Abelmoschus manihot polysaccharide fortifies intestinal mucus barrier to alleviate intestinal inflammation by modulating Akkermansia muciniphila abundance.

The intestinal mucus barrier is an important line of defense against gut pathogens. Damage to this barrier brings bacteria into close contact with the epithelium, leading to intestinal inflammation. Therefore, its restoration is a promising strategy for alleviating intestinal inflammation. This study showed that Abelmoschus manihot polysaccharide (AMP) fortifies the intestinal mucus barrier by increasing mucus production, which plays a crucial role in the AMP-mediated amelioration of colitis. IL-10-deficient mouse models demonstrated that the effect of AMP on mucus production is dependent on IL-10. Moreover, bacterial depletion and replenishment confirmed that the effects of AMP on IL-10 secretion and mucus production were mediated by Akkermansia muciniphila. These findings suggest that plant polysaccharides fortify the intestinal mucus barrier by maintaining homeostasis in the gut microbiota. This demonstrates that targeting mucus barrier is a promising strategy for treating intestinal inflammation.

Genome- and Toxicology-Based Safety Assessment of Probiotic Akkermansia muciniphila ONE Isolated from Humans.

In this study, the genome of Akkermansia muciniphila ONE (designated AKK ONE) was sequenced, assembled, and analyzed. In addition, the safety of this strain was further evaluated by toxicological studies. The results showed that the AKK ONE genome is contained on a single chromosome with a total length of 2,817,524 bp and an average GC content of 55.48%. In total, 2411, 1131, 1168, 1745, and 1402 genes were annotated to the NR, GO, KEGG, COG, and SwissProt database, respectively. Potential resistance genes, adeF, tetW, ANT(3″)-IIa, and aadA1 were detected. AKK ONE was sensitive to ampicillin, ceftriaxone, cefotaxime, meropenem, tetracycline, and chloramphenicol and resistant to moxifloxacin. No potential virulence-related genes were detected. The PathogenFinder database analysis showed that AKK ONE was a non-potential human pathogen. This strain had good gastroenteric fluid tolerance and a weak ability to colonize the gut. No test item-related adverse effects were observed in the acute and subchronic toxicity test. AKK ONE did not display mutagenic activity either. This strain did not change the hematological and clinical biochemical parameters of mice. The weights of the organs were not affected by AKK ONE treatment. These results support that AKK ONE is safe for use as a probiotic at a dose of 8.28 × 109 CFU/kg bw/day.

Akkermansia muciniphila and its outer membrane protein Amuc_1100 prevent high-fat diet-induced nonalcoholic fatty liver disease in mice.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. A. muciniphila and its outer membrane protein Amuc_1100 ameliorate metabolic disorders, enteritis, depression, and other diseases in mice. The NAFLD mouse model was established by feeding a high-fat diet (HFD) for 10 weeks. To assess the effect of A. muciniphila and Amuc_1100 on NAFLD, we used atorvastatin, a common lipid-lowering drug, as a positive control. A. muciniphila and Amuc_1100 significantly reduced body weight and serum ALT and AST levels, and improved serum lipid levels in NAFLD mice, which had similar effects to Ator. In addition, A. muciniphila and Amuc_1100 decreased the concentration of LPS in the serum and upregulated the mRNA expression of the colonic tight junction proteins. In the liver, A. muciniphila and Amuc_1100 significantly reduced the mRNA expression levels of nodular receptor protein 3 (NLRP3) and Toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB), and the protein and mRNA expression levels inflammatory cytokines. At the genus level, Amuc_1100 treatment significantly reduced the abundance of Coriobacteriaceae_UCG-002 produced by the HFD. The abundances of Blautia, norank_f__Ruminococcaceae, Lachnoclostridium, GCA-900066575 and Lachnospiraceae_UCG-006 increased dramatically. Together, A. muciniphila and Amuc_1100 alleviate HFD-induced NAFLD by acting on the gut-liver axis and regulating gut microbes.

Administration of A. muciniphila ameliorates pulmonary arterial hypertension by targeting miR-208a-3p/NOVA1 axis.

Pulmonary arterial hypertension (PH) is a chronic disease induced by a progressive increase in pulmonary vascular resistance and failure of the right heart function. A number of studies show that the development of PH is closely related to the gut microbiota, and lung-gut axis might be a potential therapeutic target in the PH treatment. A. muciniphila has been reported to play a critical role in treating cardiovascular disorders. In this study we evaluated the therapeutic effects of A. muciniphila against hypoxia-induced PH and the underlying mechanisms. Mice were pretreated with A. muciniphila suspension (2 × 108 CFU in 200 µL sterile anaerobic PBS, i.g.) every day for 3 weeks, and then exposed to hypoxia (9% O2) for another 4 weeks to induce PH. We showed that A. muciniphila pretreatment significantly facilitated the restoration of the hemodynamics and structure of the cardiopulmonary system, reversed the pathological progression of hypoxia-induced PH. Moreover, A. muciniphila pretreatment significantly modulated the gut microbiota in hypoxia-induced PH mice. miRNA sequencing analysis reveals that miR-208a-3p, a commensal gut bacteria-regulated miRNA, was markedly downregulated in lung tissues exposed to hypoxia, which was restored by A. muciniphila pretreatment. We showed that transfection with miR-208a-3p mimic reversed hypoxia-induced abnormal proliferation of human pulmonary artery smooth muscle cells (hPASMCs) via regulating the cell cycle, whereas knockdown of miR-208a-3p abolished the beneficial effects of A. muciniphila pretreatment in hypoxia-induced PH mice. We demonstrated that miR-208a-3p bound to the 3'-untranslated region of NOVA1 mRNA; the expression of NOVA1 was upregulated in lung tissues exposed to hypoxia, which was reversed by A. muciniphila pretreatment. Furthermore, silencing of NOVA1 reversed hypoxia-induced abnormal proliferation of hPASMCs through cell cycle modulation. Our results demonstrate that A. muciniphila could modulate PH through the miR-208a-3p/NOVA1 axis, providing a new theoretical basis for PH treatment.

Akkermansia muciniphila, which is enriched in the gut microbiota by metformin, improves cognitive function in aged mice by reducing the proinflammatory cytokine interleukin-6.

BACKGROUND: Metformin, a type 2 diabetes treatment, improves the cognitive function of aged mice; however, whether the protective effects of metformin on cognitive function in aged mice are associated with the gut microbiome is poorly understood. Although some studies suggest that the gut microbe composition influences cognitive function and that manipulating the gut microbiota might protect against age-related cognitive dysfunction, there is no direct evidence to validate that the gut microbiota mediates the effect of metformin on cognitive improvement. RESULTS: In this study, we show that the gut microbiota is altered by metformin, which is necessary for protection against ageing-associated cognitive function declines in aged mice. Mice treated with antibiotics did not exhibit metformin-mediated cognitive function protection. Moreover, treatment with Akkermansia muciniphila, which is enriched by metformin, improved cognitive function in aged mice. Mechanistically, A. muciniphila decreased pro-inflammatory-associated pathways, particularly that of the pro-inflammatory cytokine interleukin (IL)-6, in both the peripheral blood and hippocampal profiles, which was correlated with cognitive function improvement. An IL-6 antibody protected cognitive function, and an IL-6 recombinant protein abolished the protective effect of A. muciniphila on cognitive function in aged mice. CONCLUSION: This study reveals that A. muciniphila, which is mediated in the gut microbiota by metformin, modulates inflammation-related pathways in the host and improves cognitive function in aged mice by reducing the pro-inflammatory cytokine IL-6. Video Abstract.

A comprehensive systematic review of the effectiveness of Akkermansia muciniphila, a member of the gut microbiome, for the management of obesity and associated metabolic disorders.

AIMS AND BACKGROUND: Obesity is recognised as a significant public health burden worldwide. Recently the cross-talk between gut microbiota and obesity has attracted much attention. To that end, Akkermansia muciniphila has been proposed as a promising microbe to manage obesity. In the present systematic review, we evaluated evidence on the effectiveness and mechanisms of action of Akkermansia muciniphila supplementation in the management of obesity. METHODS: Electronic databases of MEDLINE, PubMed, Scopus, Web of Science, and Google Scholar were searched thought March 2020 to identify relevant published articles, and eligible articles were systematically reviewed. RESULTS AND CONCLUSIONS: Fifteen studies were included in the present study. Findings from the present review, which included human and animal (rodent) models support the effectiveness of Akkermansia supplementation as a novel therapeutic approach for the management of obesity and metabolic complications associated with obesity. However, future clinical trials are warranted to verify these outcomes.

Postnatal intestinal mucosa and gut microbial composition develop hand in hand: A mouse study.

BACKGROUND: During the early postnatal life, gut microbiota development experiences dynamic changes in their structural and functional composition. The postnatal period is the critical window to develop a host defense mechanism. The maturation of intestinal mucosal barrier integrity is one of the essential defense mechanisms to prevent the entry of pathogens. However, the co-development of intestinal microbial colonization, formation of barrier integrity, and intestinal epithelial cell layer is not entirely understood. METHODS: We studied the gut microbial composition and diversity using 16S rRNA marker gene-based sequencing in mice to understand postnatal age-dependent association kinetics between gut microbial and intestinal development. Next, we assessed the intestinal development by in vivo gut permeability assay, mRNA gene expression of different tight junction proteins and intestinal epithelial cell markers, goblet cells population, villus length, and cecal IgA quantification. RESULTS: Our results showed a significant shift in gut microbial structural and functional composition from postnatal day 14 onwards with early life Proteobacteria abundance. Relative abundance of Verrucomicrobia was maximum at postnatal day 14 and showed a gradual decrease over time. We also observed an age-dependent biphasic pattern in barrier integrity improvement and differentiation of intestinal epithelial cells (IECs). A significant improvement in barrier integrity between days 1 and 7 showed the host factor contribution, while that beyond day 14 revealed an association with changes in microbiota composition. Our temporal correlation analysis associated Bacteroidetes phylum with the mucosal barrier formation during postnatal development. CONCLUSIONS: The present study revealed the importance and interplay of host factors and the microbiome in gut development and intestinal mucosal homeostasis.

Outer membrane protein Amuc_1100 of Akkermansia muciniphila alleviates antibiotic-induced anxiety and depression-like behavior in mice.

Akkermansia muciniphila is present in the mucus layer of its host gut, and its outer membrane protein Amuc_1100 has a significant ameliorative effect on metabolic disorders and emotional memory aspects of enteritis, obesity, depression, and anxiety in the host. Antibiotics affect gut microbial composition, leading to imbalance and behavioral changes in the gut-brain axis, while probiotics have a protective effect against behavioral changes caused by gut flora disorders. In the present study, a depressed mouse model using a broad-spectrum cocktail mixture resulted in increased anxiety and depression-like behavior, decreased serum and hippocampal levels of 5-hydroxytryptamine (5-HT), and increased serum corticosterone (cort) levels. After application of A. muciniphila and Amuc_1100, anxiety and depression-like behavior in antibiotic-treated mice were significantly alleviated. In addition, the brain derived neurotrophic factor / Tropomyosin receptor kinase B (BDNF/TrkB) signaling pathway was altered, glial fibrillary acidic protein (GFAP) expression increased, and c-Fos protein expression decreased in the hippocampus of antibiotic-treated mice. After treatment with A. muciniphila and Amuc_1100, BDNF and TrkB levels were restored in the hippocampus and cortex. These results suggest that A. muciniphila and Amuc_1100 may alleviate antibiotic-induced anxiety and depression by affecting the BDNF/TrkB signaling pathway.

Effects of ginsenoside compound K on colitis-associated colorectal cancer and gut microbiota profiles in mice.

Background: Ginsenoside compound K (GC-K), generated from ginseng saponins bioconverted by gut microbiota, has potential anti-colorectal cancer (CRC) effects. Meanwhile, GC-K may interact with gut microbiota, playing important roles in the occurrence and development of CRC. However, the effects of gut microbiota on the preventive and therapeutic effects of GC-K in CRC remain to be elucidated. Methods: The anti-CRC effects of GC-K were evaluated in an azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colitis-associated CRC Balb/c mice model under the dosage of 30 and 60 mg/kg. Stool samples were collected during the experiments for profiling gut microbiota by 16S rRNA sequencing. Correlative analysis between gut microbiota and anti-CRC effect of GC-K was also assessed. Finally, the anti-CRC effect of Akkermansia muciniphila (A. muciniphila) was validated in CRC cell lines. Results: GC-K could significantly suppress tumor growth in vivo at the dosage of 60 mg/kg without exogenous interference of gut microbiota. Moreover, dysbiosis of gut microbiota was observed in the CRC model group, which could be recovered by GC-K treatment. In particular, A. muciniphila, which could inhibit the proliferation of HCT-116, HT-29, and LOVO cells, was significantly up-regulated by GC-K. Conclusions: GC-K was verified to suppress the tumor growth of AOM/DSS-induced colitis-associated CRC through the modulation of gut microbiota, partially by up-regulation of A. muciniphila.

Akkermansia muciniphila and its outer membrane protein Amuc_1100 prophylactically attenuate 5-fluorouracil-induced intestinal mucositis.

5-Fluorouracil (5-FU) is a chemotherapy drug used to treat tumors. Previous studies have shown that Akkermansia muciniphila (A. muciniphila) and its outer membrane protein, Amuc_1100, alleviate dextran sodium sulfate (DSS)-induced colitis in mice. We investigated the effects of both A. muciniphila and Amuc_1100 on 5-FU-induced intestinal mucosal damage in mice. C57BL/6 mice were gavaged with A. muciniphila or Amuc_1100 daily before, during, and after 5-FU injection for a total of 14 days. By evaluating diarrheal toxicity scores, body weight changes, colonic anatomy images, and histopathology of intestinal injury in these mice, we found that A. muciniphila and Amuc_1100 alleviated 5-FU-induced intestinal mucositis. Quantitative polymerase chain reaction assays of intestinal cytokine mRNA levels demonstrated that both A. muciniphila and Amuc_1100 attenuated the upregulation of intestinal Tumor Necrosis Factor-α (TNF-α) and interleukin-6 (IL-6) induced by 5-FU treatment. In addition, they both reduced 5-FU-induced the NLR family pyrin domain containing 3 (NLRP3) inflammatory vesicle activation. Furthermore, by monitoring the mRNA expression of tight junction proteins in the intestine, we found that A. muciniphila and Amuc_1100 were capable of restoring the damaged intestinal barrier. Notably, A. muciniphila and Amuc_1100 also played a role in altering the structure of the intestinal microbial community. The present study revealed the protective role of both A. muciniphila and Amuc_1100 in the intestinal mucositis caused by 5-FU, providing new insights into treatment options.

Interrelationship of Gut Microbiota, Obesity, Body Composition and Insulin Resistance in Asians with Type 2 Diabetes Mellitus.

Metabolic syndrome (MS) has been an important health issue in the world, and insulin resistance (IR) is one of the characteristics of MS, increasing the risk for the onset and poor prognosis of type 2 diabetes mellitus (T2D). However, the interactional effect of obesity or abnormal body composition on the correlation between gut microbiota and IR in T2D patients is not well-explored. This cross-sectional study used a body composition monitor to evaluate lean tissue mass and fat tissue mass. IR was calculated using homeostatic model assessment-insulin resistance (HOMA-IR). Eight pairs of 16S rRNA gene primers specific to Firmicutes, Bacteroidetes, Clostridium leptum group, Faecalibacteriumprausnitzii, B acteroides, Bifidobacterium, Akkermansia muciniphila, and Escherichia coli were utilized to measure their abundance by qPCR. One hundred and fifty-four T2D patients were enrolled and stratified by the median HOMA-IR (2.5) and body mass index (BMI) of 25 kg/m2. A lower abundance of A. muciniphila was found in T2D patients with high HOMA-IR and BMI respectively. HOMA-IR and BMI had a synergistic effect on the reduction of the abundance of A. muciniphila. After adjusting metabolic factors, the low abundance of A. muciniphila significantly increased the risk for greater severity of IR. Furthermore, the negative correlation between A. muciniphila and IR was only found in T2D patients with high lean tissue. In conclusion, decreased abundance of fecal A. muciniphila enhanced the severity of IR in Asians with T2D, especially those having lean mass, and this significant relationship was independent of obesity.

A. muciniphila Supplementation in Mice during Pregnancy and Lactation Affects the Maternal Intestinal Microenvironment.

During pregnancy and lactation, considerable factors that affect the maternal microbiome are associated with the advancement of numerous diseases, which can potentially affect offspring health. Probiotics have shown potential for the maintenance of microbiota homeostasis of mothers in this period. The specific objective of this study was to investigate whether the application of Akkermansia muciniphila (A. muciniphila) during pregnancy and lactation impacts maternal and offspring health. Here we show that dams fed with A. muciniphila is safe, enhances the intestinal barrier and alters gut microbiota composition and diversity at the end of lactation, including the significant enrichment of A. muciniphila and Ruminococcus_1 in offspring from probiotic-fed dams. However, compared with the control group, the fecal metabolites of the A. muciniphila group only changed slightly. Additionally, A. muciniphila supplementation did not significantly increase the abundance of A. muciniphila in the fecal microbiota of offspring mice. Compared with the control group, the fecal metabolic profile of three-week-old offspring of mice fed with A. muciniphila were significantly changed, containing the D-glutamine and D-glutamate metabolism pathways. These results provided evidence that A. muciniphila supplementation in mice during pregnancy and lactation is safe and seemed to have a more beneficial effect on dams. In the future, using probiotics to regulate maternal microbiomes during pregnancy and lactation could be shown to have a more lasting and beneficial effect.

Inulin Improves Diet-Induced Hepatic Steatosis and Increases Intestinal Akkermansia Genus Level.

Hepatic steatosis is characterized by triglyceride accumulation within hepatocytes in response to a high calorie intake, and it may be related to intestinal microbiota disturbances. The prebiotic inulin is a naturally occurring polysaccharide with a high dietary fiber content. Here, we evaluate the effect of inulin on the intestinal microbiota in a non-alcoholic fatty liver disease model. Mice exposed to a standard rodent diet or a fat-enriched diet, were supplemented or not, with inulin. Liver histology was evaluated with oil red O and H&E staining and the intestinal microbiota was determined in mice fecal samples by 16S rRNA sequencing. Inulin treatment effectively prevents liver steatosis in the fat-enriched diet group. We also observed that inulin re-shaped the intestinal microbiota at the phylum level, were Verrucomicrobia genus significantly increased in the fat-diet group; specifically, we observed that Akkermansia muciniphila increased by 5-fold with inulin supplementation. The family Prevotellaceae was also significantly increased in the fat-diet group. Overall, we propose that inulin supplementation in liver steatosis-affected animals, promotes a remodeling in the intestinal microbiota composition, which might regulate lipid metabolism, thus contributing to tackling liver steatosis.

Characteristics of an In Vitro Mesenteric Lymph Node Cell Suspension Model and Its Possible Association with In Vivo Functional Evaluation.

In a previous study, we uncovered three immune-responsive patterns of gut microbes using an in vitro mesenteric lymph node cell suspension model, abbreviated as the MLN model hereafter. We used Akkermansia muciniphila and Clostridium butyricum as the first group directly inducing an immune response, Bifidobacterium sp. and Bacteroides sp. as the second group evoking an immune response with the help of stimuli (anti-CD3 and anti-CD28 antibodies), and Lactobacillus sp. as the third group blunting the immune response with or without stimuli. Our group previously clarified the immune-activation characteristics of A. muciniphila and linked its in vivo immune induction effect in GF and SPF mice under homeostasis. In the present study, we supplemented the characteristics of C. butyricum and B. bifidum in the in vitro MLN model and addressed the specific elements of the model. Finally, we used an in vivo TNBS-challenge model to show the functional differences between these species with different response patterns in vitro. The results showed that C. butyricum and B. bifidum evoked an immune response in vitro in a dose-dependent and strain-unique manner. Although TLR2, rather than TLR4, is indispensable for immune activation in the present in vitro model, it may not involve interaction between TLR2 and bacterial ligands. Like the PBMC model, the present in vitro MLN model is highly dependent on cell resources and should be given more attention when used to conduct a quantitative comparison. Finally, a mixture of two strong immunogenic strains, A. muciniphila and C. butyricum, significantly increased the mortality of TNBS-challenged (2,4,6-trinitrobenzene sulfonic acid, TNBS) mice, indicating a possible link between the in vitro MLN model and in vivo functional evaluation. However, more evidence is needed to clarify the associations and underlying mechanisms.

Candida tropicalis Infection Modulates the Gut Microbiome and Confers Enhanced Susceptibility to Colitis in Mice.

BACKGROUND & AIMS: We previously showed that abundance of Candida tropicalis is significantly greater in Crohn's disease patients compared with first-degree relatives without Crohn's disease. The aim of this study was to determine the effects and mechanisms of action of C tropicalis infection on intestinal inflammation and injury in mice. METHODS: C57BL/6 mice were inoculated with C tropicalis, and colitis was induced by administration of dextran sodium sulfate in drinking water. Disease severity and intestinal permeability subsequently were evaluated by endoscopy, histology, quantitative reverse-transcription polymerase chain reaction, as well as 16S ribosomal RNA and NanoString analyses (NanoString Technologies, Seattle, WA). RESULTS: Infected mice showed more severe colitis, with alterations in gut mucosal helper T cells (Th)1 and Th17 cytokine expression, and an increased frequency of mesenteric lymph node-derived group 2 innate lymphoid cells compared with uninfected controls. Gut microbiome composition, including changes in the mucin-degrading bacteria, Akkermansia muciniphila and Ruminococcus gnavus, was altered significantly, as was expression of several genes affecting intestinal epithelial homeostasis in isolated colonoids, after C tropicalis infection compared with uninfected controls. In line with these findings, fecal microbiome transplantation of germ-free recipient mice using infected vs uninfected donors showed altered expression of several tight-junction proteins and increased susceptibility to dextran sodium sulfate-induced colitis. CONCLUSIONS: C tropicalis induces dysbiosis that involves changes in the presence of mucin-degrading bacteria, leading to altered tight junction protein expression with increased intestinal permeability and followed by induction of robust Th1/Th17 responses, which ultimately lead to an accelerated proinflammatory phenotype in experimental colitic mice.

Serum metabolite profiling yields insights into health promoting effect of A. muciniphila in human volunteers with a metabolic syndrome.

Reduction of A. muciniphila relative abundance in the gut microbiota is a widely accepted signature associated with obesity-related metabolic disorders. Using untargeted metabolomics profiling of fasting plasma, our study aimed at identifying metabolic signatures associated with beneficial properties of alive and pasteurized A. muciniphila when administrated to a cohort of insulin-resistant individuals with metabolic syndrome. Our data highlighted either shared or specific alterations in the metabolome according to the form of A. muciniphila administered with respect to a control group. Common responses encompassed modulation of amino acid metabolism, characterized by reduced levels of arginine and alanine, alongside several intermediates of tyrosine, phenylalanine, tryptophan, and glutathione metabolism. The global increase in levels of acylcarnitines together with specific modulation of acetoacetate also suggested induction of ketogenesis through enhanced ß-oxidation. Moreover, our data pinpointed some metabolites of interest considering their emergence as substantial compounds pertaining to health and diseases in the more recent literature.

Akkermansia muciniphila: is it the Holy Grail for ameliorating metabolic diseases?

The increasing prevalence of metabolic diseases has become a severe public health problem. Gut microbiota play important roles in maintaining human health by modulating the host's metabolism. Recent evidences demonstrate that Akkermansia muciniphila is effective in improving metabolic disorders and is thus considered as a promising "next-generation beneficial microbe". In addition to the live A. muciniphila, similar or even stronger beneficial effects have been observed in pasteurized A. muciniphila and its components, including the outer membrane protein Amuc_1100, A. muciniphila-derived extracellular vesicles (AmEVs), and secreted protein P9. Hence, this paper presents a systemic review of recent progress in the effects and mechanisms of A. muciniphila and its components in the treatment of metabolic diseases, including obesity, type 2 diabetes mellitus, cardiovascular disease, and nonalcoholic fatty liver disease, as well as perspectives on its future study.

Alterations in the Gut Microbiome in the Progression of Cirrhosis to Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality worldwide. While cirrhosis is the main risk factor for HCC, the factors influencing progression from cirrhosis to HCC remain largely unknown. Gut microbiota plays a key role in liver diseases; however, its association with HCC remains elusive. This study aimed to elucidate microbial differences between patients with HCC-associated cirrhosis (HCC-cirrhosis) and cirrhotic patients without HCC and healthy volunteers and to explore the associations between diet, lifestyle, and the microbiome of these patients. Fecal samples and food frequency questionnaires were collected from 95 individuals (30 HCC-cirrhosis patients, 38 cirrhotic patients without HCC, and 27 age- and body mass index [BMI]-matched healthy volunteers). 16S rRNA gene sequencing was performed. Bacterial richness in cirrhosis and HCC-cirrhosis patients was significantly lower than in healthy controls. The HCC-cirrhosis group was successfully classified with an area under the curve (AUC) value of 0.9 based on the dysbiotic fecal microbial signature. The HCC-cirrhosis group had a significant overrepresentation of Clostridium and CF231 and reduced Alphaproteobacteria abundance compared to cirrhotic patients without HCC. Patients with HCC-cirrhosis who were overweight displayed significantly decreased bacterial richness and altered microbiota composition compared to their normal-weight counterparts. There was a significant correlation in the HCC-cirrhosis group between intake of artificial sweeteners and the presence of Akkermansia muciniphila A unique microbial signature was observed in patients with HCC-cirrhosis, irrespective of cirrhosis stage, diet, or treatment. BMI, dietary sugar, and artificial sweeteners were significantly associated with alterations in the microbiome of HCC-cirrhosis patients. However, the increased abundance of Clostridium and CF231 observed in HCC-cirrhosis patients was not influenced by environmental factors, implying that this change was due to development of HCC.IMPORTANCE Development of hepatocellular carcinoma in patients with cirrhosis is associated with alterations in intestinal microbiota, including an escalation of dysbiosis and reduced bacterial richness. This study demonstrates that reduced bacterial richness and dysbiosis escalate with the progression of cirrhosis from compensated to decompensated cirrhosis and to HCC-associated cirrhosis (HCC-cirrhosis). Moreover, we report for the first time the effect of environmental factors on HCC-cirrhosis. Excess weight was associated with increased dysbiosis in patients with HCC compared to their normal-weight counterparts. Moreover, fatty liver, consumption of artificial sweeteners, and high-sugar foods were associated with altered microbial composition, including altered levels of Akkermansia muciniphila in HCC-cirrhosis. We have successfully determined that levels of Alphaproteobacteria and the two genera CF231 and Clostridium are significantly altered in cirrhotic patients who develop hepatocellular carcinoma, independently of cirrhosis severity and dietary habits.

Matrix metalloproteinase MMP9 maintains epithelial barrier function and preserves mucosal lining in colitis associated cancer.

In colitis associated cancer (CAC), chronic inflammation exposes the epithelial mucosal defensive lining to inflammatory mediators such as cytokines and anti-microbial peptides (AMPs) causing the dysbiosis of microbiota population and the dysregulation of immune response. Matrix Metalloproteinases (MMPs) are zinc dependent endopeptidases which mediate inflammation, tissue remodeling, and carcinogenesis. MMP9 is undetectable in healthy tissue, although highly upregulated during inflammation and cancer. We have previously shown that MMP9 plays a protective role in CAC opposite to its conventional role of acute inflammation and cancer mediator. In this study, we investigated the mechanistic role of MMP9 in preserving the epithelial mucosal integrity to suppress the progression of tumor microenvironment in CAC. We used transgenic mice constitutively expressing MMP9 in colonic epithelium (TgM9) as an in vivo model and intestinal cell line CaCo2BBE as an in vitro model. We induced CAC with three cycles of dextran sodium sulfate (DSS). We observed that MMP9 expression in colonic epithelium maintains the microbiota. We also observed that MMP9 mediates pro-inflammatory cytokine levels and AMPs but suppresses IL-22 resulting in lower levels of REG3-g and S100A8 AMPs. We also found that MMP9 maintains an efficient barrier function and the integrity of tight junctions. We also observed increased levels of mucin and intestinal trefoil factor among TgM9 mice in CAC. We also found that MMP9 expressing CaCo2BBE cells had increased expressions of EGFR and nuclear transcription factor- specificity protein 1 (Sp1). These data imply that MMP9 acts as a tumor suppressor in CAC by sustaining the epithelial mucosal integrity due to the activation of EGFR-Sp1 signaling pathway.