Mouse Gastrointestinal Tract Research Articles

Isolation of Myenteric and Submucosal Plexus from Mouse Gastrointestinal Tract and Subsequent Co-Culture with Small Intestinal Organoids.

Intestinal homeostasis results from the proper interplay among epithelial cells, the enteric nervous system (ENS), interstitial cells of Cajal (ICCs), smooth muscle cells, the immune system, and the microbiota. The disruption of this balance underpins the onset of gastrointestinal-related diseases. The scarcity of models replicating the intricate interplay between the ENS and the intestinal epithelium highlights the imperative for developing novel methods. We have pioneered a sophisticated tridimensional in vitro technique, coculturing small intestinal organoids with myenteric and submucosal neurons. Notably, we have made significant advances in (1) refining the isolation technique for culturing the myenteric plexus, (2) enhancing the isolation of the submucosal plexus-both yielding mixed cultures of enteric neurons and glial cells from both plexuses, and (3) subsequently co-culturing myenteric and submucosal neurons with small intestinal organoids. This co-culture system establishes neural innervations with intestinal organoids, allowing for the investigation of regulatory interactions in the context of gastrointestinal diseases. Furthermore, we have developed a method for microinjecting the luminal space of small intestinal organoids with fluorescently labeled compounds. This technique possesses broad applicability such as the assessment of intestinal permeability, transcytosis, and immunocytochemical and immunofluorescence applications. This microinjection method could be extended to alternative experimental setups, incorporating bacterial species, or applying treatments to study ENS-small intestinal epithelium interactions. Therefore, this technique serves as a valuable tool for evaluating the intricate interplay between neuronal and intestinal epithelial cells (IECs) and shows great potential for drug screening, gene editing, the development of novel therapies, the modeling of infectious diseases, and significant advances in regenerative medicine. The co-culture establishment process spans twelve days, making it a powerful asset for comprehensive research in this critical field.

Short communication: Metabolic synthesis of vitamin D2 by the gut microbiome

The origin of vitamin D2 in herbivorous animals was investigated in vivo in sheep and in bovine as well as mouse gastrointestinal tracts. A high concentration of 25-hydroxyvitamin D2 in blood plasma of sheep both in summer and winter appeared to be incompatible with the undetectable level of vitamin D2 in the pasture on which the sheep were grazing. Studies with bovine rumen contents from a cow grazing the same pasture as the sheep, demonstrated an increased concentration of vitamin D2 on anaerobic incubation in a ‘Rusitec’ artificial rumen, which was further enhanced when cellulose powder was added as a fermentation substrate. The colon contents of mice that were fed from weaning on a vitamin D-free diet were found to contain vitamin D2. The results of these comparative studies in 3 animal species indicated that vitamin D2 was being generated by microbial anaerobic metabolism in the gastrointestinal tract.

403 Epithelial hypoxia maintains colonization resistance against Candida albicans

OBJECTIVES/GOALS: Antibiotic treatment sets the stage for intestinal domination by Candida albicanswhich is necessary for development of invasive disease, but the resources driving this bloom remain poorly defined. We sought to determine these factors in order to design novel prophylaxis strategies for reducing gastrointestinal (GI) colonization. METHODS/STUDY POPULATION: We initially developed a generalizable framework, termed metabolic footprinting to determine the metabolites C. albicanspreferentially uses in the mouse GI tract. After identifying the metabolites C. albicansutilizes, we usedin vitro growth assays in the presence and absence of oxygen to validate out metabolomics findings. We next determined if a probiotic E. coli that utilizes oxygen would reduce C. albicanscolonization compared to a mutant E. coli that could not respire oxygen. Finding that oxygen was a necessary resource, we utilized germ-free mice to determine if Clostridiaspp. known to reduce GI oxygen would prevent C. albicanscolonization. Lastly, we sought to see if 5-aminosalicylic acid (5-ASA) could prevent C. albicanscolonization. RESULTS/ANTICIPATED RESULTS: We found that C. albicans preferentially utilizes simple carbohydrates including fructo-oligosaccharides (e.g., 1-kestose), disaccharides (e.g., β-gentiobiose), and alcoholic sugars (e.g., sorbitol) and is able to grow in vitro on minimal media supplemented with either of these nutrients. However, in the hypoxic environment that is found in the “healthy” colon, C. albicans cannot utilize these nutrients. We next found that pre-colonization in a mouse model with a probiotic E. coli significantly reduced C. albicanscolonization, but the mutant E. coli had no effect on colonization. We next showed that Clostridia supplementation restored GI hypoxia and reduced C. albicanscolonization. Remarkably, we found that 5-ASA significantly reduced GI colonization of C. albicans. DISCUSSION/SIGNIFICANCE: We have shown that C. albicans requires oxygen to colonize the GI tract. Importantly, we found that 5-ASA can prevent an antibiotic mediated bloom of C. albicans by restoring GI hypoxia, which warrants additional studies to determine if 5-ASA can be used as an adjunctive prophylactic treatment in high risk patients.

An M cell-targeting recombinant L. lactis vaccine against four H. pylori adhesins

The acidic environment and enzyme degradation lead to oral vaccines often having little immune effect. Therefore, it is an attractive strategy to study an effective and safe oral vaccine delivery system that can promote gastrointestinal mucosal immune responses and inhibit antigen degradation. Moreover, the antigens uptake by microfold cells (M cells) is the determining step in initiating efficient immune responses. Therefore, M cell-targeting is one promising approach for enhancing oral vaccine potency. In the present study, an M cell-targeting L. lactis surface display system (plSAM) was built to favor the multivalent epitope vaccine antigen (FAdE) to achieve effective gastrointestinal mucosal immunity against Helicobacter pylori. Therefore, a recombinant Lactococcus lactic acid vaccine (LL-plSAM-FAdE) was successfully prepared, and its immunological properties and protective efficacy were analyzed. The results showed that LL-plSAM-FAdE can secretively express the recombinant proteins SAM-FAdE and display the SAM-FAdE on the bacterial cell surface. More importantly, LL-plSAM-FAdE effectively promoted the phagocytosis and transport of vaccine antigen by M cells in the gastrointestinal tract of mice, and simulated high levels of cellular and humoral immune responses against four key H. pylori adhesins (Urease, CagL, HpaA, and Lpp20) in the gastrointestinal tract, thus enabling effective prevention of H. pylori infection and to some extent eliminating H. pylori already present in the gastrointestinal tract.Key points• M-cell-targeting L. lactis surface display system LL- plSAM was designed• This system displays H. pylori vaccine-promoted phagocytosis and transport of M cell• A promising vaccine candidate for controlling H. pylori infection was verified

Fluorescent Labeling and Imaging of IL-22 mRNA-Loaded Lipid Nanoparticles.

Lipid nanoparticle (LNP)-based drug delivery systems (DDSs) are widely recognized for their ability to enhance efficient and precise delivery of therapeutic agents, including nucleic acids like DNA and mRNA. Despite this acknowledgment, there is a notable knowledge gap regarding the systemic biodistribution and organ accumulation of these nanoparticles. The ability to track LNPs in vivo is crucial for understanding their fate within biological systems. Fluorescent labeling of LNPs facilitates real-time tracking, quantification, and visualization of their behavior within biological systems, providing valuable insights into biodistribution, cellular uptake, and the optimization of drug delivery strategies. Our prior research established reversely engineered LNPs as an exceptional mRNA delivery platform for treating ulcerative colitis. This study presents a detailed protocol for labeling interleukin-22 (IL-22) mRNA-loaded LNPs, their oral administration to mice, and visualization of DiR-labeled LNPs biodistribution in the gastrointestinal tract using IVIS spectrum. This fluorescence-based approach will assist researchers in gaining a dynamic understanding of nanoparticle fate in other models of interest. Key features • This protocol is developed to assess the delivery of IL-22 mRNA to ulcerative colitis sites using lipid nanoparticles. • This protocol uses fluorescent DiR dye for imaging of IL-22 mRNA-loaded lipid nanoparticles in the gastrointestinal tract of mice. • This protocol employs the IVIS spectrum for imaging.

Surface functionalization affects the retention and bio-distribution of orally administered mesoporous silica nanoparticles in a colitis mouse model

Besides the many advantages of oral drug administration, challenges like premature drug degradation and limited bioavailability in the gastro-intestinal tract (GIT) remain. A prolonged residence time in the GIT is beneficial for enhancing the therapeutic outcome when treating diseases associated with an increased intestinal clearance rate, like inflammatory bowel disease (IBD). In this study, we synthesized rod-shaped mesoporous silica nanoparticles (MSNs) functionalized with polyethylene glycol (PEG) or hyaluronic acid (HA) and investigated their bio-distribution upon oral administration in vivo. The negatively charged, non-toxic particles showed different accumulation behavior over time in healthy mice and in mice with dextran sulfate sodium (DSS)-induced intestinal inflammation. PEGylated particles were shown to accumulate in the lower intestinal tract of healthy animals, whereas inflammation promoted retention of HA-functionalized particles in this area. Overall systemic absorption was low. However, some particles were detected in organs of mice with DSS-induced colitis, especially in the case of MSN-PEG. The in vivo findings were connected to surface chemistry-related differences in particle adhesion on Caco-2/Raji and mucus-producing Caco-2/Raji/HT29 cell co-culture epithelial models in vitro. While the particle adhesion behavior in vivo was mirrored in the in vitro results, this was not the case for the resorption results, suggesting that the in vitro model does not fully reflect the erosion of the inflamed epithelial tissue. Overall, our study demonstrates the possibility to modulate accumulation and retention of MSNs in the GIT of mice with and without inflammation through surface functionalization, which has important implications for the formulation of nanoparticle-based delivery systems for oral delivery applications.

Genome-wide screens reveal shared and strain-specific genes that facilitate enteric colonization by Klebsiella pneumoniae.

Klebsiella pneumoniae is a common cause of difficult-to-treat infections due to its propensity to express resistance to many antibiotics. For example, carbapenem-resistant K. pneumoniae has been named an urgent threat by the United States Centers for Disease Control and Prevention. Gastrointestinal colonization in patients with K. pneumoniae has been linked to subsequent infection, making it a key process to control in the prevention of multidrug-resistant infections. However, the bacterial factors which contribute to K. pneumoniae colonization are not well understood. Additionally, individual strains exhibit large amounts of genetic diversity, begging the question of whether some colonization factors are strain dependent. This study identifies the enteric colonization factors of three classical strains using transposon mutant screens to define a core colonization program for K. pneumoniae as well as detecting strain-to-strain differences in colonization strategies.

Dual-Targeted Nanoparticle-in-Microparticle System for Ulcerative Colitis Therapy.

Conventional oral therapy for ulcerative colitis (UC) is associated with premature release or degradation of drugs in the harsh gastrointestinal environment, resulting in reduced therapeutic effectiveness. Consequently, the present study aims to develop a dual-targeted delivery system with a nanoparticle-in-microparticle (nano-in-micro) structure. The prepared Asiatic Acid-loaded delivery system (AA/CDM-BT-ALG) has pH-sensitive properties. Cellular uptake evaluation confirms that nanoparticles exhibit targeted absorption by macrophages and Caco-2 cells through mannose (Man) receptor and biotin-mediated endocytosis, respectively. Therefore, this mechanism effectively enhances intracellular drug concentration. Additionally, the biodistribution study conducted on the gastrointestinal tract of mice indicates that the colon of the microspheres group shows higher fluorescence intensity with longer duration than the other groups. This finding indicates that the microspheres exhibit selective accumulation in areas of colon inflammation. In vivo experiments in colitis mice showed that AA/CDM-BT-ALG significantly alleviates the histopathological characteristics of the colon, reduced neutrophil, and macrophage infiltration, and decreases pro-inflammatory cytokine expression. Furthermore, the effect of AA/CDM-BT-ALG on colitis is validated to be closely related to the TLR4/MyD88/NF-κB signaling pathway. The present findings suggest that the development of a dual-targeted delivery system is accomplished effectively, with the potential to serve as a drug-controlled release system for treatingUC.

Bioluminescent monitoring of recombinant lactic acid bacteria and their products.

Lactic acid bacteria constitute a genetically diverse group of microorganisms with significant roles in the food industry, biotechnology, agriculture, and medicine. A core understanding of bacterial physiology in diverse environments is crucial to select and develop bacteria for industrial and medical applications. However, there is a lack of versatile tools to track (recombinant) protein production in lactic acid bacteria. In this study, we adapted a peptide-based bioluminescent tagging system that is functional across multiple genera and species. This system enables tracking of tagged proteins both in vitro and in situ, while it also can be used to enumerate recombinant bacteria from the mouse gastrointestinal tract with accuracy comparable to that of conventional plate counts. Our work expands the lactic acid bacteria genetic toolbox and will facilitate researchers in industry and academia with opportunities to monitor microbes and proteins under different physiologically relevant conditions.

Influence of bisphenol A and its analog bisphenol S on cocaine- and amphetamine-regulated transcript peptide-positive enteric neurons in the mouse gastrointestinal tract.

Bisphenol A (BPA) is used in large quantities for the production of plastics and is present in various everyday objects. It penetrates living organisms and shows multidirectional adverse influence on many internal organs. For this reason, BPA is often replaced in plastic production by other substances. One of them is bisphenol S (BPS), whose effects on the enteric nervous system (ENS) have not been explained. Therefore, the present study compares the influence of BPA and BPS on the number of enteric neurons immunoreactive to cocaine-and amphetamine-regulated transcript (CART) peptide located in the ENS of the stomach, jejunum and colon with the use of double immunofluorescence method. The obtained results have shown that both bisphenols studied induced an increase in the number of CART-positive enteric neurons, and the severity of changes depended on the type of enteric ganglion, the dose of bisphenols and the segment of the digestive tract. The most visible changes were noted in the myenteric ganglia in the colon. Moreover, in the colon, the changes submitted by BPS are more noticeable than those observed after BPA administration. In the stomach and jejunum, bisphenol-induced changes were less visible, and changes caused by BPS were similar or less pronounced than those noted under the impact of BPA, depending on the segment of the gastrointestinal tract and ganglion type studied. The results show that BPS affects the enteric neurons containing CART in a similar way to BPA, and the BPS impact is even stronger in the colon. Therefore, BPS is not neutral for the gastrointestinal tract and ENS.

Ret deficiency decreases neural crest progenitor proliferation and restricts fate potential during enteric nervous system development

The receptor tyrosine kinase RET plays a critical role in the fate specification of enteric neural crest-derived cells (ENCDCs) during enteric nervous system (ENS) development. RET loss of function (LoF) is associated with Hirschsprung disease (HSCR), which is marked by aganglionosis of the gastrointestinal (GI) tract. Although the major phenotypic consequences and the underlying transcriptional changes from Ret LoF in the developing ENS have been described, cell type- and state-specific effects are unknown. We performed single-cell RNA sequencing on an enriched population of ENCDCs from the developing GI tract of Ret null heterozygous and homozygous mice at embryonic day (E)12.5 and E14.5. We demonstrate four significant findings: 1) Ret-expressing ENCDCs are a heterogeneous population comprising ENS progenitors as well as glial- and neuronal-committed cells; 2) neurons committed to a predominantly inhibitory motor neuron developmental trajectory are not produced under Ret LoF, leaving behind a mostly excitatory motor neuron developmental program; 3) expression patterns of HSCR-associated and Ret gene regulatory network genes are impacted by Ret LoF; and 4) Ret deficiency leads to precocious differentiation and reduction in the number of proliferating ENS precursors. Our results support a model in which Ret contributes to multiple distinct cellular phenotypes during development of the ENS, including the specification of inhibitory neuron subtypes, cell cycle dynamics of ENS progenitors, and the developmental timing of neuronal and glial commitment.

Design of SC PEP with enhanced stability against pepsin digestion and increased activity by machine learning and structural parameters modeling

Prolyl endopeptidases from Sphingomonas capsulata (SC PEP) has attracted much attention as promising oral therapy candidate for celiac sprue, however, its low stability in the gastric environment leads to unsatisfactory clinical results. Therefore, improving its stability against pepsin digestion at low pH is crucial for clinical applications, but challenging. In this study, machine learning and physical parameter model were combined to design SC PEP mutants. After iterations, 20 mutants had higher hydrolysis activity in stomach environment, which was up to 14.1-fold compared with wild-type SC PEP. Mutant M24 involving stable and active mutations and pegylated M24 (M24-PEG) had higher activity of hydrolyzing immunogen in bread than wild-type SC PEP in vitro and in vivo, and residual immunogens in simulated gastric environment were only 1/8 and 1/10 of that in the wild-type SC PEP group. The total residual immunogens in the gastrointestinal tract of mice in the M24 and M24-PEG groups were <20 ppm, reaching the standard of non-toxic food. Our results indicate that the combination of M24 (or M24-PEG) with EP-B2 may be a promising candidate for celiac disease, and the strategies developed in this study provide a paradigm for the design of SC PEP stability mutants.

Effect of Secretion Efficiency of Mutant KRAS Neoantigen by Lactococcus lactis on the Immune Response of a Mucosal Vaccine Delivery Vehicle Targeting Colorectal Cancer.

Colorectal cancer (CRC) is often caused by mutations in the KRAS oncogene, making KRAS neoantigens a promising vaccine candidate for immunotherapy. Secreting KRAS antigens using live Generally Recognized as Safe (GRAS) vaccine delivery hosts such as Lactococcus lactis is deemed to be an effective strategy in inducing specific desired responses. Recently, through the engineering of a novel signal peptide SPK1 from Pediococcus pentosaceus, an optimized secretion system was developed in the L. lactis NZ9000 host. In this study, the potential of the L. lactis NZ9000 as a vaccine delivery host for the production of two KRAS oncopeptides (mutant 68V-DT and wild-type KRAS) through the use of the signal peptide SPK1 and its mutated derivative (SPKM19) was investigated. The expression and secretion efficiency analyses of KRAS peptides from L. lactis were performed in vitro and in vivo in BALB/c mice. Contradictory to our previous study using the reporter staphylococcal nuclease (NUC), the yield of secreted KRAS antigens mediated by the target mutant signal peptide SPKM19 was significantly lower (by ~1.3-folds) compared to the wild-type SPK1. Consistently, a superior elevation of IgA response against KRAS aided by SPK1 rather than mutant SPKM19 was observed. Despite the lower specific IgA response for SPKM19, a positive IgA immune response from mice intestinal washes was successfully triggered following immunization. Size and secondary conformation of the mature proteins are suggested to be the contributing factors for these discrepancies. This study proves the potential of L. lactis NZ9000 as a host for oral vaccine delivery due to its ability to evoke the desired mucosal immune response in the gastrointestinal tract of mice.

Bacterial community mapping of the AIA mouse gastrointestinal tract under dietary intervention with skipjack tuna (Katsuwonus pelamis) elastin peptides

BackgroundOur previous study demonstrated that a dietary intervention with skipjack tuna (Katsuwonus pelamis) elastin peptides alleviated adjuvant-induced arthritis (AIA) and affected the gut microbiota of AIA mice. However, whether the faecal microbiota serves as a surrogate to reflect the dynamics of the gut microbiota has not been determined. MethodsUsing 16 S rRNA sequencing technology, the present study investigated the microbiota in three segments (stomach, colon and caecum) of mice to comprehensively define microbial dysregulation and regulation related to rheumatoid arthritis (RA). ResultsThe results showed that the composition and distribution of the microbiota in the stomach, colon and caecum were significantly different depending on their intrinsic environment and their functions, and the colon possessed the richest and most diverse microbiota structure. The gut microbiota of AIA mice showed ecological dysbiosis, and a dietary intervention with skipjack tuna elastin peptides improved this negative effect, particularly the changes in the gastric microbiota, which were most obvious at RA onset and remission. ImplicationThe results not only indicate the spatial heterogeneity of the gut microbiota in terms of composition and distribution, but also indicate the potential of the application of elastin peptides for the alleviation of RA.

AQP1 in the Gastrointestinal Tract of Mice: Expression Pattern and Impact of AQP1 Knockout on Colonic Function.

Aquaporin 1 (AQP1) is one of thirteen known mammalian aquaporins. Its main function is the transport of water across cell membranes. Lately, a role of AQP has been attributed to other physiological and pathological functions including cell migration and peripheral pain perception. AQP1 has been found in several parts of the enteric nervous system, e.g., in the rat ileum and in the ovine duodenum. Its function in the intestine appears to be multifaceted and is still not completely understood. The aim of the study was to analyze the distribution and localization of AQP1 in the entire intestinal tract of mice. AQP1 expression was correlated with the hypoxic expression profile of the various intestinal segments, intestinal wall thickness and edema, as well as other aspects of colon function including the ability of mice to concentrate stools and their microbiome composition. AQP1 was found in a specific pattern in the serosa, the mucosa, and the enteric nervous system throughout the gastrointestinal tract. The highest amount of AQP1 in the gastrointestinal tract was found in the small intestine. AQP1 expression correlated with the expression profiles of hypoxia-dependent proteins such as HIF-1α and PGK1. Loss of AQP1 through knockout of AQP1 in these mice led to a reduced amount of bacteroidetes and firmicutes but an increased amount of the rest of the phyla, especially deferribacteres, proteobacteria, and verrucomicrobia. Although AQP-KO mice retained gastrointestinal function, distinct changes regarding the anatomy of the intestinal wall including intestinal wall thickness and edema were observed. Loss of AQP1 might interfere with the ability of the mice to concentrate their stool and it is associated with a significantly different composition of the of the bacterial stool microbiome.

Therapeutic Oral Application of Carvacrol Alleviates Acute Campylobacteriosis in Mice Harboring a Human Gut Microbiota.

Human Campylobacter jejuni infections are rising globally. Since antibiotics are usually not indicated in acute campylobacteriosis, antibiotic-independent intervention measures are desirable. The phenolic compound carvacrol constitutes a promising candidate molecule given its antimicrobial and immune-modulatory features. To test the disease-alleviating effects of oral carvacrol treatment in acute murine campylobacteriosis, IL-10-/- mice harboring a human gut microbiota were perorally infected with C. jejuni and treated with carvacrol via the drinking water. Whereas C. jejuni stably established in the gastrointestinal tract of mice from the placebo cohort, carvacrol treatment resulted in lower pathogen loads in the small intestines on day 6 post infection. When compared to placebo, carvacrol ameliorated pathogen-induced symptoms including bloody diarrhea that was accompanied by less distinct histopathological and apoptotic cell responses in the colon. Furthermore, innate and adaptive immune cell numbers were lower in the colon of carvacrol- versus placebo-treated mice. Notably, carvacrol application dampened C. jejuni-induced secretion of pro-inflammatory mediators in intestinal, extra-intestinal and systemic organs to naive levels and furthermore, resulted in distinct shifts in the fecal microbiota composition. In conclusion, our preclinical placebo-controlled intervention study provides evidence that therapeutic carvacrol application constitutes a promising option to alleviate campylobacteriosis in the infected vertebrate host.

Mesoporous Silica Particles Retain Their Structure and Function while Passing through the Gastrointestinal Tracts of Mice and Humans.

Mesoporous silica particles (MSPs) can be used as food additives, clinically for therapeutic applications, or as oral delivery vehicles. It has also been discussed to be used for a number of novel applications including treatment for diabetes and obesity. However, a major question for their possible usage has been if these particles persist structurally and retain their effect when passing through the gastrointestinal tract (GIT). A substantial breaking down of the particles could reduce function and be clinically problematic for safety issues. Hence, we investigated the biostability of MSPs of the SBA-15 kind prepared at large scales (100 and 1000 L). The MSPs were orally administered in a murine model and clinically in humans. A joint extraction and calcination method was developed to recover the MSPs from fecal mass, and the MSPs were characterized physically, structurally, morphologically, and functionally before and after GIT passage. Analyses with N2 adsorption, X-ray diffraction, electron microscopy, and as a proxy for general function, adsorption of the enzyme α-amylase, were conducted. The adsorption capacity of α-amylase on extracted MSPs was not reduced as compared to the pristine and control MSPs, and adsorption of up to 17% (w/w) was measured. It was demonstrated that the particles did not break down to any substantial degree and retained their function after passing through the GITs of the murine model and in humans. The fact the particles were not absorbed into the body was ascribed to that they were micron-sized and ingested as agglomerates and too big to pass the intestinal barrier. The results strongly suggest that orally ingested MSPs can be used for a number of clinical applications.

Immunostaining patterns reveal potential morphogenetic role of Toll-like receptors 4 and 7 in the development of mouse respiratory system, liver and pancreas.

Toll-like receptors (TLRs) are the mammalian ortholog of Drosophila melanogaster protein Toll, originally identified for its involvement in embryonic development. In mammals, TLRs are mainly known for their ability to recognize pathogen- or damage-associated molecular patterns and, consequently, to initiate the immune response. However, it is becoming clear that TLRs can play a role also in mammal embryo development. We have previously described TLR4 and TLR7 expression in developing mouse peripheral nervous system and gastrointestinal tract. In the present study, we extended the investigation of TLR4 and TLR7 to the respiratory system and to the two main accessory organs of the digestive system, the liver and pancreas. TLR4 and TLR7 immunostaining was performed on mouse conceptuses collected at different stages, from E12 to E18. TLR4 and TLR7 immunoreactivity was evident in the embryo pancreas and liver at E12, while, in the respiratory apparatus, appeared at E14 and E17, respectively. Although further studies are required to elucidate the specific role of these TLRs in embryo development, the differential spatiotemporal TLR4 and TLR7 appearance may suggest that TLR expression in developing embryos is highly regulated for a possible their direct involvement in the formation of the organs and in the acquisition of immune-related features in preparation for the birth.

The Gut Microbiome Influences the Gene Expression Profile of Neuroendocrine and Enteroendocrine Systems

Objective/Hypothesis: The gut microbiome influences metabolism, but the underlying mechanisms are not completely understood. We hypothesized that the gut microbiome modulates metabolism by regulating the expression of key neuroendocrine and enteroendocrine genes such as neuropeptide Y (Npy) which is a potent brain orexigenic peptide and G-protein-coupled receptor-17 (Gpr17) which decreases incretin secretion in the gut.&#x0D; Approach: The gene expression profiles in mouse hypothalamus and gastrointestinal tract segments (duodenum, jejunum, ileum, and colon) were compared from germ-free (GF) mice (n=9 females, n=9 males) and conventional mice (n=9 females, n=9 males). Gene transcription was determined through total RNA yield extracted from the various tissues using qRT-qPCR. The data was analyzed using two-way ANOVA and unpaired t-test.&#x0D; Results: We found that RNA yield in brain and gut tissues differed in a sex and microbiome dependent manner. The medial basal hypothalamus of the female germ-free mice had lower RNA yield compared with conventional female mice (p=0.0082). In the intestine, the RNA concentrations of the germ-free mice showed a decrease from the proximal to distal gut, however, an opposite trend was observed in the conventional mice with an overall increase of concentration from the proximal to distal gut. This trend may reflect the high concentration of the microbiota in the distal gut. Regarding gene expression of the neuroendocrine and enteroendocrine systems, Npy had lower expression in the medial basal hypothalamus (MBH) of conventional males (p=0.004), Gpr17 showed moderate duodenal expression in conventional mice (p=0.029), and Gpr17 showed increased duodenal expression in germ-free females (p=0.044).&#x0D; Summary/Conclusions: With enriched microbiota in the ileum and colon, our results indicate that gut microbiota increase host gene transcription in the distal gut segments. Future studies will aim at investigating the mechanistic link between the microbiome and regulation of gene expression in neuroendocrine and enteroendocrine tissues.

Spatial distribution of gut microbiota in mice during the occurrence and remission of hyperuricemia.

Previous studies have shown that anserine can alleviate hyperuricemia by changing the fecal microbiota of hyperuricemic mice. However, the fecal microbiota could not fully represent the distribution of the whole gut microbiota. Knowing the spatial distribution of the gastrointestinal tract microbiota is therefore important for understanding its action in the occurrence and remission of hyperuricemia. This study provides a comprehensive map of the most common bacterial communities that colonize different parts of the mouse gastrointestinal tract (stomach, duodenum, ileum, cecum, and colon) using a modern methodological approach. The stomach, colon, and cecum showed the greatest richness and diversity in bacterial species. Three clusters of bacterial populations were observed along the digestive system: (1) in the stomach, (2) in the duodenum and ileum, and (3) in the colon and cecum. A high purine solution changed the composition and abundance of the digestive tract microbiota, and anserine relieved hyperuricemia by restoring the homeostasis of the digestive tract microbiota, especially improving the abundance of probiotics in the digestive tract. This could be the starting point for further research on the regulation of hyperuricemia by gut microbiota with the ultimate goal of promoting health and welfare. © 2022 Society of Chemical Industry.