Fut2 deficiency aggravates chronic colitis through 2-oxindole-AHR mediated cGAS-STING pathway.

OBJECTIVE: This study aims to disclose how loss of fucosyltransferase 2 (Fut2) impacts intestinal inflammation through cGAS-STING pathway that is closely associated with gut microbiota, and which microbial metabolite improves colitis in Fut2 deficiency. METHODS: Chronic colitis was induced in intestinal epithelial Fut2 knock out mice (Fut2△IEC), whose intestinal inflammation and activity of cGAS-STING pathway were evaluated. 16S rRNA sequencing and metabolomics were performed using intestinal samples. 2-oxindole was used to treat RAW264.7 cells and Fut2△IEC mice with colitis (Fut2△IEC-DSS) to investigate the effect of 2-oxindole on cGAS-STING response and intestinal inflammation. RESULTS: Fut2 loss exacerbated chronic colitis in mice, manifested by declined body weight, reduced colon length, increased disease activity index (DAI) and more colon injury in Fut2△IEC-DSS mice compared with WT-DSS (wild type mice with colitis). Lack of Fut2 promoted activation of cGAS-STING pathway. Fut2 deficiency had a primary impact on colonic microbiota, as shown by alteration of microbial diversity and structure, as well as decreased Lactobacillus. Metabolic structure and tryptophan metabolism in colonic luminal microbiota were also influenced by Fut2 loss. Fut2 deficiency also led to decreased levels of aryl hydrocarbon receptor (AHR) and its ligand 2-oxindole derived from tryptophan metabolism. 2-oxindole compromised cGAS-STING response through activating AHR in macrophages, and protected against intestinal inflammation and overactive cGAS-STING pathway in Fut2△IEC-DSS mice. CONCLUSION: Fut2 deficiency promotes cGAS-STING pathway through suppressing 2-oxindole-AHR axis, ultimately facilitating the susceptibility to chronic colitis.

Modulation of the microbiota across different intestinal segments by Rifaximin in PI-IBS mice.

BACKGROUND: Rifaximin has been increasingly applied in irritable bowel syndrome (IBS) treatment. Whether there were differences in the effects of rifaximin on microbiota from different intestinal segments, especially the small intestine where rifaximin predominantly acted, has not been confirmed. METHODS: In this study, we used Trichinella spiralis infection to induce post infectious irritable bowel syndrome (PI-IBS) and measured visceral sensitivity of mice by means of abdominal withdrawal reflex (AWR) tests to colorectal distention (CRD). We compared the effects of rifaximin on the composition of ileal, colonic mucosal and fecal microbiota in PI-IBS mice. RESULTS: Rifaximin significantly reduced AWR scores and increased pain threshold in PI-IBS mice, and this effect was associated with the change in the relative abundance of ileal mucosal microbiota. Rifaximin could obviously decrease ileum mucosal microbiota alpha diversity assessed by Shannon microbial diversity index. Meanwhile, the analysis of beta diversity and relative abundance of microbiota at phylum, family and genus levels showed that rifaximin could improve the microbiota structure of ileal mucosa. However, for colonic mucosal and fecal microbiota, this effect of rifaximin was not obvious. Rifaximin could reshape the correlation of genera between different intestinal segments. CONCLUSION: Rifaximin improved visceral hypersensitivity in PI-IBS mice. Rifaximin mainly affected ileal mucosal microbiota, and its improvement effect on IBS might be closely related to the improvement of ileal microbiota structure.

Baseline gut microbial profiles are associated with the efficacy of Bacillus subtilis and Enterococcus faecium in IBS-D.

OBJECTIVE: Little is known about association between the efficacy of probiotics and baseline gut microbiota in irritable bowel syndrome (IBS). We aimed to explore gut microbiota in diarrhea-predominant IBS (IBS-D) and whether baseline gut microbiota was related to the efficacy of Bacillus subtilis and Enterococcus faecium (BE). METHODS: This study recruited 19 healthy controls (HC) and 50 IBS-D patients, among whom 19 patients were administrated 500 mg BE orally three times daily for 2 weeks. Clinical data and fecal samples were collected from patients before and after treatment. 16S rRNA sequencing was performed to obtain fecal bacterial data. RESULTS: There was no significant difference of alpha diversity, beta diversity, profiles of microbial phyla and genera between HC and IBS. BE improved IBS-SSS (IBS severity scoring system) and stool consistency, and altered Enterococcus, Blautia, Lachnoclostridium and Fusobacterium without significant impact on microbial structure in IBS-D. Notably, baseline fecal bacterial composition differed between non-responders and responders to BE concerning abdominal pain and bloating, with Atopobium, Pyramidobacter, Ruminococcus gnavus and Peptostreptococcus enriched in responders in terms of abdominal pain. There was reduced abundance of Prevotella, Ruminococcaceae UCG, Eubacterium eligens, Faecalibacterium and Eubacterium coprostanoligenes in responders compared with non-responders. Furthermore, BE increased beneficial bacteria including Faecalibacterium, Blautia and Butyricicoccus, decreased Lachnoclostridium and Bilophila, and influenced some microbial metabolic pathways in responders, such as mineral absorption, metabolism of arachidonic acid, d-arginine, D-ornithine, phenylalanine and vitamin B6. CONCLUSION: Baseline fecal microbiome is associated with the efficacy of BE in attenuating abdominal pain and bloating in IBS-D.

Promoting Effect of L-Fucose on the Regeneration of Intestinal Stem Cells through AHR/IL-22 Pathway of Intestinal Lamina Propria Monocytes.

The recovery of the intestinal epithelial barrier is the goal for curing various intestinal injurious diseases, especially IBD. However, there are limited therapeutics for restoring intestinal epithelial barrier function in IBD. The stemness of intestinal stem cells (ISCs) can differentiate into various mature intestinal epithelial cells, thus playing a key role in the rapid regeneration of the intestinal epithelium. IL-22 secreted by CD4+ T cells and ILC3 cells was reported to maintain the stemness of ISCs. Our previous study found that L-fucose significantly ameliorated DSS-induced colonic inflammation and intestinal epithelial injury. In this study, we discovered enhanced ISC regeneration and increased intestinal IL-22 secretion and its related transcription factor AHR in colitis mice after L-fucose treatment. Further studies showed that L-fucose promoted IL-22 release from CD4+ T cells and intestinal lamina propria monocytes (LPMCs) via activation of nuclear AHR. The coculture system of LPMCs and intestinal organoids demonstrated that L-fucose stimulated the proliferation of ISCs through an indirect manner of IL-22 from LPMCs via the IL-22R-p-STAT3 pathway, and restored TNF-α-induced organoid damage via IL-22-IL-22R signaling. These results revealed that L-fucose helped to heal the epithelial barrier by accelerating ISC proliferation, probably through the AHR/IL-22 pathway of LPMCs, which provides a novel therapy for IBD in the clinic.

L-Fucose inhibits the progression of cholangiocarcinoma by causing microRNA-200b overexpression.

BACKGROUND: Cholangiocarcinoma (CCA) is a malignant biliary tract tumor with an extremely poor prognosis. There is an urgent demand to explore novel therapeutic strategies. L-fucose has been confirmed to participate in anti-inflammation and antitumor activities. However, the effect of L-fucose on the progression of CCA has not been well investigated. This study aimed to determine whether L-fucose induced the inhibition of CCA and its possible mechanism. METHODS: The anti-growth activity was determined using Cell Counting Kit-8 assay, colony formation assays, Annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) assay, and cell cycle analysis. The anti-metastasis activity was determined by wound healing, transwell, and invasion assays. The anti-angiogenesis activity was determined by tube formation and transwell assays. MicroRNAs that may be involved in the L-fucose-induced CCA inhibition was analyzed using bioinformatics methods. The preclinical therapeutic efficacy was mainly estimated by ultrasound in xenograft nude mouse models. Differences were analyzed via Student's t test or one-way analysis of variance. RESULTS: L-Fucose induced apoptosis and G0/G1 cell cycle arrest, inhibited cell epithelial-mesenchymal transition of CCA cells, and additionally inhibited tube formation of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner, leading to a decrease in cell proliferation, metastasis, and angiogenesis. Mechanistically, L-fucose induced microRNA-200b (miR-200b) upregulation, and mitogen-activated protein kinase 7 (MAPK7) downregulation was found to be targeted by miR-200b, with decreased cell proliferation and metastasis. Additionally, phosphorylated signal transducer and activator of transcription 3 was found to be downregulated after L-fucose treatment. Finally, in vivo experiments in CCA xenograft models also confirmed the antitumor properties of L-fucose. CONCLUSION: L-Fucose inhibited the progression of CCA via the miR-200b/MAPK7 and signal transducer and activator of transcription 3 signaling pathways.

Understanding of the Site-Specific Microbial Patterns towards Accurate Identification for Patients with Diarrhea-Predominant Irritable Bowel Syndrome.

Fecal microbial community could not fully represent the intestinal microbial community. However, most studies analyzing diarrhea-dominant irritable bowel syndrome (IBS-D) were mainly based on fecal samples. We aimed to characterize the IBS-D microbial community patterns using samples at multiple intestinal sites. This study recruited 74 IBS-D patients and 20 healthy controls (HC). 22.34%, 8.51%, 14.89%, and 54.26% of them contributed to one, two, three, and four sites: duodenal mucosa (DM), duodenal lumen (DL), rectal mucosa (RM), and rectal lumen (RL) of intestinal samples, respectively. Then 16S rRNA gene analysis was performed on these 283 samples. The result showed that IBS-D microbial communities have specific patterns at each intestinal site differing from that of HC. Across hosts and sites, Bacillus, Burkholderia, and Faecalibacterium were the representative genera in duodenum of IBS-D, duodenum of HC, and rectum of HC, respectively. Samples from mucosa and lumen in rectum were highly distinguishable, regardless of IBS-D and HC. Additionally, IBS-D patients have lower microbial co-abundance network connectivity. Moreover, RM site-specific biomarker: Bacteroides used alone or together with Prevotella and Oscillospira in RM showed outstanding performance in IBS-D diagnosis. Furthermore, Bacteroides and Prevotella in RM were strongly related to the severity of abdominal pain, abdominal discomfort, and bloating in IBS-D patients. In summary, this study also confirmed fecal microbial community could not fully characterize intestinal microbial communities. Among these site-specific microbial communities, RM microbial community would be more applicable in the diagnosis of IBS-D. IMPORTANCE Microbial community varied from one site to another along the gastrointestinal tract, but current studies about intestinal microbial community in IBS-D were mainly based on fecal samples. Based on 283 intestinal samples collected from DM, DL, RM, and RL of HC and IBS-D, we found different intestinal sites had their site-specific microbial patterns in IBS-D. Notably, RM site-specific microbes Bacteroides, Prevotella, and Oscillospira could be used to discriminate IBS-D from HC accurately. Our findings could help clinicians realize the great potential of the intestinal microbial community in RM for better diagnosis of IBS-D patients.

Gut microbiota-mediated lysophosphatidylcholine generation promotes colitis in intestinal epithelium-specific Fut2 deficiency.

BACKGROUND AND AIMS: Previous study disclosed Fucosyltransferase 2 (Fut2) gene as a IBD risk locus. This study aimed to explore the mechanism of Fut2 in IBD susceptibility and to propose a new strategy for the treatment of IBD. METHODS: Intestinal epithelium-specific Fut2 knockout (Fut2△IEC) mice was used. Colitis was induced by dextran sulfate sodium (DSS). The composition and diversity of gut microbiota were assessed via 16S rRNA analysis and the metabolomic findings was obtained from mice feces via metabolite profiling. The fecal microbiota transplantation (FMT) experiment was performed to confirm the association of gut microbiota and LPC. WT mice were treated with Lysophosphatidylcholine (LPC) to verify its impact on colitis. RESULTS: The expression of Fut2 and α-1,2-fucosylation in colonic tissues were decreased in patients with UC (UC vs. control, P = 0.036) and CD (CD vs. control, P = 0.031). When treated with DSS, in comparison to WT mice, more severe intestinal inflammation and destructive barrier functions in Fut2△IEC mice was noted. Lower gut microbiota diversity was observed in Fut2△IEC mice compared with WT mice (p < 0.001). When exposed to DSS, gut bacterial diversity and composition altered obviously in Fut2△IEC mice and the fecal concentration of LPC was increased. FMT experiment revealed that mice received the fecal microbiota from Fut2△IEC mice exhibited more severe colitis and higher fecal LPC concentration. Correlation analysis showed that the concentration of LPC was positively correlated with four bacteria-Escherichia, Bilophila, Enterorhabdus and Gordonibacter. Furthermore, LPC was proved to promote the release of pro-inflammatory cytokines and damage epithelial barrier in vitro and in vivo. CONCLUSION: Fut2 and α-1,2-fucosylation in colon were decreased not only in CD but also in UC patients. Gut microbiota in Fut2△IEC mice is altered structurally and functionally, promoting generation of LPC which was proved to promote inflammation and damage epithelial barrier.

Mucosal-Associated Microbiota Other Than Luminal Microbiota Has a Close Relationship With Diarrhea-Predominant Irritable Bowel Syndrome.

Studies have linked dysbiosis of gut microbiota to irritable bowel syndrome (IBS). However, dysbiosis only referring to structural changes without functional alteration or focusing on luminal microbiota are incomplete. To fully investigate the relationship between gut microbiota and clinical symptoms of Irritable Bowel Syndrome with Diarrhea (IBS-D), fecal samples, and rectal mucosal biopsies were collected from 69 IBS-D patients and 20 healthy controls (HCs) before and during endoscopy without bowel preparation. 16S rRNA genes were amplified and sequenced, and QIIME pipeline was used to process the composition of microbial communities. PICRUSt was used to predict and categorize microbial function. The composition of mucosa-associated microbiota (MAM) was significantly different in IBS-D patients compared to HCs; while no difference in luminal microbiota (LM). MAM, but not LM, was significantly positively correlated with abdominal pain and bloating. A greater number of MAM functional genes changed in IBS-D patients than that of LM compared with HCs. Metabolic alteration in MAM not in LM was related to abdominal pain and bloating. There was a close relationship between the composition and function of MAM and clinical symptoms in IBS-D patients which suggests the important role of MAM in pathogenesis and therapies in IBS-D and it should be highlighted in the future.

Gut fungal dysbiosis and altered bacterial-fungal interaction in patients with diarrhea-predominant irritable bowel syndrome: An explorative study.

BACKGROUND: Little is known about intestinal fungi in IBS patients whose gut bacteria have been investigated a lot. In order to explore causal relationship between IBS and gut mycobiome, and use gut fungi to diagnose or even treat IBS, further characterization of it in IBS is required. METHODS: Fifty-five diarrhea-predominant IBS (D-IBS) patients fulfilling Rome III criteria, and 16 healthy controls (HC) were recruited. Fresh fecal samples were collected and used for 16s rRNA and ITS2 high-throughput sequencing. Diversity and composition of gut bacteria and fungi, as well as bacterial-fungal interactions in D-IBS patients, were characterized. Specific fungal taxa differentiating D-IBS from HC were recognized by LEfSe and RandomForest methods, and their association with clinical symptoms was assessed by Spearman's correlation. RESULTS: Diarrhea-predominant irritable bowel syndrome patients showed abnormal (IBS-dysbiosis) or normal (HC-like IBS) fecal bacterial structure and diversity compared with healthy controls. However, fecal fungal signatures differed absolutely between D-IBS and HC, which indicated a more susceptible alteration of gut fungi than bacteria in D-IBS. Fecal fungi showed significant correlations with IBS symptoms, especially Mycosphaerella, Aspergillus, Sporidiobolus, and Pandora which were identified to potentially differentiate D-IBS from HC. Moreover, compared with HC there were markedly declined bacterial-fungal interactions in D-IBS, in which Candida changed from negative to positive correlations with bacteria, and Eurotium changed from positive correlations to irrelevance, while Debaryomyces gained negative correlations with bacteria. CONCLUSIONS: Gut fungal dysbiosis and altered bacterial-fungal interactions were present in patients with D-IBS, and gut fungi could be used to diagnose D-IBS.

Fecal bacteria can predict the efficacy of rifaximin in patients with diarrhea-predominant irritable bowel syndrome.

OBJECTIVE: Rifaximin for treating diarrhea-predominant irritable bowel syndrome (IBS-D) by regulating intestinal microbiota has been studied and recommended. In this study, we tried to investigate the effect of rifaximin on different components of intestinal microbiota and explore which component of gut microbiota can predict the efficacy of rifaximin in IBS-D. METHODS: Healthy controls (HC) and IBS-D patients meeting the Rome III criteria were recruited, and IBS-D patients were orally administered 400 mg rifaximin three times daily for 2 weeks. Subjects were tested for small intestinal bacterial overgrowth (SIBO), their symptoms were recorded, and fecal and rectal mucosal samples were collected before and after treatment. Fecal and rectal mucosal bacterial data were obtained via 16S rRNA sequencing, and fecal fungal data were obtained via ITS2 sequencing. RESULTS: IBS-D patients were divided into two subgroups based on fecal bacterial composition, IBS1 (patients whose fecal bacterial composition were different from HC) and IBS0 (patients whose fecal bacterial profiles were similar to HC). Rifaximin increased fecal Bifidobacterium and decreased E. coli and Enterobacter in IBS1 patients. Although rectal mucosal bacteria and fecal fungi were not significantly altered in all patients after rifaximin intervention, rifaximin enhanced the connections among fecal bacteria, mucosal bacteria and fecal fungi in IBS1 patients. Compared with IBS0, we surprisingly found rifaximin ameliorated abdominal symptoms of IBS1 much better. Receiver operating curve analysis revealed patients whose fecal microbial dysbiosis indices (MDI) were higher than -3.006 could be diagnosed as IBS1. CONCLUSION: Fecal bacterial dysbiosis could be a biomarker for rifaximin treatment for IBS-D.

Probiotic mixtures with aerobic constituent promoted the recovery of multi-barriers in DSS-induced chronic colitis.

AIMS: Gut microbiota has been closely linked to the mucosal immune and been regarded as a reliable target for intestinal inflammation. This study aimed to explore the therapeutic roles of probiotic mixtures of Bifidobacterium infantis, Lactobacillus acidophilus, Enterococcus faecalis with (quadruple probiotics, P-qua) or without (triple probiotics, P-tri) aerobic Bacillus cereus in colitis, focusing on the multiple barrier functions. MATERIALS AND METHODS: Chronic colitis was induced by dextran sulfate sodium (DSS) in C57BL/6 mice. The probiotic mixtures P-qua or P-tri was gavage administrated respectively, while fecal microbiota transplantation (FMT) as a positive control. The intestinal inflammation and functions of multiple barriers were assessed, including the mucus barrier, epithelial barrier and endothelial barrier known as gut-vascular barrier (GVB). Altered composition and diversity in gut microbiota were observed via sequencing analysis. KEY FINDINGS: Both P-qua and P-tri relieved the intestinal inflammation and improved the functions of multiple barriers with increased integrity of mucous layer, enhanced transepithelial electrical resistance, declined epithelial and endothelial permeability to macromolecules in DSS-colitis. Aerobe-contained P-qua revealed a more active role in barrier recovering relative to P-tri, while FMT as a positive control seemed to get better results than pure probiotics. Indeed, P-qua was effective in rebuilding the structure and diversity of gut flora in DSS-colitis, especially increased abundance of Bifidobacterium, Akkermansia, Lactobacillus and Bacteroides. SIGNIFICANCE: Aerobe-contained P-qua was a powerful adjuvant therapy for chronic colitis, via restoring the intestinal microflora and recovering the multi-barriers in the inflamed gut.

Duodenal and rectal mucosal microbiota related to small intestinal bacterial overgrowth in diarrhea-predominant irritable bowel syndrome.

BACKGROUND AND AIM: Small intestinal bacterial overgrowth (SIBO) has been proposed as an etiologic factor in irritable bowel syndrome, particularly the diarrhea-predominant subtype (IBS-D). We aimed to identify potential intestinal microbial pattern in IBS-D patients with SIBO. METHODS: Diarrhea-predominant irritable bowel syndrome patients fulfilling Rome III criteria were recruited and randomly divided into an exploratory cohort (57 cases) and a validation cohort (20 cases). SIBO was identified according to standard glucose hydrogen breath test. For 16S rRNA gene sequencing, samples of duodenal mucosa, duodenal fluid, rectal mucosa, and fresh feces were collected and performed. The α and ß diversity, as well as differences in microbial composition and function, in SIBO+ and SIBO- IBS-D subjects were evaluated. RESULTS: The microbial diversity and composition obviously differed between SIBO+ and SIBO- IBS-D in duodenal and rectal mucosa but not in duodenal fluid and fresh feces. For rectal mucosal microbiota, it displayed markedly reduced aerobe and Gram-negative bacteria and increased facultative anaerobe and Gram-positive bacteria, moreover, altered functions of microbial metabolism in SIBO+ IBS-D. Significantly higher rectal mucosa-related microbial dysbiosis index was observed in SIBO+ IBS-D, and a cut-off value at -0.37 had a sensitivity of 56.55% and specificity of 90.91% to identify the SIBO in IBS-D subjects. CONCLUSIONS: Mucosal microbiota, rather than luminal bacteria, has a more apparent dysbiosis in SIBO+ IBS-D patients relative to those without SIBO. Rectal mucosa-associated microbiota may act as a potential predictor of SIBO in IBS-D patients.