Modulation of Systemic Immune Responses through Commensal Gastrointestinal Microbiota

Colonization of the gastrointestinal (GI) tract is initiated during birth and continually seeded from the individual’s environment. Gastrointestinal microorganisms play a central role in developing and modulating host immune responses and have been the subject of investigation over the last decades. Animal studies have demonstrated the impact of GI tract microbiota on local gastrointestinal immune responses; however, the full spectrum of action of early gastrointestinal tract stimulation and subsequent modulation of systemic immune responses is poorly understood. This study explored the utility of an oral microbial inoculum as a therapeutic tool to affect porcine systemic immune responses. For this study a litter of 12 pigs was split into two groups. One group of pigs was inoculated with a non-pathogenic oral inoculum (modulated), while another group (control) was not. DNA extracted from nasal swabs and fecal samples collected throughout the study was sequenced to determine the effects of the oral inoculation on GI and respiratory microbial communities. The effects of GI microbial modulation on systemic immune responses were evaluated by experimentally infecting with the pathogen Mycoplasma hyopneumoniae. Coughing levels, pathology, toll-like receptors 2 and 6, and cytokine production were measured throughout the study. Sequencing results show a successful modulation of the GI and respiratory microbiomes through oral inoculation. Delayed type hypersensitivity responses were stronger (p = 0.07), and the average coughing levels and respiratory TNF-α variance were significantly lower in the modulated group (p<0.0001 and p = 0.0153, respectively). The M. hyopneumoniae infection study showed beneficial effects of the oral inoculum on systemic immune responses including antibody production, severity of infection and cytokine levels. These results suggest that an oral microbial inoculation can be used to modulate microbial communities, as well as have a beneficial effect on systemic immune responses as demonstrated with M. hyopneumoniae infection.     

Chronic HIV Infection Enhances the Responsiveness of Antigen Presenting Cells to Commensal Lactobacillus

Chronic immune activation despite long-term therapy poses an obstacle to immune recovery in HIV infection. The role of antigen presenting cells (APCs) in chronic immune activation during HIV infection remains to be fully determined. APCs, the frontline of immune defense against pathogens, are capable of distinguishing between pathogens and non-pathogenic, commensal bacteria. We hypothesized that HIV infection induces dysfunction in APC immune recognition and response to some commensal bacteria and that this may promote chronic immune activation. Therefore we examined APC inflammatory cytokine responses to commensal lactobacilli. We found that APCs from HIV-infected patients produced an enhanced inflammatory response to Lactobacillus plantarum WCFS1 as compared to APCs from healthy, HIV-negative controls. Increased APC expression of TLR2 and CD36, signaling through p38-MAPK, and decreased expression of MAP kinase phosphatase-1 (MKP-1) in HIV infection was associated with this heightened immune response. Our findings suggest that chronic HIV infection enhances the responsiveness of APCs to commensal lactobacilli, a mechanism that may partly contribute to chronic immune activation.     

The toxicity of Bacteroides thetaiotaomicron cell-surface antigens to chicken embryos.

The toxicity of B. thetaiotaomicron lipopolysaccharides and capsular antigen to 11-day-old chicken embryos was examined. CELD50 (chicken embryo LD50) of these preparations were determined. All examined preparations showed the lethal activity for chicken embryos. The toxicity of lipopolysaccharides was much higher than the toxicity of capsular antigen.     

多形拟杆菌对糖尿病模型小鼠的影响

目的研究多形拟杆菌(BT)干预糖尿病模型小鼠后对血糖、体重和C肽的影响。方法 (1)用四氧嘧啶(200 mg/kg腹腔注射)制备糖尿病模型小鼠,并分成四组:空白组(n=10)、空白给菌组(n=10)、四氧嘧啶糖尿病模型组(n=11)和模型给菌组(n=11),15 d。(2)BHI血琼脂培养基培养ATCC 29148标准菌株,比浊法测定混悬菌液数量。(3)用多形拟杆菌菌液干预空白给菌组和模型给菌组小鼠,观察15 d中四组小鼠的体重、空腹血糖水平的变化;实时荧光定量PCR测定小鼠肠道内多形拟杆菌的数量;运用酶联免疫法(ELISA)测定血清中C肽的水平。结果 (1)给予菌悬液后,空白给菌组与空白组相比,BT在第3天就可以定植并维持到第15天。与模型组相比,模型给菌组在第15天时可以定植;(2)线性回归相关性分析显示,肠道内多形拟杆菌与体重呈负性相关(r=-0.70,P0.05);与空腹血糖(FPG)呈正性相关(r=0.71,P0.05);与C肽呈负性相关(r=-0.62,P0.05);与胰岛素抵抗指数(HOMA-IR)呈正性相关(r=0.55,P0.05);与胰岛素分泌指数(HOMA-IS)呈负性相关(r=-0.43,P0.05)。结论 (1)外源性灌胃给予多形拟杆菌可以在肠道内定植。(2)肠道内多形拟杆菌的数量变化与糖尿病有相关性。     

Genetic Control of Immune Responses to a Synthetic Fimbrial Antigen of Actinobacillus actinomycetemcomitans

The incidence of infection by Actinobacillus actinomycetemcomitans, one of the important pathogens in human periodontal diseases, has been reported to be associated with racial background and genetic factors. We attempted to determine the genetic regulation of immune responses to A. actinomycetemcomitans fimbriae, an attachment factor, using various inbred strains of mice. For this purpose, we synthesized an oligopeptide antigen using the amino acid sequence of the fimbriae and conjugated this antigen to branched lysine polymer resin beads. After immunization with the synthetic A. actinomycetemcomitans fimbrial antigen, serum antibody levels and the delayed-type hypersensitivity (DTH) reaction to the antigen were measured by enzyme-linked immunosorbent assay (ELISA) and footpad swelling responses, respectively. The strains of mice found to be high-IgG responders to the antigen were B10.HTT, B10.RIII, B10A (5R) and B10.S (9R). These results indicate that mice with E_β^s: E_α^k, E_β^r: E_α^r and E_β^b: E_α^k respond strongly to the synthetic peptide. All of the high-IgG responders showed a high DTH response. A cell transfer experiment confirmed that CD4 T cells mediated with a DTH response to the synthetic peptide. Thus, the results of this study demonstrate that the immune responses to A. actinomycetemcomitans fimbriae are genetically controlled.     

Specificity of a Bacteroides thetaiotaomicron Marker for Human Feces

A bacterial primer set, known to produce a 542-bp amplicon specific for Bacteroides thetaiotaomicron, generated this product in PCR with 1 ng of extracted DNA from 92% of 25 human fecal samples, 100% of 20 sewage samples, and 16% of 31 dog fecal samples. The marker was not detected in 1 ng of fecal DNA from 61 cows, 35 horses, 44 pigs, 24 chickens, 29 turkeys, and 17 geese.     

Plasmacytoid Dendritic Cells Mediate Anti-inflammatory Responses to a Gut Commensal Molecule via Both Innate and Adaptive Mechanisms

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Bacterial Infection Disrupts Clock Gene Expression to Attenuate Immune Responses

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Influenza Antigen Engineering Focuses Immune Responses to a Subdominant but Broadly Protective Viral Epitope

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Characterization of a pyridine nucleotide-nonspecific glutamate dehydrogenase from Bacteroides thetaiotaomicron.

An oxidized nicotinamide adenine dinucleotide phosphate/oxidized nicotinamide adenine dinucleotide (NADP+/NAD+) nonspecific L-glutamate dehydrogenase from Bacteroides thetaiotaomicron was purified 40-fold (NADP+ or NAD+ activity) over crude cell extract by heat treatment, (NH4)2SO2 fractionation, diethylaminoethyl-cellulose, Bio-Gel A 1.5m, and hydroxylapatite chromatography. Both NADP+- and NAD+-dependent activities coeluted from all chromatographic treatments. Moreover, a constant ratio of NADP+/NAD+ specific activities was demonstrated at each purification step. Both activities also comigrated in 6% nondenaturing polyacrylamide gels. Affinity chromatography of the 40-fold-purified enzyme using Procion RED HE-3B gave a preparation containing both NADP+- and NAD+-linked activities which showed a single protein band of 48,5000 molecular weight after sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The dual pyridine nucleotide nature of the enzyme was most readily apparent in the oxidative direction. Reductively, the enzyme was 30-fold more active with reduced NADP than with reduced NAD. Nonlinear concave 1/V versus 1/S plots were observed for reduced NADP and NH4Cl. Salts (0.1 M) stimulated the NADP+-linked reaction, inhibited the NAD+-linked reaction, and had little effect on the reduced NADP-dependent reaction. The stimulatory effect of salts (NADP+) was nonspecific, regardless of the anion or cation, whereas the degree of NAD+-linked inhibition decreased in the order to I- greater than Br- greater than Cl- greater than F-. Both NADP+ and NAD+ glutamate dehydrogenase activities were also detected in cell extracts from representative strains of other bacteroides deoxyribonucleic acid homology groups.     

Engineering Bacteroides thetaiotaomicron to produce non-native butyrate based on a genome-scale metabolic model-guided design

Bacteroides thetaiotaomicron represents a major symbiont of the human gut microbiome that is increasingly viewed as a promising candidate strain for microbial therapeutics. Here, we engineer B. thetaiotaomicron for heterologous production of non-native butyrate as a proof-of-concept biochemical at therapeutically relevant concentrations. Since B. thetaiotaomicron is not a natural producer of butyrate, we heterologously expressed a butyrate biosynthetic pathway in the strain, which led to the production of butyrate at the final concentration of 12 mg/L in a rich medium. Further optimization of butyrate production was achieved by a round of metabolic engineering guided by an expanded genome-scale metabolic model (GEM) of B. thetaiotaomicron. The in silico knock-out simulation of the expanded model showed that pta and ldhD were the potent knock-out targets to enhance butyrate production. The maximum titer and specific productivity of butyrate in the pta-ldhD double knockout mutant increased by nearly 3.4 and 4.8 folds, respectively. To our knowledge, this is the first engineering attempt that enabled butyrate production from a non-butyrate producing commensal B. thetaiotaomicron. The study also highlights that B. thetaiotaomicron can serve as an effective strain for live microbial therapeutics in human.     

The cell-surface components of Bacteroides thetaiotaomicron as coating antigens in ELISA test

Cell-surface antigens were extracted out of three Bacteroides thetaiotaomicron strains of different origin. Lipopolysaccharides, their fractions, L1 preparations and capsular antigen were obtained. All substances were tested as coating antigens in ELISA test against antibacterial rabbit immune sera. The highest absorbances were observed with lipopolysaccharides (LPS), their polysaccharide fractions (PS) and capsular material (CPS). Lipopolysaccharides after nuclease treatment (N-LPS), free from nucleic acids, were more active than crude phenol-water extracts (PW-LPS).     

A Cross-Reactive Monoclonal Antibody to Nematode Haemoglobin Enhances Protective Immune Responses to Nippostrongylus brasiliensis

Background

Nematode secreted haemoglobins have unusually high affinity for oxygen and possess nitric oxide deoxygenase, and catalase activity thought to be important in protection against host immune responses to infection. In this study, we generated a monoclonal antibody (48Eg) against haemoglobin of the nematode Anisakis pegreffii, and aimed to characterize cross-reactivity of 4E8g against haemoglobins of different nematodes and its potential to mediate protective immunity against a murine hookworm infection.

Methodology/Principal Findings

Immunoprecipitation was used to isolate the 4E8g-binding antigen in Anisakis and Ascaris extracts, which were identified as haemoglobins by peptide mass fingerprinting and MS/MS. Immunological cross-reactivity was also demonstrated with haemoglobin of the rodent hookworm N. brasiliensis. Immunogenicity of nematode haemoglobin in mice and humans was tested by immunoblotting. Anisakis haemoglobin was recognized by IgG and IgE antibodies of Anisakis-infected mice, while Ascaris haemoglobin was recognized by IgG but not IgE antibodies in mouse and human sera. Sequencing of Anisakis haemoglobin revealed high similarity to haemoglobin of a related marine nematode, Psuedoterranova decipiens, which lacks the four –HKEE repeats of Ascaris haemoglobin important in octamer assembly. The localization of haemoglobin in the different parasites was examined by immunohistochemistry and associated with the excretory-secretary ducts in Anisakis, Ascaris and N. brasiliensis. Anisakis haemoglobin was strongly expressed in the L3 stage, unlike Ascaris haemoglobin, which is reportedly mainly expressed in adult worms. Passive immunization of mice with 4E8g prior to infection with N. brasiliensis enhanced protective Th2 immunity and led to a significant decrease in worm burdens.

Conclusion

The monoclonal antibody 4E8g targets haemoglobin in broadly equivalent anatomical locations in parasitic nematodes and enhances host immunity to a hookworm infection.     

An anaerobic bacterium,Bacteroides thetaiotaomicron,uses a consortium of enzymes to scavenge hydrogen peroxide

Obligate anaerobes are periodically exposed to oxygen, and it has been conjectured that on such occasions their low‐potential biochemistry will predispose them to rapid ROS formation. We sought to identify scavenging enzymes that might protect the anaerobe Bacteroides thetaiotaomicron from the H₂O₂ that would be formed. Genetic analysis of eight candidate enzymes revealed that four of these scavenge H₂O₂ in vivo: rubrerythrins 1 and 2, AhpCF, and catalase E. The rubrerythrins served as key peroxidases under anoxic conditions. However, they quickly lost activity upon aeration, and AhpCF and catalase were induced to compensate. The AhpCF is an NADH peroxidase that effectively degraded low micromolar levels of H₂O₂, while the catalytic cycle of catalase enabled it to quickly degrade higher concentrations that might arise from exogenous sources. Using a non‐scavenging mutant we verified that endogenous H₂O₂ formation was much higher in aerated B. thetaiotaomicron than in Escherichia coli. Indeed, the OxyR stress response to H₂O₂ was induced when B. thetaiotaomicron was aerated, and in that circumstance this response was necessary to forestall cell death. Thus aeration is a serious threat for this obligate anaerobe, and to cope it employs a set of defences that includes a repertoire of complementary scavenging enzymes.     

IL-17作为分子佐剂增强蛋白疫苗细胞免疫应答的研究

目的:为了进一步增强重组蛋白质疫苗的细胞免疫应答,利用重组的IL-17作为分子佐剂,与卵清蛋白(ovalbumin,OVA)一起免疫小鼠,研究IL-17作为分子佐剂对适应性免疫的影响,探索IL-17对蛋白疫苗诱导的免疫反应,特别是细胞免疫应答的影响.方法:用OVA作为特异性蛋白疫苗,与不同剂量的IL-17联合免疫C57...     

The Ocular Conjunctiva as a Mucosal Immunization Route: A Profile of the Immune Response to the Model Antigen Tetanus Toxoid

Background

In a quest for a needle-free vaccine administration strategy, we evaluated the ocular conjunctiva as an alternative mucosal immunization route by profiling and comparing the local and systemic immune responses to the subcutaneous or conjunctival administration of tetanus toxoid (TTd), a model antigen.

Materials and methods

BALB/c and C57BL/6 mice were immunized either subcutaneously with TTd alone or via the conjunctiva with TTd alone, TTd mixed with 2% glycerol or TTd with merthiolate-inactivated whole-cell B. pertussis (wBP) as adjuvants. Mice were immunized on days 0, 7 and 14 via both routes, and an evaluation of the local and systemic immune responses was performed two weeks after the last immunization. Four weeks after the last immunization, the mice were challenged with a lethal dose (2 × LD₅₀) of tetanus toxin.

Results

The conjunctival application of TTd in BALB/c mice induced TTd-specific secretory IgA production and skewed the TTd-specific immune response toward a Th1/Th17 profile, as determined by the stimulation of IFNγ and IL-17A secretion and/or the concurrent pronounced reduction of IL-4 secretion, irrespective of the adjuvant. In conjunctivaly immunized C57BL/6 mice, only TTd administered with wBP promoted the establishment of a mixed Th1/Th17 TTd-specific immune response, whereas TTd alone or TTd in conjunction with glycerol initiated a dominant Th1 response against TTd. Immunization via the conjunctiva with TTd plus wBP adjuvant resulted in a 33% survival rate of challenged mice compared to a 0% survival rate in non-immunized animals (p<0.05).

Conclusion

Conjunctival immunization with TTd alone or with various adjuvants induced TTd-specific local and systemic immune responses, predominantly of the Th1 type. The strongest immune responses developed in mice that received TTd together with wBP, which implies that this alternative route might tailor the immune response to fight intracellular bacteria or viruses more effectively.