ABSTRACT
In cancer and infections, self-renewing stem-like CD8+ T cells mediate the response of immunotherapies and replenish terminally exhausted T cells and effector-like T cells. However, the programs governing the lineage choice in chimeric antigen receptor (CAR) T cells are unclear. Here, by simultaneously profiling single-cell chromatin accessibility and transcriptome in the same CAR T cells, we identified heterogeneous chromatin states within CD8+ T cell subsets that foreshadowed transcriptional changes and were primed for regulation by distinct transcription factors. Transcription factors that controlled each CD8+ T cell subset were regulated by high numbers of enhancers and positioned as hubs of gene networks. FOXP1, a hub in the stem-like network, promoted expansion and stemness of CAR T cells and limited excessive effector differentiation. In the effector network, KLF2 enhanced effector CD8+ T cell differentiation and prevented terminal exhaustion. Thus, we identified gene networks and hub transcription factors that controlled the differentiation of stem-like CD8+ CAR T cells into effector or exhausted CD8+ CAR T cells.
Subject(s)
CD8-Positive T-Lymphocytes , Transcription Factors , Transcription Factors/genetics , T-Lymphocyte Subsets , Cell Differentiation , ChromatinABSTRACT
During chronic infection and cancer, a self-renewing CD8+ T cell subset maintains long-term immunity and is critical to the effectiveness of immunotherapy. These stem-like CD8+ T cells diverge from other CD8+ subsets early after chronic viral infection. However, pathways guarding stem-like CD8+ T cells against terminal exhaustion remain unclear. Here, we show that the gene encoding transcriptional repressor BACH2 is transcriptionally and epigenetically active in stem-like CD8+ T cells but not terminally exhausted cells early after infection. BACH2 overexpression enforced stem-like cell fate, whereas BACH2 deficiency impaired stem-like CD8+ T cell differentiation. Single-cell transcriptomic and epigenomic approaches revealed that BACH2 established the transcriptional and epigenetic programs of stem-like CD8+ T cells. In addition, BACH2 suppressed the molecular program driving terminal exhaustion through transcriptional repression and epigenetic silencing. Thus, our study reveals a new pathway that enforces commitment to stem-like CD8+ lineage and prevents an alternative terminally exhausted cell fate.
Subject(s)
Arenaviridae Infections/metabolism , Basic-Leucine Zipper Transcription Factors/metabolism , CD8-Positive T-Lymphocytes/metabolism , Cell Differentiation , Epigenesis, Genetic , Precursor Cells, T-Lymphoid/metabolism , Transcription, Genetic , Animals , Arenaviridae Infections/genetics , Arenaviridae Infections/immunology , Arenaviridae Infections/virology , Basic-Leucine Zipper Transcription Factors/deficiency , Basic-Leucine Zipper Transcription Factors/genetics , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/virology , Cell Lineage , Cells, Cultured , Chronic Disease , Disease Models, Animal , Host-Pathogen Interactions , Lymphocytic choriomeningitis virus/immunology , Lymphocytic choriomeningitis virus/pathogenicity , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Precursor Cells, T-Lymphoid/immunology , Precursor Cells, T-Lymphoid/virology , Signal TransductionABSTRACT
Cytokine expression during T cell differentiation is a highly regulated process that involves long-range promoter-enhancer and CTCF-CTCF contacts at cytokine loci. Here, we investigated the impact of dynamic chromatin loop formation within the topologically associating domain (TAD) in regulating the expression of interferon gamma (IFN-γ) and interleukin-22 (IL-22); these cytokine loci are closely located in the genome and are associated with complex enhancer landscapes, which are selectively active in type 1 and type 3 lymphocytes. In situ Hi-C analyses revealed inducible TADs that insulated Ifng and Il22 enhancers during Th1 cell differentiation. Targeted deletion of a 17 bp boundary motif of these TADs imbalanced Th1- and Th17-associated immunity, both in vitro and in vivo, upon Toxoplasma gondii infection. In contrast, this boundary element was dispensable for cytokine regulation in natural killer cells. Our findings suggest that precise cytokine regulation relies on lineage- and developmental stage-specific interactions of 3D chromatin architectures and enhancer landscapes.
Subject(s)
CCCTC-Binding Factor , Cell Differentiation , Interferon-gamma , Interleukin-22 , Interleukins , Th1 Cells , Animals , CCCTC-Binding Factor/metabolism , CCCTC-Binding Factor/genetics , Th1 Cells/immunology , Mice , Cell Differentiation/immunology , Interferon-gamma/metabolism , Binding Sites , Interleukins/metabolism , Interleukins/genetics , Enhancer Elements, Genetic/genetics , Mice, Inbred C57BL , Chromatin/metabolism , Toxoplasmosis/immunology , Toxoplasmosis/parasitology , Toxoplasmosis/genetics , Gene Expression Regulation , Toxoplasma/immunology , Cytokines/metabolism , Cell Lineage , Th17 Cells/immunologyABSTRACT
Depleting regulatory T cells (Treg cells) to counteract immunosuppressive features of the tumor microenvironment (TME) is an attractive strategy for cancer treatment; however, autoimmunity due to systemic impairment of their suppressive function limits its therapeutic potential. Elucidating approaches that specifically disrupt intratumoral Treg cells is direly needed for cancer immunotherapy. We found that CD36 was selectively upregulated in intrautumoral Treg cells as a central metabolic modulator. CD36 fine-tuned mitochondrial fitness via peroxisome proliferator-activated receptor-ß signaling, programming Treg cells to adapt to a lactic acid-enriched TME. Genetic ablation of Cd36 in Treg cells suppressed tumor growth accompanied by a decrease in intratumoral Treg cells and enhancement of antitumor activity in tumor-infiltrating lymphocytes without disrupting immune homeostasis. Furthermore, CD36 targeting elicited additive antitumor responses with anti-programmed cell death protein 1 therapy. Our findings uncover the unexplored metabolic adaptation that orchestrates the survival and functions of intratumoral Treg cells, and the therapeutic potential of targeting this pathway for reprogramming the TME.
Subject(s)
CD36 Antigens/immunology , Neoplasms/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Apoptosis/immunology , CD36 Antigens/deficiency , CD36 Antigens/genetics , Cell Line, Tumor , Female , Homeostasis/immunology , Humans , Immunotherapy , Lipid Metabolism/genetics , Lymphocytes, Tumor-Infiltrating/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neoplasms/metabolism , Neoplasms/pathology , PPAR-beta/immunology , Signal Transduction/immunology , T-Lymphocytes, Regulatory/metabolism , T-Lymphocytes, Regulatory/pathology , Tumor Microenvironment/immunologyABSTRACT
Progenitor-like CD8+ T cells mediate long-term immunity to chronic infection and cancer and respond potently to immune checkpoint blockade. These cells share transcriptional regulators with memory precursor cells, including T cell-specific transcription factor 1 (TCF1), but it is unclear whether they adopt distinct programs to adapt to the immunosuppressive environment. By comparing the single-cell transcriptomes and epigenetic profiles of CD8+ T cells responding to acute and chronic viral infections, we found that progenitor-like CD8+ T cells became distinct from memory precursor cells before the peak of the T cell response. We discovered a coexpression gene module containing Tox that exhibited higher transcriptional activity associated with more abundant active histone marks in progenitor-like cells than memory precursor cells. Moreover, thymocyte selection-associated high mobility group box protein TOX (TOX) promoted the persistence of antiviral CD8+ T cells and was required for the programming of progenitor-like CD8+ T cells. Thus, long-term CD8+ T cell immunity to chronic viral infection requires unique transcriptional and epigenetic programs associated with the transcription factor TOX.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Gene Expression Regulation , Homeodomain Proteins/genetics , Infections/etiology , Single-Cell Analysis , Animals , Biomarkers , Chromatin Immunoprecipitation , Epigenesis, Genetic , Gene Expression Profiling , High-Throughput Nucleotide Sequencing , Homeodomain Proteins/metabolism , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Immunologic Memory , Infections/metabolism , Lymphocytic Choriomeningitis/immunology , Lymphocytic Choriomeningitis/virology , Lymphocytic choriomeningitis virus/immunology , Mice , Time Factors , TranscriptomeABSTRACT
In the version of this article initially published, the bars were not aligned with the data points or horizontal axis labels in Fig. 5d, and the labels along each horizontal axis of Fig. 5j-l indicating the presence (+) or absence (-) of doxycycline (Dox) were incorrectly included with the labels below that axis. Also, the right vertical bar above Fig. 7b linking 'P = 0.0001' to the key was incorrect; the correct comparison is αPD-1 versus Dox + αPD-1. Similarly, the right vertical bar above Fig. 7e linking 'P = 0.0002' to the key was incorrect; the correct comparison is αPD-1 versus Rosig + αPD-1. The errors have been corrected in the HTML and PDF versions of the article.
ABSTRACT
Immune checkpoint blockade therapy has shifted the paradigm for cancer treatment. However, the majority of patients lack effective responses due to insufficient T cell infiltration in tumors. Here we show that expression of mitochondrial uncoupling protein 2 (UCP2) in tumor cells determines the immunostimulatory feature of the tumor microenvironment (TME) and is positively associated with prolonged survival. UCP2 reprograms the immune state of the TME by altering its cytokine milieu in an interferon regulatory factor 5-dependent manner. Consequently, UCP2 boosts the conventional type 1 dendritic cell- and CD8+ T cell-dependent anti-tumor immune cycle and normalizes the tumor vasculature. Finally we show, using either a genetic or pharmacological approach, that induction of UCP2 sensitizes melanomas to programmed cell death protein-1 blockade treatment and elicits effective anti-tumor responses. Together, this study demonstrates that targeting the UCP2 pathway is a potent strategy for alleviating the immunosuppressive TME and overcoming the primary resistance of programmed cell death protein-1 blockade.
Subject(s)
Antineoplastic Agents, Immunological/therapeutic use , Melanoma, Experimental/immunology , Skin Neoplasms/immunology , Tumor Microenvironment/immunology , Uncoupling Protein 2/immunology , Animals , Antineoplastic Agents, Immunological/pharmacology , CD8-Positive T-Lymphocytes/immunology , Cell Line, Tumor , Dendritic Cells/immunology , Drug Resistance, Neoplasm/immunology , Female , Humans , Immunotherapy/methods , Interferon Regulatory Factors/immunology , Interferon Regulatory Factors/metabolism , Melanoma, Experimental/blood supply , Melanoma, Experimental/drug therapy , Melanoma, Experimental/mortality , Mice, Inbred C57BL , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/immunology , Skin Neoplasms/blood supply , Skin Neoplasms/drug therapy , Skin Neoplasms/mortality , Survival Analysis , Treatment Outcome , Uncoupling Protein 2/genetics , Uncoupling Protein 2/metabolismABSTRACT
MicroRNAs are important regulators of immune responses. Here, we show miR-221 and miR-222 modulate the intestinal Th17 cell response. Expression of miR-221 and miR-222 was induced by proinflammatory cytokines and repressed by the cytokine TGF-ß. Molecular targets of miR-221 and miR-222 included Maf and Il23r, and loss of miR-221 and miR-222 expression shifted the transcriptomic spectrum of intestinal Th17 cells to a proinflammatory signature. Although the loss of miR-221 and miR-222 was tolerated for maintaining intestinal Th17 cell homeostasis in healthy mice, Th17 cells lacking miR-221 and miR-222 expanded more efficiently in response to IL-23. Both global and T cell-specific deletion of miR-221 and miR-222 rendered mice prone to mucosal barrier damage. Collectively, these findings demonstrate that miR-221 and miR-222 are an integral part of intestinal Th17 cell response that are induced after IL-23 stimulation to constrain the magnitude of proinflammatory response.
Subject(s)
Inflammation/immunology , Interleukin-23/metabolism , Intestinal Mucosa/immunology , MicroRNAs/genetics , Th17 Cells/immunology , Animals , Feedback, Physiological , Mice , Mice, Inbred C57BL , Mice, Knockout , Proto-Oncogene Proteins c-maf/metabolism , Receptors, Interleukin/metabolism , Signal Transduction , Transforming Growth Factor beta/metabolismABSTRACT
Activated T cells engage aerobic glycolysis and anabolic metabolism for growth, proliferation, and effector functions. We propose that a glucose-poor tumor microenvironment limits aerobic glycolysis in tumor-infiltrating T cells, which suppresses tumoricidal effector functions. We discovered a new role for the glycolytic metabolite phosphoenolpyruvate (PEP) in sustaining T cell receptor-mediated Ca(2+)-NFAT signaling and effector functions by repressing sarco/ER Ca(2+)-ATPase (SERCA) activity. Tumor-specific CD4 and CD8 T cells could be metabolically reprogrammed by increasing PEP production through overexpression of phosphoenolpyruvate carboxykinase 1 (PCK1), which bolstered effector functions. Moreover, PCK1-overexpressing T cells restricted tumor growth and prolonged the survival of melanoma-bearing mice. This study uncovers new metabolic checkpoints for T cell activity and demonstrates that metabolic reprogramming of tumor-reactive T cells can enhance anti-tumor T cell responses, illuminating new forms of immunotherapy.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Melanoma/immunology , Melanoma/therapy , Monitoring, Immunologic , Phosphoenolpyruvate/metabolism , Tumor Microenvironment , Animals , Calcium/metabolism , Endoplasmic Reticulum/metabolism , Glycolysis , Hexokinase/metabolism , Immunotherapy , Mice , NFATC Transcription Factors/metabolism , Receptors, Antigen, T-Cell/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Signal Transduction , Transforming Growth Factor beta/immunologyABSTRACT
Innate immune responses rely on rapid and precise gene regulation mediated by accessibility of regulatory regions to transcription factors (TFs). In natural killer (NK) cells and other innate lymphoid cells, competent enhancers are primed during lineage acquisition, and formation of de novo enhancers characterizes the acquisition of innate memory in activated NK cells and macrophages. Here, we investigated how primed and de novo enhancers coordinate to facilitate high-magnitude gene induction during acute activation. Epigenomic and transcriptomic analyses of regions near highly induced genes (HIGs) in NK cells both in vitro and in a model of Toxoplasma gondii infection revealed de novo chromatin accessibility and enhancer remodeling controlled by signal-regulated TFs STATs. Acute NK cell activation redeployed the lineage-determining TF T-bet to de novo enhancers, independent of DNA-sequence-specific motif recognition. Thus, acute stimulation reshapes enhancer function through the combinatorial usage and repurposing of both lineage-determining and signal-regulated TFs to ensure an effective response.
Subject(s)
Enhancer Elements, Genetic/genetics , Enhancer Elements, Genetic/immunology , Killer Cells, Natural/immunology , Transcription Factors/genetics , Transcription Factors/immunology , Animals , Chromatin/genetics , Chromatin/immunology , Female , Gene Expression/genetics , Gene Expression/immunology , Gene Expression Profiling/methods , Gene Expression Regulation/genetics , Gene Expression Regulation/immunology , Immunity, Innate/genetics , Immunity, Innate/immunology , Macrophages/immunology , Mice , Mice, Inbred C57BL , Toxoplasma/immunology , Toxoplasmosis/genetics , Toxoplasmosis/immunologyABSTRACT
Innate lymphocytes maintain tissue homeostasis at mucosal barriers, with group 2 innate lymphoid cells (ILC2s) producing type 2 cytokines and controlling helminth infection. While the molecular understanding of ILC2 responses has advanced, the complexity of microenvironmental factors impacting ILC2s is becoming increasingly apparent. Herein, we used single-cell analysis to explore the diversity of gene expression among lung lymphocytes during helminth infection. Following infection, we identified a subset of ILC2s that preferentially expressed Il5-encoding interleukin (IL)-5, together with Calca-encoding calcitonin gene-related peptide (CGRP) and its cognate receptor components. CGRP in concert with IL-33 and neuromedin U (NMU) supported IL-5 but constrained IL-13 expression and ILC2 proliferation. Without CGRP signaling, ILC2 responses and worm expulsion were enhanced. Collectively, these data point to CGRP as a context-dependent negative regulatory factor that shapes innate lymphocyte responses to alarmins and neuropeptides during type 2 innate immune responses.
Subject(s)
Inflammation/immunology , Lymphocytes/immunology , Nippostrongylus/physiology , Receptors, Calcitonin Gene-Related Peptide/metabolism , Strongylida Infections/immunology , Animals , Cells, Cultured , Cytokines/metabolism , Immunity, Innate , Interleukin-33/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Neuropeptides/metabolism , Receptors, Calcitonin Gene-Related Peptide/genetics , Single-Cell Analysis , Th2 Cells/immunology , Transplantation ChimeraABSTRACT
CD4+ T helper (Th) differentiation is regulated by diverse inputs, including the vitamin A metabolite retinoic acid (RA). RA acts through its receptor RARα to repress transcription of inflammatory cytokines, but is also essential for Th-mediated immunity, indicating complex effects of RA on Th specification and the outcome of the immune response. We examined the impact of RA on the genome-wide transcriptional response during Th differentiation to multiple subsets. RA effects were subset-selective and were most significant in Th9 cells. RA globally antagonized Th9-promoting transcription factors and inhibited Th9 differentiation. RA directly targeted the extended Il9 locus and broadly modified the Th9 epigenome through RARα. RA-RARα activity limited murine Th9-associated pulmonary inflammation, and human allergic inflammation was associated with reduced expression of RA target genes. Thus, repression of the Th9 program is a major function of RA-RARα signaling in Th differentiation, arguing for a role for RA in interleukin 9 (IL-9) related diseases.
Subject(s)
Hypersensitivity/immunology , Lung/physiology , Pneumonia/immunology , Retinoic Acid Receptor alpha/metabolism , T-Lymphocytes, Helper-Inducer/physiology , Animals , Epigenetic Repression , HEK293 Cells , Humans , Hypersensitivity/genetics , Interleukin-9/metabolism , Lung/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Pneumonia/genetics , Retinoic Acid Receptor alpha/genetics , Signal Transduction , Transcription, Genetic , Tretinoin/metabolismABSTRACT
Interferon gamma (IFN-γ), critical for host defense and tumor surveillance, requires tight control of its expression. Multiple cis-regulatory elements exist around Ifng along with a non-coding transcript, Ifng-as1 (also termed NeST). Here, we describe two genetic models generated to dissect the molecular functions of this locus and its RNA product. DNA deletion within the Ifng-as1 locus disrupted chromatin organization of the extended Ifng locus, impaired Ifng response, and compromised host defense. Insertion of a polyA signal ablated the Ifng-as1 full-length transcript and impaired host defense, while allowing proper chromatin structure. Transient knockdown of Ifng-as1 also reduced IFN-γ production. In humans, discordant expression of IFNG and IFNG-AS1 was evident in memory T cells, with high expression of this long non-coding RNA (lncRNA) and low expression of the cytokine. These results establish Ifng-as1 as an important regulator of Ifng expression, as a DNA element and transcribed RNA, involved in dynamic and cell state-specific responses to infection.
Subject(s)
Gene Expression Regulation/immunology , Immunologic Memory , Infections/immunology , Interferon-gamma/immunology , RNA, Untranslated/immunology , T-Lymphocytes/immunology , Animals , Chromatin/genetics , Chromatin/immunology , Female , Gene Knockdown Techniques , Infections/genetics , Infections/pathology , Interferon-gamma/genetics , Mice , RNA, Untranslated/genetics , T-Lymphocytes/pathologyABSTRACT
Exorbitant sustained inflammation is closely linked to inflammation-associated disorders, including cancer. The initiation of gastrointestinal cancers such as colorectal cancer (CRC) is frequently accelerated by uncontrollable chronic inflammation which is triggered by excessive activation of nuclear factor kappa-B (NF-κB) signaling. Linear ubiquitin chains play an important role in activating canonical NF-κB pathway. The only known E3 complex, linear ubiquitin assembly complex (LUBAC) is responsible for the synthesis of linear ubiquitin chains, thus leading to the activation of NF-κB axis and promoting the development of inflammation and inflammation-associated cancers. We report here cyclophilin J (CYPJ) is a negative regulator of the LUBAC. The N-terminus of CYPJ binds to the second Npl4-like zinc finger (NZF2) domain of HOIP and the ubiquitin-like (UBL) domain of SHARPIN to disrupt the interaction between HOIP and SHARPIN and thus restrains linear ubiquitin chain synthesis and NF-κB activation. Cypj-deficient mice are highly susceptible to dextran sulfate sodium (DSS)-induced colitis and DSS plus azoxymethane (AOM)-induced colon cancer. Moreover, CYPJ expression is induced by hypoxia. Patients with high expression of both CYPJ and hypoxia-inducible factor-1α (HIF-1α) have longer overall survival and progression-free survival. These results implicate CYPJ as an unexpected robust attenuator of inflammation-driven tumorigenesis that exerts its effects by controlling linear ubiquitin chain synthesis in NF-κB signal pathway.
ABSTRACT
BACKGROUND: The effects and risks of endovascular thrombectomy 6 to 24 hours after stroke onset due to basilar-artery occlusion have not been extensively studied. METHODS: In a trial conducted over a 5-year period in China, we randomly assigned, in a 1:1 ratio, patients with basilar-artery stroke who presented between 6 to 24 hours after symptom onset to receive either medical therapy plus thrombectomy or medical therapy only (control). The original primary outcome, a score of 0 to 4 on the modified Rankin scale (range, 0 to 6, with a score of 0 indicating no disability, 4 moderately severe disability, and 6 death) at 90 days, was changed to a good functional status (a modified Rankin scale score of 0 to 3, with a score of 3 indicating moderate disability). Primary safety outcomes were symptomatic intracranial hemorrhage at 24 hours and 90-day mortality. RESULTS: A total of 217 patients (110 in the thrombectomy group and 107 in the control group) were included in the analysis; randomization occurred at a median of 663 minutes after symptom onset. Enrollment was halted at a prespecified interim analysis because of the superiority of thrombectomy. Thrombolysis was used in 14% of the patients in the thrombectomy group and in 21% of those in the control group. A modified Rankin scale score of 0 to 3 (primary outcome) occurred in 51 patients (46%) in the thrombectomy group and in 26 (24%) in the control group (adjusted rate ratio, 1.81; 95% confidence interval [CI], 1.26 to 2.60; P<0.001). The results for the original primary outcome of a modified Rankin scale score of 0 to 4 were 55% and 43%, respectively (adjusted rate ratio, 1.21; 95% CI, 0.95 to 1.54). Symptomatic intracranial hemorrhage occurred in 6 of 102 patients (6%) in the thrombectomy group and in 1 of 88 (1%) in the control group (risk ratio, 5.18; 95% CI, 0.64 to 42.18). Mortality at 90 days was 31% in the thrombectomy group and 42% in the control group (adjusted risk ratio, 0.75; 95% CI, 0.54 to 1.04). Procedural complications occurred in 11% of the patients who underwent thrombectomy. CONCLUSIONS: Among patients with stroke due to basilar-artery occlusion who presented 6 to 24 hours after symptom onset, thrombectomy led to a higher percentage with good functional status at 90 days than medical therapy but was associated with procedural complications and more cerebral hemorrhages. (Funded by the Chinese National Ministry of Science and Technology; BAOCHE ClinicalTrials.gov number, NCT02737189.).
Subject(s)
Arterial Occlusive Diseases , Basilar Artery , Endovascular Procedures , Stroke , Thrombectomy , Humans , Arterial Occlusive Diseases/complications , Arterial Occlusive Diseases/drug therapy , Arterial Occlusive Diseases/mortality , Arterial Occlusive Diseases/surgery , Basilar Artery/drug effects , Basilar Artery/surgery , Brain Ischemia/drug therapy , Brain Ischemia/etiology , Brain Ischemia/mortality , Brain Ischemia/surgery , Disability Evaluation , Endovascular Procedures/adverse effects , Endovascular Procedures/methods , Fibrinolytic Agents/adverse effects , Fibrinolytic Agents/therapeutic use , Intracranial Hemorrhages/chemically induced , Intracranial Hemorrhages/etiology , Recovery of Function , Stroke/drug therapy , Stroke/etiology , Stroke/mortality , Stroke/surgery , Thrombectomy/adverse effects , Thrombectomy/methods , Time Factors , Treatment OutcomeABSTRACT
BACKGROUND: Ruptured atherosclerotic plaques often precipitate severe ischemic events, such as stroke and myocardial infarction. Unraveling the intricate molecular mechanisms governing vascular smooth muscle cell (VSMC) behavior in plaque stabilization remains a formidable challenge. METHODS: In this study, we leveraged single-cell and transcriptomic datasets from atherosclerotic plaques retrieved from the gene expression omnibus (GEO) database. Employing a combination of single-cell population differential analysis, weighted gene co-expression network analysis (WGCNA), and transcriptome differential analysis techniques, we identified specific genes steering the transformation of VSMCs in atherosclerotic plaques. Diagnostic models were developed and validated through gene intersection, utilizing the least absolute shrinkage and selection operator (LASSO) and random forest (RF) methods. Nomograms for plaque assessment were constructed. Tissue localization and expression validation were performed on specimens from animal models, utilizing immunofluorescence co-localization, western blot, and reverse-transcription quantitative-polymerase chain reaction (RT-qPCR). Various online databases were harnessed to predict transcription factors (TFs) and their interacting compounds, with determination of the cell-specific localization of TF expression using single-cell data. RESULTS: Following rigorous quality control procedures, we obtained a total of 40,953 cells, with 6,261 representing VSMCs. The VSMC population was subsequently clustered into 5 distinct subpopulations. Analyzing inter-subpopulation cellular communication, we focused on the SMC2 and SMC5 subpopulations. Single-cell subpopulation and WGCNA analyses revealed significant module enrichments, notably in collagen-containing extracellular matrix and cell-substrate junctions. Insulin-like growth factor binding protein 4 (IGFBP4), apolipoprotein E (APOE), and cathepsin C (CTSC) were identified as potential diagnostic markers for early and advanced plaques. Notably, gene expression pattern analysis suggested that IGFBP4 might serve as a protective gene, a hypothesis validated through tissue localization and expression analysis. Finally, we predicted TFs capable of binding to IGFBP4, with Krüppel-like family 15 (KLF15) emerging as a prominent candidate showing relative specificity within smooth muscle cells. Predictions about compounds associated with affecting KLF15 expression were also made. CONCLUSION: Our study established a plaque diagnostic and assessment model and analyzed the molecular interaction mechanisms of smooth muscle cells within plaques. Further analysis revealed that the transcription factor KLF15 may regulate the biological behaviors of smooth muscle cells through the KLF15/IGFBP4 axis, thereby influencing the stability of advanced plaques via modulation of the PI3K-AKT signaling pathway. This could potentially serve as a target for plaque stability assessment and therapy, thus driving advancements in the management and treatment of atherosclerotic plaques.
Subject(s)
Insulin-Like Growth Factor Binding Protein 4 , Kruppel-Like Transcription Factors , Myocytes, Smooth Muscle , Plaque, Atherosclerotic , Animals , Humans , Male , Gene Expression Profiling , Gene Regulatory Networks , Insulin-Like Growth Factor Binding Protein 4/metabolism , Insulin-Like Growth Factor Binding Protein 4/genetics , Kruppel-Like Transcription Factors/metabolism , Kruppel-Like Transcription Factors/genetics , Multiomics , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/metabolism , Plaque, Atherosclerotic/metabolism , Plaque, Atherosclerotic/genetics , Plaque, Atherosclerotic/pathology , Single-Cell Analysis , Transcriptome , RatsABSTRACT
Tolerance to endotoxins that is triggered by prior exposure to Toll-like receptor (TLR) ligands provides a mechanism with which to dampen inflammatory cytokines. The receptor-interacting protein RIP140 interacts with the transcription factor NF-κB to regulate the expression of genes encoding proinflammatory cytokines. Here we found lipopolysaccharide stimulation of kinase Syk-mediated tyrosine phosphorylation of RIP140 and interaction of the NF-κB subunit RelA with RIP140. These events resulted in more recruitment of the E3 ligase SCF to tyrosine-phosphorylated RIP140, which degraded RIP140 to inactivate genes encoding inflammatory cytokines. Macrophages expressing nondegradable RIP140 were resistant to the establishment of endotoxin tolerance for specific 'tolerizable' genes. Our results identify RelA as an adaptor with which SCF fine tunes NF-κB target genes by targeting the coactivator RIP140 and show an unexpected role for RIP140 degradation in resolving inflammation and endotoxin tolerance.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Endotoxins/immunology , Immune Tolerance/immunology , Inflammation/immunology , NF-kappa B/metabolism , Nuclear Proteins/metabolism , Adaptor Proteins, Signal Transducing/immunology , Animals , Chromatin Immunoprecipitation , Gene Knockdown Techniques , Immunoblotting , Inflammation/metabolism , Mice , Mice, Transgenic , NF-kappa B/immunology , Nuclear Proteins/immunology , Nuclear Receptor Interacting Protein 1 , SKP Cullin F-Box Protein Ligases/immunology , SKP Cullin F-Box Protein Ligases/metabolism , Signal Transduction/immunology , Ubiquitin-Protein Ligases/immunology , Ubiquitin-Protein Ligases/metabolismABSTRACT
BACKGROUND: Aberrant Derlin-1 (DERL1) expression is associated with an overactivation of p-AKT, whose involvement in breast cancer (BRCA) development has been widely speculated. However, the precise mechanism that links DERL1 expression and AKT activation is less well-studied. METHODS: Bioinformatic analyses hold a promising approach by which to detect genes' expression levels and their association with disease prognoses in patients. In the present work, a dual-luciferase assay was employed to investigate the relationship between DERL1 expression and the candidate miRNA by both in vitro and in vivo methods. Further in-depth studies involving immunoprecipitation-mass spectrum (IP-MS), co-immunoprecipitation (Co-IP), as well as Zdock prediction were performed. RESULTS: Overexpression of DERL1 was detected in all phenotypes of BRCA, and its knockdown showed an inhibitory effect on BRCA cells both in vitro and in vivo. The Cancer Genome Atlas (TCGA) database reported that DERL1 overexpression was correlated with poor overall survival in BRCA cases, and so the quantification of DERL1 expression could be a potential marker for the clinical diagnosis of BRCA. On the other hand, miR-181c-5p was downregulated in BRCA, suggesting that its overexpression could be a potent therapeutic route to improve the overall survival of BRCA cases. Prior bioinformatic analyses indicated a somewhat positive correlation between DERL1 and TRAF6 as well as between TRAF6 and AKT, but not between miR-181c-5p and DERL1. In retrospect, DERL1 overexpression promoted p-AKT activation through K63 ubiquitination. DERL1 was believed to directly interact with the E3 ligase TRAF6. As Tyr77Ala or Tyr77Ala/Gln81Ala/Arg85Ala/Val158Ala attempts to prevent the interaction between DERL1 and TRAF domain of TRAF6, resulted in a significant reduction in K63-ubiquitinated p-AKT production. However, mutations in Gln81Ala, Arg85Ala, or Val158Ala could possibly interrupt with these processes. CONCLUSIONS: Our data confirm that mediation of the miR-181c-5p/DERL1 pathway by TRAF6-linked AKT K63 ubiquitination holds one of the clues to set our focus on toward meeting the therapeutic goals of BRCA.
ABSTRACT
Innate resistance to Candida albicans in mucosal tissues requires the production of interleukin-17A (IL-17A) by tissue-resident cells early during infection, but the mechanism of cytokine production has not been precisely defined. In the skin, we found that dermal γδ T cells were the dominant source of IL-17A during C. albicans infection and were required for pathogen resistance. Induction of IL-17A from dermal γδ T cells and resistance to C. albicans required IL-23 production from CD301b(+) dermal dendritic cells (dDCs). In addition, we found that sensory neurons were directly activated by C. albicans. Ablation of sensory neurons increased susceptibility to C. albicans infection, which could be rescued by exogenous addition of the neuropeptide CGRP. These data define a model in which nociceptive pathways in the skin drive production of IL-23 by CD301b(+) dDCs resulting in IL-17A production from γδ T cells and resistance to cutaneous candidiasis.