Characteristic And It‘s Mechanism Of Multiple Effects Of Intestinal Microbiota In Small Intestine Allotransplantation And Autotransplantation

AimsIntestinal transplantation(ITx)represents the only effective therapeutic strategy for patients with intestinal failure(IF)who have developed life-threatening complications related to long-term total parenteral nutrition(TPN).Recently,despite impressive advances in immunosuppression,there are still about 30-40% of adult ITx recipients inevitably develop an episode of allograft rejection within 1-year post-transplantation.Unlike transplantation of parenchymal organs(aminotransferases for liver and creatinine for kidney),there is no such special biomarker as indicators for intestinal allograft rejection.Therefore,researching the new targeted biomarker for intestine allograft rejection has become a hot spot in exploration of ITx specific immune tolerance,which also represents the direction for clinical development of ITx in the future.Short bowel syndrome(SBS)induced by intestine ischemic diseases,are the most commonly causes among ITx adult candidates.Patients in whom ischemic intestine tissue cannot be effectively treated progressed to IF,and of which these patients ultimately undergo transplantation depends on the success or failure of rescuing their ischemic intestine tissue.Because of its insidious onset,rapid progression and lack of special biomarkers,leading obstacle to clinical practitioners to find effective therapeutic time windows,thus the diagnosis of intestine ischemic diseases was usually delayed,rendering treatment more intractable.The intestinal autotransplantation(IATx)provides the chance for the researches of ischemic bowel diseases,of which the most crucial pathological process is intestine ischemic reperfusion injury(IIRI).Therefore,researches focus on IIRI in IATx patients not only provide new biomarkers for the prognosis or treatment of IATx,but possibly give new ideas for the treatment of ischemic bowel disease or ITx.There are over 1000 types microbiota inhabiting the complex human gut ecosystem and most type of these species are bacteria.During last decade,researches based on the high-throughput sequencing technologies indicated that these bacteria are not only crucial to maintain health in humanbeing,but also related to the occurrence and progression in plenty of diseases.Therefore,researchers suggest that intestinal flora,as a novel biological marker,provides a new therapeutic target for the treatment of many intestinal diseases.However,current studies show that the changes and functions of intestinal flora in living related ITx still remain unclear,and researches related to intestinal flora of IATx are rarely reported.Thus,this study will explore the structural and functional changes of intestinal flora during the perioperative period of ITx and IATx patients,aiming to reveal the potential mechanisms of intestinal flora in living related ITx and IATx.Methods1.Gut microbiota and living related intestine allogeneic transplantation1.1 Patients cohortTwelve living related ITx patients were conducted in our programs between April 2015 and November 2019 at department of digestive diseases,Xijing Hospital.Patients were divided into 6 groups: Pre-ITx(ITx BF),Post-ITx week 1st(ITx AF1),week 2nd(ITx AF2),week 3rd(ITx AF3),week 4th(ITx AF4),and the point prior to discharge(ITx AF5)were defined as the specimen collection point for subsequent analyses.1.2 16 s r DNA sequence analysis and microbial metabolites detectionTo reveal the gut bacterial community composition in ITx patients during perioperative period,microbial DNA was applied to amplify the V3-V4 region of the 16 S r DNA.Basebuilding was conducted using an Ion Plus Fragment Library Kit 48 rxns.An Ion S5?XLwas selected for high-throughput sequencing according to standard protocols.DNA was extracted from ITx patients by using phenol/chloroform extraction method,and detected by q-PCR assay.Flagellin,peptidoglycan and other bacteria related indicators;downstream bacterial related receptors NOD1,NOD2,TLR4 and TLR5;and cytokines IL-2,IL-6,TNF-α and IL-10 collected from blood of ITx patients were also detected.1.3 Blood microbial related substances and immune cell analysisThe laboratory results for CRP,PCT,immune cells and cytokines were collected from clinical reports and transformed into analyzable data.Perioperative monocyte,eosinophil,neutrophil,lymphocyte,and basophil counts were presented based on the CD45+ cells.Cytotoxic T lymphocyte,T helper cell,natural killer cell and B cell counts are presented relative to the frequency of lymphocyte cells,revealing perioperative immune cell changes in living related ITx patients.2.Gut microbiota and intestine autotransplantation2.1 Specimen collection and intestinal flora analysisFive transplants were conducted between November 2017 and March 2019 at department of digestive diseases,Xijing Hospital.Patients were divided into 6 groups,which was similar to the previous grouping strategy: IATx BF,IATx AF1,IATx AF2,IATx AF3,IATx AF4,IATx AF5.Methods of specimen collection and intestinal microbiota sequencing were the same as described above in 1.1.2.2 Rats IIRI model and bacterial metabolite detectionSuperior mesenteric artery(SMA)was clipped for 1 h and reperfusion for 23 h to establish rats IIRI model.Once the changes of the gut flora were determined,the targeted metabolism of SCFAs was measured.The protective effects of SCFAs Ac and But that were significantly down regulated induced by IIRI were detected,and the relationship between the protective effects and intestinal bacterial structure as well as the overall metabolic changes of bacterial community was then explored.2.3 Protective effects of SCFA But on OGD IEC-6 cellsThe oxygen glucose deprivation(OGD)model that oxygen glucose deprivation 6 h and reoxygenation for 18 h on IEC-6 cells was established to simulate IIRI in vitro.The protective effects of But on OGD IEC-6 cells was detected by cell stability and cell survival rate experiments.Combined transcriptome and metabolic analysis was established to reveal the potential protective mechanism of But on OGD damaged IEC-6 cells.According to the results of sequencing combined analysis,q-PCR and western-blot were applied to detect the expression level of NLRP3,and the expression levels of downstream cytokine IL-18 and related receptor TLR4.The binding site between But and NLRP3 PYD functional structure area,and the mechanism of But affecting activation of NLRP3 were then explored.The regulation of But on NLRP3 was further detected in IIRI intestinal tissue,and the regulatory effects of But on NLRP3 were also detected by in situ hybridization and immunofluorescence.Results1.Gut microbiota and living related intestine allotransplantation1.1.Characteristic of gut microbiota in IF patientsIn IF patients,the intestinal flora structure consists mainly of proteobacteria and firmicutes,which account for more than 98% of the total bacteria,while the relative abundance of bacteroidetes,actinobacteria and fusobacteria was far below the normal abundance.1.2 Intestinal flora of ITx patients changes during perioperative periodIntestinal flora significantly changes in the perioperative period of ITx patients.We found that proteobacteria was one of the most significantly changed bacteria,which was upregulated within 2nd weeks post-ITx,but down-regulated at 3rd weeks.Firmicutes were also significantly down-regulated after ITx,reaching the lowest level at the 3rd week post-ITx.Bacteroidetes were also significantly up-regulated post-ITx.Moreover,we found that the α diversity of gut microbiota significantly decreased in 1st and 2nd week post-ITx,hitting bottom at 2nd week and β diversity appeared significant differences at 1st week post-ITx and the differences keeping until 4th week post-ITx.1.3 Functional changes of intestinal flora in ITx patientsTax4Fun functional analysis suggested that microbial functions such as bacterial motility,bacterial chemotaxis and LPS biosynthesis significantly changed in postoperative period in ITx patients.According to further verification,we found that,in ITx patients,translocation of 16 s r DNA,flagellum protein,and LPS significantly increased.Microbial pattern recognition receptors(PRRs)such as the TLR4,NOD2 were also increased posttransplantation.Moreover,further results suggested that downstream inflammatory cytokines such as proinflammatory cytokines IFN-γ,IL-6 and IL-8 were also significantly up-regulated at 1th week post-ITx.Furthermore,we found that,due to the immunosuppressant,B cells were significantly suppressed post-ITx,while cytotoxic T lymphocytes(CTL)cells were significantly increase at 1st week post-ITx.NK cells were significantly raised from 1st to 4th week post-ITx.Further results showed that immune regulatory cytokines,such as IL-10 and TGF-β,were synchronously changed following these immune cells.2.Gut microbiota and intestine autotransplantation2.1 The abundance of gut microbiota significantly changes in IATx patientsThe composition of intestinal flora of IATx patients before transplantation was: firmicutes 69.5%,proteobacteria 19.8%,bacteroidetes 10.4%,actinobacteria 0.27%,etc.Bacteroidetes were up-regulated and firmicutes down-regulated after IATx,and the ratio of bacteroidetes and firmicutes was about 1 at 5th week post-IATx.2.2 IIRI relates to the changes of gut microbiota in IATxFurther results suggested that IIRI caused significant changes in intestinal flora in rats.Firmicutes,proteobacteria and actinobacteria were significantly increased in the IIRI injury(IR)group compared with the control group.Structural changes of intestinal flora often lead to changes in microbial metabolites.Therefore,we conducted targeted metabolic detection of microbial metabolites SCFAs,and found that butyrate(But)and acetate(Ac)were significantly down-regulated in IIRI group,compared with control group.2.3 But alleviates IIRI induced gut microflora structure and metabolic disordersFurther results suggested that But protected IIRI induced intestinal tissue damage and may alleviate IIRI induced intestinal microbiota structural disorder,suggesting that the protective effects may be related to intestinal microbiota.According to the metabolome results in microbiota,we found that But induces changes in intestinal microbiota metabolites.According to further tests in vitro,we found that But showed protective effects on OGD IEC-6 cells.Then,based on the combined analysis of transcriptome and metabolome,we found that But may have a regulatory effect on the key protein NLRP3.But significantly downregulated overexpression of NLRP3 in OGD damaged IEC-6 cells and inhibited the expression level of ASC.Docking results showed that But could bind the PYD structure of NLRP3 protein,changing the conformation of NLRP3,thus may disturb NLRP3 binding to ASC.Further results in vivo suggested that But could also alleviate IIRI induced NLRP3 and TLR4 overexpression in intestine tissue.ConclusionThe intestinal flora of living related ITx patients changes from 1 week pre-ITx to 5th week post-ITx,and some of these microbiota such as proteobacteria and firmicutes,dramatically changes in the acute rejection period of 1st and 2nd weeks post-ITx.The structure changes of these flora further induce changes in their biological functions such as bacterial chemotaxis,flagellar assembly and LPS biosynthesis,leading to further changes in microbial metabolites,and thus regulate the expression of relevant inflammatory cytokines,and finally induce significant changes in the composition of immune cells from the innate immune system in ITx patients.These results suggest that the immune-related inflammatory injury regulated by gut mocrobiota may provide a new biological target for the prediction and intervention of immune rejection in ITx patients.The intestinal flora of IATx patients changed significantly during perioperative period,which may relate to IIRI.In rats,IIRI induces alteration of intestinal flora resulted in significant changes of metabolites SCFAs such as Ac and But.Supplementation of Ac and But significantly alleviates intestinal tissue damage induced by IIRI.But also significantly alleviates OGD induced IEC-6 cells damage based on the key protein NLRP3 that played a crucial role between gut flora and intestinal epithelial cells.In all,But may interferes with the assembly of NLRP3-inflammasome to block the activation of inflammatory response,and protects the integrity of intestinal mucosa,inhibits the migration of 16 S r DNA of intestinal flora,thus blocks the contact of PAMPs with intestinal epithelial cells.These results suggest that But,a microbial metabolite SCFA,protects against IIRI and improve the structure gut microbiota,which may provide a potential biological target for IATx treatment or intestine ischemic diseases.