| Background and ObjectiveCoronary heart disease(CHD)is the leading cause of mortality among all chronic diseases.The development of percutaneous coronary intervention(PCI)and advancement in medical therapy have reduced the occurrence of major adverse cardiovascular events(MACE),however,optimal prevention has not been achieved.The underlying mechanisms of acute coronary syndrome(ACS)developed from stable atherosclerosis need to be further investigated.Recent studies reported that gut microbiome is associated with various heart-metabolic diseases such as diabetes,metabolic syndrome,and atherosclerosis.Gut microbiota refers to flora of bacteria,viruses,archaea,protozoans,and fungi that coinhabit in human gut.Disruption of microbiome homeostasis can affect host metabolism and immunity.The effects of gut microbiota on immunity in ACS remains unclear.ACS is considered as a complex inflammation and immunomodulation process,with participation of adaptive immunity and inflammatory cytokines.Increasing body of evidences showed that the number of pro-inflammatory CD4+T cells subtype increased,and the imbalance between T helpers cells 17(Thl7)and CD4+CD25+regulatory T cells(Treg)occurred in ACS.We proposed a hypothesis that the effect of microbiome on inflammation in ACS is through the immunomodulation of CD4+T cells polarization.In addition,we studied the molecular mechanisms of dysregulation of CD4+T cells in ACS,which remain largely unknown.The overall objective of this study was to evaluate the association among microbiome,adaptive immunity,and the occurrence of ACS.The first part examined the associations of microbiome with inflammation and immunity in ACS using 16S rDNA Sequences.The second part evaluated the effects of metabolites on CD4+T cells polarization using liquid chromatography-mass spectrometry(LC-MS).And the third part,an in-vitro experiment,was designed to determine molecular pathway of Trimethylamine N-oxide(TMAO)on the polarization of CD4+T cells.MethodsFirst Part:From October 2017 to October 2018,we prospectively and consectively enrolled 100 patients with ACS,50 patients with stable angina(SCAD),and 50 subjects without coronary stenosis confirmed by coronary angiography(Control).The blood and fecal samples of all participants were collected.The gut microbiome profile was analyzed via 16S rDNA sequences and compared among the three groups.The frequencies of CD4+T cells subpopulaltion including Th17 and Treg were detected via flow cytometry.The association between the gut microbiome and Treg/Th17 ratio was examined.Second Part:40 patients were randomly selected with the ratio of 2:1:1 among the three groups.LC-MS was used to detect the plasma metabolites profile.The difference of the metabolites profile and its association with Treg/Th17 ratio was analyzed.Third Part:In vitro study,the effect of TMAO on the proliferation and differentiation of CD4+T cells was evaluated.First,the different concentrations of TMAO were used to stimulate the CD4+T cells to dectect whether TMAO has an effect on the viability of T cells.The concentrations were as follows:0?mol/l,50?mol/l,100?mol/l,200?mol/l,300?mol/l,600?mol/l,and 900?mol/l TMAO.Furthermore,Flow cytometry was used to dectect Treg and Th17 differentiation and ELISA was used to dectect the secretion level of Interleukin-10(IL-10)and Interleukin-17A(IL-17A),whiche are the representative cytokines in Treg and Th17 separately.Finally,Western blot was used to detect the expression of Forkshead box P3(Fox P3),Retinoid-related orphan nuclear receptor yt(RORyt),and the activation of Akt/mammalian target of rapamycin(mTOR)pathway.ResultsFirst Part:Compared with the SCAD group and Control group,the relative abundance of Blautia(0.13±0.11 vs.0.09±0.07 vs.0.06±0.03,P=0.011)and Bifidobacterium(0.12±0.1 vs.0.06±0.08 vs.0.02±0.01,P=0.02)was significantly lower in ACS group,while the Fusobacterium(0.0029±0.005 vs.0.0005±0.001 vs.0.0004±0.0005,P=0.022),Enterococcus(0.16±0.04 vs.0.02±0.005 vs.0.006±0.002,P=0.025),Weissela(0.13±0.04 vs.0.02±0.007 vs.0.006±0.003,P=0.03),and Klebsiella(0.042±0.02 vs.0.036±0.01 vs.0.01v0.004,P=0.04)were significantly higher.The imbalance of CD4+T cells was also observed in ACS group.The level of Treg(1.88±1.04%vs.2.9±1.35%vs.4.4±2.6%,P<0.001)was lower while Th17 higher(3.1±1.7%vs.1.9±0.7%vs.1.6±0.9%,P<0.001)in ACS group,which results in a significantly lower ratio of Treg/Th17.Furthermore,Treg/Th17 was negatively associated with the abundance of Enterococcus(r=-0.35,P=0.001),Weissela(r=-0.3,P=0.007)and Klebsiella(r=-0.34,P=0.001),and positively associated with Bifidobacterium(r=0.25,P=0.03).Second Part:The plasma metabolites profile in ACS group was significantly different from SCAD group and Control group.ACS patients had higher concentration of L-kynurenine(Kyn)and Trimethylamine N-oxide(TMAO)and lower concentration of Indole compared with SCAD and Control group.Kyn was partly and Indole was solely derived from the microbiome metabolism through tryptophan metabolism pathway,while TMAO was totally dependent on the microbiome metabolism.In the tryptophan pathway,Indole was positively correlated with Treg(r=0.61,P<0.001)and Treg/Th17(r=0.476,P=0.008),while Kyn was positively correlated with Th17(r=0.445,P=0.045),which suggesting an imbalance between the pro-and anti-inflammatory process in ACS.Besides,TMAO was positively correlated with Th17(r=0.491,P=0.03)and negatively correlated with Treg/Th17(r=-0.411,P=0.024),which may represent another pro-inflammatory mechanism in ACS.Third Part:TMAO reduced the dencity of Treg while enhanced the differentiation of Th17.Consistent with this trend,the expression of FoxP3 was reduced while RORyt was enhanced significantly.Furthermore,the enhancement of the phosphorylation of Akt and mTOR indicated TMAO may influence the imbalance of Treg/Thl7 via Akt/mTOR pathway.Conclusions1.Dysbiosis of gut microbiome in ACS was significantly associated with the imbalance of CD4+T cells subpopulations with increased Th17 and decreased Treg,which suggested that the effect of gut microbiome in ACS may be mediated by inflammation.2.The interaction between gut microbiome and host inflammation process is likely through the metabolites,among which Indole,L-kynurenine and TMAO were significantly associated with CD4+T cell subpopulations.3.The microbiome-derived metabolite TMAO significantly effected the polarization of CD4+T cells and increased the differentiation of CD4+T towards pro-inflammatory Th 17 cells.The underlying mechanism is likely the activation of Akt/mTOR pathway. |
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