Recent advances in omics technology have shown that dysbiosis of gut microbiota contributes to chronic gut inflammation and can be closely associated with onset, disease severity, and poor response to conventional treatment of IBD, resulting in UC(Ananthakrishnan 2020; Gisbert and Chaparro 2020). However, the relationship between mucosal microbiota and UC still remains unclear. In the present study, we explored the pattern of gut microbiota composition using 16s RNA sequencing and host gene expression using RNA sequencing in the colonic mucosa of patients with UC and NC. Using a computing method, we identified the relationship between microbes and host genes in the inflamed mucosa of patients with UC to understand the role of dysbiosis in the pathogenesis of UC.
Our results revealed that microbial diversity was not significantly different between UC and normal controls. This result is not in line with those of previous studies. In previous stool-based metagenomic studies, microbial species richness of UC patients was reduced compared with that of normal groups with correlation of the degree of severity and disease extent(Lopez-Siles, et al. 2017; Shin, et al. 2023). Metagenomic analysis of stool samples is a noninvasive method without patient damage during sample collection, and the sample can supply a large amount of microbiota. However, in the case of IBD, the quality of stool can be poor for analysis, and microbial diversity can affect the consistency of stools; the stool of Bristol scale 3–4 has higher diversity than that of 5–6 (Nishihara, et al. 2021; Vandeputte, et al. 2016). Furthermore, environmental factors, including dietary patterns and drug history, can affect metagenomic results.(Ahn and Hayes 2021) On the other hand, although the analysis of microbe from colonic mucosa has limitations in the context of relative low diversity compared to stool analysis, it can reveal the pattern of microbes that have a direct effect on the mucosa with chronic inflammation with minimal bias during sample acquisition in each sample.
The purpose of our study was to identify microbes in the colonic mucosa associated with UC development and to identify the differences in genetic changes in the colonic mucosa according to the expression of microbes to characterize microbes that distinguish UC patients and identify related genes. The microbiota adherent to the inflamed mucosa can be considered to have a significant effect on the maintenance of intestinal integrity, leading to chronic and uncontrolled gut inflammation.(Zheng, et al. 2020). In recent reports, a decrease in microbial diversity was found in patients with relapsed or refractory UC in the late stages of the disease(Basha, et al. 2022; Schirmer, et al. 2018). We characterized the bacterial composition of the mucosal microbiota of patients with UC at different taxonomic levels. At the phylum level, both Bacteroidetes and Firmicutes were significantly abundant in UC patients compared with their level in NC, whereas the abundance of Proteobacteria was high in NC. Previous studies have shown that Proteobacteria are abundant in IBD patients, especially in patients with severe and aggressive CD. This discrepancy may be due to the differences in our study designs, such as in the analysis of samples and characteristics of the enrolled patients. In this study, we enrolled naïve patients with severe UC and a short duration of illness. Therefore, we hypothesized that change in mucosal microbial diversity does not occur during this short duration of UC. Further research with a larger sample size is required.
We explored candidate species and genera that could discriminate between UC patients and NC. We found that the genera Lactobacillus, Neisseria, and Faecalicatena were abundant in UC patients. At the species level, Lactobacillus ruminis, Neisseria subflava and Faecalicaten aortica were abundant in UC, whereas three species (Alistipes putredinis, Bacteroides coporocola and Bacteroides plebeius) were abundant in NC. Lactobacillus has been shown to be able to improve mucosal integrity by reducing gut inflammation, increasing mucus production, and improving the barrier function of the gut lining.(Dempsey and Corr 2022; Martin, et al. 2019). In addition, Lactobacillus can have anti-inflammatory and immune-modulating effects. There have been a number of studies that have investigated the effects of Lactobacillus-based probiotics on a healthy gut. (Hemarajata and Versalovic 2013; Kristensen, et al. 2016). Our research, which showed an increase in Lactobacillus in UC patients, may be contrary to previous reports. However, a previous study has shown a higher abundance of Lactobacillus groups in both fecal and mucosal samples in patients with active IBD. Higher Lactobacillus abundance is closely related to the clinical course, including non-remission to both immunomodulators and anti-TNF-α. (Wang, et al. 2014) Therefore, it is still unclear whether the increase in Lactobacillus is a compensatory response to the decrease in other gut bacteria, a true pathogen, or simply a bystander phenomenon. On integrating the gut mucosal microbiome and host gene expression profiles, we observed several correlations between the mucosal microbiome and the differentially expressed host genes in UC. We found that Lactobacillus was highly abundant in UC patients and strongly correlated with REG4, a highly expressed gene associated with epithelial inflammation and causing chronic inflammation in patients with IBD(Cao, et al. 2016). Furthermore, we found a strong negative correlation between Lactobacillus and PTGDR. However, the effect of prostaglandin D on chronic intestinal inflammation remains unclear. The metabolite of PDG2, 15d PGJ2, plays an anti-inflammatory role through PPARG in the colonic mucosa by downregulating the infiltration of inflammatory cells, particularly infiltration into the mucosa in acute colitis.(Li, et al. 2019) Furthermore, PDG2 activation was observed in the colonic mucosa of patients with IBD (Le Loupp, et al. 2015). Taken together, our results showed that a higher abundance of Lactobacillus may be associated with the regulation of host genes causing chronic inflammation in the colic epithelium in UC. This result suggests that Lactobacillus administration as a probiotic should be performed more carefully in patients with active or early phases of UC. Further research is needed to clarify the mechanism of action of Lactobacillus in the chronic intestinal epithelium. Non-gonorrhoeic Neisseria is an oral-resident microbe that is less abundant in patients with UC. Our results showing a high abundance of Neisseria in UC patients were contrary to the results of previous studies. (Hirano, et al. 2018). We hypothesized that this opposite result may be due to the characteristics of the enrolled UC patients, shorter duration of disease, and active disease state. However, further research with a larger sample size is needed to validate our results.
Although, 16s rRNA sequencing has a limitation with regard to the accuracy of taxonomic resolution to species, we characterized the species that were depleted in UC patients and the species-host gene associations. Bacteroides coprocola was positively correlated to NBR1 and CAMK2D and inversely proportional to HLA-G and CFAP44, whereas Parabacteroides merdae was proportional to TBX101 and inversely proportional to RRAGA. Previous studies have reported that Bacteroides play a role in ameliorating intestinal inflammation by regulating inflammatory cells((Hooper, et al. 2001; Kelly, et al. 2004; Rios-Covian, et al. 2017; Wrzosek, et al. 2013). It has been reported that the level of Bacteroides increases in the endoscopic remission group of UC patients and the genus functions to coordinate the immune response to suppress chronic inflammation.(Nomura, et al. 2021) The correlation that we observed was consistent with previous reports of the modulatory effect of Bacteroides on chronic inflammation in IBD. Nevertheless, our study had some limitations. First, in our study, bowel preparation agents for colonoscopy may have influenced the composition of the intestinal microflora. The consistency and quality of each sample can be better maintained than with stool samples, but some microbes that are not tightly attached to the colonic epithelium may be lost during bowel preparation. Second, the sample size was relatively small, and samples were obtained only from the sigmoid colon. Therefore, changes in the abundance of microorganisms in colon segments were not fully evaluated. Third, this was a cross-sectional study performed at the initial diagnosis stage. Therefore, changes in the microbiome and genes and microbes related to prognosis during UC treatment were not identified. Fourth, we analyzed the mucosal microbiome using 16s rRNA sequencing. 16S rRNA sequencing is a powerful and effective tool for identifying microbial communities, but it is not as accurate as whole genome sequencing (shotgun sequencing) for determining species taxonomic resolution. Previous studies showed that although 16S rRNA sequencing and full-length sequencing both detected similar patterns in the microbial community, 16S rRNA sequencing was less accurate at the species level, with only 78% of the sequences being classified correctly (Bharti and Grimm 2021; Kim, et al. 2022; Peterson, et al. 2021; Shin, et al. 2023). Taxonomic assignment of species is more useful and crucial than that at the genus level in the clinical field. Therefore, further studies using new omics technologies, including shotgun and other culture-omics, should be performed to validate our results.
In summary, we identified a taxonomic difference between UC patients and NC and characterized the interactions and correlations between colonic epithelial microbes and host genes in UC patients. Our results provide a basis for the role of the microbiota of colonic mucosa in the initial development of UC, and the integrated analysis of the microbe-host gene can serve as a biomarker for diagnosis and treatment target in patients with UC.