ABSTRACT
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. The host-derived oxidation products were found to decrease levels of Bacteroidaceae and overall microbiota metabolic potential, while increasing levels of proinflammatory Enterobacteriaceae and lipopolysaccharide in bacterial cultures, reflecting shifts that occur in vivo in inflammation. Spent microbiota media induced elevated intracellular mucin levels and reduced intestinal monolayer integrity as reflected in transepithelial 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. This 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.
Subject(s)
Gastrointestinal Microbiome , Microbiota , Animals , Bacteria , Cytokines , Humans , Inflammation , MammalsABSTRACT
Myeloid cells play critical and diverse roles in mammalian physiology, including tissue development and repair, innate defense against pathogens, and generation of adaptive immunity. As cells that show prolonged recruitment to sites of injury or pathology, myeloid cells represent therapeutic targets for a broad range of diseases. However, few approaches have been developed for gene editing of these cell types, likely owing to their sensitivity to foreign genetic material or virus-based manipulation. Here we describe optimized strategies for gene disruption in primary myeloid cells of human and murine origin. Using nucleofection-based delivery of Cas9-ribonuclear proteins (RNPs), we achieved near population-level genetic knockout of single and multiple targets in a range of cell types without selection or enrichment. Importantly, we show that cellular fitness and response to immunological stimuli is not significantly impacted by the gene editing process. This provides a significant advance in the study of myeloid cell biology, thus enabling pathway discovery and drug target validation across species in the field of innate immunity.
Subject(s)
CRISPR-Cas Systems/genetics , Gene Knockout Techniques , Gene Transfer Techniques , Myeloid Cells/metabolism , Animals , Cells, Cultured , Dendritic Cells/metabolism , Gene Deletion , Gene Editing , Genetic Engineering , Genome , Humans , Macrophages/metabolism , Mice , Monocytes/metabolism , Phagocytosis , Phenotype , RNA, Guide, Kinetoplastida/genetics , Ribonucleoproteins/metabolism , Viruses/metabolismABSTRACT
Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of incompletely understood pathophysiology predominantly affecting premature infants. While NEC is associated with microbial invasion of intestinal tissues, and mucus modulates interactions between microbes and underlying tissues, variations in mucus barrier properties with NEC-associated risk factors have not been investigated. This study explored differences in mucus composition (total protein, DNA, mucin content, sialic acid, and immunoregulatory proteins), as well as structural and transport properties, assessed by tracking of particles and bacteria (E. coli and E. cloacae) with developmental age and exposure to NEC stressors in Sprague Dawley rats. Early developmental age (5 day old) was characterized by a more permeable mucus layer relative to 21 day old pups, suggesting immaturity may contribute to exposure of the epithelium to microbes. Exposure to NEC stressors was associated with reduced mucus permeability, which may aid in survival. Feeding with breastmilk as opposed to formula reduces incidence of NEC. Thus, NEC-stressed (N-S) rat pups were orally dosed with breastmilk components lysozyme (N-S-LYS) or docosahexaenoic acid (N-S-DHA). N-S-LYS and N-S-DHA pups had a less permeable mucus barrier relative to N-S pups, which suggests the potential of these factors to strengthen the mucus barrier and thus protect against disease.
Subject(s)
Aging/pathology , Docosahexaenoic Acids/administration & dosage , Docosahexaenoic Acids/therapeutic use , Enterocolitis, Necrotizing/drug therapy , Mucus/metabolism , Muramidase/administration & dosage , Muramidase/therapeutic use , Stress, Physiological , Administration, Oral , Animals , DNA/metabolism , Docosahexaenoic Acids/pharmacology , Enterobacter cloacae/physiology , Enterocolitis, Necrotizing/microbiology , Escherichia coli/physiology , Fucose/metabolism , Ileum/pathology , Ileum/ultrastructure , Immunoglobulin G/metabolism , Mucins/metabolism , Mucus/drug effects , Muramidase/pharmacology , N-Acetylneuraminic Acid/metabolism , Permeability , Polyethylene Glycols/chemistry , Rats, Sprague-Dawley , Stress, Physiological/drug effectsABSTRACT
Mucus constitutes a protective layer which coats the gastrointestinal tract, controlling interactions of both commensal and pathogenic microbes with underlying tissues. Changes to the mucus barrier, for example due to altered mucin expression or external stimuli, may impact interactions with microbes and thus potentially contribute to altered gut homeostasis, onset of inflammation, or pathogen invasion. Food-associated stimuli, including lipids, have been shown to change mucus barrier properties and reduce transport of model drug carriers through mucus. Here, we explore the impact of lipids, specifically triglycerides in a model intestinal medium mimicking a fed state, on Escherichia coli (E. coli) transport through mucus by directly imaging swimming patterns and analyzing associated changes in mucus structure. Lipids in model fed state intestinal contents reduced E. coli speed and track linearity within mucus. These changes may be due in part to changes in molecular interactions within the mucus network as well as crowding of the mucus network by lipid emulsion droplets, which visibly stay intact in the mucus gel. In addition, observed physical interactions between bacteria and lipid structures may impact microbial speed and trajectories. As lipids are normal food components and thus represent safe, mild stimuli, these results support exploration of lipid-based strategies to alter the mucus barrier to control interactions with microbes and potentially prevent microbial invasion of underlying epithelium.
Subject(s)
Escherichia coli/physiology , Lipids/pharmacology , Locomotion/drug effects , Animals , Escherichia coli/drug effects , Lipid Droplets/chemistry , Lipids/chemistry , Microscopy, Video , Mucus/chemistry , Mucus/metabolism , SwineABSTRACT
The consumption of generally regarded as safe emulsifiers has increased, and has been associated with an increased prevalence of inflammatory bowel and metabolic diseases, as well as an altered microbiome. The mucus barrier, which selectively controls the transport of particulates and microorganisms to the underlying epithelial layer, has been previously shown to be altered by dietary salts and lipids. However, the potential impact of emulsifiers on the protective mucus barrier, its permeability, and associated structural changes are not clear. In this study, we analyzed changes in the mucus barrier to both passively diffusing nanoparticles and actively swimming E. coli upon exposure to two emulsifiers, carboxymethylcellulose (CMC) and polysorbate 80 (Tween). When exposed to CMC, mucus pore size decreased, which resulted in significantly slower E. coli speed and particle diffusion rates through mucus. Tween exposure minimally impacted mucus microstructure and particle diffusion, but increased E. coli speed in mucus. Moreover, both emulsifiers appeared to alter mucus amount and thickness in rat intestinal tissue and mucus-producing cell cultures. These results indicate that acute exposure to emulsifiers impacts barrier and structural properties of intestinal mucus, modulating interactions between intestinal lumen contents, microbes, and underlying tissue, which may contribute to development of intestinal inflammation.
Subject(s)
Carboxymethylcellulose Sodium/pharmacology , Emulsifying Agents/pharmacology , Intestinal Mucosa/metabolism , Polysorbates/pharmacology , Tight Junctions/physiology , Animals , Biological Transport/physiology , Carboxymethylcellulose Sodium/adverse effects , Cell Line , Emulsifying Agents/adverse effects , Escherichia coli/genetics , HT29 Cells , Humans , Intestinal Mucosa/ultrastructure , Male , Mucus/metabolism , Nanoparticles/metabolism , Polysorbates/adverse effects , Rats , Rats, Wistar , SwineABSTRACT
Gut-on-a-chip in vitro modeling is an emerging field, as the human gut epithelium and gut microbiome have been recently identified as novel drug targets for a wide variety of diseases. Realistic in vitro gut models require a variety of precise environmental cues, such as chemical and gas gradients, in combination with substrates like mucus that support the growth of microbial communities. This technical brief describes a microfluidic architecture capable of developing a physiologically relevant oxygen gradient that emulates the oxygen profile proximal to the epithelial inner lining of the human colon. The device generates stable and repeatable defined oxygen gradients from 0% to 4 % partial pressure O2 over a length scale of hundreds of microns, and was applied to study the effects of oxygenation on the structure of native mucus that lines the colon wall. Using simulation as a design tool for hybrid gas-liquid microfluidic devices enables on-chip creation of defined, physiologically oxygen gradients. These microfluidic architectures have powerful potential applications for gut physiology, including providing optimal oxygenation conditions for the culture of mammalian epithelial cells in the gut lining, as well as creating a realistic mimic of the oxygen gradient found in the intestinal lumen for complex microbiome cultures.
Subject(s)
Colon/chemistry , Colon/physiology , Lab-On-A-Chip Devices , Oxygen/metabolism , Humans , Models, Biological , Mucus/chemistry , Partial PressureABSTRACT
Mucus is a complex hydrogel that acts as a natural barrier to drug delivery at different mucosal surfaces including the respiratory, gastrointestinal, and vaginal tracts. To elucidate the role mucus plays in drug delivery, different in vitro, in vivo, and ex vivo mucus models and techniques have been utilized. Drug and drug carrier diffusion can be studied using various techniques in either isolated mucus gels or mucus present on cell cultures and tissues. The species, age, and potential disease state of the animal from which mucus is derived can all impact mucus composition and structure, and therefore impact drug and drug carrier diffusion. This review provides an overview of the techniques used to characterize drug and drug carrier diffusion, and discusses the advantages and disadvantages of the different models available to highlight the information they can afford.
Subject(s)
Diffusion , Models, Biological , Mucus/metabolism , Pharmaceutical Preparations/metabolism , Animals , Drug Delivery Systems , Humans , Mucus/chemistry , Particle Size , Pharmaceutical Preparations/chemistryABSTRACT
This article presents an investigation on the effectiveness of magnesium and its alloys as a novel class of antibacterial and biodegradable materials for ureteral stent applications. Magnesium is a lightweight and biodegradable metallic material with beneficial properties for use in medical devices. Ureteral stent is one such example of a medical device that is widely used to treat ureteral canal blockages clinically. The bacterial colony formation coupled with the encrustation on the stent surface from extended use often leads to clinical complications and contributes to the failure of indwelling medical devices. We demonstrated that magnesium alloys decreased Escherichia coli viability and reduced the colony forming units over a 3-day incubation period in an artificial urine (AU) solution when compared with currently used commercial polyurethane stent. Moreover, the magnesium degradation resulted in alkaline pH and increased magnesium ion concentration in the AU solution. The antibacterial and degradation properties support the potential use of magnesium-based materials for next-generation ureteral stents. Further studies are needed for clinical translation of biodegradable metallic ureteral stents.
Subject(s)
Alloys/chemistry , Anti-Bacterial Agents/chemistry , Biocompatible Materials/chemistry , Magnesium/chemistry , Stents/microbiology , Ureter/microbiology , Alloys/pharmacology , Anti-Bacterial Agents/pharmacology , Biocompatible Materials/pharmacology , Escherichia coli/drug effects , Escherichia coli Infections/prevention & control , Humans , Magnesium/pharmacology , Urinary Catheters/microbiology , Urine/chemistryABSTRACT
Bacterial infection often causes clinical complications and failure of indwelling medical devices. This is a major problem of current ureteral stents, which are used clinically to treat the blockage of ureteral canals. This study investigates the effectiveness and applicability of magnesium as a novel biodegradable ureteral stent material that has inherent antimicrobial properties. Incubating Escherichia coli with the magnesium samples showed a decrease in the bacterial cell density as compared with the currently used commercial polyurethane stent. Magnesium degradation in the immersion solutions (artificial urine, luria bertani broth, and deionized water) resulted in an alkaline pH shift. Antimicrobial and biodegradation properties of magnesium make it an attractive alternative as next-generation ureteral stent material.