A Study Of The Role And Mechanism Of Gut Microbiome In Abdominal Aortic Aneurysm

Background:Abdominal aortic aneurysm(AAA)is a localized permanent expansion of the abdominal aorta that exceeds the normal vessel diameter by 50%or has a diameter greater than 3 cm.AAA is highly insidious,persistently progressive,and dangerous,with a mortality rate of more than 80%after rupture,posing a serious threat to people’s life and health.Currently,there is a lack of effective methods to treat and delay the progression of AAA,except for surgery.However,surgery is accompanied by a high rupture and reintervention rate.Inflammation plays an essential role in the pathogenesis of AAA.Various types of inflammatory cells infiltrating in the aortic wall can secrete various inflammatory factors and matrix metalloproteinases,which induce the apoptosis of vascular smooth muscle cells(VSMCs)and degradation of the extracellular matrix,promoting AAA development.Some studies discovered that B cell depletion is protective against AAA formation in Ang Ⅱ-and elastase-induced AAA mice,but other studies have not supported this finding.Obviously,our understanding of the pathogenesis of AAA is therefore still in its infancy,which requires further investigation.Neutrophils are the most abundant immune cells in the human body and can form neutrophil extracellular traps(NETs),which fulfill an important role in both immunity and inflammation.Abnormal activation of neutrophils and NETs can exacerbate inflammation and lead to the death of endothelial cells or VSMCs.There is a protective effect of reducing neutrophil infiltration on AAA.NETs level increased both in human and mice tissues.NETs promote AAA by activating plasmacytoid dendritic cells to generate type Ⅰ IFN,while NETs inhibitors significantly weaken the formation of AAA in mice.Nevertheless,the cause and mechanism of abnormal NETs formation in the pathogenesis of AAA are still poorly understood.The human intestine is the home for innumerable microbes,and the metabolites produced by these gut microbes play a pivotal role in the development of cardiovascular diseases by influencing host functions.For example,B.fragilis exerts anti-inflammatory effects by regulating the differentiation and function of Th17 and Treg cells through the production of short-chain fatty acids(SCFAs).Also,it is involved in high salt-induced hypertension by regulating corticosterone metabolism.A recent study revealed intestinal flora dysbiosis exists in Ang Ⅱ-induced AAA mice.H.hathewayi can significantly elevate serum taurine levels and reduce the formation and rupture of intracranial aneurysms in mice.However,the causal relationship and mechanism linking gut microbes and AAA remain unexplored,and there is limited knowledge regarding the association between the gut microbiome,neutrophils,and NETs.Part Ⅰ A study of the alterations of gut microbiota composition,function and metabolite profile in patients with abdominal aortic aneurysmObjectives:1.To reveal the gut microbial composition and its characteristic alterations in AAA patients using metagenomic sequencing technique;2.To elucidate the characteristic changes in gut microbiota metabolism of AAA patients using metabolomics technique;3.To identify differential gut microbes and metabolites that are closely correlated with AAA conditions and to evaluate their diagnostic potential.Methods:1.Clinical sample collectionThis study enrolled 33 AAA patients(AAA group)who were admitted to Qilu Hospital of Shandong University and 31 healthy volunteers(Healthy group)underwent physical examinations at the hospital’s health examination center.Participants signed informed consent forms,and their clinical data,fresh fecal samples,and plasma samples were collected.2.Gut metagenomic sequencing and data analysisMicrobial genomic DNA was extracted from fecal samples and subjected to metagenomic sequencing using the Illumina high-throughput sequencing platform.Software like Kraken2 and Salmon were used to obtain information on gut microbial species,genes,and metabolic pathways,as well as changes in their relative abundance.Subsequently,α and β-diversity,differential,and correlation analysis were performed.3.Metabolite detectionMetabolites extracted from feces were employed for untargeted metabolomic analysis using liquid chromatography-tandem mass spectrometry(LC-MS/MS);The SCFAs from fecal and plasma samples were analyzed qualitatively and quantitatively by gas chromatography-mass spectrometry(GC-MS);The LAL method was adopted to detect endotoxin levels in feces and plasma.4.Integration of multi-omics data for screening AAA characteristic species and metabolitesThe quantitative data matrices of metagenomic sequencing data,metabolomics data,and clinical indexes from the healthy persons and AAA patients were analyzed integratively.With correlation analysis as the leading method,bioinformatics methods,such as dimension reduction analysis,multivariate analysis,and feature screening,were combined to screen gut microbes and metabolites closely related to AAA.5.Establishment and evaluation of an AAA diagnostic model based on changes in gut microbial abundanceA random forest model was used to identify gut microbial biomarkers that effectively differentiated healthy persons and AAA patients,with their diagnostic potential assessed via receiver operating characteristic(ROC)curve analysis.Results:1.Comparison of demographic and clinical characteristics of AAA patients(AAA)and healthy control(Healthy)Aside from AAA,there were no significant differences in the demographic data and clinical indicators between the two groups after adjustment,eliminating the influence of confounding factors on the difference in gut microbes between groups.2.Gut microbiota dysbiosis in AAA patients,and changes in the abundance of multiple intestinal bacteria are significantly associated with AAA conditionβ-diversity reveals significant changes in the structure and composition of gut microbial communities in AAA patients,with a clear separation from the healthy group.Concurrently,the abundance of various gut microbes in AAA patients also significantly altered.R.intestinalis,the highly abundant butyric acid producer in the human intestine,were markedly decreased in the AAA group,and it was significantly negatively correlated with AAA diameter;While some intestinal bacteria such as Klebsiella spp.,the producer of LPS,were significantly increased in the intestine of AAA patients and significantly positively correlated with the AAA diameter.3.Significant alterations in symbiotic networks and functions of gut microbes in AAA patientsThe negative correlation between the probiotic Roseburia intestinalis and the harmful bacteria Klebsiella spp.and Enterobacter spp.existing in Healthy group was lost in AAA group.The function of gut microbiome was also significantly altered in the AAA patients.Correlation analysis indicated that Klebsiella spp.were positively correlated with key enzymes genes for LPS synthesis,such as lpxA,while R.intestinalis was positively correlated with with the key enzyme phosphate butyryltransferase for butyrate production.The abundance of Klebsiella.spp and key enzyme genes(lpxA,lpxC,lpxD)regulating LPS production were also significantly increased in the AAA group.The ability of R.intestinalis to produce butyric acid was also confirmed by targeted metabolomics of its supernatant.4.Gut microbiota metabolic profiles are significantly altered and associated with condition in AAA patientsUntargeted metabolomics analysis showed significant metabolic abnormalities in the gut microbiota of AAA patients.For example,compared with Healthy group,beneficial metabolites such as γ-glutamylcysteine(γ-GC)and niacin were significantly decreased in the intestine of AAA patients,while LPS levels were significantly elevated in both the intestine and plasma of AAA patients.Targeted metabolomics analysis showed that the levels of butyric acid in the intestine and plasma of AAA patients were remarkably decreased and strongly positively correlated with intestinal R.intestinalis abundance,but strongly negatively correlated with AAA diameter,suggesting that R.intestinalis might inhibit the AAA development by producing butyric acid that enters the circulation via intestinal absorption.5.The gut microbiota has great potential as a diagnostic marker for AAAThe random forest model identified a characteristic group of 30 gut microbes,including R.intestinalis,which could serve as markers to accurately identify AAA patients with an 88.9%accuracy rate.Moreover,their diagnostic efficacy for AAA was significantly stronger than clinical indicators such as blood pressure.Plasma butyrate levels also accurately identified AAA.These findings suggest that gut microbiota and their metabolites have the potential to serve as diagnostic or non-invasive screening indicators for AAA.Conclusions:1.Significantly altered intestinal microbial composition in AAA patients,with a significant decrease in the butyric acid producer R.intestinalis.2.Significantly altered gut microbial function and metabolic profile characteristics of AAA patients,with a significant decrease in butyric acid content.3.Decreased abundance of R.intestinalis and reduced butyric acid content in AAA patients’ intestines are closely associated with the AAA diameter.4.The characteristic changes in gut microbiota have the potential for diagnosing AAA.Part Ⅱ A study of the role and mechanism of gut microbiome dysbiosis in the pathogenesis of AAAObjectives:1.To investigate the causal relationship between gut microbiota and AAA’s onset using fecal microbiota transplantation(FMT)in two types of AAA mouse models;2.To study the mechanism of gut microbiota in the onset of AAA in two types of AAA mouse models;3.To identify the role of neutrophils in gut microbiota-regulated AAA’s occurence by depleting neutrophils in two types of AAA mouse models.Methods:1.Investigation of the causal relationship between gut microbiota and AAA onset through FMTAfter gut microbiota depletion by antibiotic cocktail,mice were transplanted with gut microbiota from healthy or AAA patient donors through FMT.Subsequently,two types of AAA mouse models were induced by Ang Ⅱ or CaCl2.Groups:Control group:No treatment given;Control+Anti.group:Antibiotic pretreatment;AAA group:AAA mice;AAA+Anti.group:Antibiotic pretreatment AAA mice;Healthy-FMT group:After antibiotic pretreatment,fecal microbiota from healthy persons were transplanted into AAA mice;AAA-FMT group:After antibiotic pretreatment,fecal microbiota from AAA patients were transplanted into AAA mice.2.Investigation of the role of neutrophils in the regulation of AAA by gut microbiotaNeutrophils in mice were depleted by incessantly intraperitoneal injection of anti-Ly6G antibody and AAA-FMT process(see as above)was conducted.Groups:IgG2a Ctrl group:AAA-FMT mice were injected with IgG2a isotype control antibody;Anti-Ly6G group:AAA-FMT mice were injected with anti-mouse Ly6G antibody.3.16S rRNA gene sequencing and analysis of mouse feces16S rRNA gene sequencing was performed on the fecal microbiota genomic DNA from the donor,recipient mice before and after antibiotic treatment,as well as before and after FMT for one week.The taxonomic classification annotations of ASV were obtained using QIIME2-Deblur and RDP classifier.PCoA analysis was performed to evaluate the effect of antibiotic and FMT.4.Histopathological examinationAbdominal aortic tissues underwent H&E staining,VVG staining,immunohistochemistry staining with α-SMA,etc.,immunofluorescence staining with NETs,etc.;whereas colon tissues underwent immunohistochemistry staining with Claudinl,etc.5.Molecular biology detectionThe levels of protein of α-SMA,SM22α,etc.extracted from abdominal aortic tissues were detected by Western Blot.The mRNA levels of TNF-α,IL-6,MCP-1,and Il-1β extracted from abdominal aortic tissues were detected by qPCR.6.Metabolite detectionThe SCFAs from feccal and plasma samples were analyzed qualitatively and quantitatively by gas chromatography-mass spectrometry(GC-MS);The LAL method was adopted to detect endotoxin levels in AAA tissues.7.Flow cytometryThe content of CD11b+Ly6G+neutrophils in the peripheral blood of mice in each group after 5 days of treatment with anti-Ly6G or IgG2a isotype control antibody were measured by flow cytometry.Results:1.Gut microbiota from AAA patients significantly promoted the development and progression of Ang Ⅱ-induced abdominal aortic aneurysms in miceThe aneurysm formation rate,rupture rate,mortality rate,and aneurysm diameter were significantly increased in AAA-FMT group in comparison to AAA+Anti.group,whereas the opposite effect was observed for Healthy-FMT group.The results of aortic wall pathology analysis showed that compared with AAA+Anti.group,the degradation of elastic fibres in the abdominal aorta was significantly enhanced.The expression of α-SMA and SM22α was obviously decreased,while the expression of osteopontin and vimentin was notably increased,indicating that the phenotypic switch from contractile to synthetic VSMCs was significantly enhanced.The levels of many proinflammatory and chemotactic cytokines(TNF-α,IL-1β,IL-6,MCP-1,CXCL1)and LPS were significantly upregulated in AAA lesions.However,the levels of butyrate in the intestine and plasma were markedly reduced.The elevated level of LPS in the tissues of AAA-FMT group may be attributed to the disruption of the intestinal barrier caused by decreased expressions of colon tight-juction protein,such as claudin-1,occludin,and ZO-1.Meanwhile,neutrophil infiltration and NETs formation in the abdominal aorta increased markedly in the AAA-FMT group compared with AAA+Anti.group.However,the above pathological changes were reduced in Healthy-FMT group.The above results suggest that the gut microbiota from AAA patients significantly promotes the pathogenesis of Ang Ⅱ-induced AAA disease in ApoE-/mice and leads to the increasing of neutrophil infiltration and NETs formation in the vascular wall.2.Gut microbiota from AAA patients significantly promoted the development and progression of CaCl2-induced abdominal aortic aneurysms in miceWe repeated most of the above experiments in CaCl2-induced wild-type AAA mice.The results were consistent with those from Ang Ⅱ-induced AAA mice,namely gut microbiota from AAA patients significantly exacerbated AAA diameter,elastic fiber destruction,VSMC phenotypic switching,the expression level of inflammatory factor,and neutrophil infiltration and NETs formation in CaCl2-induced wild-type AAA mice,while gut microbiota from healthy persons showed the opposite pattern.All these results suggest that the abnormal gut microbiota is an important aetiological factor of AAA,and its mechanism may be attributed to the effect on neutrophil infiltration and NETs formation.3.Neutrophil depletion reduced the promoting effect of gut microbiota from AAA patients on the conditions of Ang Ⅱ-and CaCl2-induced AAA miceAnti-Ly6G antibody were continuously intraperitoneally injected to deplete neutrophils in Ang Ⅱ-induced AAA-FMT mice.Results showed that neutrophil depletion significantly reduced the contribution of gut microbiota from AAA patients to AAA development,namely decreased the rates of incidence,rupture and mortality,maximal aneurysm diameter,and elastin degradation,indicating that neutrophils played causal roles in AAA mediated by gut microbiota dysbiosis.The same results were confirmed in the CaCl2-induced AAA mouse model.These suggest that gut microbiota dysbiosis promotes the development and progression of AAA by affecting neutrophil function.Conclusion:1.Gut microbiota dysbiosis promotes the development and progression of AAA conditions in AAA mice and is an important cause for AAA;2.Gut microbiota dysbiosis promotes various pathological findings such as infiltration of neutrophils and formation of NETs in the arterial wall of AAA mice;3.Neutrophils play a key role in the development and progression of AAA mediated by the gut microbiota,which is an important mechanism for the pathogenesis of AAA regulated by the gut microbiota.Part Ⅲ A study of the role and mechanism of Roseburia intestinalis and its metabolite butyrate in the pathogenesis of AAAObjective:1.To study the role of R.intestinalis and its metabolite butyrate in the development of AAA;2.To find out the molecular mechanism of R.intestinalis and its metabolite butyrate in regulating neutrophil infiltration and NETs formation.Methods:1.Study design and grouping at animal level1.1 To study the role of R.intestinalis and butyrate in the pathogenesis of AAAMice were given sodium butyrate via intragastric administration,or colonized with R.intestinalis after gut microbiota clearance using antibiotics.Subsequently,the AAA mouse model was induced with CaCl2 treatment,allowing for the observation of the effects of sodium butyrate and R.intestinalis on AAA development.Groups:Control group;Control+Anti.group;AAA group;A A A+Anti.group;AAA+R.I.group;AAA+NaBu group.1.2 To investigate the role of R.intestinalis and butyrate in the development of AAA regulated by intestinal microorganisms independent of LPSTLR4-KO mice were pretreated with antibiotics,followed by FMT with fecal bacterial solution from AAA patients,and AAA was induced using CaCl2(TLR4-KO AAA-FMT).Meanwhile,R.intestinalis or sodium butyrate were administered to mice to investigate their impact on gut microbiome-regulated TLR4-KO AAA condition.Groups:WT group;AAA group;TLR4-KO AAA group;TLR4-KO AAA-FMT group;TLR4-KO AAA-FMT+R.I.group;TLR4-KO AAA-FMT+NaBu group.1.3 To study the mechanism of R.intestinalis and butyrate in AAA pathogenesisMice were injected with empty vector(Ctrl)and NOX2 expressing adeno-associated virus vector(NOX2)in tail vein and gavaged with R.intestinalis(need antibiotic pretreatment)or sodium butyrate,and then AAA was induced by CaCl2 to observe whether NOX2 overexpression could attenuate the effect of R.intestinalis or sodium butyrate on AAA mice.Groups:AAA group;AAA+Ctrl group;AAA+NOX2 group;AAA+Ctrl+R.I.group;AAA+Ctrl+NaBu group;AAA+NOX2+R.I.group;AAA+NOX2+NaBu group.2.Study design and grouping at cell level2.1 To determine the effects of different concentrations of NaBu on LPS-O128-induced NETsLPS-O128(15μg/mL)+NaBu(0μM,1μM,10μM,100μM,500μM,1mM).2.2 To identify the mechanism of NaBu to inhibit the LPS-O128-induced NETsGroups:(1)PBS;(2)PMA(100nM);(3)LPS-O128(15μg/mL);(4)LPS-O128(15μg/mL)+GSK2795039(25μM);(5)LPS-O128+NaBu(100μM);(6)LPS-O128(15μg/mL)+GSK2795039(25μM)+NaBu(100μM).2.3 To identify the mechanism of NaBu to inhibit the TNF-α-induced NETsGroups:(1)PBS;(2)PMA(100nM);(3)TNF-α(50ng/mL);(4)TNF-α(50ng/mL)+GSK2795039(25μM);(5)TNF-α(50ng/mL)+NaBu(100μM);(6)TNF-α(50ng/mL)+GSK2795039(25μM)+NaBu(100μM).3.Extraction,culture and purity determination of human peripheral blood neutrophilsNeutrophils from healthy people’s peripheral blood were extracted and purified by Lymphoprep separation medium and cultured in RPMI-1640 medium.The purity of neutrophils was assayed by Wright-Giemsa staining and CD66b flow cytometry.4.Histopathological examinationAbdominal aortic tissues underwent H&E staining,VVG staining,immunohistochemistry staining with α-SMA,etc.,immunofluorescence staining with SM22α,NETs,etc.;whereas colon tissues underwent immunohistochemistry staining with Claudin1,etc.5.Molecular biology detectionThe level of protein expression of α-SMA,etc.extracted from abdominal aortic tissues and NOX2 from neutrophils were detected by Western blot.The mRNA levels of TNF-α,etc.extracted from abdominal aortic tissues were detected by qPCR.6.Metabolite detectionThe SCFAs from feccal and plasma samples were analyzed qualitatively and quantitatively by gas chromatography-mass spectrometry(GC-MS);The LAL method was adopted to detect endotoxin levels in AAA tissues.Results:1.R.intestinalis and sodium butyrate significantly alleviate pathological changes in AAA mice,such as neutrophil infiltration and NETs formation in the abdominal aorta,thereby improving AAA.PCR results showed that the abundance of R.intestinalis was significantly reduced in the intestine of AAA patients.Both R.intestinalis and sodium butyrate significantly reduced the AAA diameter and intestinal barrier damage of AAA mice,and significantly decreased the levels of LPS,TNF-α,IL-1β,IL-6,MCP-1,and inhibited the expression levels of MMP2/9 and the degradation of elastic fibers in the abdominal aorta.Meanwhile,they significantly upregulated the expression of α-SMA and SM22α and downregulated the expression of osteopontin and vimentin,thereby inhibiting the switch of VSMCs from contractile phenotype to the synthetic phenotype.In addition,they also significantly inhibited neutrophil infiltration and NETs formation in the abdominal aortic wall of AAA mice.2.TLR4 knockout does not affect the improvement of mouse AAA conditions by R.intestinalis and sodium butyrateTLR4 knockout mice showed a significant attenuation of AAA diameter and other pathological findings,suggesting that LPS promotes the development of AAA.However,even after blocking the effect of LPS,R.intestinalis and sodium butyrate still significantly reverse the adverse effect of AAA patients’ microbiota on the condition of CaCl2-induced TLR4-KO AAA mice,namely reduced the pathological changes of AAA such as AAA diameter,inflammatory factor levels,elastic fiber degradation and neutrophil infiltration,and NETs formation.This suggests that the protective effects of these two are not entirely dependent on the reduction of LPS.3.Sodium butyrate inhibits NETs formation by suppressing NOX2 expression in neutrophils,thereby alleviating AAAIn vitro,human peripheral blood neutrophils from healthy individuals were treated with LPS-O128 or TNF-α to mimic the effects of inflammatory conditions on NETs formation.The results showed that NOX2 inhibitor or sodium butyrate could significantly reduce NETs formation induced by LPS-O128 or TNF-α,and they also had an obvious synergistic effect.Sodium butyrate significantly reduced NOX2 expression level in the LPS-or TNF-α-induced neutrophils.In vivo,in CaCl2-induced AAA mice,R.intestinalis or sodium butyrate significantly reduced NOX2 expression level in neutrophils infiltrating in the abdominal aorta tissue of mice.NOX2 overexpression also significantly attenuated the effects of R.intestinalis and butyrate in alleviating AAA lesions,namely NOX2 overexpression aggravated AAA diameter,elastic fiber destruction,neutrophil infiltration and NETs formation.These results all suggested that sodium butyrate inhibited NETs formation under an inflammatory state by suppressing neutrophil NOX2 expression.Conclusion:1.R.intestinalis and butyrate can improve AAA by inhibiting neutrophil infiltration and NETs formation.2.Butyrate inhibits NETs formation by down-regulating the expression level of NOX2 in neutrophils.