The relationship of epigenetic processes and the intestinal microbiota may serve as an essential role in elevating the bisulfite sequencing potentials in discovering host-microbiota interactions in germfree (GF) and conventional mice. The previous studies have established that the microbiota regulates a large proportion of the intestinal epithelial transcriptome in the adult host. However, microbial effects on DNA methylation and gene expression during early postnatal development are still poorly understood. In recent years, the number of studies investigating the impact of the gut microbiome in colorectal cancer (CRC) has risen sharply. As a result, we now know that various microbes (and microbial communities) are found more frequently in the stool and mucosa of individuals with CRC than healthy controls, including in the primary tumors themselves and even in distant metastases. We took albino mice and reared them under laboratory conditions. After 16 weeks of rearing, mice were slaughtered, and DNA extraction was performed later on. Bisulfite sequencing was done under controlled environmental conditions to unveil the role of sequencing in determining the host-microbiota interactions. The study results showed a strong host-microbiota interaction in GF mice as it significantly affects lipid metabolism, inflammation, carcinogenic, and postnatal development.
A germfree murine (GFM) animal is defined as a rat or mouse that has been maintained free from demonstrable microbial associates such as bacteria, viruses, fungi, and parasites throughout its life. Its sterile characteristics make it a unique animal model for exploring the relationship between the intestinal microbiota and the host. Using this model system, we can demonstrate the physiological and pathological effects of the intestinal microbiota on the host. Moreover, it is possible to investigate the roles of certain important microbes by manipulating GFM. The term gnotobiology is used to refer to studies of this kind. It is detected that the human microbiota possesses 1014 bacterial cells, a number 10 times greater than the total number of human cells (Bäckhed, 2009).
The first germfree (GF) animals were developed as far back as the 1800s by aseptic cesarean section, and the methodology used in the generation of GF mice remains unchanged mainly today. Evidence of grossly abnormal enteric plexus architecture and size in GF rats has been reported. Moreover, GF rats have been used to demonstrate the impact of the microbiota on migrating myoelectric complex activity. However, no change in ENS neurochemistry was observed following the colonization of these animals. More recently, the early postnatal developmental trajectory, neurochemical profile, and function of the ENS have been investigated in GF mice. In the context of my enteric nerve fiber density, a GF environment significantly decreased the development of the enteric neural network in a region-specific manner on postnatal day 3 relative to both offspring born in a specific pathogen-free (SPF) environment or to dams colonized with a simplified microbiota (Collins
While our knowledge of the links between physiological functions and microbiota are growing constantly, notably thanks to the comparison between GF animals and conventionally raised conspecifics, recent studies indicate that the nature of the microbiota plays a role in the observed effects on animal physiology. One of the most striking examples comes from the studies of metabolic disorders. For example, transplantation into mice of fecal microbiota from human twins discordant for obesity induces different metabolic phenotypes (Ridaura
Microbial communities are related to changes in gut morphology, physiology, and biochemistry. Microbes ferment polysaccharides and proteins, produce vitamins, and metabolize bile acids, affecting enterohepatic circulation and nutrient absorption. However, the implication of gut microbiota on host energy homeostasis remains elusive (Bäckhed
In humans, the population of microorganisms is 10 times higher than host cells, which code a hundred times more genes than that of the human genes with 500–1000 different types and account for 30–50% of the total weight of feces. The gut microbes synthesize many essential vitamins for the development of the host immune system and hypothalamic-pituitary-adrenal (HPA) system and SE protein residues to formulate the non-essential amino acids and promote the absorption of mineral elements (Sudo
In 1991, Dr. Margulis and Fester introduced the concept of the “holobiont.” This term is defined as the association of the host and its entire microbial community, including transient and stable members. The human microbiome is the assemblage of all microbiota that reside in tissues and biofluids, including the saliva, oral mucosa, skin, mammary glands, uterus, placenta, seminal fluid, ovarian follicles, airways, and gastrointestinal tract. It was often proposed that in the human body, bacteria outnumber human cells by a ratio of at least 10:1. However, in 2016, the ratio was revised and now it is estimated to be closer to 1:1. The vast majority of commensal bacteria reside in the gastrointestinal tract followed by the skin. The cecum and the colon are the dominant contributors to the total bacterial population within the gastrointestinal tract. As a result, the microbiota of the gastrointestinal tract and its interplay with systemic health are the most extensively documented.
So far, various animals have been employed as essential models to evaluate microbial functions or the effects of drugs or toxic materials on host health. Among those animal models, zebrafish and conventional mice own numerous advantages, such as having a small body, short lifespan, and high similarity to the human genome (Feitsma and Cuppen, 2008). Thus, those wild and transgenic zebrafish models have been applied to study human diseases and toxicology of environmental contaminants such as inorganic (e.g., heavy metals) and organic pollutants (e.g., endocrine disruptors), by evaluating the biochemical markers or endpoints related to acute toxicity, behavior, cell death, and transcriptional gene expression of signaling (Yang
In recent years, GF or gnotobiotic animal models including mice, zebrafish, and swine are being employed as essential tools for studying the functional interactions of gut microbes in host health. Gut microbes assist in developing the mucosal barrier involved in food digestion and stimulate the immune system, which disruption can induce obesity, diabetes, cancer, and other human diseases (Wang and Donovan, 2015).
The
Cancer is a disease initiated and progresses (through processes including tissue invasion and metastasis) through changes in the genome and epigenome. Hence, to establish a direct, causal connection between the gut microbiome and colorectal cancer (CRC) development, we must determine whether and how microbes alter mutation rates, gene methylation, chromatin structure, and/or non-coding RNA expression in CECs. Several epidemiological studies have associated specific bacteria in the gut with tumors characterized by DNA hypermethylation or specific mutational patterns (Burns
Bisulfite genomic sequencing is regarded as a gold standard technology for the detection of DNA methylation because it provides a qualitative, quantitative, and efficient approach to identify 5-methylcytosine at single base-pair resolution. This method was first introduced by Frommer
There are several advantages attributed to bisulfite sequencing in GF and conventional mice to reveal host-microbiota interactions. These include bisulfite-based DNA methylation analysis which has more quantitative accuracy, detection sensitivity, high efficiency, and a broad spectrum for sample analysis. As a fundamental method of DNA methylation analysis, bisulfite genomic sequencing has been widely used in various research and clinical settings. To optimize the final results of the bisulfite genomic sequencing protocol, numerous modifications have been explored and have significantly improved the sensitivity and accuracy in this procedure (Hajkova
Gnotobiotic mice (from the Greek gnotos for known and bios for life) refer to mice in which every microorganism present is defined. GF mice are one class of gnotobiotic animals, but mice associated with defined bacterial communities (e.g., altered Schaedler flora) are also considered gnotobiotic. Gnotobiotic animals are kept in isolators for long-term maintenance of their microbiological status, although various caging systems can allow for alternate husbandry solutions.
Conventional is an often used but poorly defined term in microbiome research. A conventional mouse is colonized with a diverse and largely undefined microbiome containing ~1011 bacteria within the gut alone. The exact microbial composition of a “conventional” mouse can vary widely, and there is no standardized “conventional microbiome.” The microbiome of a wild mouse differs widely from that of laboratory mice, which themselves can have very different microbiomes depending on how and where they are raised. A conventionalized mouse generally refers to a GF mouse that is colonized with the fecal or intestinal microbiome of a non-germ-free mouse.
Modern laboratory mice are often referred to as SPF, meaning that a defined list of mouse pathogens is excluded from these animal colonies. These specified pathogens vary between institutions, making SPF itself a vague term. Vendors often raise mice at defined health standards that provide various levels of exclusion for pathogens, opportunistic bacteria, and commensals. Apart from these excluded microorganisms, the exact microbiome of an SPF mouse is not generally known or controlled and is determined by the husbandry methods established for each health standard.
Male Albino mice (170–200 g) were purchased from Experimental Animal Market (Hong Kong Inc.) and were reared under controlled conditions (23 ± 2oC and 12 h/12 h light/dark cycle). The mice were fed with standard food and water
Mice undergo a slightly different process due to lacking an egg life stage. To create a GF mouse, an embryo was created through
A sampling of intestines was performed on ice. For ileum samples, the distal 8 cm the small intestine was used, colon was sampled in whole. Both ileum and colon were rinsed with 0.9 % NaCl solution to remove intestinal contents and subsequently snap frozen in liquid nitrogen.
To prepare the library for bisulfite sequencing, we fragmented the genomic DNA of all the tissue samples to an average size of 100–300 bp through sonication (Covaris, Massachusetts, USA). The genomic fragments were end-repaired and TrueSeq methylated adaptors were ligated to their ends. Adaptor ligated genomic fragments were treated with sodium bisulfite as described in the previous study. Library preparation and sequencing were performed to generate 90-nt long reads in paired-end mode with sufficient sequencing depth (>30×) through HiSeq-2000 platform (Illumina, San Diego, USA).
The adaptor sequences and low-quality reads were removed from the raw reads using NGSQC Toolkit (v2.3) at default parameters. Duplicated reads were filtered out by mapping on the rice genome (MSU v7.0) using Bismark (v0.8) under default parameters. The efficiency of bisulfite conversion was estimated by mapping high-quality filtered reads on rice chloroplast genome. More than 99% of the cytosines in the chloroplast genome were converted to thymine(s), indicating a very high efficiency of bisulfite conversion in our experiments. The mCs in mice genome were identified based on ≤0.001
Total RNA was extracted from total tissue using the RNeasy Mini Kit (Qiagen). The purity and integrity of the RNA were assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies). For real-time PCR, 2 mg RNA was transcribed into cDNA using the reverse transcription system from Promega. Real-time quantitative RT-PCR analysis was performed using the Light Cycler LC 480 (Roche). Relative quantification was carried out using the LightCycler 480 SW 1.5.1 (Roche). GAPDH was used as a reference gene, relative mRNA expression values were calculated using delta-delta threshold cycle (Ct) analysis (Langmann
Stock solutions of phenylephrine, acetylcholine, and sodium nitroprusside were prepared freshly in sterile and distilled water.
The statistical procedures used included Student’s unpaired t-tests and one- or two-way analysis of variance (ANOVA). All analyses were performed using data analysis software GraphPad Prism 5.0 (USA, CA). The number (
Here, we observed for the 1st time that resistance arteries [
Rearing cage for mice under laboratory conditions
The stress-strain relationship is non-linear; therefore [
Germfree and conventional mice models
However, there was a distinct change in the type of remodeling between the sexes [
Bisulfite sequencing and vascular microbial properties in Conv and GF mice. Conv: Conventional, GF: Germfree
Total heart (A) left (LV, B) and right (RV, C) ventricles [
Emax to phenylephrine in resistance arteries of Conv and GF mice.
Postnatal development and the microbiota affect the DNA methylation profile. (a) Multidimensional scaling analysis plot displaying the overall methylation profiles. (b) Venn plots showing the number of differentially methylated sites between CONV-R and GF at the three developmental stages. Note the high number of differentially methylated sites at W1. (c) Number of hypo- and hypomethylated sites among all DMPs (CONV-R vs. GF) for each developmental stage. (d) Expression of Dnmt3a and Tet3 genes, which function in
Lipids were quantified by electrospray ionization tandem mass spectrometry in positive ion mode as described previously. A precursor ion scan of m/z 184 specific for phosphocholine-containing lipids was used for PC (Liebisch
PE-based plasmalogens were analyzed according to the principles described by Zemski-Berry. Free cholesterol (FC) and cholesteryl ester (CE) were quantified using a fragment ion of m/z 369 after selective derivatization of FC using acetyl chloride (Liebisch
Over the past decade, our understanding of gut microbiota and its products in various diseases such as inflammatory bowel disease and metabolic disease has increased, and more recent reports have revealed the gut microbiota contributes to cardiovascular and renal diseases. In the contemporary era, given the rise of antibiotic-resistant microorganisms, increased number of immune compromised patients, and lack of new antimicrobial medications, researching pathogenic microbes remain highly relevant.
Furthermore, recent research strongly suggests that commensal [
Bisulfite sequencing efficiency in terms of host-microbiota interactions
Commensal gut microbiota perform a variety of functions that are important to the host. Perhaps, one of the most apparent functions of gut microbiota is to help the host digest plant-derived complex carbohydrates and generates energy in short-chain fatty acids. Gut microbiota are also essential for the source of biotin, Vitamins K and B12, and essential amino acids. All these elements are essential for the well-being of the host. Therefore, based on these premises, we questioned whether the lack of microbiota would disrupt the host vascular homeostasis, specifically if it would induce changes in vascular function in young mice relative to Conv mice, and if these changes, if any, would be in a sex-specific manner.
The study of germfree and conventional mice thus insights to the host microbiota interactions.
Appreciation is growing for the role of non-dietary, environmental factors in obesity, including early-life events that impact intestinal microbes and regulate the host epigenome. However, specific microbiota-regulated targets that influence the obese phenotype are currently unknown. This can this be studied by comparing bisulphite sequencing of germfree and conventional mice. Inorder to elucidate the interactions among the microbiota, immune system, and epigenome in the context of obesity to facilitate future development of dietary strategies that modulate gut bacteria to prevent disease.
National Institutes of Health, United States.