Journal: mBio

Article Title: ENO2 regulates CD4 + T cell pyroptosis via mitochondrial ROS to drive immunological non-response in HIV infection

doi: 10.1128/mbio.01702-25

Figure Lengend Snippet: ENO2 and ENO3 may be the key metabolic regulatory genes in regulating cellular pyroptosis. ( A ) Analyze the difference in transcript levels between PD-1 + and PD-1 − CD4 + T cells by analyzing with GSE17606 , and the filtering parameters were set to adj. P < 0.05, |fold change| > 1.5, and the differentially expressed genes were screened out, and unsupervised hierarchical clustering analysis and heatmap plotting were applied with the MeV software. ( B ) Analyze the downregulated genes for KEGG signaling pathway analysis using the DAVID website. ( C ) Venn diagrams of the intersections of genes involved in the amino acid biosynthesis, glycolysis, and HIF-1 signaling pathways. ( D ) Sorting purity of PD-1 + and PD-1 − CD4 + T cells by flow sorter. ( E ) Validation of the mRNA levels of PD-1 + and PD-1 − CD4 + T cell glycolytic enzymes ENO2 and ENO3 (IR: n = 3). Data are analyzed by paired t -test in panel E . ** P < 0.01.

Article Snippet: ENO2 was inhibited by the addition of 10 μM ENO2 inhibitor ENOblock (MCE), and anti-CD3/CD28 Dynabeads (Gibco) were added and incubated at 37°C for 24 h for flow cytometry.

Techniques: Software, Protein-Protein interactions, Biomarker Discovery

Journal: mBio

Article Title: ENO2 regulates CD4 + T cell pyroptosis via mitochondrial ROS to drive immunological non-response in HIV infection

doi: 10.1128/mbio.01702-25

Figure Lengend Snippet: ENO2 was found to be a key metabolic regulatory molecule, which could regulate pyroptosis through screening and verification. ( A ) Difference in ENO2 mRNA expression levels between CD4 + T cells of IR and INR patients (IR: n = 12, INR: n = 12). ( B ) Correlation between ENO2 mRNA expression levels of CD4 + T cells and CD4 + T cell counts of ART patients (IR: n = 12, INR: n = 12). ( C ) Difference in ENO3 mRNA expression levels between CD4 + T cells of IR and INR patients (IR: n = 8, INR: n = 8). ( D ) Correlation between ENO3 mRNA expression levels of CD4 + T cells and CD4 + T cell counts of ART patients (IR: n = 8, INR: n = 8). ( E ) Correlation analysis between CD4 + T cell ENO2 and caspase-1 mRNA expression level in HIV-infected patients after ART (ART: n = 67). ( F ) Correlation analysis between CD4 + T cell ENO2 and NLRP3 mRNA expression level in HIV-infected patients after ART (ART: n = 67). ( G ) Correlation analysis between CD4 + T cell ENO2 and IL-1β mRNA expression level in HIV-infected patients after ART (ART: n = 67). ( H ) Correlation analysis between CD4 + T cell ENO2 and IL-18 mRNA expression level in HIV-infected patients after ART (ART: n = 67). ( I ) Detection of caspase-1 expression on CD4 + T cells 24 h after addition of the ENO2 inhibitor ENOblock in HIV-infected patients after ART, with typical flow graphs on the left and a statistical graph of percent change in caspase-1 levels on the right ( n = 17). ( J ) Caspase-1 expression on CD4 + T cells was assayed after knockdown of ENO2 by siENO2 in HIV-infected patients after ART, with typical flow graphs on the left and a statistical graph of the percent change in caspase-1 levels on the right ( n = 6). Data are analyzed by Mann-Whitney U -test in panels A and C . Spearman correlation analysis in panels B, D and E–H . Wilcoxon signed-rank test in panels I and J . **** P < 0.0001, ** P < 0.01, and * P < 0.05. ART, HIV patients after antiretroviral therapy.

Article Snippet: ENO2 was inhibited by the addition of 10 μM ENO2 inhibitor ENOblock (MCE), and anti-CD3/CD28 Dynabeads (Gibco) were added and incubated at 37°C for 24 h for flow cytometry.

Techniques: Expressing, Infection, Knockdown, MANN-WHITNEY

Journal: mBio

Article Title: ENO2 regulates CD4 + T cell pyroptosis via mitochondrial ROS to drive immunological non-response in HIV infection

doi: 10.1128/mbio.01702-25

Figure Lengend Snippet: ENO2 regulates CD4 + T cell mitochondrial metabolism. ( A ) Volcano plot of ENO2 low and ENO2 high differentially expressed genes, adj. P < 0.05. ( B ) Gene ontology enrichment analysis of ENO2 low and ENO2 high differentially expressed genes. ( C ). Typical OCR graphs of negatively selected CD4 + T cells after the addition of 10 µM ENOblock and CD3/CD28 stimulation. ( D ) Statistical graph of key parameters after addition of 10 µM ENOblock. Basal respiration, ATP production, maximal respiration, and spare respiration were significantly reduced ( n = 4). ( E and F ) Negative selection of CD4 + T cells from HIV-infected patients PBMC after ART, 10 µM ENOblock was added, and changes in the level of mitochondrial depolarization and mitochondrial membrane potential were detected after 24 h of incubation, with a typical flow graph on the left and a statistic of the percentage of depolarization and the membrane potential MFI statistic on the right ( n = 9). ( G and H ) Negatively selected CD4 + T cells from PBMC of HIV-infected patients after ART, mitochondrial ROS were detected 24 h after the addition of 10 µM ENOblock, typical flow chart on the left, and percentage of ROS, MFI statistics on the right (IR: n = 9, INR: n = 1). ( I and J ) HIV-infected PBMC negatively selected CD4 + T cells after ART were added with 100 pm siENO2, a typical flow diagram was on the left, and the right was the mitochondrial ROS percentage statistic and mitochondrial ROS MFI statistic graph ( n = 13). Data are analyzed by Ratio paired t -test in panel D . Wilcoxon signed-rank test in panels E–J . ** P < 0.01 and * P < 0.05.

Article Snippet: ENO2 was inhibited by the addition of 10 μM ENO2 inhibitor ENOblock (MCE), and anti-CD3/CD28 Dynabeads (Gibco) were added and incubated at 37°C for 24 h for flow cytometry.

Techniques: Selection, Infection, Membrane, Incubation

Journal: mBio

Article Title: ENO2 regulates CD4 + T cell pyroptosis via mitochondrial ROS to drive immunological non-response in HIV infection

doi: 10.1128/mbio.01702-25

Figure Lengend Snippet: Phosphoenolpyruvate supplementation was found to partially restore mitochondrial oxidative phosphorylation, reduce mitochondrial ROS level, and reduce pyroptosis in CD4 + T cells. ( A and B ) Negatively selected HIV-infected CD4 + T cells after ART were incubated with 10 µM ENOblock with or without 10 µM PEP for 24 h to detect mitochondrial respiratory capacity OCR (IR: n = 3, INR: n = 1). ( C and D ) Negatively selected ART-treated HIV-infected CD4 + T cells, 10 µM ENOblock was added, incubated with or without 10 µM PEP for 24 h to detect mitochondrial ROS (IR: n = 10, INR: n = 3) and levels of pyroptosis (IR: n = 20), typical flow charts are shown on the left, and the statistical graph of the percentage is shown on the right. ( E ) Caspase-1 expression on CD4 + T cells was assayed after knockdown of ENO2 in HIV-infected patients after ART with siENO2, accompanied by exogenous supplementation of PEP, with typical flow charts on the left and a statistical plot of the percent change in caspase-1 levels on the right ( n = 7). Data are analyzed by RM one-way ANOVA test in panel B ; Friedman test in panels C–E . **** P < 0.0001, *** P < 0.001, ** P < 0.01, and * P < 0.05.

Article Snippet: ENO2 was inhibited by the addition of 10 μM ENO2 inhibitor ENOblock (MCE), and anti-CD3/CD28 Dynabeads (Gibco) were added and incubated at 37°C for 24 h for flow cytometry.

Techniques: Phospho-proteomics, Infection, Incubation, Expressing, Knockdown

Journal: mBio

Article Title: ENO2 regulates CD4 + T cell pyroptosis via mitochondrial ROS to drive immunological non-response in HIV infection

doi: 10.1128/mbio.01702-25

Figure Lengend Snippet: Schematic diagram of the effect of ENO2 on pyroptosis of CD4 + T cells by regulating mitochondrial ROS.

Article Snippet: ENO2 was inhibited by the addition of 10 μM ENO2 inhibitor ENOblock (MCE), and anti-CD3/CD28 Dynabeads (Gibco) were added and incubated at 37°C for 24 h for flow cytometry.

Techniques: