The impact of Akkermansia muciniphila and its extracellular vesicles in the regulation of serotonergic gene expression in a small intestine of mice.

OBJECTIVES: A better understanding of host-microbe interactions as a cross-talk between the gastrointestinal (GI) tract and the gut microbiota can help treat and prevent GI disorders by improving the maintenance of GI homeostasis. The gut microbiota can affect signaling molecules, such as serotonin, which regulates endocrine systems through the GI tract. Moreover, studying the effects of gut microbiota in the small intestine on the human GI tract health is pivotal. METHODS: Male C57BL/6J mice (n = 30, 10 mice per group) were orally gavaged with 200 µL of PBS (control group); mice in group II were orally gavaged with 109 colony-forming units (CFU)/200 µL of viable A. muciniphila, suspended in PBS (A. muciniphila group); and mice in group III were orally gavaged with 10 µg of protein/200 µL of EVs (A. muciniphila-EV group) once daily for four weeks. The gene expression of serotonin system-related genes (Slc6a4, Tph1, Mao, Htr3, Htr4, and Htr7) was examined by quantitative real-time PCR (qPCR) method. RESULTS: Based on the results, A. muciniphila significantly affected the mRNA expression of genes related to the serotonin system (Tph1, Mao, Htr3B, and Htr7) in the duodenum and (Htr3B, Htr4 and Htr7) in the ileum of mice (P < 0.05). Moreover, A. muciniphila-derived EVs affected the expression of major genes related to the serotonin system (Tph1, slc6a4a, Mao, Htr3B, Htr4, and Htr7) in the duodenum and ileum of mice (P < 0.05). CONCLUSIONS: The present findings may pave the way for further investigation of the effects of strain-specific probiotics on the serotonergic system, which is currently in its infancy.

Effects of Akkermansia muciniphila and Faecalibacterium prausnitzii on serotonin transporter expression in intestinal epithelial cells.

PURPOSE: The highest level of peripheral serotonin in the body can be found in the gastrointestinal (GI) tract as its reservoir. There is complete interaction between human gastrointestinal microbiota and serotonin system. Serotonin in the GI is transferred by serotonin transporters (SERTs), which play a crucial role in the bioavailability of serotonin in the GI. SERT impairment is associated with the pathology of GI disorders. It is known that intestinal microbiota can regulate the SERT function. Therefore, it may be useful to regulate of SERT expression by modulation of microbiota and improvement of intestinal motility and GI sensation. In this study, we aimed to evaluate the effects of two next-generation probiotics, including Akkermansia muciniphila and Faecalibacterium prausnitzii, and their supernatants on SERT gene expression in human epithelial colorectal adenocarcinoma cells (Caco-2). METHODS: The Caco-2 cells were treated with multiplicity of infection (MOI) ratio of 100 of A. muciniphila and F. prausnitzii, as well as their supernatants. After 24 h, SERT gene expression was examined by quantitative real-time polymerase chain reaction (qRT-PCR) assay. RESULTS: A. muciniphila up-regulated the SERT mRNA level by 3.01 folds, compared to the control group. F. prausnitzii, similar to A. muciniphila, increased the expression of SERT gene in Caco-2 cells by 3.43 folds (P < 0.001). Moreover, the supernatants of A. muciniphila and F. prausnitzii significantly up-regulated the expression of SERT gene in the cell line by 2.4 and 5.7 folds, respectively, compared to the control group (P < 0.001). CONCLUSIONS: The present results showed that A. muciniphila and F. prausnitzii, as well as their supernatants, increased the expression of SERT gene in Caco-2 cells. Therefore, they might be helpful in the microbiota-modulating treatment of inflammatory bowel diseases.

Effect of Akkermansia muciniphila, Faecalibacterium prausnitzii, and Their Extracellular Vesicles on the Serotonin System in Intestinal Epithelial Cells.

The gastrointestinal (GI) tract is an essential reservoir of serotonin or 5-hydroxytryptamine (5-HT), which possesses a set of bacterial species communities. Intestinal microbiota has the ability to modulate the host's serotonin system. In this regard, we evaluated the effect of Akkermansia muciniphila and Faecalibacterium prausnitzii along with their extracellular vesicles (EVs) on serotonin system-related genes in human epithelial colorectal adenocarcinoma (Caco-2) cells. The differentiated Caco-2 cells were treated with A. muciniphila and F. prausnitzii with the multiplicity of infection ratio of 1 and 10 and the EV concentration of 1 µg/mL and 50 µg/mL, respectively. After 24 h, the serotonin level was quantified using an ELISA kit and also the gene expression of serotonin system-related genes was examined using the quantitative real-time PCR method. According to the results, treatment with A. muciniphila and F. prausnitzii-derived EVs increased the serotonin level, while none of the bacteria could affect the serotonin level in the Caco-2 cells. Both bacteria had significant effects on the mRNA expression of serotonin system-related genes in the Caco-2 cells. Moreover, we observed that A. muciniphila and F. prausnitzii-derived EVs could impact the expression of major genes involved in the serotonin system. Our findings showed that A. muciniphila and F. prausnitzii along with their EVs could modulate serotonin system-related genes; hence, they may be useful in microbiota modulation therapies to maintain the homeostasis of the serotonin system.

Comparative effects of alive and pasteurized Akkermansia muciniphila on normal diet-fed mice.

Recently, Akkermansia muciniphila an anaerobic member of the gut microbiota, has been proposed as a next-generation probiotic. The aim of this study was evaluation of the effect of alive and pasteurized A. muciniphila on health status, intestinal integrity, immune response, lipid metabolism, and gut microbial composition in normal-diet fed mice as well as direct effects of the bacterium on Caco-2 cell line. A total of 30 mice were distributed into three different groups, control, alive, and pasteurized A. muciniphila-treated group. After acclimation, control and treatment groups were administrated with PBS and 109 CFU/200µL of bacterial suspension for 5 weeks, respectively. Besides, Caco-2 separately exposed to alive, pasteurized A. muciniphila and PBS for 24 h. The results showed that administration of A. muciniphila leads to reduction in body, liver, and white adipose weight. Histology data revealed both treatments had no adverse effects in colon, liver, and adipose tissues as well as induced better gut structure. Moreover, biochemical parameters and inflammatory biomarkers in plasma demonstrated that pasteurized A. muciniphila had more pronounce effect. Furthermore, alive A. muciniphia had better effects on the modulation of gene expression related to fatty acid synthesis, energy homeostasis, and immune response in the liver; meanwhile, these effects in the adipose was more in the pasteurized A. muciniphila administration. More importantly, the improvement of gut health by enhancing strengthen intestinal integrity and maintaining immune homeostasis was seen in both treatments; notably, pasteurized A. muciniphila had more effective. Similarly, treatment with the pasteurized form more effectively upregulated tight junction and regulated immune response-related genes in Caco-2 cell line. Both treatments triggered the improvement of microbiota communities, particularly the alive form. Therefore, both forms of A. muciniphila could modulate lipid and immune homeostasis, improved some gut microbiota, and promoted the overall health, while all these effects were dominantly observed in pasteurized form. In conclusion, pasteurized A. muciniphila can be considered as new medical supplement to maintain health state and prevent diseases in normal mice through different mechanisms.

Modulation of serotonin signaling/metabolism by Akkermansia muciniphila and its extracellular vesicles through the gut-brain axis in mice.

Several studies have reported that the host-microbe interactions in the gut modulate the host serotonin or 5-hydroxytryptamine (5-HT) system. Here, we evaluated the effects of Akkermansia muciniphila and its extracellular vesicles (EVs) on genes pertaining to the serotonergic system in the colon and hippocampus of mice. Male C57BL/6J mice were administered viable A. muciniphila and its EVs for 4 weeks. The serotonin levels in the colon, hippocampus, and serum of mice, as well as the human colon carcinoma cells (Caco-2), were measured by ELISA assays. Also, the effects of A. muciniphila and its EVs on the expression of serotonin system genes in the colon and hippocampus were examined. A. muciniphila and its EVs may have a biological effect on the induction of serotonin levels in the colon and hippocampus of mice. Also, EVs increased the serotonin level in the Caco-2 cell line. In contrast, both treatments decreased the serotonin level in the serum. Both the bacterium and its EVs had significant effects on the mRNA expression of genes, involved in serotonin signaling/metabolism in the colon and hippocampus of mice. Moreover, A. muciniphila and its EVs affected the mRNA expression of inflammatory cytokines (Il-10 and Tnf-α) in the colon, however, there is no significant difference in inflammatory cell infiltrate in the histopathology of the colon. The presence of A. muciniphila and its EVs in the gut promotes serotonin concentration, they also affect serotonin signaling/metabolism through the gut-brain axis and may be considered in new therapeutic strategies to ameliorate serotonin-related disorders.

Akkermansia muciniphila-Derived Extracellular Vesicles as a Mucosal Delivery Vector for Amelioration of Obesity in Mice.

Recent evidence suggests that probiotics can restore the mucosal barrier integrity, ameliorate inflammation, and promote homeostasis required for metabolism in obesity by affecting the gut microbiota composition. In this study, we investigated the effect of Akkermansia muciniphila and its extracellular vesicles (EVs) on obesity-related genes in microarray datasets and evaluated the cell line and C57BL/6 mice by conducting RT-PCR and ELISA assays. A. muciniphila-derived EVs caused a more significant loss in body and fat weight of high-fat diet (HFD)-fed mice, compared with the bacterium itself. Moreover, treatment with A. muciniphila and EVs had significant effects on lipid metabolism and expression of inflammatory markers in adipose tissues. Both treatments improved the intestinal barrier integrity, inflammation, energy balance, and blood parameters (i.e., lipid profile and glucose level). Our findings showed that A. muciniphila-derived EVs contain various biomolecules, which can have a positive impact on obesity by affecting the involved genes. Also, our results showed that A. muciniphila and its EVs had a significant relationship with intestinal homeostasis, which highlights their positive role in obesity treatment. In conclusion, A. muciniphila-derived EVs can be used as new therapeutic strategies to ameliorate HFD-induced obesity by affecting various mechanisms.

Microbiological Analysis of Hemodialysis Water in a Developing Country.

Microbiological control of hemodialysis fluid is important for the prevention of hemodialysis-associated illness. Bacterial populations inhabiting a distribution system for hemodialysis water were studied over a 4 month period in five hospitals (one in Tehran, and the others at Alborz). All the samples from the four hospitals at Alborz had colony counts of ≥100 CFU/ml, which at different points of sampling were higher than the maximum recommended values. A total of 80 samples taken at different points in each hospital's hemodialysis distribution system were collected, and 229 planktonic bacteria isolated on R2A medium. No growth was detected by culturing the samples on Blood agar or Mueller-Hinton agar, according to routine procedures currently used in the five hospitals. A representative of isolates from each of 45 different morphotypes were identified using 16S RNA sequencing. A diverse bacterial community, containing predominantly gram-positive members of Kocuria, Arthrobacter and Staphylococcus and Mycobacterium, was detected. Bacteria from the genera Acinetobacter, Burkholderia, Halomonas, Herbaspirillum, Pseudomonas, and Sphingomonas were identified, which has been described in the build-up of biofilms. Some of the species reported here may represent a health risk to patients receiving hemodialysis treatment. In conclusion, it is recommended that standard protocols for evaluation of microbial contamination be used for regular monitoring and identification of culturable bacteria.