An In Vitro Intestinal Model Captures Immunomodulatory Properties of the Microbiota in Inflammation

Abstract BackgroundThere is a growing appreciation for the significance of the gut microbiome in health and disease. Specifically, considerable effort has been put forth to understand mechanisms by which the microbiota modulates and responds to inflammation. Here, we explored whether oxidation metabolites produced by the host during inflammation, sodium nitrate and trimethylamine oxide, impact the composition of a human stool bacterial population in a gut simulator. We then assessed whether an immune-competent in vitro intestinal model responded differently to spent medium from bacteria exposed to these cues compared to spent medium from a control bacterial population. ResultsThe host-derived oxidation products were found to decrease levels of Bacteroidaceae and overall microbiota metabolic potential, while increasing levels of pro-inflammatory Enterobacteriaceae and lipopolysaccharide in bacterial cultures, reflecting shifts that occur in vivo in inflammation. Spent medium, with or without sodium nitrate and trimethylamine oxide, induced elevated intracellular mucin levels and reduced intestinal monolayer integrity as reflected in trans-epithelial electrical resistance relative to fresh medium controls. However, multiplexed cytokine analysis revealed markedly different cytokine signatures from intestinal cultures exposed to spent medium with added oxidation products relative to spent control medium, while cytokine signatures of cultures exposed to fresh media were similar regardless of addition of host-derived cues. Further, the presence of immune cells in the intestinal model was required for this differentiation of cytokine signatures. ConclusionsThis study indicates that simple in vitro immune-competent intestinal models can capture bacterial-mammalian cross-talk in response to host-derived oxidation products and supports utility of these systems for mechanistic studies of interactions between the gut microbiome and host in inflammation.

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Plant-Derived Extracellular Vesicles: Current Findings,Challenges, and Future Applications

Membranes ◽

10.3390/membranes11060411 ◽

2021 ◽

Vol 11 (6) ◽

pp. 411

Author(s):

Nader Kameli ◽

Anya Dragojlovic-Kerkache ◽

Paul Savelkoul ◽

Frank R. Stassen

Keyword(s):

Human Health ◽

Extracellular Vesicles ◽

Preliminary Results ◽

In Vivo Experiments ◽

Health And Disease ◽

Conducting Research ◽

Protocol Standardization ◽

Insight Into

In recent years, plant-derived extracellular vesicles (PDEVs) have gained the interest of many experts in fields such as microbiology and immunology, and research in this field has exponentially increased. These nano-sized particles have provided researchers with a number of interesting findings, making their application in human health and disease very promising. Both in vitro and in vivo experiments have shown that PDEVs can exhibit a multitude of effects, suggesting that these vesicles may have many potential future applications, including therapeutics and nano-delivery of compounds. While the preliminary results are promising, there are still some challenges to face, such as a lack of protocol standardization, as well as knowledge gaps that need to be filled. This review aims to discuss various aspects of PDEV knowledge, including their preliminary findings, challenges, and future uses, giving insight into the complexity of conducting research in this field.

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Xylitol enhances synthesis of propionate in the colon via cross-feeding of gut microbiota

Microbiome ◽

10.1186/s40168-021-01029-6 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Shasha Xiang ◽

Kun Ye ◽

Mian Li ◽

Jian Ying ◽

Huanhuan Wang ◽

...

Keyword(s):

Gut Microbiome ◽

Lactobacillus Reuteri ◽

Carbon Sources ◽

Short Chain Fatty Acids ◽

Microbial Composition ◽

Key Enzymes ◽

Rrna Sequencing ◽

Phosphate Acetyltransferase

Abstract Background Xylitol, a white or transparent polyol or sugar alcohol, is digestible by colonic microorganisms and promotes the proliferation of beneficial bacteria and the production of short-chain fatty acids (SCFAs), but the mechanism underlying these effects remains unknown. We studied mice fed with 0%, 2% (2.17 g/kg/day), or 5% (5.42 g/kg/day) (weight/weight) xylitol in their chow for 3 months. In addition to the in vivo digestion experiments in mice, 3% (weight/volume) (0.27 g/kg/day for a human being) xylitol was added to a colon simulation system (CDMN) for 7 days. We performed 16S rRNA sequencing, beneficial metabolism biomarker quantification, metabolome, and metatranscriptome analyses to investigate the prebiotic mechanism of xylitol. The representative bacteria related to xylitol digestion were selected for single cultivation and co-culture of two and three bacteria to explore the microbial digestion and utilization of xylitol in media with glucose, xylitol, mixed carbon sources, or no-carbon sources. Besides, the mechanisms underlying the shift in the microbial composition and SCFAs were explored in molecular contexts. Results In both in vivo and in vitro experiments, we found that xylitol did not significantly influence the structure of the gut microbiome. However, it increased all SCFAs, especially propionate in the lumen and butyrate in the mucosa, with a shift in its corresponding bacteria in vitro. Cross-feeding, a relationship in which one organism consumes metabolites excreted by the other, was observed among Lactobacillus reuteri, Bacteroides fragilis, and Escherichia coli in the utilization of xylitol. At the molecular level, we revealed that xylitol dehydrogenase (EC 1.1.1.14), xylulokinase (EC 2.7.1.17), and xylulose phosphate isomerase (EC 5.1.3.1) were key enzymes in xylitol metabolism and were present in Bacteroides and Lachnospiraceae. Therefore, they are considered keystone bacteria in xylitol digestion. Also, xylitol affected the metabolic pathway of propionate, significantly promoting the transcription of phosphate acetyltransferase (EC 2.3.1.8) in Bifidobacterium and increasing the production of propionate. Conclusions Our results revealed that those key enzymes for xylitol digestion from different bacteria can together support the growth of micro-ecology, but they also enhanced the concentration of propionate, which lowered pH to restrict relative amounts of Escherichia and Staphylococcus. Based on the cross-feeding and competition among those bacteria, xylitol can dynamically balance proportions of the gut microbiome to promote enzymes related to xylitol metabolism and SCFAs.

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Glycoside Hydrolases Degrade Polymicrobial Bacterial Biofilms in Wounds

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01998-16 ◽

2016 ◽

Vol 61 (2) ◽

Cited By ~ 43

Author(s):

Derek Fleming ◽

Laura Chahin ◽

Kendra Rumbaugh

Keyword(s):

Glycoside Hydrolase ◽

Bacterial Population ◽

Chronic Wounds ◽

Glycoside Hydrolases ◽

Bacterial Biofilms ◽

Planktonic Cells ◽

Content Type ◽

Biomass Dissolution

ABSTRACT The persistent nature of chronic wounds leaves them highly susceptible to invasion by a variety of pathogens that have the ability to construct an extracellular polymeric substance (EPS). This EPS makes the bacterial population, or biofilm, up to 1,000-fold more antibiotic tolerant than planktonic cells and makes wound healing extremely difficult. Thus, compounds which have the ability to degrade biofilms, but not host tissue components, are highly sought after for clinical applications. In this study, we examined the efficacy of two glycoside hydrolases, α-amylase and cellulase, which break down complex polysaccharides, to effectively disrupt Staphylococcus aureus and Pseudomonas aeruginosa monoculture and coculture biofilms. We hypothesized that glycoside hydrolase therapy would significantly reduce EPS biomass and convert bacteria to their planktonic state, leaving them more susceptible to conventional antimicrobials. Treatment of S. aureus and P. aeruginosa biofilms, grown in vitro and in vivo, with solutions of α-amylase and cellulase resulted in significant reductions in biomass, dissolution of the biofilm, and an increase in the effectiveness of subsequent antibiotic treatments. These data suggest that glycoside hydrolase therapy represents a potential safe, effective, and new avenue of treatment for biofilm-related infections.

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DNAJB6b is Downregulated in Synucleinopathies

Journal of Parkinson s Disease ◽

10.3233/jpd-202512 ◽

2021 ◽

pp. 1-13

Author(s):

Jonas Folke ◽

Sertan Arkan ◽

Isak Martinsson ◽

Susana Aznar ◽

Gunnar Gouras ◽

...

Keyword(s):

Neurodegenerative Diseases ◽

Substantia Nigra Pars Compacta ◽

Endogenous Protein ◽

Highly Sensitive ◽

Brain Material ◽

Health And Disease ◽

Isoform B

Background: α-synuclein (α-syn) aggregation contributes to the progression of multiple neurodegenerative diseases. We recently found that the isoform b of the co-chaperone DNAJB6 is a strong suppressor of a-syn aggregation in vivo and in vitro. However, nothing is known about the role of the endogenous isoform b of DNAJB6 (DNAJB6b) in health and disease, due to lack of specific antibodies. Objective: Here we generated a novel anti-DNAJB6b antibody to analyze the localization and expression this isoform in cells, in tissue and in clinical material. Methods: To address this we used immunocytochemistry, immunohistochemistry, as well as a novel quantitative DNAJB6 specific ELISA method. Results: The endogenous protein is mainly expressed in the cytoplasm and in neurites in vitro, where it is found more in dendrites than in axons. We further verified in vivo that DNAJB6b is expressed in the dopaminergic neurons of the substantia nigra pars compacta (SNpc), which is a neuronal subpopulation highly sensitive to α-syn aggregation, that degenerate to a large extend in patients with Parkinson’s disease (PD) and multiple system atrophy (MSA). When we analyzed the expression levels of DNAJB6b in brain material from PD and MSA patients, we found a downregulation of DNAJB6b by use of ELISA based quantification. Interestingly, this was also true when analyzing tissue from patients with progressive supranuclear palsy, a taupathic atypical parkinsonian disorder. However, the total level of DNAJB6 was upregulated in these three diseases, which may indicate an upregulation of the other major isoform of DNAJB6, DNAJB6a. Conclusion: This study shows that DNAJB6b is downregulated in several different neurodegenerative diseases, which makes it an interesting target to further investigate in relation to amyloid protein aggregation and disease progression.

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Abstract 064: The Lipoxigenase Inhibitor Nordihydroguaiaretic Acid Prevents the Development of Hypoxia-Induced Pulmonary Hypertension in Mice

Circulation Research ◽

10.1161/res.113.suppl_1.a064 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Andrea Iorga ◽

Gabriel Wong ◽

Denise Mai ◽

Jingyuan Li ◽

Salil Sharma ◽

...

Keyword(s):

Pulmonary Hypertension ◽

Smooth Muscle ◽

Smooth Muscle Actin ◽

Nordihydroguaiaretic Acid ◽

Treated Group ◽

Oxidation Products ◽

Lipoxygenase Inhibitor ◽

Human Pulmonary Artery

Pulmonary hypertension (PH) is a chronic lung disease characterized by progressively elevated pulmonary arterial pressures and severe pulmonary vascular remodeling resulting from interactions between oxidized lipoprotein deposition and increased endothelial proliferation. Previously we have shown increased plasma levels of biological oxidation products such as hydroxyoctadecadienoic acids (HODEs) and hydroxyeicosatetraenoic acids (HETEs) in the rat monocrotaline model of PH. Here we investigated the role of HETEs and HODEs in the development of PH and whether their inhibition with the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) attenuates the progression of PH. Mice were placed in a hypoxic chamber with O2 concentrations of ≤10% for 21 days and either left untreated to develop PH (n=7) or treated with NDGA daily (10mg/kg/day, i.p., n=4) from day 1. Direct RV catheterization was terminally performed to record RV pressure (RVP). Pulmonary arteriolar thickening and oxidized lipid deposition were assessed by staining lung sections with Masson’s Trichrome or with α-smooth muscle actin and E-06 (marker for oxidized low-density lipoproteins). In vitro, human pulmonary artery smooth muscle cell (hPASMC) proliferation was assessed by MTT assays in the absence or presence of 12-HETE (100ng/ml), 9-HODE (1µg/ml) and 13-HODE (1µg/ml) alone or together with NDGA (10, 25 and 50µM). In-vitro, HETE/HODE treatment increased hPASMC proliferation ~ 2-fold when compared to untreated cells and NDGA significantly inhibited the proliferative effects of all three oxidized lipids. In-vivo, NDGA treatment prevented the development of PH. RVP was lower in the NDGA-treated group vs. the PH group (24.01±1.39mmHg vs. 36.91±5.74mmHg, p<0.05) and was comparable to control normoxic mice (20.93±2.52mmHg). RV hypertrophy index was significantly elevated in the PH mice versus control mice (0.38±0.03 vs. 0.28±0.02 (p<0.001), while NDGA treatment completely prevented the development of RV hypertrophy (0.28±0.04). Lung sections demonstrated arteriolar thickening and E-06 positive deposits in the PH group, which was prevented by NDGA therapy. We conclude that oxidized fatty acid deposition and accumulation might play a role in the development of PH.

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The Effects of AHCC®, a Standardized Extract of Cultured Lentinura edodes Mycelia, on Natural Killer and T Cells in Health and Disease: Reviews on Human and Animal Studies

Journal of Immunology Research ◽

10.1155/2019/3758576 ◽

2019 ◽

Vol 2019 ◽

pp. 1-7 ◽

Cited By ~ 3

Author(s):

Min Sun Shin ◽

Hong-Jai Park ◽

Takahiro Maeda ◽

Hiroshi Nishioka ◽

Hajime Fujii ◽

...

Keyword(s):

T Cells ◽

Immune System ◽

Natural Killer ◽

Animal Studies ◽

Lentinula Edodes ◽

Traditional Medicines ◽

Immune Mediated ◽

Health And Disease

Mushrooms have been used for various health conditions for many years by traditional medicines practiced in different regions of the world although the exact effects of mushroom extracts on the immune system are not fully understood. AHCC® is a standardized extract of cultured shiitake or Lentinula edodes mycelia (ECLM) which contains a mixture of nutrients including oligosaccharides, amino acids, and minerals obtained through liquid culture. AHCC® is reported to modulate the numbers and functions of immune cells including natural killer (NK) and T cells which play important roles in host defense, suggesting the possible implication of its supplementation in defending the host against infections and malignancies via modulating the immune system. Here, we review in vivo and in vitro effects of AHCC® on NK and T cells of humans and animals in health and disease, providing a platform for the better understanding of immune-mediated mechanisms and clinical implications of AHCC®.

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Monitoring food digestion with magnetic resonance techniques

Proceedings of The Nutrition Society ◽

10.1017/s0029665120007867 ◽

2020 ◽

pp. 1-11

Author(s):

Paul A. M. Smeets ◽

Ruoxuan Deng ◽

Elise J. M. van Eijnatten ◽

Morwarid Mayar

Keyword(s):

Magnetic Resonance ◽

Food Systems ◽

Chemical Exchange ◽

Quantitative Information ◽

Model Systems ◽

Chemical Exchange Saturation Transfer ◽

Food Properties ◽

Health And Disease

This review outlines the current use of magnetic resonance (MR) techniques to study digestion and highlights their potential for providing markers of digestive processes such as texture changes and nutrient breakdown. In vivo digestion research can be challenging due to practical constraints and biological complexity. Therefore, digestion is primarily studied using in vitro models. These would benefit from further in vivo validation. NMR is widely used to characterise food systems. MRI is a related technique that can be used to study both in vitro model systems and in vivo gastro-intestinal processes. MRI allows visualisation and quantification of gastric processes such as gastric emptying and coagulation. Both MRI and NMR scan sequences can be configured to be sensitive to different aspects of gastric or intestinal contents. For example, magnetisation transfer and chemical exchange saturation transfer can detect proton (1H) exchange between water and proteins. MRI techniques have the potential to provide molecular-level and quantitative information on in vivo gastric (protein) digestion. This requires careful validation in order to understand what these MR markers of digestion mean in a specific digestion context. Combined with other measures they can be used to validate and inform in vitro digestion models. This may bridge the gap between in vitro and in vivo digestion research and can aid the optimisation of food properties for different applications in health and disease.

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Image-based modelling of skeletal muscle oxygenation

Journal of The Royal Society Interface ◽

10.1098/rsif.2016.0992 ◽

2017 ◽

Vol 14 (127) ◽

pp. 20160992 ◽

Cited By ~ 7

Author(s):

B. Zeller-Plumhoff ◽

T. Roose ◽

G. F. Clough ◽

P. Schneider

Keyword(s):

Skeletal Muscle ◽

Blood Vessel ◽

Ex Vivo ◽

Imaging Techniques ◽

Muscle Oxygenation ◽

Skeletal Muscle Oxygenation ◽

Health And Disease ◽

Vessel Networks

The supply of oxygen in sufficient quantity is vital for the correct functioning of all organs in the human body, in particular for skeletal muscle during exercise. Disease is often associated with both an inhibition of the microvascular supply capability and is thought to relate to changes in the structure of blood vessel networks. Different methods exist to investigate the influence of the microvascular structure on tissue oxygenation, varying over a range of application areas, i.e. biological in vivo and in vitro experiments, imaging and mathematical modelling. Ideally, all of these methods should be combined within the same framework in order to fully understand the processes involved. This review discusses the mathematical models of skeletal muscle oxygenation currently available that are based upon images taken of the muscle microvasculature in vivo and ex vivo . Imaging systems suitable for capturing the blood vessel networks are discussed and respective contrasting methods presented. The review further informs the association between anatomical characteristics in health and disease. With this review we give the reader a tool to understand and establish the workflow of developing an image-based model of skeletal muscle oxygenation. Finally, we give an outlook for improvements needed for measurements and imaging techniques to adequately investigate the microvascular capability for oxygen exchange.

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The Glucocorticoid Receptor in Cardiovascular Health and Disease

Cells ◽

10.3390/cells8101227 ◽

2019 ◽

Vol 8 (10) ◽

pp. 1227 ◽

Cited By ~ 7

Author(s):

Liu ◽

Zhang ◽

Knight ◽

Goodwin

Keyword(s):

Glucocorticoid Receptor ◽

Cardiovascular System ◽

Gene Networks ◽

Cardiovascular Health ◽

Critical Role ◽

Glucocorticoid Signaling ◽

Health And Disease ◽

Nuclear Receptor Family

The glucocorticoid receptor is a member of the nuclear receptor family that controls many distinct gene networks, governing various aspects of development, metabolism, inflammation, and the stress response, as well as other key biological processes in the cardiovascular system. Recently, research in both animal models and humans has begun to unravel the profound complexity of glucocorticoid signaling and convincingly demonstrates that the glucocorticoid receptor has direct effects on the heart and vessels in vivo and in vitro. This research has contributed directly to improving therapeutic strategies in human disease. The glucocorticoid receptor is activated either by the endogenous steroid hormone cortisol or by exogenous glucocorticoids and acts within the cardiovascular system via both genomic and non-genomic pathways. Polymorphisms of the glucocorticoid receptor are also reported to influence the progress and prognosis of cardiovascular disease. In this review, we provide an update on glucocorticoid signaling and highlight the critical role of this signaling in both physiological and pathological conditions of the cardiovascular system. With increasing in-depth understanding of glucocorticoid signaling, the future is promising for the development of targeted glucocorticoid treatments and improved clinical outcomes.

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Chlorogenic Acid as a Model Compound for Optimization of an In Vitro Gut Microbiome-Metabolism Model

Proceedings ◽

10.3390/proceedings2019011031 ◽

2019 ◽

Vol 11 (1) ◽

pp. 31 ◽

Cited By ~ 2

Author(s):

Olivier Mortelé ◽

Elias Iturrospe ◽

Annelies Breynaert ◽

Christine Lammens ◽

Xavier Basil Britto ◽

...

Keyword(s):

Chlorogenic Acid ◽

Gut Microbiome ◽

Model Compound ◽

In Vitro Models ◽

Cytochrome P450 Enzyme ◽

Flight Mass Spectrometry ◽

P450 Enzyme ◽

Analytical Phase

It has been believed that the metabolism of xenobiotics occurred mainly by the cytochrome P450 enzyme system in the liver. However, recent data clearly suggest a significant role for the gut microbiota in the metabolism of xenobiotic compounds. This microbiotic biotransformation could lead to differences on activation, inactivation and possible toxicity of these compounds. In vitro models are generally used to study the colonic biotransformation as they allow easy dynamic and multiple sampling over time. However, to ensure this accurately mimics communities in vivo, the pre-analytical phase requires optimization. Chlorogenic acid, a polyphenolic compound abundantly present in the human diet, was used as a model compound to optimize a ready-to-use gut microbiome biotransformation platform. Samples of the in vitro gastrointestinal dialysis-model with colon stage were analyzed by liquid chromatography coupled to high resolution time-of-flight mass spectrometry. Complementary screening approaches were also employed to identify the biotransformation products.

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