Background
Ulcerative colitis (UC) is a chronic, spontaneous inflammatory bowel disease affecting the innermost mucosa lining of the colon and rectum [
1]. Extensive studies over the past decade have demonstrated that environmental factors, especially bacterial microflora, genetic and immunological factors play a substantial role in the pathogenesis of UC [
2].
The dysbiosis of gut microbiota plays crucial roles in the pathogenesis of ulcerative colitis [
3‐
5]. In mice with ulcerative colitis induced by DSS, the population of intestinal microflora,
Bacteroides distasonis, Clostridium spp., and
Clostridium difficile, increased significantly [
6]. The relative shifts in abundant 16S rRNA (gene) phylotypes of
Akkermansia muciniphila and
Enterobacteriaceae are associated with the disease activity index of UC in DSS-induced UC mouse model.
Bifidobacterium and the
Lactobacillus group were increased in active IBD patients, while the population of butyric acid-producing bacteria decreased to some extent [
7].
The intestinal epithelium and the tight junctions form a barrier that prevents permeation of pathogens from the luminal environment into the mucosal tissues and circulatory system. The integrity and normal regeneration capability of the mucosal surface epithelium are the structural basis of the intestinal mucosal barrier. The intestinal mucosal epithelium consists of absorptive enterocytes, goblet cells, and Paneth cells. Goblet cells secrete highly glycosylated gel-forming mucins that form a hydrophilic mucus on the epithelial layer. Due to the structural features and negative surface charge, mucin is able to trap and wrap up bacteria. The chemical groups exposed on mucin surface share similar structures with the intestinal epithelium, which facilitates the recognition and adhesion of bacteria. In addition, mucin binds to adhesion molecule on the intestinal epithelium, which can competitively inhibit the binding of harmful bacteria to adhesion molecules and subsequently inhibit the colonization of bacteria. The density of mucus is higher in the inner layer and decreases gradually toward the intestinal lumen. The outer mucus layer is loose and becomes habitat for commensal bacteria. If colonic epithelium is not covered by mucin, colitis will occur as a result of host immune response to invading bacteria [
8]. Current studies of gut microbiota in UC use luminal content samples frequently. However, the luminal content flora can only partially reflect the alteration of gut microbiota. Research into the alternation of mucus layer flora under pathological conditions has been sparse. In this study, we established a DSS-induced mice UC model. Next, we conducted high throughput sequencing of the outer mucus layer flora, the inner mucus layer flora, and the luminal content flora. We then analyzed the differential counts of flora under pathological conditions in an attempt to understand the role of microbiota in the pathogenesis of UC.
Methods
Experimental animals and establishment of UC model
Thirty 9-week old male SPF C57BALB/c mice weighing 22 ± 2 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China; License #: SCXK (JING) 2012–0001). They were housed 3–5 mice per cage in an SPF animal facility of Institute of Science and Technology, National Health and Family Planning Commission of the People’s Republic of China (SYKX (JIGN) 2016–0010). The animals were randomly divided into a normal control group (
n = 10) or UC model group (
n = 20). A UC mouse model was established using the method reported by Benoit Chassaing [
9]. Control mice received sterile drinking water throughout the study. UC model group mice were exposed to three cycles of DSS treatment. During the first cycle, animals were orally treated with 2.5% DSS (molecular weight 36–50kD; MP Biomedicals, US) for 7 days followed by 7 days of normal drinking water. During the second cycle, animals were orally treated with 2.0% DSS for 7 days followed by 7 days of normal drinking water. During the third cycle, animals were orally treated with 2.5% DSS for 2 days, then with 2.0% DSS for 5 days, followed by 2 days of normal drinking water. After the UC model was established, the animals were sacrificed by cervical dislocation and colons were excised and prepared for histopathological examination. The experimental protocols were performed after approval and in accordance with the guidelines set by the Ethical Committee of Minzu University of China (Protocol number: 201702).
Isolation of intestinal content, an external mucus layer, and internal mucus layer
Intestinal segments were opened longitudinally. The contents were gently picked away using forceps until no visible particles remained. The intestinal content was placed in a 2 ml sterile cryopreservation tube and kept in liquid nitrogen. The outer mucus layer was gently scraped off, placed in 2 ml sterile cryopreservation tube and kept in liquid nitrogen. The rest of the colon tissue (inner mucus layer) was rinsed with PBS, minced, placed in a 2-ml sterile cryopreservation tube, and kept in liquid nitrogen.
Genomic DNA of luminal content-, inner and outer mucosa-associated flora were extracted from using a fecal DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd.; product number: DP304).
High-throughput sequencing of 16S rRNA amplicons
The purity and concentration of extracted genomic DNA were as determined using agarose gel electrophoresis. Then the genome DNA sample was diluted to 1 mg/μl with sterile water for PCR amplification. The primers were 341F: CCTAYGGGRBGCASCAG; and 806R: GGACTACNNGGGTATCTAAT. PCR reaction (30 μL) consisted of Phusion Master Mix (2×), 15 μl; each of the primer (2 μM), 3 μl; gDNA (1 ng/μL), 10 μL (5–10 ng); H2O, 2 μL. The thermal cycles included predenaturation at 98 °C for 1 min, and 30 cycles of 98 °C for 10s, 50 °C for 30s, 72 °C for 30s; and followed by a final extension at 72 °C for 5 min. PCR product was analyzed using 2% agarose gel electrophoresis. According to concentrations, the PCR products were mixed in the same concentration, then mixed with 1 × TAE buffer and purified using 2% agarose gel. PCR products with the size of 400–450 bp were recovered from the gel and further purified using GeneJet Gel Extraction Kit (Thermo Fisher Scientific). DNA libraries were generated using the NEB Next® Ultra™ DNA Library Prep Kit for Illumina (New England Biolabs, US) according to the manufacturer’s instructions. After Qubit quantification and quality control, these libraries were sequenced on the Illumina HiSeq system. We yielded 6,130,487 raw reads. After adaptor clipping, decloning, and rejecting low quality sequencing, 4,907,035 clean reads were obtained. Each read had Q20 > 98% and Q30 ≥ 97%.
After trimming and quality filtering of the raw data, we obtained a clean dataset for construction of molecular operational taxonomic units (OTUs). Representative OTU was annotated to species, then the corresponding species information and species abundance information was delineated. Then the relative OTUs abundance, alpha diversity metrics, and Venn diagram were analyzed to obtain the information about bacterial species richness, proportional abundances of species (evenness), and OTUs differed or shared between groups. By constructing a phylogenetic tree using multiple sequence alignment of OTUs, we assessed microbial community structure differences between different samples and groups for analysis using ordination approaches, such as PCoA, PCA, or NMDS.
The microbiota composition and diversity were analyzed using MetaStat, LefSe, multivariate analysis of variance, and spatial statistics. Generalized linear mixed modeling was performed using the onset of UC as the dependent variable and repeat measurements of OTU abundances of luminal contents flora, inner mucus layer flora, and outer mucus layer flora as independent variables. The results showed that the distribution of 3862 OTUs in each layer was not repeated measurement data. Therefore, multiple logistic regression was used to analyze the relationship between UC onset and OTU abundance in each layer.
Discussion
Among various chemically induced colitis models, DSS-induced colitis model is widely used because of its simplicity and many similarities with human ulcerative colitis. DSS carries a highly negative charge contributed by sulfate groups, is toxic to the colonic epithelia, and induces erosions that ultimately compromise barrier integrity resulting in increased colonic epithelial permeability [
6,
9]. Further, its anticoagulant property aggravates intestinal bleeding. For unknown reasons, DSS-induced extensive pathology is confined to the large intestine, specifically the distal colon where an enormous number of microorganisms live. The mechanism by which DSS passes through mucosal epithelial cells remains unclear, but a recent publication suggests that DSS induces colitis in mice by forming nano-lipocomplexes with medium-chain-length fatty acids in the colon [
10].
In the large intestine (colon) the mucus is continuous and has two layers. Tight stacking of polymeric glycoproteins adheres firmly to the epithelium forms a compact inner layer that is largely sterile [
11]. Following proteolytic dispersion of mucin polymers, the outer layer is looser and contains commensal microbiota [
12]. Only some bacterial species have a sufficient repertoire of genome-encoded catabolic glycosidic enzymes to break down complex mucus glycans, while most bacteria are asaccharolytic and use mucus glycoprotein as a source of carbon [
13]. For these mucolytic bacteria, mucus is a potentially distinct microbiological niche based on the available carbon source [
14]. The inner mucus layer is dense and impenetrable to bacteria, which allows it to protect the colonic epithelium. However, many microbial metabolites can enter the host, including short chain fatty acids that feed host epithelial cells and regulate immunity [
15,
16]. This suggests that the bacteria residing in the colonic mucus microbiota niche contribute to the protective effects of the mucus barrier [
17]. Under normal conditions, intestinal flora does not trigger inflammation at colonic mucosa. When the host is under stress or under some pathological conditions, the balance is perturbed, and harmful bacteria in the intestine or opportunistic pathogens may colonize another niche, invade the intestinal epithelium, and trigger inflammation. The tight junction proteins joining adjacent enterocytes are crucial for the maintenance of epithelial barrier integrity. During inflammatory processes, the tight junction proteins can be disrupted, allowing free passage of luminal contents into the lamina propria [
18,
19].
The colonic mucus layers consist of polymeric sheets of Muc2 mucin and other bioactive molecules synthesized and secreted by goblet cells. Colonic commensal bacteria do not trigger excessive host immune responses. Colonic inner mucus layer separate intestinal epithelial cells from most of the bacteria, but the inner mucus layer is not sterile. The inner mucus layer comes into contact with a small number of bacteria. However, long-term exposure to bacteria can trigger a severe immune response; this immune response can affect the secretion of MUC2 mucin and the characteristics and function of the inner mucus layer [
20]. In the case of ulcerative colitis, the thickness of the colonic mucus layer will decrease. In this way, bacteria can penetrate the inner mucus layer, reach the epithelium, and trigger severe colonic immune response. The dysbiosis of gut flora, together with an impaired intestinal clearance of bacteria, enhances the invasiveness of pathogens, disrupts the intestinal immune response, accelerates the intestinal inflammatory response and eventually leads to ulcerative colitis [
21,
22].
The imbalance between beneficial and potentially harmful gut bacteria induces UC. However, studies of the locations of such dysbiosis have been lacking. In this study, we found that, in the control group, the luminal flora differed greatly from the inner mucus flora. In the UC model group, the difference between the inner mucus layer flora and the outer mucus layer flora and the difference between luminal flora and inner mucus flora were significant. These results indicated that the microbiota community structures of the inner and outer mucus layers were different in UC model mice. The different microbiota composition in inner and outer mucus layers may contribute greatly to the onset of UC. The luminal and outer layer flora were always in a dynamic state of flux. The dysbiosis of the inner and outer mucus layers is closely associated with the onset of UC. We also found that the flora of the outer mucus layers of control animals differed greatly from those of UC animals. However, the inner mucus layer flora and luminal flora did not differ between normal and UC animals. In addition, more bacteria in the outer mucus layer showed differences in abundance and structure relative to the inner mucus layer. Similar results were obtained from logistic regression analysis of the relationship between UC and OTU of different layers. These results indicated that dysbiosis occurred mainly in the outer mucus layer in UC animals and the mucus microbes are very active and competitive between different species. This may be due to the physiological functions of the outer mucus layer. The outer mucus layer is a very challenging habitat as it is undergoing rapid turnover with a time frame of several hours. As a result, microbes need to be fit enough to be renewed at the same rate while competing with each other for resources to persist in this stressful niche [
14]. When the symbiotic niche is perturbed, some microbes will rapidly colonize and become dominant. The colonized microbes will reside in the outer mucus layer, produce protease, and degrade the MUC2 polymer [
23], which leads to the invasion and direct contact of microbes with colonic epithelium. According to Venn diagram and MetaStat analysis, we found that the microbiota composition changed in UC mice and the flora change in the luminal sample could not reflect the flora change in the entire intestinal ecosystem. The flora of the mucus layers needs to be investigated as well. The luminal content sample has been used for intestinal microbiota analysis because it is less likely to be contaminated by alien species and is easy to collect. However, research into the correlation between dysbiosis and diseases shows that mucus flora usually displays significant alterations in species richness while luminal content flora does not, which means that much less information is derived from luminal content flora than from mucus flora [
24]. Our results are consistent with this report, and the flora of the outer layer of mucus changed more significantly than either inner layer flora or luminal flora. In this way, the dynamic changes of mucus flora are of great importance regarding the characteristic microbiota changes in IBD.
About 99% of the normal human gut microbiota comprises of four phyla, namely Firmicutes (including the majority of Clostridium XIX and IV groups), Bacteroidetes (account for 90% of gut microbiota), Proteobacteria and Actinobacteria [
25]. However, the analysis of mucosally associated bacteria showed enrichment of Streptococcal and Lactobacillus spp. (Bacillus subgroup of Firmicutes) [
26]. Investigations have shown a relative decrease in the bacterial phyla Firmicutes and Bacteroidete, while an increase in Proteobacteria and Actinobacteria were observed with mucosal inflammation [
27]. The research into the abundance of these bacteria in each mucus layers has been lacking. In this study, we found new phyla Proteobacteria, Firmicutes, Fusobacteria, and Bacteroidetes invaded into the outer mucus layer of UC model mice. We also observed invasion of phyla Proteobacteria, Gemmatimonadetes, Chloroflexi, Firmicutes, Verrucomicrobia, Bacteroidetes, Nitrospirae, Thaumarchaeota, Planctomycetes, and Actinobacteria into the inner mucus layer in UC mice. The invasion of these microbes may explain the alteration in microbiota composition here observed in UC mice. Bacteria of the phyla Actinobacteria and Proteobacteria are the most abundant in human and mouse colonic mucosa [
13,
28]. Bacteroidaceae and Prevotellaceae families of phylum Bacteroidetes are abundant in mouse colonic mucosa. Our study showed similar results. Bacteroides species can break down a broad array of dietary polysaccharides and thus occupy a metabolic niche in which a variety of polysaccharides exist as sources of carbon [
29]. When large numbers of glycanphiles, such as Bacteroides species, invade the inner mucus layer [
30], they degrade glycan and spread with mucus. Because of gradual degradation, the inner mucus layer becomes thinner or even develops exposed patches. Then more bacteria or other microbes penetrate the inner mucus layer and adhere to the intestinal epithelial cells. When the bacterial metabolites are recognized by host intestinal epithelial cells and the immune system, a cascade of host immune responses will be triggered [
26].