Akkermansia Muciniphila Ameliorates Intestinal Inflammation Through Stimulation Of Intestinal Stem Cells Proliferation

There is huge commensal microbiota in the animal intestinal tracts.Studies have shown that there is a communication interaction between intestinal microbiota and intestinal innate immunity,and their dynamic balance affects the development of animals.The intestinal microbiota is rich in species,which is composed of 1013-14 kinds of microorganisms.The composition of intestinal microbiota is universal,specific and variable.The universality means that the same genus animals have similar flora components at the same age.The specificity means that each animal has a unique composition of intestinal flora.The variability means that intestinal flora will be affected by age,diet,drugs,disease and other factors.There is an intestinal mucosal barrier composed of intestinal flora,mucus,intestinal epithelial cells and submucosal immune cells,which can deal with the complex and changeable intestinal environment to ensure intestine health.As a new type of probiotics,A.muciniphila has a significant effect o improving metabolism and reducing obesity,but its role in intestinal inflammation remains to be explored.1 Akkermansia muciniphila reduced the symptoms of intestinal inflammationIn our previous research,Lactobacillus reuteri significantly increased the number of A.muciniphila in the intestinal microbiota when repairing intestinal inflammation caused by 3.5%dextran sodium sulfate（DSS）.Unlike traditional probiotics,A.muciniphila is gram-negative bacteria.Five-week-old male C57BL/6 mice were respectively gavaged by 105CFU,106CFU,107CFU,and 108CFU A.muciniphila for one week,and no significant difference was found in body weight and colon length.Orally administered of 107CFU A.muciniphila,the level of LPS was the lowest.The intestinal morphology performed normally when treated with 105CFU and 106CFU A.muciniphila,while the colon showed light inflammation at high concentraton of A.muciniphila.However,the crypt depth increased significantly when orally gavaged with 106CFU A.muciniphila.Based on these results,106CFU A.muciniphila was selected as the better treatment concentration for subsequent experiments.In order to investigate the role of A.muciniphila in repairing intestinal inflammation,five-week-old C57BL/6 mice were selected as to oral gavage A.muciniphila and heat-killed A.muciniphila（106CFU）for 14 days,and given 3.5%DSS with drinking water on the 7th day to cause intestinal inflammation.With the treatment of 3.5%DSS,the weight growth rate of the mice was decreased,the colon length was significantly shortened,and the colon lost normal tissue morphology.ELISA detected the concentration of inflammatory factors in the colon and blood,and found that the colon inflammatory factor IL-1β（interleukin 1β）and LPS in the blood were significantly increased.The intestinal stem cells will accelerate the proliferation and differentiation to repair the damaged intestinal epithelial cell when the intestine injured.However,immunofluorescence showed that the PCNA+ positive cells（proliferating cells）in the crypt decreased sharply when treated with DSS.With the protection of A.especially heat-killed A.muciniphila,the colonic pathological morphology,colonic tissue morphological damage,and PCNA+ cell numbers were significantly improved.The above results proved that A.muciniphila can relieve DSS-induced inflammation and maintain the intestinal health.It has been proved that A.muciniphila could also ameliorate S.pullorum-induced intestinal inflammation in chicks.In order to further investigate the mechanism of A.muciniphila protective effect,we used membrane protein Amuc₁100 perform the same experiment to comfirm its protective function.The results showed that Amuc₁100 can maintain the normal weight of inflammatory mice,the colon length is not affected by DSS,the LPS expression level is reduced,the colon can maintain normal tissue morphology,and the number of PCNA+cells in the intestinal crypts significantly increases.In a word,A.muciniphila can play a key role in alleviating DSS-induced colon inflammation,and its membrane protein Amuc₁100 also has similar functions.It is speculated that A.muciniphila protected intestine through its membrane protein Amuc₁100.2 Akkermansia muciniphila stimulated IL-10 expressiom to regulate the wnt/β-catenin signal pathwayA.muciniphila and its membrane protein Amuc₁100 can protect intestinal inflammatory,but its underlying mechanism remains unknown.Intestinal inflammation is closely related to intestinal immunity,so we detected the colonic immune cells changes,especially CD4+FOXP3+Treg cells and CD4+IL-17A+Th17 cells.Flow cytometry showed that A.muciniphila could significantly increase the number of Treg cells at the lamina propria and increase the expression of the corresponding cytokine IL-10 in the colon.The expression of Lgr5+stem cells and Bmil+ stem cells was remarkably improved,and the transcription levels of wnt3a and Cyc were conspicuously increased.The experiment further explored the changes in intestinal immunity and stem cell proliferation when mice treated with Amuc₁100.The results showed that the number of Treg cells increased significantly and the number of Th17 cells increased slightly,while the expression of IL-10 increased and the expression of IL-17A decreased.The wnt/β-catenin signal pathway was activated and Lgr5+stem cell expression increased with the Amuc₁100 treatment.Above results proved that A.muciniphila relieved DSS-induced inflammation by its membrane Amuc₁100 through regulating the proportion of lamina propria lymphocytes.Amuc₁100 treatment increased the number of Treg cells to secrete IL-10,and activated wnt/β-catenin signal pathway to promote damaged intestinal cells renewal.So Amuc₁100 could enable mice to maintain a normal state during intestinal inflammation.3 DC activated by Amuc₁100 to promote Treg cell differentiation and facilitated stem cell proliferation by IL-10The membrane protein Amuc₁100 can induce T cell differentiation in vivo,but the underlying mechanism needs further investigation.Amuc₁100 was co-incubated with naive dendritic cells,and then denditic cells were sitmulated to mature with high expression of CD86 and CD 103.It was reported that CD103+ dendritic cells can induce T cells to differentiate into Treg cells.Therefore,the matured dendritic cells were incubated with naive T cells and their differentiation was detected by flow cytometry.The results showed that naive T cells had a tendency to differentiate into Treg cells when co-cultured with dendritic cells.The corresponding cytokines IL-10 and IL-17 in the supernatant of the co-culture system were found to have an upward tendency.In order to further explore the role of these two cytokines,IL-10 and IL-17A were incubated with intestinal organoids which were induce inflammation by TNF-α（tumor damage factor α）.The intestinal organoids morphology was shown that IL-10 pretreatment could relieve the organoid damage.The wnt/β-catenin signal pathway was activated and the expression of Lgr5+ stem cells and Bmil+ stem cells also increased.The results proved that the membrane protein Amuc₁100 can promote T cells to differentiate into Treg cells by stimulating the maturation of dendritic cells.Treg cells secreted IL-10 to activate the wnt/β-catenin signal pathway,and induce intestinal stem cell proliferation to repair intestinal damage.