and previous studies have suggested an association between genetic variation in innate immunity genes and non‐Hodgkin lymphoma (NHL). We evaluated NHL risk associated with genetic variation in pattern‐recognition genes using data from a case–control study of NHL conducted in Connecticut women. Single nucleotide polymorphisms (SNPs) in 27 pattern‐recognition genes were genotyped in 432 Caucasian incident NHL cases and 494 frequency‐matched controls. Unconditional logistic regression was used to compute odds ratios (ORs) for NHL and common NHL subtypes in relation to individual SNPs and haplotypes. A gene‐based analysis that adjusted for the number of tagSNPs genotyped in each gene showed a significant association with overall NHL for the MBP gene (P = 0.028), with the diffuse large B‐cell lymphoma (DLBCL) subtype for the MASP2 gene (P = 0.011), and with the follicular lymphoma (FL) subtype for DEFB126 (P = 0.041). A SNP‐based analysis showed that MBP rs8094402 was associated with decreased risks of overall NHL (allele risk OR = 0.72, P‐trend = 0.0018), DLBCL (allele risk OR = 0.72, P‐trend = 0.036), and FL (allele risk OR = 0.67, P‐trend = 0.021), while MASP2 rs12711521 was associated with a decreased risk of DLBCL (allele risk OR = 0.57, P‐trend = 0.0042). We also observed an increased risk of FL for DEFB126 rs6054706 (allele risk OR = 1.39, P‐trend = 0.033). Our results suggest that genetic variation in pattern‐recognition genes is associated with the risk of NHL or specific NHL subtypes, but these preliminary findings require replication in larger studies. Mol. Mutagen. 2013. © 2012 Wiley Periodicals, Inc.     

Galectin‐3 is an amplifier of the interleukin‐1β‐mediated inflammatory response in corneal keratinocytes

Interleukin‐1β (IL‐1β) is a potent mediator of innate immunity commonly up‐regulated in a broad spectrum of inflammatory diseases. When bound to its cell surface receptor, IL‐1β initiates a signalling cascade that cooperatively induces the expression of canonical IL‐1 target genes such as IL‐8 and IL‐6. Here, we present galectin‐3 as a novel regulator of IL‐1β responses in corneal keratinocytes. Using the SNAP‐tag system and digitonin semi‐permeabilization, we show that recombinant exogenous galectin‐3 binds to the plasma membrane of keratinocytes and is internalized into cytoplasmic compartments. We find that exogenous galectin‐3, but not a dominant negative inhibitor of galectin‐3 polymerization lacking the N‐terminal domain, exacerbates the response to IL‐1β by stimulating the secretion of inflammatory cytokines. The activity of galectin‐3 could be reduced by a novel d ‐galactopyranoside derivative targeting the conserved galactoside‐binding site of galectins and did not involve interaction with IL‐1 receptor 1 or the induction of endogenous IL‐1β. Consistent with these observations, we demonstrate that small interfering RNA‐mediated suppression of endogenous galectin‐3 expression is sufficient to impair the IL‐1β‐induced secretion of IL‐8 and IL‐6 in a p38 mitogen‐activated protein kinase‐independent manner. Collectively, our findings provide a novel role for galectin‐3 as an amplifier of IL‐1β responses during epithelial inflammation through an as yet unidentified mechanism.     

Staphylococcus aureus convert neonatal conventional CD4+ T cells into FOXP3+ CD25+ CD127low T cells via the PD‐1/PD‐L1 axis

The gut microbiota provides an important stimulus for the induction of regulatory T (Treg) cells in mice, whether this applies to newborn children is unknown. In Swedish children, Staphylococcus aureus has become a common early colonizer of the gut. Here, we sought to study the effects of bacterial stimulation on neonatal CD4⁺ T cells for the induction of CD25⁺ CD127^low Treg cells in vitro. The proportion of circulating CD25⁺ CD127^low Treg cells and their expression of FOXP3, Helios and CTLA‐4 was examined in newborns and adults. To evaluate if commensal gut bacteria could induce Treg cells, CellTrace violet‐stained non‐Treg cells from cord or peripheral blood from adults were co‐cultured with autologous CD25⁺ CD127^low Treg cells and remaining mononuclear cells and stimulated with S. aureus. Newborns had a significantly lower proportion of CD25⁺ CD127^low Treg cells than adults, but these cells were Helios⁺ and CTLA‐4⁺ to a higher extent than in adults. FOXP3⁺ CD25⁺ CD127^low T cells were induced mainly in neonatal CellTrace‐stained non‐Treg cells after stimulation with S. aureus. In cell cultures from adults, S. aureus induced CD25⁺ CD127^low T cells only if sorted naive CD45RA⁺ non‐Treg cells were used, but these cells expressed less FOXP3 than those induced from newborns. Sorted neonatal CD25⁺ CD127^low T cells from S. aureus‐stimulated cultures were still suppressive. Finally, blocking PD‐L1 during stimulation reduced the induction of FOXP3⁺ CD25⁺ CD127^low T cells. These results suggest that newborns have a higher proportion of circulating thymically derived Helios⁺ Treg cells than adults and that S. aureus possess an ability to convert neonatal conventional CD4⁺ T cells into FOXP3⁺ CD25⁺ CD127^low Treg cells via the PD‐1/PD‐L1 axis.     

Interferon‐γ enhances both the anti‐bacterial and the pro‐inflammatory response of human mast cells to Staphylococcus aureus

Human mast cells (huMCs) are involved in both innate and adaptive immune responses where they release mediators including amines, reactive oxygen species (ROS), eicosanoids and cytokines. We have reported that interferon‐γ (IFN‐γ) enhances FcγR‐dependent ROS production. The aim of this study was to extend these observations by investigating the effect of IFN‐γ on the biological responses of huMCs to Staphylococcus aureus. We found that exposure of huMCs to S. aureus generated intracellular and extracellular ROS, which were enhanced in the presence of IFN‐γ. IFN‐γ also promoted bacteria killing, β‐hexosaminidase release and eicosanoid production. Interferon‐γ similarly increased expression of mRNAs encoding CCL1 to CCL4, granulocyte–macrophage colony‐stimulating factor (GM‐CSF), tumour necrosis factor‐α and CXCL8 in S. aureus‐stimulated huMCs. The ability of IFN‐γ to increase CXCL8 and GM‐CSF protein levels was confirmed by ELISA. Fibronectin or a β₁ integrin blocking antibody completely abrogated IFN‐γ‐dependent S. aureus binding and reduced S. aureus‐dependent CXCL8 secretion. These data demonstrate that IFN‐γ primes huMCs for enhanced anti‐bacterial and pro‐inflammatory responses to S. aureus, partially mediated by β₁ integrin.     

NOD1 in the modulation of host–microbe interactions and inflammatory bone resorption in the periodontal disease model

Periodontitis is a chronic inflammatory condition characterized by destruction of non‐mineralized and mineralized connective tissues. It is initiated and maintained by a dysbiosis of the bacterial biofilm adjacent to teeth with increased prevalence of Gram‐negative microorganisms. Nucleotide‐binding oligomerization domain containing 1 (NOD1) is a member of the Nod‐like receptors (NLRs) family of proteins that participate in the activation of the innate immune system, in response to invading bacteria or to bacterial antigens present in the cytoplasm. The specific activating ligand for NOD1 is a bacterial peptidoglycan derived primarily from Gram‐negative bacteria. This study assessed the role of NOD1 in inflammation‐mediated tissue destruction in the context of host–microbe interactions. We used mice with whole‐genome deletion of the NOD1 gene in a microbe‐induced periodontitis model using direct injections of heat‐killed Gram‐negative or Gram‐negative/Gram‐positive bacteria on the gingival tissues. In vitro experiments using primary bone‐marrow‐derived macrophages from wild‐type and NOD1 knockout mice provide insight into the role of NOD1 on the macrophage response to Gram‐negative and Gram‐negative/Gram‐positive bacteria. Microcomputed tomography analysis indicated that deletion of NOD1 significantly aggravated bone resorption induced by Gram‐negative bacteria, accompanied by an increase in the numbers of osteoclasts. This effect was significantly attenuated by the association with Gram‐positive bacteria. In vitro, quantitative PCR arrays indicated that stimulation of macrophages with heat‐killed Gram‐negative bacteria induced the same biological processes in wild‐type and NOD1‐deficient cells; however, expression of pro‐inflammatory mediators was increased in NOD1‐deficient cells. These results suggest a bone‐sparing role for NOD1 in this model.     

Influence of NKG2C gene deletion and CCR5Δ32 in Pre‐eclampsia—Approaching the effect of innate immune gene variants in pregnancy

Pre‐eclampsia (PE) is a hypertensive disorder that affects an important number of pregnant women worldwide. The exact causes of PE remain poorly understood. However, inflammation and deregulation of innate immune cells, such as natural killer (NK) cells, contribute to PE pathogenesis. Besides, the mother's genetic background also impacts on PE susceptibility. Thus, genetic variants that potentially modify the behaviour of inflammatory cells may help us to understand the causes of PE. Variants of genes encoding NKG2C (expressed in NK cells) and C–C chemokine receptor type 5 (CCR5) (expressed mainly in leucocytes) are important targets in the study of gestational disorders. In this context, we evaluated the impact of both NKGC2 gene deletion and CCR5Δ32 gene variant on PE susceptibility in a population sample from central‐southeast Brazil composed by 369 women (156 with PE and 213 healthy pregnant women). No statistically significant association between the NKG2C gene deletion and susceptibility to PE was observed. However, taking into consideration the important role of NK cells in pregnancy, the influence of NKG2C gene deletion on PE pathogenesis should not be ruled out and deserves further studies in populations with different genetic/ethnic backgrounds. In addition, our results regarding CCR5Δ32 corroborate previous data from our group approaching a distinct cohort and reinforce CCR5Δ32 as a protective factor against PE development (p < 0.05).     

The Drosophila IMD pathway in the activation of the humoral immune response

The IMD pathway signaling plays a pivotal role in the Drosophila defense against bacteria. During the last two decades, significant progress has been made in identifying the components and deciphering the molecular mechanisms underlying this pathway, including the means of bacterial sensing and signal transduction. While these findings have contributed to the understanding of the immune signaling in insects, they have also provided new insights in studying the mammalian NF-κB signaling pathways. Here, we summarize the current view of the IMD pathway focusing on how it regulates the humoral immune response of Drosophila.