The Study On The Effect Of Interleukin 4 Induced 1(IL4IL) On The T Cell Function In HIV Infection And Related Mechanism

Objective:Human immunodeficiency virus(HIV)can destroy the body’s immune system and cause a series of immunodeficiency-related symptoms,eventually developing into acquired immunodeficiency syndrome(AIDS).The application of antiretroviral therapy(ART)has dramatically reduced the morbidity and mortality of HIV infections.However,HIV infection still brings a huge burden to global health due to the complex transmission and poor immune reconstruction after treatment.Therefore,exploring the mechanism affecting the progression and prognosis of HIV infection is of great significance for the control and treatment of HIV infection.As one of the most important immune cells in the body,the changes of T cell number and function have an important impact on the disease process of HIV infection.HIV-specific CD8+T cell response can effectively prevent the spread of HIV-infected cells and control the occurrence of HIV viremia.At the same time,CD4+T can activate helper CD8+T cells after HIV infection.The number and function of CD4+T cells play a vital role in the position of CD8+T cells.Many studies have pointed out that persistent infection of HIV would lead to T cell exhaustion,impaired T cell proliferation and cytolytic potential.And these deficiencies of T cells in HIV infection can not be fully reversed by ART treatment.The dysfunction of T cells is closely related to the disease progression of HIV infection and immune reconstruction after therapy.In addition,studies also pointed out that T cell dysfunction caused by HIV infection was related to the occurrence of immune remodeling inflammatory syndrome(IRIS).T cell dysfunction in HIV infection is related to persistent antigen stimulation,chronic inflammation,cell-intrinsic defects,and so on.But the underlying mechanism still needs to be further explored.Therefore,a comprehensive understanding of the influencing factors and related mechanisms of the changes in the number and function of T cells after HIV infection is an essential basis for controlling HIV infection and improving prognosis.IL4I1 is a secretory L-phenylalanine oxidase produced by myeloid and antigen-presenting cells and helper T cell(Th)17 cells.The studies have shown that IL4I1 participates in the fine regulation of B cell and T cell acquired immune response.IL4I1 plays an essential role in the acquired immune response of mice and humans in promoting immature CD4+T cells differentiate into regulatory T cells and inhibiting T cell proliferation and cytokine production.However,the effect and related mechanism of IL4I1 on HIV-infected T cells have not been reported yet.A full understanding of the relationship between HIV infection,IL4IL,and T cells can provide new ideas and targets for further improving the disease control and prognosis of HIV infection.Methods:1.Patient selectionIn this study,32 HIV-negative controls(NC),42 HIV-infected persons who received ART treatment(ART),and 34 ART-naive HIV-infected persons(HIV)were recruited for PCR and ELISA assays to explore the relationship between IL4I1 and HIV infection.Subsequently,we collected 50 ART people to analyze the effect of IL4I1 on the function of HIV-infected T cells,12 ART people for transcriptome data collection.Finally,we collected 60 samples from the ART population to analyze the mechanism of the effect of IL4I1 on the function of T cells infected by HIV.The participants in this study were all from the first affiliated Hospital of China Medical University.Before the sample collection,the subjects were informed of the experimental aim of the sample and signed the informed consent form.This study has been approved by the Ethics Committee of China Medical University.2.Peripheral blood mononuclear cells(PBMC)separation and T cell sorting peripheral blood mononuclear cells(PBMC)were isolated by density gradient centrifugation.T cells were sorted using STEMCELL negative sorting kit with magnetic poles,and the sorting purity was more than 96%.3.Flow cytometry of intracellular cytokineThe sorted T cells were cultured with CD3/28 dynabeads for 24h.GolgiStop was added at 18h.After T cell culture,using LIVE/DEADTM Fixable Aqua Dead Cell Stain Kit to avoid the effect of dead cells,and then CD4-APC-Cy7 and CD8-PerCP-Cy5.5 were added to mark the surface of the cells.After the cells were permeated with Cytofix/Cytoperm Soln Kit,we added 3μLintracellular cytokine-related flow cytometry antibodies for intracellular staining.Data analysis is carried out on Flowjo v.10.3.4.Apoptosis analysisAfter 48h of stimulation,we first mark cells with CD4-APC-Cy7 and CD8-FITC.After washing the cells,the apoptotic cells were labeled with AnnexinV and 7AAD dyes in the presence of Binding Buffer.The cells were detected on the BD LSRII flow cytometer within 1h after the stain.5.Cell proliferation analysisThe negatively selected cells were washed and re-suspended in PBS.After labeling with Cell Trace Violet(CTV)at a final concentration of 5 μM,the cells were re-suspended in R10 medium and stimulated with CD3/28 dynabeads.The cells were cultured at 37℃,5%CO2 incubator for 120h.After the culture,the cell proliferation capacity was detected on the BD LSRII flow cytometer.6.Cell RNA extraction and quantitative real-time PCRThe total RNA in the cell was extracted with the RNeasy Plus Mini Kit kit.The reverse transcription and PCR amplification of RNA were performed according to the reagent instructions of PrimeScript RT reagent Kit and TB Green ?Premix Ex Taq Ⅱ of TaKaRa Company.The results were calculated by 2-ΔΔCT.7.Enzyme Linked Immunosorbent Assay(ELISA)The culture supernatant was collected,and the content of IL4I1 in the culture supernatant was detected by Human IL4I1 DuoSet ELISA kit.The absorbance detection wavelength range was 450nm,and the concentration IL4I1 was calculated by a four-parameter fitting curve.8.Mitochondrial respiration and glycolysisAccording to the manufacture instructions,we detected the function of mitochondrial respiration and glycolysis with XFp Cell Mito Stress Test Kit and XFp Glycolysis Stress Test Kit on the Seahorse XF HS Mini analyzer.The drug concentrations used in the experiment were 1.5 μM oligomycin(Oligo),0.5 μM mitochondrial oxidative phosphate uncoupling agent(FCCP),0.5 μM rotenone/antimycin A(R&aa),10mM glucose and 50mM 2-deoxyglucose(2-DG).9.Flow cytometric detection of cell mitochondrial related markersThe cells’ mitochondrial mass and membrane potential were labeled with 50nM MitoTracker Green FM and 25nM MitoTracker Orange CMTMRos in the culture medium at 37℃ for 30 minutes,respectively.After incubation,cells were washed with pre-cooled PBS three times,and the LIVE/DEADTM Fixable Aqua Dead Cell Stain Kit was used to avoid the effect of dead cells.10.The detection of reactive oxygen species(ROS)After the cells were treated,the reactive oxygen species produced by the cells were stained with CellROX ?GreenReagent reagent.Adding 0.4μLCellROX ?GreenReagent to the 96-well plate culture medium,the cells were incubated at 37℃ for 30 minutes.After incubation,the cells were re-suspended in PBS and detected by flow cytometry.11.Data enrichment analysis and drawingThe differentially expressed genes were filtered according to the P-value and the logFC.The enrichment analysis was conducted by gene ontology(Gene Ontology,GO)and KEGG signal pathway on the DAVID website(https://david.ncifcrf.gov),and the enrichment results of GO and KEGG were visualized by image GP(http://www.ehbio.com/ImageGP/index.php/Home/Index/index.html).12.Protein-protein interaction network analysis(PPI)Protein-protein interaction network analysis(PPI)was analyzed on STRING data platform(https://cn.string-db.org/).The data obtained from STRING were imported into Cytoscape to draw the protein interaction network.The hub genes in the protein interaction network were screened by MCODE and cytoHubba plugins in Cytoscape.13.GSEA gene set enrichment analysisAccording to the standard requirements of GSEA software,data of gene expression matrix in the different groups were imported into GSEA4.1.0 software to set enrichment regulation according to the experimental needs,selected the database used for enrichment,and ran the software for enrichment analysis.14.Statistical analysisMann Whitney test was used to analyze the difference of IL4I1 expression and secretion among different groups.The Spearman correlation coefficient analyzed the correlation between IL4I1 and CD4 count and viral load.The paired t-test analyzed the effect of IL4I1 on cell function and the related mechanism.All statistical analyses and drawings are carried out on GraphPad Prism v8.0 software.The data of each group were expressed in terms of Mean ±SD.P<0.05 was considered to be statistically significant.*for P<0.05,**for P<0.01,***for P<0.001.Results:1.The expression and secretion of IL4I1 in HIV-infected patients and immune non-responders increased,and it was related to the disease progression of HIV infectionGEO data and PCR results showed that the expression of IL4I1 was up-regulated after HIV infection.ELISA data also showed that the cells of HIV infection and immune non-responders had a stronger ability to secrete IL4I1.By analyzing the correlation between the expression and secretion of IL4I1 and the CD4 count and viral load of HIV-infected patients,it was found that IL4I1 was negatively correlated with CD4 count and positively correlated with viral load.2.IL4I1 promotes the apoptosis and dysfunction of T cells in HIV infectionsIt was confirmed by cell experiments in vitro that IL4I1 could significantly promote the apoptosis of T cells in HIV-infected patients and inhibit the secretion of IL2,IFN-y,and other effector factors in CD4+T cells infected by HIV.At the same time,it affected the cytotoxic function by down-regulating the secretion of granzyme B and perforin in CD8+T cells.3.IL4I1 can significantly affect the gene expression profile of CD8+T cells in HIV infectionsThe differentially expressed genes from RNA-seq were obtained by setting the filter condition of P<0.05,FC>1.2.After the screening,97 differentially expressed genes were obtained in CD4+T cells,including 29 up-regulated and 68 down-regulated genes.In CD8+T cells,there were 255 down-regulated and 420 up-regulated,with a total of 675 differentially expressed genes.Based on the unsupervised hierarchical clustering analysis of the differential genes,we find that the cell samples of different treatments are clustered into a group first,which shows the similarity of the data.This phenomenon is more evident in the results of CD8+T cells.The results showed that the treatment of IL4I1 significantly changed the gene expression profile of CD8+T cells.4.IL4I1 down-regulates oxidative phosphorylation of CD8+T cells in HIV infectionsThe differential genes obtained in CD8+T cells were analyzed by gene ontology(GO)and KEGG signal pathways.The results showed that the up-regulated genes obtained by IL4I1-treated CD8+T cells were mainly concentrated in the biological processes of apoptosis and senescence.In contrast,the down-regulated genes were enriched in the electron transport chain,oxidative phosphorylation,and other signal pathways.The oxygen consumption rate(Oxygen Consumption Rate,OCR)was measured by Seahorse cell energy metabolism analyzer combined with Cell Mito Stress Test Kit to reflect the level of oxidative phosphorylation of cells.The results showed that the level of oxidative phosphorylation of cells decreased after the addition of IL4I1.Combined with the enrichment analysis results of GO_BP and KEGG,we believe that IL4I1 may affect the oxidative phosphorylation of HIV-infected T cells and then affect the function and apoptosis of T cells.5.Inhibition of oxidative phosphorylation can promote apoptosis and functional dysfunction of CD8+T cells in HIV infectionsInhibit pyruvate kinase(PK)with sodium oxalate(OXA)to down-regulate oxidative phosphorylation.Observe the effect of down-regulation of oxidative phosphorylation on cell function.The results showed that the apoptosis percentage of CD8+T cells increased significantly after the addition of sodium oxalate.The secretion of perforin,granzyme B,and IFN-γ in CD8+T cells were also inhibited.6.IL4I1 down-regulated the expression of genes related encoding mitochondrial proteinsThe protein interaction network of differential genes in CD8+T cells showed that mitochondrial-related proteins had a more vigorous interaction index.The results of GSEA enrichment analysis showed that multiple mitochondrial-related signaling pathways were enriched in the cell groups without IL4I1 treatment.Combined protein interaction network analysis and GSEA enrichment results showed that IL41 down-regulated the expression of related genes encoding mitochondrial proteins.7.IL4I1 increases mitochondrial mass and the production of reactive oxygen speciesThe mitochondrial mass(MM)and mitochondrial membrane potential(MMP)were detected by MitoTracker Green FM and MitoTracker Orange.The results showed that mitochondria mass(MM)increased after IL4I1 treatment.At the same time,CellROX?Green reagent was used to detect reactive oxygen species(ROS)production.The results show that IL4I1 can increase ROS generation.8.IL4I1 increased mitochondrial depolarization and down-regulated the expression of mitochondrial transcription factor A in CD8+T cells in HIV infectionsWhen mitochondria are damaged,there will be a group of cells with high expression of mitochondrial mass(MM)and low expression of mitochondrial membrane potential(MMP),called depolarized mitochondria(MMPlow/MMhigh).This group of mitochondria usually means the damage of mitochondria and the decrease of mitochondrial activity per unit mass.Our results show that IL4I1 can significantly increase the proportion of depolarized mitochondria.At the same time,mitochondrial transcription factor A(TFAM),as a mitochondrial DNA transcription factor,was significantly down-regulated after IL4I1 treatment.9.Inhibition of mitochondrial electron transport chain can down-regulate the effect of CD8+T cells in HIV infectionsThe inhibitor of the electron transport chain complex was added to the experimental system to interfere with the energy supply of mitochondria and detect its effect on cell function.The results showed that after the addition of oligomycin(Oligo),an inhibitor of mitochondrial electron transport respiratory complex V,doxorubicin(antimycin A),rotenone(rotenone),and metformin(metformin),the inhibitor of mitochondrial electron transport complex III and I,the secretion of effector factors of CD8+T cells in HIV infections was significantly decreased,indicating that the decrease of the activity of mitochondrial electron transport chain would damage the effect function of CD8+T cells in HIV infections.It is further confirmed that IL4I1 down-regulates oxidative phosphorylation by damaging mitochondria,which leads to the dysfunction of CD8+T cells in HIV infections.10.IL41 enhanced the phosphorylation level of the p38 MAPK signaling pathway of CD8+T cells in HIV infectionsThe phosphorylation of the Akt,mTOR,and MAPK signal pathway was detected by flow cytometry.The results showed that IL4I1 could significantly up-regulate the phosphorylation level of the p38 MAPK signal pathway but had no significant effect on the phosphorylation of Akt,mTOR,and S6.According to the above results,we believe that IL4I1 causes mitochondrial damage through the p38 MAPK signal pathway and then down-regulates oxidative phosphorylation of CD8+T cells in HIV infection.Conclusion:1.The first part of the study confirmed that the expression and the cell secretion of IL4I1 increased in patients with HIV infection and immune non-responders.IL4I1 was positively correlated with viral load and negatively correlated with CD4+T count.It is confirmed that IL4I1 is related to the disease progression of HIV infection.By co-culturing IL4I1 with T cells,we confirmed that IL4I1 could promote the dysfunction and apoptosis of HIV-infected T cells but had no significant effect on T cell proliferation.2.The second part of the study showed that IL4I1 inhibited the oxidative phosphorylation of CD8+T cells in HIV infections.Down-regulation of oxidative phosphorylation can promote the apoptosis and inhibit the cellular function of CD8+T cells in HIV infections.3.The results of the third part show that IL4I1 can down-regulate the expression of mitochondrial electron transport chain-related genes and mitochondrial transcription factor A by up-regulating p38 MAPK signal pathway,resulting in the increase of mitochondrial mass,depolarization,and reactive oxygen species,resulting in apoptosis and abnormal function of CD8+T cells in HIV infections.