Regulation of Serum Amyloid A3 (SAA3) in Mouse Colonic Epithelium and Adipose Tissue by the Intestinal Microbiota

The gut microbiota has been proposed as an environmental factor that affects the development of metabolic and inflammatory diseases in mammals. Recent reports indicate that gut bacteria-derived lipopolysaccharide (LPS) can initiate obesity and insulin resistance in mice; however, the molecular interactions responsible for microbial regulation of host metabolism and mediators of inflammation have not been studied in detail. Hepatic serum amyloid A (SAA) proteins are markers and proposed mediators of inflammation that exhibit increased levels in serum of insulin-resistant mice. Adipose tissue-derived SAA3 displays monocyte chemotactic activity and may play a role in metabolic inflammation associated with obesity and insulin resistance. To investigate a potential mechanistic link between the intestinal microbiota and induction of proinflammatory host factors, we performed molecular analyses of germ-free, conventionally raised and genetically modified Myd88−/− mouse models. SAA3 expression was determined to be significantly augmented in adipose (9.9±1.9-fold; P<0.001) and colonic tissue (7.0±2.3-fold; P<0.05) by the presence of intestinal microbes. In the colon, we provided evidence that SAA3 is partially regulated through the Toll-like receptor (TLR)/MyD88/NF-kappaB signaling axis. We identified epithelial cells and macrophages as cellular sources of SAA3 in the colon and found that colonic epithelial expression of SAA3 may be part of an NF-kappaB-dependent response to LPS from gut bacteria. In vitro experiments showed that LPS treatments of both epithelial cells and macrophages induced SAA3 expression (27.1±2.5-fold vs. 1.6±0.1-fold, respectively). Our data suggest that LPS, and potentially other products of the indigenous gut microbiota, might elevate cytokine expression in tissues and thus exacerbate chronic low-grade inflammation observed in obesity.     

Evidence for a core gut microbiota in the zebrafish

Experimental analysis of gut microbial communities and their interactions with vertebrate hosts is conducted predominantly in domesticated animals that have been maintained in laboratory facilities for many generations. These animal models are useful for studying coevolved relationships between host and microbiota only if the microbial communities that occur in animals in lab facilities are representative of those that occur in nature. We performed 16S rRNA gene sequence-based comparisons of gut bacterial communities in zebrafish collected recently from their natural habitat and those reared for generations in lab facilities in different geographic locations. Patterns of gut microbiota structure in domesticated zebrafish varied across different lab facilities in correlation with historical connections between those facilities. However, gut microbiota membership in domesticated and recently caught zebrafish was strikingly similar, with a shared core gut microbiota. The zebrafish intestinal habitat therefore selects for specific bacterial taxa despite radical differences in host provenance and domestication status.     

Moderate exercise ameliorates osteoarthritis by reducing lipopolysaccharides from gut microbiota in mice

Lipopolysaccharides (LPSs) released by gut microbiota are correlated with the pathophysiology of osteoarthritis (OA). Exercise remodels the composition of gut microbiota. The present study investigated the hypothesis that wheel-running exercise prevents knee OA induced by high-fat diet (HFD) via reducing LPS from intestinal microorganisms. Male C57BL/6 J mice were treated with sedentary or wheel-running exercise, standard diet (13.5% kcal) or HFD (60% kcal), berberine or not according to their grouping. Knee OA severity, blood and synovial fluid LPS, cecal microbiota, and TLR4 and MMP-13 expression levels were determined. Our findings reveal that HFD treatment decreased gut microbial diversity. Increase in endotoxin-producing bacteria, decrease in gut barrier-protecting bacteria, high LPS levels in the blood and synovial fluid, high TLR4 and MMP-13 expression levels, and severe cartilage degeneration were observed. By contrast, voluntary wheel running caused high gut microbial diversity. The gut microbiota were reshaped, LPS levels in the blood and synovial fluid and TLR4 and MMP-13 expression levels were low, and cartilage degeneration was ameliorated. Berberine treatment reduced LPS levels in the samples, but decreased the diversity of intestinal flora with similar changes to that caused by HFD. In conclusion, unlike taking drugs, exercising can remodel gut microbial ecosystems, reduce the circulating levels of LPS, and thereby contribute to the relief of chronic inflammation and OA. Our findings showed that moderate exercise is a potential therapeutic approach for preventing and treating obesity-related OA.     

Antibiotic growth promoter olaquindox increases pathogen susceptibility in fish by inducing gut microbiota dysbiosis

Low dose antibiotics have been used as growth promoters in livestock and fish. The use of antibiotics has been associated with reduced pathogen infections in livestock. In contrast, antibiotic growth promoter has been suspected of leading to disease outbreaks in aquaculture. However, this phenomenon is circumstantial and has not been confirmed in experimental conditions. In this study,we showed that antibiotic olaquindox increased the susceptibility of zebrafish to A. hydrophila infection. Olaquindox led to profound alterations in the intestinal microbiota of zebrafish, with a drastic bloom of Enterobacter and diminishing of Cetobacterium. Moreover, the innate immune responses of zebrafish were compromised by olaquindox(P0.05). Transfer of microbiota to GF zebrafish indicated that while the immuo-suppression effect of olaquindox is a combined effect mediated by both OLA-altered micro biota and direct action of the antibiotic(P0.05), the increased pathogen susceptibility was driven by the OLA-altered microbiota and was not dependent on direct antibiotic effect. Taken together, these data indicate that low level of OLA induced gut microbiota dysbiosis in zebrafish, which led to increased pathogen susceptibility.     

Amelioration of non-alcoholic steatohepatitis by Qushi Huayu decoction is associated with inhibition of the intestinal mitogen-activated protein kinase pathway

BackgroundGut microbiota is increasingly recognized as the key participant in the pathogenesis of non-alcoholic fatty liver disease (NAFLD) by translocation of its products, such as lipopolysaccharide (LPS), via the dysfunctional intestinal barrier. Qushi Huayu decoction (QHD), a traditional Chinese medicine, is developed specially for NAFLD and used in clinic in China for more than a decade and previously found to ameliorate non-alcoholic steatohepatitis (NASH) induced by high-fat diet (HFD) in mice accompanied with inhibited metabolic endotoxemia and hepatic LPS signalling.PurposeTo investigate the mechanism of LPS gut-leakage inhibition by QHD in NASH.MethodsEffects of QHD on gut microbioa and intestinal barrier were evaluated in NASH induced by HFD in mice. 16S rRNA sequencing is employed to analyse the gut microbiota composition. To identify the potential signalling pathway responsible for tight junction regulation, the colonic phosphoprotein profile is screened via the Phospho Explorer Antibody Array and verified in NASH, intestinal barrier dysfunctional mouse and Caco-2 cells.ResultsQHD ameliorates NASH accompanied with regulating the gut microbiota composition, protecting intestinal tight junctions and inhibiting LPS gut-leakage without decreasing the abundance of identified Gram-negative bacteria. The validated data of phosphorylated proteins suggested that mitogen-activated protein kinase (MAPK) pathway is predominantly responsible for the colonic tight junction regulation by QHD.ConclusionQHD inhibits LPS gut-leakage in NASH, which is associated with downregulation of intestinal MAPK pathway.     

Growth, metastasis, and expression of CCL2 and CCL5 by murine mammary carcinomas are dependent upon Myd88

Previously we reported that lipopolysaccharide (LPS) treatment of murine mammary carcinomas resulted in decreased growth of the tumors. Here we show the decreased growth following LPS treatment was mediated through effects downstream of TLR4 and Myd88. Perhaps more notably, simply reducing TLR4 or Myd88 levels was sufficient to slow tumor growth rates. Moreover, reduced levels of Myd88 correlated with a significant reduction in lung metastasis as well as decreased CCL2 and CCL5 expression. To determine whether inhibiting Myd88 function could also alter tumor growth and chemokine expression we used a Myd88 homodimerization inhibitory peptide. Indeed, inhibiting Myd88 function in four different murine mammary carcinomas as well as the human breast cancer cell line MDA-MB-231 led to decreased growth as well as CCL2 and CCL5 expression. These data imply that Myd88 is important for growth and metastasis of breast cancer, and expression of at least two proinflammatory chemokines.     

Alkaline phosphatase: keeping the peace at the gut epithelial surface

Vertebrate intestinal surfaces are in constant contact with a vast consortium of commensal bacteria. To preserve mutually beneficial host-microbial relationships, gut epithelia have evolved strategies to limit the proinflammatory potential of resident gut microbes. In this issue of Cell Host & Microbe, Bates and colleagues report that intestinal alkaline phosphatase, whose expression is induced during establishment of the microbiota, dephosphorylates lipopolysaccharide and promotes mucosal tolerance to commensal bacteria in zebrafish.     

Probiotics modulate gut microbiota and improve insulin sensitivity in DIO mice

Obesity and type 2 diabetes are characterized by subclinical inflammatory process. Changes in composition or modulation of the gut microbiota may play an important role in the obesity-associated inflammatory process. In the current study, we evaluated the effects of probiotics (Lactobacillus rhamnosus, L. acidophilus and Bifidobacterium bifidumi) on gut microbiota, changes in permeability, and insulin sensitivity and signaling in high-fat diet and control animals. More importantly, we investigated the effects of these gut modulations on hypothalamic control of food intake, and insulin and leptin signaling. Swiss mice were submitted to a high-fat diet (HFD) with probiotics or pair-feeding for 5 weeks. Metagenome analyses were performed on DNA samples from mouse feces. Blood was drawn to determine levels of glucose, insulin, LPS, cytokines and GLP-1. Liver, muscle, ileum and hypothalamus tissue proteins were analyzed by Western blotting and real-time polymerase chain reaction. In addition, liver and adipose tissues were analyzed using histology and immunohistochemistry. The HFD induced huge alterations in gut microbiota accompanied by increased intestinal permeability, LPS translocation and systemic low-grade inflammation, resulting in decreased glucose tolerance and hyperphagic behavior. All these obesity-related features were reversed by changes in the gut microbiota profile induced by probiotics. Probiotics also induced an improvement in hypothalamic insulin and leptin resistance. Our data demonstrate that the intestinal microbiome is a key modulator of inflammatory and metabolic pathways in both peripheral and central tissues. These findings shed light on probiotics as an important tool to prevent and treat patients with obesity and insulin resistance.     

七鳃鳗myd88基因的生物学特性及其下游分子的表达模式分析

髓样分化因子88 (Myeloid differentiation factor 88, MYD88)是Toll样受体(Toll-like receptor, TLR)信号通路的关键接头分子, 在先天性免疫和适应性免疫中都起到重要作用。为了揭示七鳃鳗Myd88的生物学功能, 研究首次从七鳃鳗(Lampetra japonica)中克隆了myd88基因, 其ORF为852 bp, 共编码283个氨基酸, 推测的分子量为32.432 kD, 等电点为6.25, 无信号肽。多重序列比对表明七鳃鳗Myd88的氨基酸序列与其他物种同源性较高, 具有高度保守的N端死亡结构域和C端的TIR结构域的Box1、Box2和Box3基序。实时荧光定量PCR分析表明: myd88基因在七鳃鳗各组织中均有低水平转录表达, 鳃中表达量最高, 其次是肌肉、髓和肾。脂多糖(LPS)体内刺激七鳃鳗后, 七鳃鳗myd88在白细胞中表达量升高最显著, 其次是在鳃中的表达量也明显升高, 表明七鳃鳗Myd88参与七鳃鳗的抗菌免疫过程。此外, LPS刺激七鳃鳗还能诱导TLR信号通路Myd88依赖途径的下游信号分子Irak1、Traf6、Ikkβ和Nfkb在各组织中的转录表达。研究结果表明七鳃鳗中可能存在TLR/Myd88信号通路, 为进一步探究该信号通路参与免疫应答的起源与进化奠定了基础。     

Distinct signals from the microbiota promote different aspects of zebrafish gut differentiation

All animals exist in intimate associations with microorganisms that play important roles in the hosts' normal development and tissue physiology. In vertebrates, the most populous and complex community of microbes resides in the digestive tract. Here, we describe the establishment of the gut microbiota and its role in digestive tract differentiation in the zebrafish model vertebrate, Danio rerio. We find that in the absence of the microbiota, the gut epithelium is arrested in aspects of its differentiation, as revealed by the lack of brush border intestinal alkaline phosphatase activity, the maintenance of immature patterns of glycan expression and a paucity of goblet and enteroendocrine cells. In addition, germ-free intestines fail to take up protein macromolecules in the distal intestine and exhibit faster motility. Reintroduction of a complex microbiota at later stages of development or mono-association of germ-free larvae with individual constituents of the microbiota reverses all of these germ-free phenotypes. Exposure of germ-free zebrafish to heat-killed preparations of the microbiota or bacterial lipopolysaccharide is sufficient to restore alkaline phosphatase activity but not mature patterns of Gal alpha1,3Gal containing glycans, indicating that the host perceives and responds to its associated microbiota by at least two distinct pathways.     

Neutrophil Elastase Alters the Murine Gut Microbiota Resulting in Enhanced Salmonella Colonization

The intestinal microbiota has been found to play a central role in the colonization of Salmonella enterica serovar Typhimurium in the gastrointestinal tract. In this study, we present a novel process through which Salmonella benefit from inflammatory induced changes in the microbiota in order to facilitate disease. We show that Salmonella infection in mice causes recruitment of neutrophils to the gut lumen, resulting in significant changes in the composition of the intestinal microbiota. This occurs through the production of the enzyme elastase by neutrophils. Administration of recombinant neutrophil elastase to infected animals under conditions that do not elicit neutrophil recruitment caused shifts in microbiota composition that favored Salmonella colonization, while inhibition of neutrophil elastase reduced colonization. This study reveals a new relationship between the microbiota and the host during infection.     

TLR4 enhances TGF-beta signaling and hepatic fibrosis

Hepatic injury is associated with a defective intestinal barrier and increased hepatic exposure to bacterial products. Here we report that the intestinal bacterial microflora and a functional Toll-like receptor 4 (TLR4), but not TLR2, are required for hepatic fibrogenesis. Using Tlr4-chimeric mice and in vivo lipopolysaccharide (LPS) challenge, we demonstrate that quiescent hepatic stellate cells (HSCs), the main precursors for myofibroblasts in the liver, are the predominant target through which TLR4 ligands promote fibrogenesis. In quiescent HSCs, TLR4 activation not only upregulates chemokine secretion and induces chemotaxis of Kupffer cells, but also downregulates the transforming growth factor (TGF)-beta pseudoreceptor Bambi to sensitize HSCs to TGF-beta-induced signals and allow for unrestricted activation by Kupffer cells. LPS-induced Bambi downregulation and sensitization to TGF-beta is mediated by a MyD88-NF-kappaB-dependent pathway. Accordingly, Myd88-deficient mice have decreased hepatic fibrosis. Thus, modulation of TGF-beta signaling by a TLR4-MyD88-NF-kappaB axis provides a novel link between proinflammatory and profibrogenic signals.     

Oral Supplementation with Non-Absorbable Antibiotics or Curcumin Attenuates Western Diet-Induced Atherosclerosis and Glucose Intolerance in LDLR?/? Mice – Role of Intestinal Permeability and Macrophage Activation

Association between circulating lipopolysaccharide (LPS) and metabolic diseases (such as Type 2 Diabetes and atherosclerosis) has shifted the focus from Western diet-induced changes in gut microbiota per se to release of gut bacteria-derived products into circulation as the possible mechanism for the chronic inflammatory state underlying the development of these diseases. Under physiological conditions, an intact intestinal barrier prevents this release of LPS underscoring the importance of examining and modulating the direct effects of Western diet on intestinal barrier function. In the present study we evaluated two strategies, namely selective gut decontamination and supplementation with oral curcumin, to modulate Western-diet (WD) induced changes in intestinal barrier function and subsequent development of glucose intolerance and atherosclerosis. LDLR−/− mice were fed WD for 16 weeks and either received non-absorbable antibiotics (Neomycin and polymyxin) in drinking water for selective gut decontamination or gavaged daily with curcumin. WD significantly increased intestinal permeability as assessed by in vivo translocation of FITC-dextran and plasma LPS levels. Selective gut decontamination and supplementation with curcumin significantly attenuated the WD-induced increase in plasma LPS levels (3.32 vs 1.90 or 1.51 EU/ml, respectively) and improved intestinal barrier function at multiple levels (restoring intestinal alkaline phosphatase activity and expression of tight junction proteins, ZO-1 and Claudin-1). Consequently, both these interventions significantly reduced WD-induced glucose intolerance and atherosclerosis in LDLR−/− mice. Activation of macrophages by low levels of LPS (50 ng/ml) and its exacerbation by fatty acids is likely the mechanism by which release of trace amounts of LPS into circulation due to disruption of intestinal barrier function induces the development of these diseases. These studies not only establish the important role of intestinal barrier function, but also identify oral supplementation with curcumin as a potential therapeutic strategy to improve intestinal barrier function and prevent the development of metabolic diseases.     

Berberine ameliorates aGVHD by gut microbiota remodelling,TLR4 signalling suppression and colonic barrier repairment for NLRP3 inflammasome inhibition

Berberine (BBR), an isoquinoline alkaloid, is used to treat gastrointestinal disorders as an herbal medicine in China. The aim of this study was to investigate the anti‐inflammatory activities of BBR in a mouse model with acute graft‐versus‐host disease (aGVHD). Mice were intravenously injected with bone marrow cells from donors combined with splenocytes to develop aGVHD. The body weight, survival rate and clinical scores were monitored. Then the levels of inflammatory cytokines, histological changes (lung, liver and colon), colonic mucosal barrier and gut microbiota were analysed. Moreover, the toll‐like receptor 4 (TLR4)/myeloid differentiation primary response gene 88 (Myd88)/nuclear factor‐κB signalling pathway, NLRP3 inflammasome and its cytokines’ expressions were determined. The results showed that the gavage of BBR lessened GVHD‐induced weight loss, high mortality and clinical scores, inhibited inflammation and target organs damages and prevented GVHD‐indued colonic barrier damage. Additionally, BBR modulated gut microbiota, suppressed the activation of the TLR4 signaling pathway and inhibited NLRP3 inflammasome and its cytokine release. This study indicated that BBR might be a potential therapy for aGVHD through NLRP3 inflammasome inhibition.     

Characterizing the Interactions between a Naturally Primed Immunoglobulin A and Its Conserved Bacteroides thetaiotaomicron Species-specific Epitope in Gnotobiotic Mice

The adaptive immune response to the human gut microbiota consists of a complex repertoire of antibodies interacting with a broad range of taxa. Fusing intestinal lamina propria lymphocytes from mice monocolonized with Bacteroides thetaiotaomicron to a myeloma fusion partner allowed us to recover hybridomas that captured naturally primed, antigen-specific antibody responses representing multiple isotypes, including IgA. One of these hybridomas, 260.8, produced a monoclonal antibody that recognizes an epitope specific for B. thetaiotaomicron isolates in a large panel of hospital- and community-acquired Bacteroides. Whole genome transposon mutagenesis revealed a 19-gene locus, involved in LPS O-antigen polysaccharide synthesis and conserved among multiple B. thetaiotaomicron isolates, that is required for 260.8 epitope expression. Mutants in this locus exhibited marked fitness defects in vitro during growth in rich medium and in gnotobiotic mice colonized with defined communities of human gut symbionts. Expression of the 260.8 epitope was sustained during 10 months of daily passage in vitro and during 14 months of monocolonization of gnotobiotic wild-type, Rag1−/−, or Myd88−/− mice. Comparison of gnotobiotic Rag1−/− mice with and without subcutaneous 260.8 hybridomas disclosed that this IgA did not affect B. thetaiotaomicron population density or suppress 260.8 epitope production but did affect bacterial gene expression in ways emblematic of a diminished host innate immune response. Our study illustrates an approach for (i) generating diagnostic antibodies, (ii) characterizing IgA responses along a continuum of specificity/degeneracy that defines the IgA repertoire to gut symbionts, and (iii) identifying immunogenic epitopes that affect competitiveness and help maintain host-microbe mutualism.     

Gut microbiota,host health,and polysaccharides

The intestinal microbiota is a complicated ecosystem that influences many aspects of host physiology (i.e. diet, disease development, drug metabolism, and regulation of the immune system). It also exhibits spatial patterning and temporal dynamics. In this review, the effects of internal and external (environmental) factors on intestinal microbiota are discussed. We describe the roles of the gut microbiota in maintaining intestinal and immune system homeostasis and the relationship between gut microbiota and diseases. In particular, the contributions of polysaccharides, as the most abundant diet components in intestinal microbiota and host health are presented. Finally, perspectives for research avenues relating to gut microbiota are also discussed.     

蜜蜂肠道菌群结构功能研究进展

在长期的共同进化中,肠道菌群与其宿主形成了紧密的联系,为宿主提供了许多有益的作用。作为一种社会性昆虫,蜜蜂的生活习性为其肠道菌群提供了良好而稳定的传播途径,因此,蜜蜂与其肠道菌群形成了一种紧密的互惠互利共生关系。近年来,随着对蜜蜂肠道菌群了解的不断加深,对蜜蜂肠道菌群功能的研究也不断深入,大量研究表明蜜蜂的肠道菌群在宿主食物的消化代谢、宿主免疫的激活和抵抗致病菌、调节宿主生理等方面都有着重要的作用,同时破坏肠道菌群的稳定对蜜蜂的健康有着明显的负面影响。本文对近年来西方蜜蜂肠道菌群功能研究进行了总结,旨在为进一步深入探索蜜蜂肠道菌群与其宿主的相互作用及在养蜂生产上应用肠道菌群防控疾病提供参考。     

模式动物在共生微生物研究中的作用

共生微生物是一类定殖于宿主体表或体内, 可执行宿主本身无法完成的功能, 并依赖于宿主所提供的生长环境的微生物。众多研究表明, 人体肠道共生微生物与免疫、营养、代谢, 甚至精神健康等生理功能密切相关, 是重要的“微生物器官”。在早期的肠道微生物研究中, 模式动物就已经作为研究工具被使用。随着肠道微生物研究的不断深入, 模式动物作为不可替代的研究对象发挥了越来越重要的作用。本综述主要对几种重要的模式动物如斑马鱼(Danio rerio)、小鼠(Mus musculus)、猪(Sus scrofa domesticus)和猕猴(Macaca mulatta)在肠道微生物研究中的应用进行了总结, 介绍了各种模式动物的发展过程及特点, 各自在应用于研究时的优缺点, 以及利用这些动物模型在共生微生物领域所取得的一些标志性的科研成果。同时, 也就近年来在共生微生物领域新兴的一些模式生物如蜜蜂(Apis)、果蝇(Drosophila)、秀丽隐杆线虫(Caenorhabditis elegans)等进行了一些探讨。旨在让该领域的研究者们了解模式动物与人体在共生微生物方面的异同, 为更好地利用这一研究工具提供参考。     

The composition of the zebrafish intestinal microbial community varies across development

The assembly of resident microbial communities is an important event in animal development; however, the extent to which this process mirrors the developmental programs of host tissues is unknown. Here we surveyed the intestinal bacteria at key developmental time points in a sibling group of 135 individuals of a model vertebrate, the zebrafish (Danio rerio). Our survey revealed stage-specific signatures in the intestinal microbiota and extensive interindividual variation, even within the same developmental stage. Microbial community shifts were apparent during periods of constant diet and environmental conditions, as well as in concert with dietary and environmental change. Interindividual variation in the intestinal microbiota increased with age, as did the difference between the intestinal microbiota and microbes in the surrounding environment. Our results indicate that zebrafish intestinal microbiota assemble into distinct communities throughout development, and that these communities are increasingly different from the surrounding environment and from one another.