The Features Of T Cell Clones And Regulation Of Vascular Endothelial Function By Let-7e In Atherosclerotic Lesions

1 IntroductionAtherosclerosis(AS)is characterized by the infiltration and accumulation of lipids and immune inflammatory cells in the artery wall.Immunoinflammatory abnormalities are pivotal factors in the pathogenesis of atherosclerosis,but the mechanism that triggers these changes has not yet been fully elucidated.T cells are the key regulators of adaptive immunity and inflammation and are closely related to the occurrence and progression of atherosclerosis.T cells are among the first cells to be recruited within the human atheroma.At the fatty streak stage prior to plaque formation,T cells have been detected in the lesions.Until recently,there were still debates on the detailed roles of T cells in atherosclerosis development.CD4+T-helper cells expressing the a(3 T cell receptor(TCR)have been mainly identified in human and murine arterial plaques.The development of atherosclerotic lesions is reduced in immunodeficient mice,and transferring CD4+T cells can reverse the atheroprotective effect of T cell defects.However,studies on the CD4-deficient ApoE-/-mice observed the opposite effect.Several other studies found no difference in atherosclerosis progression between immune-competent and immune-deficient mice fed a high-fat diet.The complexity and diversity of different T cell subsets/clones in the development of atherosclerosis may contribute to these controversies.These results indicated that the roles of T cells in AS development are much more complex than we originally thought.Thus,revealing the composition and profiling of T cell clones in patients with AS at a high-resolution may provide information that could help elucidate the abovementioned complicated questions.The T cell immune repertoire includes all the different subtypes of T cell clones within a certain scope,and its diversity and compositional characteristics directly reflect the state of the immune response.The T cell immune repertoire is significantly altered in many diseases.Thus,immune repertoire sequencing(IR-Seq)based on NGS has been widely used in the study of immune repertoire.This technology can simultaneously identify millions of different T or B cell clones at single-base resolution,which makes it possible to accurately evaluate the diversity of the immune system.The present study contributed to the understanding of the roles of T cells in the development of atherosclerotic cardiovascular disease and the mechanism of the abnormal immune/inflammatory response in atherosclerosis.2 Objectives(1)To analyze the composition and molecular characteristics of the TCR?repertoire in the peripheral blood and plaques of patients with atherosclerosis,including clonotype frequency,CDR3 length distribution,CDR3 polymorphism and V/D/J gene utilization using the powerful IR-Seq technology.(2)To reveal the features and drift of T cell clones in the peripheral blood and plaques of patients with AS compared with normal subjects.3 Methods3.1 Tissue/blood samplesAll coronary artery tissue samples were collected from normal autopsy specimens and specimens with atherosclerotic plaques.Samples of peripheral blood(5 mL each)from 55 patients who met the diagnostic criteria of acute coronary syndrome(ACS)were obtained by venipuncture.Peripheral blood samples from56 healthy subjects were obtained as controls.Peripheral blood mononuclear cells(PBMCs)were isolated from the peripheral blood by Ficoll density gradient centrifugation.3.2 Genomic DNA isolationThe genomic DNA of the PBMCs and tissues was purified using a genomic DNA extraction kit.The same amount of gDNA from each sample in the corresponding group was mixed and comprised the sample pools of ACS blood,normal blood and atherosclerotic plaques.3.3 multiplex polymerase chain reactionsThe rearranged TCR-? CDR3 regions were amplified from genomic DNA by multiplex polymerase chain reaction(PCR)with a specifically designed set of primers for the TCR? CDR3 V and J regions.3.4 High-throughput sequencingThe products of the multiplex PCR were purified.Then,the DNA fragments were subjected to library construction.Subsequently,the library quality(insert fragment length and library concentration)was evaluated by the Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System.After bridge PCR amplification,the pair-end 101-bp sequencing was performed on Hiseq2000 sequencing platform.3.5 Bioinformatics AnalysisThe raw sequencing data were filtered according to the strict criteria.After filtration,sequence alignment was performed using miXCR,which can adjust the data errors introduced by sequencing and PCR and identify different CDR3 sequences with expression information.This software also provided the amino acid sequences of the CDR3 regions,and the numbers and frequencies of the corresponding readings,indels,and V,D and J genes.Other analyses,such as frequency interval distribution,cumulative frequency distribution and VDJ utilization,were conducted using homemade scripts and Microsoft Excel software.The diversity index,including Shannon's index,Simpson's index,and the Berger-Parker index,was calculated using the diversity calculator from the BPMSG website.3.6 Real-time PCRThe purified gDNA from each group was quantified by a Nanodrop 2000.Then,real-time PCR was performed using SYBR Green Master Mix on a Bio-Rad CFX96.?-actin was used as an internal control.3.7 Statistical AnalysisThe data were presented as the mean+SEM and compared using two-tailed Student's t-tests or one-way analysis of variance with appropriate post-tests.P<0.05 was considered statistically significant.The statistical analyses were conducted with GraphPad Prism software and Excel.4 Results4.1The abnormal types and frequency distributions of T cell clones in patients with ASThe unique clonotypes of the T cells at the amino acid and nucleotide levels were significantly higher in the peripheral blood of healthy subjects than in the peripheral blood and plaques of patients with ACS.Moreover,the sum of the frequency of the top 1000 T cell clones in AS plaques was significantly higher than that in the peripheral blood of both normal subjects and patients with ACS.The number of T cell clones in AS plaques was significantly higher than that in the peripheral blood of normal subjects and patients with ACS in certain frequency intervals(0.1-0.4%).Moreover,fewer T cell clones had a higher cumulative frequency in the AS plaques.4.2 T cell clone lengths at the nucleic acid and amino acid levelsAt the amino acid and nucleotide levels,the CDR3 length of the common clones in all three groups showed a normal distribution.The frequencies of the common T cell clones with 11-16 amino acids were significantly higher in the AS plaques than in the peripheral blood of the normal subjects and patients with ACS.Moreover,the frequencies of common T cell clones with certain nucleotide lengths in the AS plaques were significantly higher than those in the other groups.4.3 The distribution characteristics of common clonesThe frequency and number of common T cell clones in the peripheral blood of normal males and female were similar and significantly higher than those in the peripheral blood of the corresponding gender of the patients with ACS.Moreover,the frequency distribution and numbers of common T cell clones in the peripheral blood of male patients with ACS were significantly different from those of female patients with ACS.Furthermore,the common T cell clones in the plaques of male patients with AS were significantly elevated.4.4The analysis of open reading frame of the T cell clonesThe proportion of in-frame T cell clones in the AS plaques was significantly higher than that in the peripheral blood of normal subjects and patients with ACS,and the proportion of out-of-frame T cell clones in the AS plaques was lower than that in the other groups.4.5 insertion and amino acid utilization of the T cell clonesThe present study showed that there was no significant difference in the nucleotide insertion in the TCR CDR3 regions of the T cell clones among all groups.The lengths of the TCR CDR3 insertions of the T cells in all groups were mainly<20 nucleotides.There were no significant differences in the utilization rates of 20 common amino acids in the TCR proteins among the individuals of all three groups.4.6 The diversity index of T cell clonesThe diversity indexes(Shannon entropy,Simpson dominance and the Berger-Parker index)of the T cell clones were significantly decreased in the AS plaques.4.7 The significant change in VDJ gene utilization of the T cell clones in the AS plaquesThe utilization of the V-J genes of the T cell repertoire in the plaques was significantly reduced.The high-frequency T cell clones in the AS plaques was significantly elevated compared to that in the other two groups,such as V20-1-J2-7 and V20-1-J2-6.The utilization rates of some V genes(such as TRBV4-1,TRBV5-4,TRBV12-4)and J genes(such as TRBJ2-5,TRBJ2-1,TRBJ1-6)in the AS plaques were significantly different from those in other two groups.4.8 PCR verification of the significantly expanded T cell clones in the AS plaquesThe result showed that the contents or frequencies of the V29-1 J2-1,V20-1 J1-6,V6-3 J2-7 and V11-2 J2-2 clones in the peripheral blood of patients with ACS were significantly higher than that of normal subjects using real-time PCR.5 Conclusion(1)The type and diversity of T cell clones in the plaques of patients with AS were significantly reduced compared to those in the peripheral blood of normal subjects and ACS patients.(2)The frequencies of the TCR clones with various lengths of the CDR3 regions were significantly different among the three groups.The common clones(especially the high-frequency common clones)in the AS plaques were elevated compared to those in the other two groups.(3)The utilization of the V and/or J genes in the T cell clones in the AS plaques was significantly decreased1 BackgroundAtherosclerosis(AS)is the main pathological basis of severe cardiovascular and cerebrovascular events such as acute myocardial infarction,and characterized by the progressive infiltration and accumulation of lipids,inflammatory cell infiltration and fibrous tissue hyperplasia in the artery wall.The pathogenesis of AS is very complex and has not yet been fully elucidated,but a large number of studies have shown that oxidized low-density lipoprotein(ox-LDL)is one of the major substances that trigger vascular inflammatory response,causing vascular endothelial injury and initiating AS.The mechanism of ox-LDL causing endothelial injury involved complex intracellular and intercellular signal transmission,including:1)ox-LDL activated T cells and other immunoinflammatory cells,and indirectly injured endothelial cells through cytotoxic effects or secreted cytokines;2)ox-LDL directly inhibited EC proliferation and migration to induce apoptosis,resulting in vascular endothelial injury after cellular uptake.3)ox-LDL could induce endothelial cell to secret inflammatory factors and adhesion molecules,resulting in endothelial injury through attracting immunoinflammatory cells.The role of ox-LDL in a single cell has been fully studied,but its effects and mechanisms in signal transduction between different cells have not been fully elucidated until now.The study contributed to find Intercellular signaling molecules and reveal the effect and mechanism of ox-LDL on communication between immune cell and endothelial cell,which contributed to the understanding of the role of ox-LDL and immune inflammation in the development of AS and provided a theoretical basis for finding effective interventions.Various hormones,cytokines,and vasoactive substances were important messenger molecules of intercellular communication.They were either directly secreted extracellularly,or transported in cell membrane structure such as microparticle and exosome,which transmitted signals and regulated the target cells function.In addition to the classical protein molecules,there were stable miRNA molecules in the extracellular environment(in plasma or serum is circulating miRNA).These extracellular miRNAs are a new class of signaling molecules with intercellular communication function that can play a regulatory role in autocrine/paracrine mode and also regulate the target cell function by circulating to distant target cells,which is a common and important way of intercellular communication.Some studies suggested that circulating miRNAs were mainly transported to outer membrane structure such as exosome and microparticle but several other studies found that circulating miRNAs were transported in the form of nucleic acid protein complex.Recent studies have shown that the transport ways of circulating miRNAs may be related to the sort of miRNA,the source of the cell and the course of the disease.But the questions about how the circulating miRNA recognized and bound to the target cell,and what was the difference and significance in the course of the disease were still unclear.These controversies and questions indicated that we had little understanding about the functions and mechanisms of circulating miRNAs as new intercellular signaling molecule.Depth study of the role and mechanism of circulating miRNA in the development of AS is helpful for us to find new biomaker of AS and therapy the clinical disease.2 Objectives(1)To find Intercellular signaling molecules and demonstrate the RNA composition of exsome secreted by CD4 + T cells using next generation sequencing;(2)To study the role of let-7e released from exosome in the regulation of HUVECs function and reveal the effect and mechanism of ox-LDL on communication between immune cell and endothelial cell.3 Methods3.1 CD4+T cell isolation and HUVECs culturePeripheral blood were collected from patients with ACS,PBMCs were isolated by Ficoll density gradient centrifugation,and high purity CD4 + T cells were isolated by magnetic beads.HUVECs were purchased from American Type Culture Collection(ATCC)and cultured in recommended conditions.3.2 Extraction of exosome from CD4 + T cellsHUVECs were treated with ox-LDL and then culture medium was harvested at 48h after treatment.The exosome precipitation was collected by Ultracentrifugation.3.3 RT-PCRTotal RNAs were prepared and quantified.The gene expression levels of let-7e were quantitatively analyzed by RT-PCR.U6 was used as an internal control.3.4 Western blotWe extracted total proteins from exosome precipitation.The expression levels of target proteins were determined by western blot assay.3.5 High-throughput sequencing Bioinformatics AnalysisTotal RNA was isolated from exosome and then,the fragments were subjected to library construction.Subsequently,the library quality(insert fragment length and library concentration)was evaluated by the Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System.After bridge PCR amplification,the pair-end 151-bp sequencing was performed on Hiseq2000 sequencing platform.The raw sequencing data were filtered according to the strict criteria.After filtration,sequence alignment was performed using Bowtie,the software could identify different RNA sequences with expression information.3.6 Immunofluorescence and apoptosis detectionFor uptake assays,HUVECs were co-cultured with exosome labeled with the dye PKH-26.The apoptosis of HUVECs was detected by Flow cytometry at 24h.4 Results4.1 The sorting purity of CD4 + T cellsAfter the CD4 + T cells were sorted by magnetic beads,the purity of CD4 +T cells was up to 98%by flow cytometry.The high purity cells were beneficial to the next experiment.4.2 exosome secretion and identificationCD4+T cells were treatment with ox-LDL which could promote the secretion of exosome into the culture medium.The exosome extracted from culture medium were detected by Electron microscopy in between 50-100nm.morever,the CD63 protein was detected in exosome by Western blot.4.3 The analysis results of exosome RNA sequencing dataSequencing results showed that exosome contained not only a large number of miRNAs,but also included many mRNAs and IncRNAs.The results of functional enrichment analysis of mRNA genes demonstrated that exosome may be involved in many biological processes,including cell apoptosis,proliferation,immune inflammation and so on.4.4 Let-7e in exosome released from CD4+T cellsRNA sequencing results revealed that exosome released from CD4 + T contained miRNAs,including let-7e which was highly expressed.Let-7e was significantly higher in CD4+T cells from AS patients than that in CD4+T cells from normal subjects using RT-PCR.Moreover,compared with CD4+T cells without ox-LDL treatment,we found the expression level of let-7e secreted by exosome was increased in CD4+T cells treated with ox-LDL.These results indicated that ox-LDL treatment can induced CD4 + T cells to secrete let-7e in the way of exosome.4.5 Endothelial cells was studied by examining the uptake of isolated exosomesHUVECs were co-cultured with exosome extracted from CD4+T cells.The results showed that the exosome secreted by CD4+T cells could be taken in HUVECs and promoted the apoptosis of HUVECs after treatment with exosome from ox-LDL-induced CD4+T cells.5 Conclusion(1)CD4 + T cells secreted exosome,which contained not only a large number of miRNAs including let-7e,but also many mRNAs and IncRNAs,into culture medium or plasma.(2)The expression of let-7e in exosome was increased in CD4+T cells treated with ox-LDL(3)The exosome released from CD4+T cells could transfer let-7e to HUVECs and affect the biological process of cells.1 BackgroundNumerous studies have demonstrated that vascular endothelial cells(ECs)are crucial in the regulation of immune and inflammatory responses.In ECs activated by inflammatory stimuli,many adhesion molecules and pro-inflammatory cytokines are up-regulated(such as ICAM1,E-selectin and IL-6)with the suppression of anti-inflammatory cytokines.The endothelial dysfunction caused by excessive and/or prolonged inflammation is one of the most important initiating events in the development of atherosclerosis and many other cardio-cerebrovascular diseases.Lots of cells and molecules are involved in the inflammatory response in ECs.Therefore,its regulation mechanisms are extremely complex and have not been fully elucidated so far.Many studies have demonstrated miRNAs as key regulators of inflammation in ECs.For example,miR-146a and miR-10a can repress inflammation by targeting several components(TRAF6/IRAK4)of the NF-kB pathway in ECs.MiR-92a promotes inflammation in ECs by targeting KLF2/KLF4 which inhibits the expression of NF-kB dependent vascular inflammatory genes.Additionally,miRNAs also directly target various adhesion molecules to play anti-inflammatory roles in activated ECs,such as miR-31 targets SELE and miR-17-3p targets ICAM1.Moreover,numerous miRNAs involved in endothelial inflammation play critical roles in many major diseases such as atherosclerosis and diabetes.Therefore,further studies are deserved to reveal more miRNAs and their action mechanisms involved in the inflammatory response of endothelial cells.The let-7 family can act as anti-inflammatory factors by targeting some pro-inflammatory factors(e.g.,let-7d targets IL-13;let-7a targets IL-6).These molecules can also act as pro-inflammatory factors by targeting some anti-inflammatory factors,including IL-10.Additionally,the let-7 family is closely associated with endothelial function,including apoptosis,angiogenesis and the endothelial-to-mesenchymal transition.Let-7e is a member of the let-7 family and also a key regulator of endothelial function and inflammation.Furthermore,let-7e expression is significantly increased in many cardiovascular diseases,including coronary heart disease.Therefore,we speculated that let-7e might perform critical roles in the regulation of the inflammatory responses of endothelial cells by directly or indirectly targeting certain inflammatory genes.However,studies of this hypothesis have not been reported until recently.2 Objective(1)To demonstrate the biological process of let-7e in endothelial cells;(2)To reveal the mechanism of let-7e regulation of endothelial function and inflammatory response.3.Methods3.1 Cell culture and treatmentHUVECs were purchased from American Type Culture Collection(ATCC)and cultured in recommended conditions.HUVECs were treated with ox-LDL(50 ug/ml)and then were harvested at 12h,24h and 48h after treatment.3.2 vectors and treatmentSiRNA and pCMV6 vectors of IkB? and wide/mutant Inc-MKI67IP-3 were obtained from GenePharma or Origene.The mimic and inhibitor of let-7e and corresponding negative controls(NC)were bought from GenePharma.The mimic/inhibitor/NC of let-7e,siRNA or different vectors,respectively,were transfected into HUVECs using lipofectamine RNAIMAX or 3000 reagent.3.3 RNA isolationTotal RNAs were isolated from HUVECs transfected with let-7e mimic or inhibitor and corresponding NC using Trizol reagent and were quantified by Nanodrop 2000 spectrophotometer and Qubit 3.0 fluorometer.3.4 MicroarrayTotal RNA were labeled with cy3 and hybridized to Agilent human LncRNA 4 x 180K microarray.Expression data of mRNAs and IncRNAs were filtered and normalized,and then the IncRNAs and mRNAs regulated by let-7e were identified using GeneSpring GX 11.5.3.5 Bioinformatics Analysis3.5.1 Enrichment analysisThe functional enrichment analysis was carried out using the Database for Annotation,Visualization and Integrated Discovery(DAVID)v6.7.3.5.2 Prediction of potential targets of let-7eLet-7e targets were predicated by Targetscan,MicroT-CDS and miRanda.The transcripts,which were oppositely expressed in let-7e mimic groups and let-7e inhibitor groups,were selected from the intersection of targets predicted by the above tools and they were regarded as potential targets of let-7e in HUVECs.3.5.3 Construction and analysis of co-expression/correlation networkBased on the predicted targets of let-7e,the co-expression/correlation network of IncRNAs potentially binding to let-7e and mRNAs potentially targeted by let-7e was constructed using CoExpress v1.5(Pearson correlation coefficient>0.99,P<0.05).Then,its topological structure and core subnetworks were analyzed using Cytoscape software.3.6 Real-time PCRTotal RNAs were prepared and quantified.The gene expression levels of let-7e,pri-let-7e and mRNAs were quantitatively analyzed by RT-PCR.3.7 ELISAThe collected culture medium was used for the assay of ICAM1,VCAM1,SELE,SELP and IL-6 by ELISA kits3.8 Western blotWe extracted Total,cytoplasmic and nuclear proteins from cultured cells.The expression levels of target proteins were determined by western blot assay.3.9 Luciferase reporter assayThe luciferase reporter vectors were constructed by cloning the target/binding sequence(or their mutants)of let-7e in IkB? 3'-UTR or Inc-MKI67IP-3 into pmirGLO Dual-Luciferase miRNA target expression vector.HUVECs,respectively,were transfected with these vectors and let-7e mimic/inhibitor or corresponding NC.The firefly and renilla luciferase activity of cell lysates was assayed using Dual-Luciferase Reporter Assay System.3.10 Statistical AnalysisP values in multiple testing were calibrated using Benjamini-Hochberg correction.Person correlation coefficients and P values were calculated using house-made scripts.Data were presented as mean+SEM and compared using two-tailed Student's t-tests or one-way analysis of variance with appropriate post-tests.P<0.05 was considered statistically significant.R package was used for all calculations.4 Results4.1 Expression profiles of mRNAs/IncRNAs regulated by let-7e and Enrichment AnalysisMicroarray analysis was performed and the results showed that let-7e profoundly affected the expression of mRNAs and IncRNAs.385 mRNAs potentially targeted by let-7e and 102 IncRNAs potentially binding to let-7e were selected and hierarchically clustered.The inhibitory effects of let-7e were very pronounced,whereas the effects of let-7e inhibitor were counter to it.The gene set enrichment analysis of these 487 transcripts showed that let-7e was involved in many important biological processes and pathways,such as immune,cell adhesion,VEGF pathway,NF-kB/MAPK pathway.Most pathways were closely associated with cell proliferation,apoptosis and inflammatory response.4.2 Co-expression/correlation network potentially targeted by let-7eThe co-expression/correlation network was constructed based on the expression data of the above mentioned 487 transcripts.The topological analysis of this large network revealed 26 essential nodes,including NFKBIB(IkB?),MAPK1,DNMT3A and TP53BP1.They were involved in the regulation of inflammation,proliferation and apoptosis.The subnetwork constituted by nodes that were significantly co-expressed with IkBp.Among these nodes,IkB? and Inc-MKI67IP-3 were most significantly regulated.4.3 Pro-inflammatory roles of let-7e in inflammation of HUVECsThe microarray data suggested that let-7e could up-regulate the expression of many crucial pro-inflammatory cytokines and adhesion molecules in HUVECs,such as IL-1B,IL-6,ICAM1,VCAM1,SELE and SELP,while let-7e inhibitor down-regulated them.Moreover,most of them were regulated by NF-kB.let-7e mimic could significantly increase their secretion and mRNA expression,while the effects of let-7e inhibitor were not significant.4.4 Let-7e exerted pro-inflammatory effects by targeting IkB? and promoting NF-kB translocationlet-7e mimic significantly reduced IkB? expression at mRNA and protein levels in HUVECs,but let-7e inhibitor acted conversely.The luciferase reporter assay also showed that let-7e mimic significantly reduced the relative luciferase activity in HUVECs transfected with Luc-IkB?-WT.Let-7e significantly down-regulated IkB? and increased the translocation of NF-kB into the nucleus.4.5 Lnc-MKI67IP-3 inhibited the pro-inflammatory effects of let-7e by functioning as a ceRNAThe predicted results indicated that let-7e could bind to Inc-MKI67IP-3.Luciferase reporter assay showed that only let-7e mimic significantly reduced the relative luciferase activity in HUVECs transfected with luc-Inc-MKI67IP-3-wt.Inc-MKI67IP-3-wt vector could attenuate the let-7e action of inhibiting IkB? expression and significantly increase the expression of IkB? mRNA and protein.Moreover,let-7e mimic significantly down-regulated Inc-MKI67IP-3 expression,while let-7e inhibitor markedly up-regulated it.4.6 Let-7e was up-regulated in ox-LDL-treated HUVECs and atherosclerotic plaquesIn HUVECs treated with oxLDL for different times,ox-LDL significantly up-regulated let-7e and pri-let-7e expression,while expressions of IkB? and Inc-MKI67IP-3 were significantly down-regulated.Moreover,expressions of let-7e,IkB? and Inc-MKI67IP-3 in human atherosclerosis plaques were similar to that in oxLDL-treated HUVECs.5 conclusion(1)By inhibiting its target gene IkB?,let-7e could promote NF-kB translocation to the nucleus and activation,enhancing inflammatory responses in ECs.(2)The study revealed let-7e as a pro-inflammatory mediator and a novel regulatory mechanism for NF-kB pathway through ceRNA crosstalk,consisting of let-7e and its target IkB? and ceRNA Inc-MKI67IP-3(3)Inc-MKI67IP-3 could antagonize the inhibitory effect of let-7e on IkB? as a ceRNA.Moreover,let-7e could down-regulate Inc-MKI67IP-3,forming a positive feedback to aggravate inflammation.(4)let-7e,Inc-MKI67IP-3 and IkB? were abnormal in oxLDL-treated HUVECs and atherosclerotic plaques.