乳酸杆菌肽聚糖对小鼠机体免疫功能的调节作用

以乳酸杆菌肽聚糖腹腔注射方式刺激小鼠,采用Affymetrix MOE430A基因芯片研究了乳酸杆菌肽聚糖对小鼠机体免疫细胞基因表达的影响,结果发现,乳酸杆菌肽聚糖可以刺激免疫调节细胞因子相关基因的表达,释放细胞免疫因子,其可能是通过激活TLR-NF-κB相关信号通路而实现的。     

植物乳杆菌NDC 75017对Caco-2细胞中il-6表达的影响

【目的】本研究通过体外实验,分析植物乳杆菌NDC75017对免疫相关基因白细胞介素6(il-6)表达的影响,并进一步揭示其机制。【方法】植物乳杆菌NDC 75017作用于Caco-2细胞0、2、4、6、8、10和12 h,采用Real Time PCR方法检测il-6基因和toll样受体2(tlr2)的表达。植物乳杆菌NDC 75017与Caco-2细胞共培养0、0.5、1、2和4 h,用western blot方法检测NF-κB的磷酸化水平;NF-κB的特异性抑制剂PDTC预处理Caco-2细胞30 min后,再加入植物乳杆菌NDC 75017作用2 h,用Real Time PCR方法检测il-6基因及tlr2的表达量。【结果】植物乳杆菌NDC 75017诱导Caco-2细胞中il-6和tlr2基因的表达,并且分别在诱导8 h和6 h时表达量达到最大。植物乳杆菌NDC 75017能够快速诱导NF-κB的磷酸化作用,在加入其特异性抑制剂PDTC后,il-6和tlr2基因的表达显著下降。【结论】植物乳杆菌NDC 75017能通过tlr2介导的NF-κB信号通路来诱导细胞因子il6短暂性的表达。     

嗜酸乳杆菌NCFM对Caco-2细胞中PTX3基因表达的影响

【目的】研究嗜酸乳杆菌NCFM对肠道上皮细胞中免疫与炎症介质因子PTX3表达的影响,并进一步揭示其调节机制。【方法】嗜酸乳杆菌NCFM与Caco-2细胞共培养0、2、4、8和12 h,提取细胞RNA,采用RealTime RT-PCR方法检测PTX3基因的表达。嗜酸乳杆菌NCFM与Caco-2细胞共培养0、0.5、1、2和4 h,提取细胞蛋白质,采用Western blot方法检测NF-κB的磷酸化水平;用NF-κB的特异性抑制剂PDTC预处理Caco-2细胞30 min,然后加入嗜酸乳杆菌NCFM作用2 h,提取细胞RNA,采用Real Time RT-PCR方法检测PTX3基因的表达。【结果】嗜酸乳杆菌NCFM与Caco-2细胞共培养后能诱导PTX3的表达,并且在共培养4 h的时候PTX3的表达量达到最大,然后逐渐下降;嗜酸乳杆菌NCFM能快速的诱导NF-κB的磷酸化,并且在加入其特异性抑制剂PDTC后,PTX3的表达显著下降。【结论】嗜酸乳杆菌NCFM作用于肠道上皮细胞后能够通过迅速激活NF-κB途径暂时性的调控PTX3的表达。     

LRP16影响电离辐射诱导的NF-κB活性

目的:研究LRP16在电离辐射激活核转录因子NF-κB信号转导通路中的作用。方法:在HeLa细胞中,分别运用双萤光素酶分析和Western印迹检测LRP16对κB-Luc报告基因及NF-κB下游靶基因表达的影响。结果:双萤光素酶实验证实LRP16过表达促进电离辐射诱导的κB-Luc活性,而抑制LRP16则降低电离辐射诱导的κB-Luc活性;Western印迹结果显示,LRP16过表达促进电离辐射诱导NF-κB的下游抗凋亡基因XIAP的表达,与之相对应的是,抑制LRP16降低电离辐射诱导NF-κB下游抗凋亡基因XIAP的表达。结论:LRP16可以调节电离辐射诱导NF-κB的转录活性,并且调控NF-κB下游抗凋亡基因XIAP的表达,为进一步阐明电离辐射激活NF-κB转录活性的分子机制奠定了基础。     

昆虫的NF-κB信号通路

昆虫NF-κB信号通路由toll和imd两条通路组成,通过转录因子NF-κB作用于靶标基因κB位点,而调节抗菌活性物质的表达。大量实验表明它能够被细菌、真菌和病毒的侵染所激活,在昆虫体液免疫中发挥着主要作用。现就昆虫的NF-κB信号通路的主要信号元件等进行综述。     

乙肝病毒X 蛋白通过激活NF-κB 信号通路上调ABCG2 的表达

目的:研究乙肝病毒X蛋白(HBx)通过核因子-κB(NF-κB)信号通路对半转运蛋白(ABCG2)的调节作用。方法:用特异性的NF-κB信号通路阻断剂PDTC阻断NF-κB信号通路,荧光双标激光扫描共聚焦显微镜观察L02细胞系转染HBx基因前后及PDTC加入前后NF-κB信号通路的激活、失活情况,同时用Real-time PCR和Western Blot技术检测转染前后及PDTC加入前后ABCG2在mRNA及蛋白水平的表达变化。结果:以L02细胞为参照,转染HBx基因后的L02-HBx细胞NF-κB信号通路被激活,ABCG2 mRNA和蛋白水平分别增加3.62±0.15和4.61±0.73倍,差异有统计学意义(P<0.05);PDTC作用24h后L02/HBx细胞NF-κB信号通路阻断,ABCG2 mRNA和蛋白表达分别为2.15±0.32倍和2.37±0.55倍,与未加入PDTC作用的L02-HBx细胞相比均有统计学意义(P<0.05)。结论:NF-κB信号通路是HBx上调ABCG2表达的途径之一。     

乙肝病毒X蛋白通过激活NF-κB信号通路上调ABCG2的表达

目的：研究乙肝病毒X蛋白（HBx）通过核因子-κB（NF-κB）信号通路对半转运蛋白（ABCG2）的调节作用。方法：用特异性的NF-κB信号通路阻断剂PDTC阻断NF-κB信号通路,荧光双标激光扫描共聚焦显微镜观察L02细胞系转染HBx基因前后及PDTC加入前后NF-κB信号通路的激活、失活情况,同时用Real-time PCR和Western Blot技术检测转染前后及PDTC加入前后ABCG2在mRNA及蛋白水平的表达变化。结果：以L02细胞为参照,转染HBx基因后的L02-HBx细胞NF-κB信号通路被激活,ABCG2 mRNA和蛋白水平分别增加3.62±0.15和4.61±0.73倍,差异有统计学意义（P〈0.05）;PDTC作用24h后L02/HBx细胞NF-κB信号通路阻断,ABCG2 mRNA和蛋白表达分别为2.15±0.32倍和2.37±0.55倍,与未加入PDTC作用的L02-HBx细胞相比均有统计学意义（P〈0.05）。结论：NF-κB信号通路是HBx上调ABCG2表达的途径之一。     

NF—κB信号通路在绿脓菌素诱导气道上皮表达IL-8的作用

探讨绿脓菌素对人气道上皮细胞株（NCI—H292细胞）表达IL-8的诱导作用及通过NF—κB信号传导通路。采用ELISA法对PCN诱导NCI-H292细胞分泌IL-8进行分析,应用westernblotting检测NF-κB的蛋白表达,观察NF—κB阻断剂对IL-8表达的影响。PCN可促进NCI-H292细胞IL-8分泌。NF—κB的阻断剂PDTC能显著抑制IL培表达（P〈0．01）。PCN可能通过NF-κB信号通路诱导呼吸道上皮细胞IL-8表达。     

烟曲霉孢子侵染小鼠骨髓源巨噬细胞的转录组研究CSCD

目的揭示小鼠骨髓源巨噬细胞(bone marrow-derived macrophages,BMMs)被烟曲霉(Aspergillus fumigatus)A1160c孢子侵染之后与未处理对照组BMMs相比表达出现差异的免疫信号通路及孢子吞噬和清除等相关基因。方法提取小鼠原代骨髓细胞并将其诱导分化成巨噬细胞。烟曲霉孢子感染并分别提取感染组和对照组BMMs的总RNA,进行RNA测序和分析比较。结果提取小鼠原代骨髓细胞并成功将其诱导分化成BMMs。BMMs在感染烟曲霉孢子后,表面受体Tlr2和C型凝集素受体Clec4e和Clec5a的转录被激活。NF-κB信号传导通路也被激活,如Il1b、Tnf、Nfkb1等炎症因子基因和Jun、Gsk3b、Tec、Cd83等相关基因转录上调。同时,Cxcl1、Cxcl2、Ccl4等多种趋化因子基因转录水平也上调。另外,活性氧产生相关基因Bax、Nos2、Glul、Nlrp3的转录水平明显升高。结论在感染烟曲霉A1160^(c)孢子过程中,BMMs通过上调Toll样受体和C型凝集素受体基因转录水平激活NF-κB等免疫信号传导通路,促进趋化因子及活性氧产生相关基因的表达,引发对烟曲霉孢子的免疫响应和清除。     

干酪乳杆菌刺激小鼠肠道的转录组学分析

干酪乳杆菌(Lactobacillus casei)刺激小鼠肠道后，利用高通量测序技术对干酪乳杆菌饲喂组和空白组小鼠的肠道组织进行分析，以期查询和验证干酪乳杆菌对肠道免疫的影响。转录组数据的生物信息学分析发现差异表达基因共751个，通过GO富集分析发现有14个基因与细胞免疫相关，聚焦在T细胞激活、细胞分化、免疫调节负调控等功能上。KEGG通路富集显示聚集在PPAR信号通路、B细胞受体信号通路和趋化因子信号通路。对基因的基本特性分析结果显示，14个基因分别定位在10条染色体上，蛋白的分子量介于11.37～83.45 kDa之间，等电点pI 4.42～11.36,均为不稳定脂溶性蛋白，并具有相关功能结构域。通过保守基序与结构分析发现Motif分布具有保守性，大部分基因含有2个内含子。qRT-PCR验证结果表明，14个基因中有6个基因(Ces1d、Lzts1、Paqr7、Aloxe3、Zbtb16、OX40)在不同时间的处理下整体表达水平较高。验证结果与转录组测序结果一致，且这些基因功能与细胞免疫相关，该结果为研究干酪乳杆菌对机体的免疫作用效果提供一定的理论依据。     

Drosophila immunity: analysis of PGRP-SB1 expression, enzymatic activity and function

Peptidoglycan is an essential and specific component of the bacterial cell wall and therefore is an ideal recognition signature for the immune system. Peptidoglycan recognition proteins (PGRPs) are conserved from insects to mammals and able to bind PGN (non-catalytic PGRPs) and, in some cases, to efficiently degrade it (catalytic PGRPs). In Drosophila, several non-catalytic PGRPs function as selective peptidoglycan receptors upstream of the Toll and Imd pathways, the two major signalling cascades regulating the systemic production of antimicrobial peptides. Recognition PGRPs specifically activate the Toll pathway in response to Lys-type peptidoglycan found in most Gram-positive bacteria and the Imd pathway in response to DAP-type peptidoglycan encountered in Gram-positive bacilli-type bacteria and in Gram-negative bacteria. Catalytic PGRPs on the other hand can potentially reduce the level of immune activation by scavenging peptidoglycan. In accordance with this, PGRP-LB and PGRP-SC1A/B/2 have been shown to act as negative regulators of the Imd pathway. In this study, we report a biochemical and genetic analysis of PGRP-SB1, a catalytic PGRP. Our data show that PGRP-SB1 is abundantly secreted into the hemolymph following Imd pathway activation in the fat body, and exhibits an enzymatic activity towards DAP-type polymeric peptidoglycan. We have generated a PGRP-SB1/2 null mutant by homologous recombination, but its thorough phenotypic analysis did not reveal any immune function, suggesting a subtle role or redundancy of PGRP-SB1/2 with other molecules. Possible immune functions of PGRP-SB1 are discussed.     

The Drosophila peptidoglycan recognition protein PGRP-LF blocks PGRP-LC and IMD/JNK pathway activation

Eukaryotic peptidoglycan recognition proteins (PGRPs) are related to bacterial amidases. In Drosophila, PGRPs bind peptidoglycan and function as central sensors and regulators of the innate immune response. PGRP-LC/PGRP-LE constitute the receptor complex in the immune deficiency (IMD) pathway, which is an innate immune cascade triggered upon Gram-negative bacterial infection. Here, we present the functional analysis of the nonamidase, membrane-associated PGRP-LF. We show that PGRP-LF acts as a specific negative regulator of the IMD pathway. Reduction of PGRP-LF levels, in the absence of infection, is sufficient to trigger IMD pathway activation. Furthermore, normal development is impaired in the absence of functional PGRP-LF, a phenotype mediated by the JNK pathway. Thus, PGRP-LF prevents constitutive activation of both the JNK and the IMD pathways. We propose a model in which PGRP-LF keeps the Drosophila IMD pathway silent by sequestering circulating peptidoglycan.     

Peptidoglycan derived from Lactobacillus rhamnosus MLGA up-regulates the expression of chicken β-defensin 9 without triggering an inflammatory response

Defensins are critical components of the innate immune system and play an important role in the integration of innate and adaptive immune responses. Although information on the immunomodulatory properties of peptidoglycan from bacteria is abundant, little is known about the β-defensin induction effect of peptidoglycan from the probiotic Lactobacillus. This study investigated the effect of intact peptidoglycan from L. rhamnosus MLGA on the induction of avian β-defensin 9 in chicken immune cells and intestinal explants. Peptidoglycan from Lactobacillus rhamnosus MLGA dose dependently promoted avian β-defensin 9 mRNA expression in chicken PBMCs, splenocytes, thymocytes, hepatocytes, and chicken embryo jejunum, ileum, and cecum explants and increased the capacity of PBMC or splenocyte lysates to inhibit the growth of Salmonella Enteritidis. In contrast to the effect of L. rhamnosus MLGA-derived peptidoglycan, peptidoglycan derived from pathogenic Staphylococcus aureus reduced avian β-defensin 9 mRNA expression in chicken PBMCs and splenocytes. The inducible effect of peptidoglycan from L. rhamnosus MLGA on avian β-defensin 9 expression in PBMCs and splenocytes was observed without activation of the expression of associated pro-inflammatory cytokines IL-1β, IL-8, and IL-12p40, whereas these cytokine expressions were suppressed by peptidoglycan hydrolysate obtained by lysozyme digestion. The results of the present study show the capability of peptidoglycan derived from L. rhamnosus MLGA to induce the antimicrobial peptide defensin while simultaneously avoiding the deleterious risks of an inflammatory response.     

Diversity of Innate Immune Recognition Mechanism for Bacterial Polymeric meso-Diaminopimelic Acid-type Peptidoglycan in Insects

In Drosophila, the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathway. The Toll signaling pathway responds mainly to the lysine-type peptidoglycan of Gram-positive bacteria and fungal β-1,3-glucan, whereas the Imd pathway responds to the meso-diaminopimelic acid (DAP)-type peptidoglycan of Gram-negative bacteria and certain Gram-positive bacilli. Recently we determined the activation mechanism of a Toll signaling pathway biochemically using a large beetle, Tenebrio molitor. However, DAP-type peptidoglycan recognition mechanism and its signaling pathway are still unclear in the fly and beetle. Here, we show that polymeric DAP-type peptidoglycan, but not its monomeric form, formed a complex with Tenebrio peptidoglycan recognition protein-SA, and this complex activated the three-step proteolytic cascade to produce processed Spätzle, a Toll receptor ligand, and induced Drosophila defensin-like antimicrobial peptide in Tenebrio larvae similarly to polymeric lysine-type peptidoglycan. Monomeric DAP-type peptidoglycan induced Drosophila diptericin-like antimicrobial peptide in Tenebrio hemocytes. In addition, both polymeric and monomeric DAP-type peptidoglycans induced expression of Tenebrio peptidoglycan recognition protein-SC2, which is DAP-type peptidoglycan-selective N-acetylmuramyl-l-alanine amidase that functions as a DAP-type peptidoglycan scavenger, appearing to function as a negative regulator of the DAP-type peptidoglycan signaling by cleaving DAP-type peptidoglycan in Tenebrio larvae. Taken together, these results demonstrate that molecular recognition mechanism for polymeric DAP-type peptidoglycan is different between Tenebrio larvae and Drosophila adults, providing biochemical evidences of biological diversity of innate immune responses in insects.     

Activation of the human complement cascade by bacterial cell walls, peptidoglycans, water-soluble peptidoglycan components, and synthetic muramylpeptides--studies on active components and structural requirements

Cell walls isolated from 29 strains of 24 gram-positive bacterial species, whose peptidoglycans belong to the group A type of Schleifer and Kandler's classification, with one exception (Arthrobacter sp.), were shown to activate the complement cascade in pooled fresh human serum mainly through the alternative pathway and partly through the classical one. The complement-activating effect of cell walls (5 species) possessing group B type peptidoglycan, except those of Corynebacterium insidiosum, was weaker than that of the walls with group A type peptidoglycan. Preparations of peptidoglycan isolated from cell walls of Staphylococcus aureus, Streptococcus pyogenes, and Lactobacillus plantarum also activated the alternative pathway of the complement cascade, but less effectively than the respective parent cell walls. A water-soluble "polymer" of peptidoglycan subunits (SEPS), which was prepared from Staphylococcus epidermidis peptidoglycans by treatment with a cross-bridge degrading endopeptidase, retained most of the complement-activating ability of the parent cell walls. A peptidoglycan "monomer," SEPS-M, which was obtained by hydrolysis of the glycan chain of SEPS with endo-N-acetylmuramidase to disaccharide units did not activate complement. In conformity with this finding, neither synthetic N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) nor MDP-L-Lys-D-Ala activated the complement cascade. Among several lipophilic derivatives of MDP, 6-O-(3-hydroxy-3-docosylhexacosanoyl)-MDP-L-Lys-D-Ala (BH48-MDP-L-Lys-D-Ala) and 6-O-(2-tetradecylhexadecanoyl)-MDP (B30-MDP) were shown to activate complement through the alternative as well as the classical pathway and exclusively through the classical pathway, respectively. The finding that a D-isoasparagine analog of B30-MDP caused the same effect as the parent molecule strongly suggests that the activation of complement by B30-MDP is different from that caused by cell wall peptidoglycans and a water-soluble "polymer" of peptidoglycan subunits.     

Downregulation of the Drosophila immune response by peptidoglycan-recognition proteins SC1 and SC2

Peptidoglycan-recognition proteins (PGRPs) are evolutionarily conserved molecules that are structurally related to bacterial amidases. Several Drosophila PGRPs have lost this enzymatic activity and serve as microbe sensors through peptidoglycan recognition. Other PGRP family members, such as Drosophila PGRP-SC1 or mammalian PGRP-L, have conserved the amidase function and are able to cleave peptidoglycan in vitro. However, the contribution of these amidase PGRPs to host defense in vivo has remained elusive so far. Using an RNA-interference approach, we addressed the function of two PGRPs with amidase activity in the Drosophila immune response. We observed that PGRP-SC1/2-depleted flies present a specific over-activation of the IMD (immune deficiency) signaling pathway after bacterial challenge. Our data suggest that these proteins act in the larval gut to prevent activation of this pathway following bacterial ingestion. We further show that a strict control of IMD-pathway activation is essential to prevent bacteria-induced developmental defects and larval death.     

Tissue- and Ligand-Specific Sensing of Gram-Negative Infection in Drosophila by PGRP-LC Isoforms and PGRP-LE

The Drosophila antimicrobial response is one of the best characterized systems of pattern recognition receptor-mediated defense in metazoans. Drosophila senses Gram-negative bacteria via two peptidoglycan recognition proteins (PGRPs), membrane-bound PGRP-LC and secreted/cytosolic PGRP-LE, which relay diaminopimelic acid (DAP)-type peptidoglycan sensing to the Imd signaling pathway. In the case of PGRP-LC, differential splicing of PGRP domain-encoding exons to a common intracellular domain-encoding exon generates three receptor isoforms, which differ in their peptidoglycan binding specificities. In this study, we used Phi31-mediated recombineering to generate fly lines expressing specific isoforms of PGRP-LC and assessed the tissue-specific roles of PGRP-LC isoforms and PGRP-LE in the antibacterial response. Our in vivo studies demonstrate the key role of PGRP-LCx in sensing DAP-type peptidoglycan-containing Gram-negative bacteria or Gram-positive bacilli during systemic infection. We also highlight the contribution of PGRP-LCa/x heterodimers to the systemic immune response to Gram-negative bacteria through sensing of tracheal cytotoxin (TCT), whereas PGRP-LCy may have a minor role in antagonizing the immune response. Our results reveal that both PGRP-LC and PGRP-LE contribute to the intestinal immune response, with a predominant role of cytosolic PGRP-LE in the midgut, the central section of endodermal origin where PGRP-LE is enriched. Our in vivo model also definitively establishes TCT as the long-distance elicitor of systemic immune responses to intestinal bacteria observed in a loss-of-tolerance model. In conclusion, our study delineates how a combination of extracellular sensing by PGRP-LC isoforms and intracellular sensing through PGRP-LE provides sophisticated mechanisms to detect and differentiate between infections by different DAP-type bacteria in Drosophila.     

Rudra interrupts receptor signaling complexes to negatively regulate the IMD pathway

Insects rely primarily on innate immune responses to fight pathogens. In Drosophila, antimicrobial peptides are key contributors to host defense. Antimicrobial peptide gene expression is regulated by the IMD and Toll pathways. Bacterial peptidoglycans trigger these pathways, through recognition by peptidoglycan recognition proteins (PGRPs). DAP-type peptidoglycan triggers the IMD pathway via PGRP-LC and PGRP-LE, while lysine-type peptidoglycan is an agonist for the Toll pathway through PGRP-SA and PGRP-SD. Recent work has shown that the intensity and duration of the immune responses initiating with these receptors is tightly regulated at multiple levels, by a series of negative regulators. Through two-hybrid screening with PGRP-LC, we identified Rudra, a new regulator of the IMD pathway, and demonstrate that it is a critical feedback inhibitor of peptidoglycan receptor signaling. Following stimulation of the IMD pathway, rudra expression was rapidly induced. In cells, RNAi targeting of rudra caused a marked up-regulation of antimicrobial peptide gene expression. rudra mutant flies also hyper-activated antimicrobial peptide genes and were more resistant to infection with the insect pathogen Erwinia carotovora carotovora. Molecularly, Rudra was found to bind and interfere with both PGRP-LC and PGRP-LE, disrupting their signaling complex. These results show that Rudra is a critical component in a negative feedback loop, whereby immune-induced gene expression rapidly produces a potent inhibitor that binds and inhibits pattern recognition receptors.