2,099 results on '"Ley, Klaus"'
Search Results
2. Dysregulated cellular metabolism in atherosclerosis: mediators and therapeutic opportunities
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Stroope, Chad, Nettersheim, Felix Sebastian, Coon, Brian, Finney, Alexandra C., Schwartz, Martin A., Ley, Klaus, Rom, Oren, and Yurdagul, Jr., Arif
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- 2024
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3. Immunotherapy for atherosclerosis by targeting pro-inflammatory T cells
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Khan, Amir and Ley, Klaus
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- 2024
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4. Distinct roles of kindlin-3 and talin-1 in neutrophil beta2 integrin clustering
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Wu, Yuanyuan, Wen, Lai, Ley, Klaus, and Fan, Zhichao
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Biochemistry and Cell Biology ,Biological Sciences ,Clinical Research ,Underpinning research ,1.1 Normal biological development and functioning ,Inflammatory and immune system ,Immunology ,Biochemistry and cell biology - Abstract
Abstract: Neutrophils are the most abundant leukocytes in human blood. Neutrophil malfunction may cause various immunological diseases. Beta2 integrins are critical for neutrophil recruitment. Kindlin-3 and talin-1 are important regulators for beta2 integrin activation. We have confirmed this using conformation-specific antibodies KIM127 and mAb24 that recognize activation epitopes in beta2 integrin. Besides activation, clustering is also critical for beta2 integrin avidity and adhesion of neutrophils. Whether kindlin-3 and talin-1 are involved in beta2 integrin clustering remains unclear. Here we used super-resolution stochastic optical reconstruction microscopy (STORM) to study their roles in beta2 integrin clustering on human neutrophil-like HL60 cells. We find that lymphocyte function-associated antigen 1 (LFA-1) and macrophage-1 antigen (Mac-1) are significantly clustered in wild-type cells after IL-8 or fMLP stimulation. Using CRISPR-Cas9-mediated kindlin-3 and talin-1 knockout HL60 cells, we found that kindlin-3 but not talin-1 are responsible for beta2 integrin clustering. Our study provides new insights into the regulation of beta2 integrin clustering and leukocyte recruitment. Supported by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute, USA (R01HL145454), and a startup fund from UConn Health.
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- 2023
5. Kindlin2 enables EphB/ephrinB bi-directional signaling to support vascular development
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Li, Wenqing, Wen, Lai, Rathod, Bhavisha, Gingras, Anne-Claude, Ley, Klaus, and Lee, Ho-Sup
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Biochemistry and Cell Biology ,Biological Sciences ,1.1 Normal biological development and functioning ,Underpinning research ,Animals ,Zebrafish ,Signal Transduction ,Receptors ,Eph Family ,Ephrin-B2 ,Ephrin-B1 ,Mammals ,Biological sciences ,Biomedical and clinical sciences - Abstract
Direct contact between cells expressing either ephrin ligands or Eph receptor tyrosine kinase produces diverse developmental responses. Transmembrane ephrinB ligands play active roles in transducing bi-directional signals downstream of EphB/ephrinB interaction. However, it has not been well understood how ephrinB relays transcellular signals to neighboring cells and what intracellular effectors are involved. Here, we report that kindlin2 can mediate bi-directional ephrinB signaling through binding to a highly conserved NIYY motif in the ephrinB2 cytoplasmic tail. We show this interaction is important for EphB/ephrinB-mediated integrin activation in mammalian cells and for blood vessel morphogenesis during zebrafish development. A mixed two-cell population study revealed that kindlin2 (in ephrinB2-expressing cells) modulates transcellular EphB4 activation by promoting ephrinB2 clustering. This mechanism is also operative for EphB2/ephrinB1, suggesting that kindlin2-mediated regulation is conserved for EphB/ephrinB signaling pathways. Together, these findings show that kindlin2 enables EphB4/ephrinB2 bi-directional signal transmission.
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- 2023
6. Human circulating CD24hi marginal zone B cells produce IgM targeting atherogenic antigens and confer protection from vascular disease
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Pattarabanjird, Tanyaporn, Nguyen, Anh Tram, McSkimming, Chantel, Dinh, Huy Q., Marshall, Melissa A., Ghosheh, Yanal, Gulati, Rishab, Durant, Chistopher, Vallejo, Jenifer, Saigusa, Ryosuke, Drago, Fabrizio, Guy, Thomas V., Premo, Katherine, Taylor, Angela M., Paul, Soumen, Kundu, Bijoy, Berr, Stuart, Gonen, Ayelet, Tsimikas, Sotirios, Miller, Yury, Pillai, Shiv, Ley, Klaus, Hedrick, Catherine C., and McNamara, Coleen A.
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- 2023
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7. Identification of human exTreg cells as CD16+CD56+ cytotoxic CD4+ T cells
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Freuchet, Antoine, Roy, Payel, Armstrong, Sujit Silas, Oliaeimotlagh, Mohammad, Kumar, Sunil, Orecchioni, Marco, Ali, Amal J., Khan, Amir, Makings, Jeffrey, Lyu, Qingkang, Winkels, Holger, Wang, Erpei, Durant, Christopher, Ghosheh, Yanal, Gulati, Rishab, Nettersheim, Felix, and Ley, Klaus
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- 2023
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8. Mild Hypoxia Accelerates Cerebral Cavernous Malformation Disease Through CX3CR1-CX3CL1 Signaling
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Frias-Anaya, Eduardo, Gallego-Gutierrez, Helios, Gongol, Brendan, Weinsheimer, Shantel, Lai, Catherine Chinhchu, Orecchioni, Marco, Sriram, Aditya, Bui, Cassandra M., Nelsen, Bliss, Hale, Preston, Pham, Angela, Shenkar, Robert, DeBiasse, Dorothy, Lightle, Rhonda, Girard, Romuald, Li, Ying, Srinath, Abhinav, Daneman, Richard, Nudleman, Eric, Sun, Hao, Guma, Monica, Dubrac, Alexandre, Mesarwi, Omar, Ley, Klaus, Kim, Helen, Awad, Issam A., Ginsberg, Mark H., and Lopez-Ramirez, Miguel Alejandro
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- 2024
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9. Neuroinflammation Plays a Critical Role in Cerebral Cavernous Malformation Disease
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Lai, Catherine Chinhchu, Nelsen, Bliss, Frias-Anaya, Eduardo, Gallego-Gutierrez, Helios, Orecchioni, Marco, Herrera, Victoria, Ortiz, Elan, Sun, Hao, Mesarwi, Omar A, Ley, Klaus, Gongol, Brendan, and Lopez-Ramirez, Miguel Alejandro
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Biomedical and Clinical Sciences ,Cardiovascular Medicine and Haematology ,Clinical Sciences ,Cerebrovascular ,Neurosciences ,Genetics ,Rare Diseases ,Brain Disorders ,2.1 Biological and endogenous factors ,Animals ,Mice ,Hemangioma ,Cavernous ,Central Nervous System ,Endothelial Cells ,Neuroinflammatory Diseases ,NF-kappa B ,NLR Family ,Pyrin Domain-Containing 3 Protein ,Proto-Oncogene Proteins ,Inflammation ,Caspases ,RNA ,astrocytes ,caspases ,cerebral cavernous malformations ,endothelial cells ,inflammasomes ,inflammation ,macrophages ,Cardiorespiratory Medicine and Haematology ,Cardiovascular System & Hematology ,Cardiovascular medicine and haematology ,Clinical sciences - Abstract
BackgroundCerebral cavernous malformations (CCMs) are neurovascular lesions caused by loss of function mutations in 1 of 3 genes, including KRIT1 (CCM1), CCM2, and PDCD10 (CCM3). CCMs affect ≈1 out of 200 children and adults, and no pharmacologic therapy is available. CCM lesion count, size, and aggressiveness vary widely among patients of similar ages with the same mutation or even within members of the same family. However, what determines the transition from quiescent lesions into mature and active (aggressive) CCM lesions is unknown.MethodsWe use genetic, RNA-sequencing, histology, flow cytometry, and imaging techniques to report the interaction between CCM endothelium, astrocytes, leukocytes, microglia/macrophages, neutrophils (CCM endothelium, astrocytes, leukocytes, microglia/macrophages, neutrophils interaction) during the pathogenesis of CCMs in the brain tissue.ResultsExpression profile of astrocytes in adult mouse brains using translated mRNAs obtained from the purification of EGFP (enhanced green fluorescent protein)-tagged ribosomes (Aldh1l1-EGFP/Rpl10a) in the presence or absence of CCM lesions (Slco1c1-iCreERT2;Pdcd10fl/fl; Pdcd10BECKO) identifies a novel gene signature for neuroinflammatory astrocytes. CCM-induced reactive astrocytes have a neuroinflammatory capacity by expressing genes involved in angiogenesis, chemotaxis, hypoxia signaling, and inflammation. RNA-sequencing analysis on RNA isolated from brain endothelial cells in chronic Pdcd10BECKO mice (CCM endothelium), identified crucial genes involved in recruiting inflammatory cells and thrombus formation through chemotaxis and coagulation pathways. In addition, CCM endothelium was associated with increased expression of Nlrp3 and Il1b. Pharmacological inhibition of NLRP3 (NOD [nucleotide-binding oligomerization domain]-' LRR [leucine-rich repeat]- and pyrin domain-containing protein 3) significantly decreased inflammasome activity as assessed by quantification of a fluorescent indicator of caspase-1 activity (FAM-FLICA [carboxyfluorescein-fluorochrome-labeled inhibitors of caspases] caspase-1) in brain endothelial cells from Pdcd10BECKO in chronic stage. Importantly, our results support the hypothesis of the crosstalk between astrocytes and CCM endothelium that can trigger recruitment of inflammatory cells arising from brain parenchyma (microglia) and the peripheral immune system (leukocytes) into mature active CCM lesions that propagate lesion growth, immunothrombosis, and bleedings. Unexpectedly, partial or total loss of brain endothelial NF-κB (nuclear factor κB) activity (using Ikkbfl/fl mice) in chronic Pdcd10BECKO mice does not prevent lesion genesis or neuroinflammation. Instead, this resulted in a trend increase in the number of lesions and immunothrombosis, suggesting that therapeutic approaches designed to target inflammation through endothelial NF-κB inhibition may contribute to detrimental side effects.ConclusionsOur study reveals previously unknown links between neuroinflammatory astrocytes and inflamed CCM endothelium as contributors that trigger leukocyte recruitment and precipitate immunothrombosis in CCM lesions. However, therapeutic approaches targeting brain endothelial NF-κB activity may contribute to detrimental side effects.
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- 2022
10. Immunodominant MHC-II (Major Histocompatibility Complex II) Restricted Epitopes in Human Apolipoprotein B
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Roy, Payel, Sidney, John, Arlehamn, Cecilia S Lindestam, Phillips, Elizabeth, Mallal, Simon, Suthahar, Sujit Silas Armstrong, Billitti, Monica, Rubiro, Paul, Marrama, Daniel, Drago, Fabrizio, Vallejo, Jenifer, Suryawanshi, Vasantika, Orecchioni, Marco, Makings, Jeffrey, Kim, Paul J, McNamara, Coleen A, Peters, Bjoern, Sette, Alessandro, and Ley, Klaus
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Autoimmune Disease ,Atherosclerosis ,Genetics ,Cardiovascular ,Clinical Research ,Biotechnology ,2.1 Biological and endogenous factors ,Aetiology ,Inflammatory and immune system ,Animals ,Apolipoproteins B ,CD4-Positive T-Lymphocytes ,Coronary Artery Disease ,Epitopes ,T-Lymphocyte ,Humans ,Interferon-gamma ,Major Histocompatibility Complex ,Mice ,Peptides ,alleles ,autoimmunity ,coronary artery disease ,peptides ,workflow ,Cardiorespiratory Medicine and Haematology ,Clinical Sciences ,Cardiovascular System & Hematology - Abstract
BackgroundCD (cluster of differentiation) 4+ T-cell responses to APOB (apolipoprotein B) are well characterized in atherosclerotic mice and detectable in humans. CD4+ T cells recognize antigenic peptides displayed on highly polymorphic HLA (human leukocyte antigen)-II. Immunogenicity of individual APOB peptides is largely unknown in humans. Only 1 HLA-II-restricted epitope was validated using the DRB1*07:01-APOB3036-3050 tetramer. We hypothesized that human APOB may contain discrete immunodominant CD4+ T-cell epitopes that trigger atherosclerosis-related autoimmune responses in donors with diverse HLA alleles.MethodsWe selected 20 APOB-derived peptides (APOB20) from an in silico screen and experimentally validated binding to the most commonly occurring human HLA-II alleles. We optimized a restimulation-based workflow to evaluate antigenicity of multiple candidate peptides in HLA-typed donors. This included activation-induced marker assay, intracellular cytokine staining, IFNγ (interferon gamma) enzyme-linked immunospot and cytometric bead array. High-throughput sequencing revealed TCR (T-cell receptor) clonalities of APOB-reactive CD4+ T cells.ResultsUsing stringent positive, negative, and crossover stimulation controls, we confirmed specificity of expansion-based protocols to detect CD4+ T cytokine responses to the APOB20 pool. Ex vivo assessment of AIM+CD4+ T cells revealed a statistically significant autoimmune response to APOB20 but not to a ubiquitously expressed negative control protein, actin. Resolution of CD4+ T responses to the level of individual peptides using IFNγ enzyme-linked immunospot led to the discovery of 6 immunodominant epitopes (APOB6) that triggered robust CD4+ T activation in most donors. APOB6-specific responding CD4+ T cells were enriched in unique expanded TCR clonotypes and preferentially expressed memory markers. Cytometric bead array analysis detected APOB6-induced secretion of both proinflammatory and regulatory cytokines. In clinical samples from patients with angiographically verified coronary artery disease, APOB6 stimulation induced higher activation and memory phenotypes and augmented secretion of proinflammatory cytokines TNF (tumor necrosis factor) and IFNγ, compared with patients with low coronary artery disease.ConclusionsUsing 3 cohorts, each with ≈20 donors, we discovered and validated 6 immunodominant, HLA-II-restricted APOB epitopes. The immune response to these APOB epitopes correlated with coronary artery disease severity.
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- 2022
11. Olfactory receptor 2 in vascular macrophages drives atherosclerosis by NLRP3-dependent IL-1 production
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Orecchioni, Marco, Kobiyama, Kouji, Winkels, Holger, Ghosheh, Yanal, McArdle, Sara, Mikulski, Zbigniew, Kiosses, William B, Fan, Zhichao, Wen, Lai, Jung, Yunmin, Roy, Payel, Ali, Amal J, Miyamoto, Yukiko, Mangan, Matthew, Makings, Jeffrey, Wang, Zhihao, Denn, Angela, Vallejo, Jenifer, Owens, Michaela, Durant, Christopher P, Braumann, Simon, Mader, Navid, Li, Lin, Matsunami, Hiroaki, Eckmann, Lars, Latz, Eicke, Wang, Zeneng, Hazen, Stanley L, and Ley, Klaus
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Atherosclerosis ,Cardiovascular ,Neurosciences ,Aging ,2.1 Biological and endogenous factors ,Aetiology ,Adult ,Aldehydes ,Animals ,Aorta ,Humans ,Inflammasomes ,Interleukin-1 ,Interleukin-1alpha ,Interleukin-1beta ,Lipid Peroxidation ,Macrophages ,Mice ,Mice ,Inbred C57BL ,Middle Aged ,NLR Family ,Pyrin Domain-Containing 3 Protein ,Oxidative Stress ,Receptors ,Odorant ,Signal Transduction ,General Science & Technology - Abstract
Atherosclerosis is an inflammatory disease of the artery walls and involves immune cells such as macrophages. Olfactory receptors (OLFRs) are G protein–coupled chemoreceptors that have a central role in detecting odorants and the sense of smell. We found that mouse vascular macrophages express the olfactory receptor Olfr2 and all associated trafficking and signaling molecules. Olfr2 detects the compound octanal, which activates the NLR family pyrin domain containing 3 (NLRP3) inflammasome and induces interleukin-1β secretion in human and mouse macrophages. We found that human and mouse blood plasma contains octanal, a product of lipid peroxidation, at concentrations sufficient to activate Olfr2 and the human ortholog olfactory receptor 6A2 (OR6A2). Boosting octanal levels exacerbated atherosclerosis, whereas genetic targeting of Olfr2 in mice significantly reduced atherosclerotic plaques. Our findings suggest that inhibiting OR6A2 may provide a promising strategy to prevent and treat atherosclerosis.
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- 2022
12. Author Correction: Combined protein and transcript single-cell RNA sequencing in human peripheral blood mononuclear cells
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Vallejo, Jenifer, Saigusa, Ryosuke, Gulati, Rishab, Suthahar, Sujit Silas Armstrong, Suryawanshi, Vasantika, Alimadadi, Ahmad, Durant, Christopher P, Ghosheh, Yanal, Roy, Payel, Ehinger, Erik, Pattarabanjird, Tanyaporn, Hanna, David B, Landay, Alan L, Tracy, Russell P, Lazar, Jason M, Mack, Wendy J, Weber, Kathleen M, Adimora, Adaora A, Hodis, Howard N, Tien, Phyllis C, Ofotokun, Igho, Heath, Sonya L, Shemesh, Avishai, McNamara, Coleen A, Lanier, Lewis L, Hedrick, Catherine C, Kaplan, Robert C, and Ley, Klaus
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Biological Sciences ,Bioinformatics and Computational Biology ,Developmental Biology ,Biological sciences - Abstract
The original article [1] contained significant errors in Fig 1A which necessitated correction; the figure has since been updated to the corrected version.
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- 2022
13. Combined protein and transcript single-cell RNA sequencing in human peripheral blood mononuclear cells
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Vallejo, Jenifer, Saigusa, Ryosuke, Gulati, Rishab, Armstrong Suthahar, Sujit Silas, Suryawanshi, Vasantika, Alimadadi, Ahmad, Durant, Christopher P, Ghosheh, Yanal, Roy, Payel, Ehinger, Erik, Pattarabanjird, Tanyaporn, Hanna, David B, Landay, Alan L, Tracy, Russell P, Lazar, Jason M, Mack, Wendy J, Weber, Kathleen M, Adimora, Adaora A, Hodis, Howard N, Tien, Phyllis C, Ofotokun, Igho, Heath, Sonya L, Shemesh, Avishai, McNamara, Coleen A, Lanier, Lewis L, Hedrick, Catherine C, Kaplan, Robert C, and Ley, Klaus
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Biological Sciences ,Bioinformatics and Computational Biology ,HIV/AIDS ,Genetics ,Clinical Research ,Human Genome ,2.1 Biological and endogenous factors ,Aetiology ,Good Health and Well Being ,Female ,Flow Cytometry ,Gene Expression Profiling ,HIV Infections ,Humans ,Leukocytes ,Mononuclear ,Sequence Analysis ,RNA ,Single-Cell Analysis ,Transcriptome ,CVD ,HIV ,scRNA-seq ,Transcriptomes ,Antibodies ,Human ,Developmental Biology ,Biological sciences - Abstract
BackgroundCryopreserved peripheral blood mononuclear cells (PBMCs) are frequently collected and provide disease- and treatment-relevant data in clinical studies. Here, we developed combined protein (40 antibodies) and transcript single-cell (sc)RNA sequencing (scRNA-seq) in PBMCs.ResultsAmong 31 participants in the Women's Interagency HIV Study (WIHS), we sequenced 41,611 cells. Using Boolean gating followed by Seurat UMAPs (tool for visualizing high-dimensional data) and Louvain clustering, we identified 50 subsets among CD4+ T, CD8+ T, B, NK cells, and monocytes. This resolution was superior to flow cytometry, mass cytometry, or scRNA-seq without antibodies. Combined protein and transcript scRNA-seq allowed for the assessment of disease-related changes in transcriptomes and cell type proportions. As a proof-of-concept, we showed such differences between healthy and matched individuals living with HIV with and without cardiovascular disease.ConclusionsIn conclusion, combined protein and transcript scRNA sequencing is a suitable and powerful method for clinical investigations using PBMCs.
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- 2022
14. Sex Differences in Coronary Artery Disease and Diabetes Revealed by scRNA-Seq and CITE-Seq of Human CD4+ T Cells
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Saigusa, Ryosuke, Vallejo, Jenifer, Gulati, Rishab, Suthahar, Sujit Silas Armstrong, Suryawanshi, Vasantika, Alimadadi, Ahmad, Makings, Jeffrey, Durant, Christopher P, Freuchet, Antoine, Roy, Payel, Ghosheh, Yanal, Pandori, William, Pattarabanjird, Tanyaporn, Drago, Fabrizio, Taylor, Angela, McNamara, Coleen A, Shemesh, Avishai, Lanier, Lewis L, Hedrick, Catherine C, and Ley, Klaus
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Genetics ,Atherosclerosis ,Human Genome ,Diabetes ,Cardiovascular ,Clinical Research ,Heart Disease - Coronary Heart Disease ,Heart Disease ,2.1 Biological and endogenous factors ,Aetiology ,Metabolic and endocrine ,Good Health and Well Being ,CD4-Positive T-Lymphocytes ,Coronary Angiography ,Coronary Artery Disease ,Diabetes Mellitus ,Female ,Humans ,Leukocytes ,Mononuclear ,Male ,Sex Characteristics ,Single-Cell Analysis ,coronary artery disease ,diabetes ,CITE-Seq ,scRNA-Seq ,PBMC ,Other Chemical Sciences ,Other Biological Sciences ,Chemical Physics - Abstract
Despite the decades-old knowledge that males and people with diabetes mellitus (DM) are at increased risk for coronary artery disease (CAD), the reasons for this association are only partially understood. Among the immune cells involved, recent evidence supports a critical role of T cells as drivers and modifiers of CAD. CD4+ T cells are commonly found in atherosclerotic plaques. We aimed to understand the relationship of CAD with sex and DM by single-cell RNA (scRNA-Seq) and antibody sequencing (CITE-Seq) of CD4+ T cells. Peripheral blood mononuclear cells (PBMCs) of 61 men and women who underwent cardiac catheterization were interrogated by scRNA-Seq combined with 49 surface markers (CITE-Seq). CAD severity was quantified using Gensini scores, with scores above 30 considered CAD+ and below 6 considered CAD-. Four pairs of groups were matched for clinical and demographic parameters. To test how sex and DM changed cell proportions and gene expression, we compared matched groups of men and women, as well as diabetic and non-diabetic subjects. We analyzed 41,782 single CD4+ T cell transcriptomes for sex differences in 16 women and 45 men with and without coronary artery disease and with and without DM. We identified 16 clusters in CD4+ T cells. The proportion of cells in CD4+ effector memory cluster 8 (CD4T8, CCR2+ Em) was significantly decreased in CAD+, especially among DM+ participants. This same cluster, CD4T8, was significantly decreased in female participants, along with two other CD4+ T cell clusters. In CD4+ T cells, 31 genes showed significant and coordinated upregulation in both CAD and DM. The DM gene signature was partially additive to the CAD gene signature. We conclude that (1) CAD and DM are clearly reflected in PBMC transcriptomes, and (2) significant differences exist between women and men and (3) between subjects with DM and non-DM.
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- 2022
15. Immune profiling and tracking of two-dimensional transition metal dichalcogenides in cells and tissues
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Gazzi, Arianna, Fusco, Laura, Orecchioni, Marco, Keshavan, Sandeep, Shin, Yuyoung, Grivel, Jean-Charles, Rinchai, Darawan, Ahmed, Eiman I., Elhanani, Ofer, Furesi, Giulia, Rauner, Martina, Keren, Leeat, Ley, Klaus, Casiraghi, Cinzia, Bedognetti, Davide, Fadeel, Bengt, and Delogu, Lucia Gemma
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- 2024
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16. Endothelial Heparan Sulfate Mediates Hepatic Neutrophil Trafficking and Injury during Staphylococcus aureus Sepsis
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Golden, Gregory J, Toledo, Alejandro Gómez, Marki, Alex, Sorrentino, James T, Morris, Claire, Riley, Raquel J, Spliid, Charlotte, Chen, Qiongyu, Cornax, Ingrid, Lewis, Nathan E, Varki, Nissi, Le, Dzung, Malmström, Johan, Karlsson, Christofer, Ley, Klaus, Nizet, Victor, and Esko, Jeffrey D
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Liver Disease ,Sepsis ,Infectious Diseases ,Hematology ,Digestive Diseases ,Emerging Infectious Diseases ,Clinical Research ,Chronic Liver Disease and Cirrhosis ,2.1 Biological and endogenous factors ,Aetiology ,Cardiovascular ,Inflammatory and immune system ,Animals ,Disease Models ,Animal ,Endothelial Cells ,Female ,Glycocalyx ,Heparitin Sulfate ,Liver ,Lung ,Male ,Mice ,Mice ,Inbred C57BL ,Neutrophil Activation ,Neutrophils ,Staphylococcus aureus ,heparan sulfate ,intravital microscopy ,liver ,neutrophils ,proteomics ,sepsis ,thrombosis ,Microbiology - Abstract
Hepatic failure is an important risk factor for poor outcome in septic patients. Using a chemical tagging workflow and high-resolution mass spectrometry, we demonstrate that rapid proteome remodeling of the vascular surfaces precedes hepatic damage in a murine model of Staphylococcus aureus sepsis. These early changes include vascular deposition of neutrophil-derived proteins, shedding of vascular receptors, and altered levels of heparin/heparan sulfate-binding factors. Modification of endothelial heparan sulfate, a major component of the vascular glycocalyx, diminishes neutrophil trafficking to the liver and reduces hepatic coagulopathy and organ damage during the systemic inflammatory response to infection. Modifying endothelial heparan sulfate likewise reduces neutrophil trafficking in sterile hepatic injury, reflecting a more general role of heparan sulfate contribution to the modulation of leukocyte behavior during inflammation. IMPORTANCE Vascular glycocalyx remodeling is critical to sepsis pathology, but the glycocalyx components that contribute to this process remain poorly characterized. This article shows that during Staphylococcus aureus sepsis, the liver vascular glycocalyx undergoes dramatic changes in protein composition associated with neutrophilic activity and heparin/heparan sulfate binding, all before organ damage is detectable by standard circulating liver damage markers or histology. Targeted manipulation of endothelial heparan sulfate modulates S. aureus sepsis-induced hepatotoxicity by controlling the magnitude of neutrophilic infiltration into the liver in both nonsterile and sterile injury. These data identify an important vascular glycocalyx component that impacts hepatic failure during nonsterile and sterile injury.
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- 2021
17. Normalization of cholesterol metabolism in spinal microglia alleviates neuropathic pain
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Navia-Pelaez, Juliana M, Choi, Soo-Ho, dos Santos Aggum Capettini, Luciano, Xia, Yining, Gonen, Ayelet, Agatisa-Boyle, Colin, Delay, Lauriane, dos Santos, Gilson Gonçalves, Catroli, Glaucilene F, Kim, Jungsu, Lu, Jenny W, Saylor, Benjamin, Winkels, Holger, Durant, Christopher P, Ghosheh, Yanal, Beaton, Graham, Ley, Klaus, Kufareva, Irina, Corr, Maripat, Yaksh, Tony L, and Miller, Yury I
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Pain Research ,Neurodegenerative ,Neurosciences ,Peripheral Neuropathy ,Genetics ,Chronic Pain ,Aetiology ,2.1 Biological and endogenous factors ,Neurological ,ATP Binding Cassette Transporter 1 ,ATP Binding Cassette Transporter ,Subfamily G ,Member 1 ,Animals ,Biological Transport ,Cell Line ,Cholesterol ,HEK293 Cells ,Humans ,Inflammation ,Male ,Membrane Microdomains ,Mice ,Mice ,Inbred C57BL ,Microglia ,Neuralgia ,Protein Binding ,Signal Transduction ,Spinal Cord ,Medical and Health Sciences ,Immunology - Abstract
Neuroinflammation is a major component in the transition to and perpetuation of neuropathic pain states. Spinal neuroinflammation involves activation of TLR4, localized to enlarged, cholesterol-enriched lipid rafts, designated here as inflammarafts. Conditional deletion of cholesterol transporters ABCA1 and ABCG1 in microglia, leading to inflammaraft formation, induced tactile allodynia in naive mice. The apoA-I binding protein (AIBP) facilitated cholesterol depletion from inflammarafts and reversed neuropathic pain in a model of chemotherapy-induced peripheral neuropathy (CIPN) in wild-type mice, but AIBP failed to reverse allodynia in mice with ABCA1/ABCG1-deficient microglia, suggesting a cholesterol-dependent mechanism. An AIBP mutant lacking the TLR4-binding domain did not bind microglia or reverse CIPN allodynia. The long-lasting therapeutic effect of a single AIBP dose in CIPN was associated with anti-inflammatory and cholesterol metabolism reprogramming and reduced accumulation of lipid droplets in microglia. These results suggest a cholesterol-driven mechanism of regulation of neuropathic pain by controlling the TLR4 inflammarafts and gene expression program in microglia and blocking the perpetuation of neuroinflammation.
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- 2021
18. Pairing of single-cell RNA analysis and T cell antigen receptor profiling indicates breakdown of T cell tolerance checkpoints in atherosclerosis
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Wang, Zhihua, Zhang, Xi, Lu, Shu, Zhang, Chuankai, Ma, Zhe, Su, Rui, Li, Yuanfang, Sun, Ting, Li, Yutao, Hong, Mingyang, Deng, Xinyi, Rafiee Monjezi, Mohammad, Hristov, Michael, Steffens, Sabine, Santovito, Donato, Dornmair, Klaus, Ley, Klaus, Weber, Christian, Mohanta, Sarajo K., Habenicht, Andreas J. R., and Yin, Changjun
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- 2023
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19. Elongated neutrophil-derived structures are blood-borne microparticles formed by rolling neutrophils during sepsis.
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Marki, Alex, Buscher, Konrad, Lorenzini, Cristina, Meyer, Matthew, Saigusa, Ryosuke, Fan, Zhichao, Yeh, Yi-Ting, Hartmann, Nadine, Dan, Jennifer M, Kiosses, William B, Golden, Gregory J, Ganesan, Rajee, Winkels, Holger, Orecchioni, Marco, McArdle, Sara, Mikulski, Zbigniew, Altman, Yoav, Bui, Jack, Kronenberg, Mitchell, Chien, Shu, Esko, Jeffrey D, Nizet, Victor, Smalley, David, Roth, Johannes, and Ley, Klaus
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Immunology ,Medical and Health Sciences - Abstract
Rolling neutrophils form tethers with submicron diameters. Here, we report that these tethers detach, forming elongated neutrophil-derived structures (ENDS) in the vessel lumen. We studied ENDS formation in mice and humans in vitro and in vivo. ENDS do not contain mitochondria, endoplasmic reticulum, or DNA, but are enriched for S100A8, S100A9, and 57 other proteins. Within hours of formation, ENDS round up, and some of them begin to present phosphatidylserine on their surface (detected by annexin-5 binding) and release S100A8-S100A9 complex, a damage-associated molecular pattern protein that is a known biomarker of neutrophilic inflammation. ENDS appear in blood plasma of mice upon induction of septic shock. Compared with healthy donors, ENDS are 10-100-fold elevated in blood plasma of septic patients. Unlike neutrophil-derived extracellular vesicles, most ENDS are negative for the tetraspanins CD9, CD63, and CD81. We conclude that ENDS are a new class of bloodborne submicron particles with a formation mechanism linked to neutrophil rolling on the vessel wall.
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- 2021
20. A CD22-Shp1 phosphatase axis controls integrin β7 display and B cell function in mucosal immunity.
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Ballet, Romain, Brennan, Martin, Brandl, Carolin, Feng, Ningguo, Berri, Jeremy, Cheng, Julian, Ocón, Borja, Alborzian Deh Sheikh, Amin, Marki, Alex, Bi, Yuhan, Abram, Clare L, Lowell, Clifford A, Tsubata, Takeshi, Greenberg, Harry B, Macauley, Matthew S, Ley, Klaus, Nitschke, Lars, and Butcher, Eugene C
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Intestinal Mucosa ,B-Lymphocytes ,Animals ,Mice ,Inbred BALB C ,Mice ,Inbred C57BL ,Mice ,Knockout ,Rotavirus ,Rotavirus Infections ,Disease Models ,Animal ,Integrins ,Integrin beta Chains ,Tissue Culture Techniques ,Signal Transduction ,Chemotaxis ,Leukocyte ,Endocytosis ,Immunity ,Mucosal ,Phosphorylation ,Female ,Male ,Protein Tyrosine Phosphatase ,Non-Receptor Type 6 ,Sialic Acid Binding Ig-like Lectin 2 ,1.1 Normal biological development and functioning ,Underpinning research ,Inflammatory and immune system ,Immunology - Abstract
The integrin α4β7 selectively regulates lymphocyte trafficking and adhesion in the gut and gut-associated lymphoid tissue (GALT). Here, we describe unexpected involvement of the tyrosine phosphatase Shp1 and the B cell lectin CD22 (Siglec-2) in the regulation of α4β7 surface expression and gut immunity. Shp1 selectively inhibited β7 endocytosis, enhancing surface α4β7 display and lymphocyte homing to GALT. In B cells, CD22 associated in a sialic acid-dependent manner with integrin β7 on the cell surface to target intracellular Shp1 to β7. Shp1 restrained plasma membrane β7 phosphorylation and inhibited β7 endocytosis without affecting β1 integrin. B cells with reduced Shp1 activity, lacking CD22 or expressing CD22 with mutated Shp1-binding or carbohydrate-binding domains displayed parallel reductions in surface α4β7 and in homing to GALT. Consistent with the specialized role of α4β7 in intestinal immunity, CD22 deficiency selectively inhibited intestinal antibody and pathogen responses.
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- 2021
21. Cell-autonomous regulation of complement C3 by factor H limits macrophage efferocytosis and exacerbates atherosclerosis
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Kiss, Máté G., Papac-Miličević, Nikolina, Porsch, Florentina, Tsiantoulas, Dimitrios, Hendrikx, Tim, Takaoka, Minoru, Dinh, Huy Q., Narzt, Marie-Sophie, Göderle, Laura, Ozsvár-Kozma, Mária, Schuster, Michael, Fortelny, Nikolaus, Hladik, Anastasiya, Knapp, Sylvia, Gruber, Florian, Pickering, Matthew C., Bock, Christoph, Swirski, Filip K., Ley, Klaus, Zernecke, Alma, Cochain, Clément, Kemper, Claudia, Mallat, Ziad, and Binder, Christoph J.
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- 2023
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22. Abstract 12560: Single Cell RNA Sequencing Signature of Cardiac Allograft Vasculopathy
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Tada, Yuko, Armstrong Suthahar, Sujit Silas, Suryawanshi, Vasantika, Wu, Amy, Wang, Erpei, Cusi, Vincenzo, Ley, Klaus F, and Kim, Paul
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- 2023
- Full Text
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23. Abstract 12179: Neutrophil-Specific Deletion of Beta2 Integrins Alleviates Myocardial Ischemia-Reperfusion Injury in Male Mice
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Liu, Wei, Cronin, Chunxia, Wen, Lai, Ley, Klaus, Liang, Bruce T, and Fan, Zhichao
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- 2023
- Full Text
- View/download PDF
24. Author Correction: Human circulating CD24hi marginal zone B cells produce IgM targeting atherogenic antigens and confer protection from vascular disease
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Pattarabanjird, Tanyaporn, Nguyen, Anh Tram, McSkimming, Chantel, Dinh, Huy Q., Marshall, Melissa A., Ghosheh, Yanal, Gulati, Rishab, Durant, Chistopher, Vallejo, Jenifer, Saigusa, Ryosuke, Drago, Fabrizio, Guy, Thomas V., Premo, Katherine, Taylor, Angela M., Paul, Soumen, Kundu, Bijoy, Berr, Stuart, Gonen, Ayelet, Tsimikas, Sotirios, Miller, Yury, Pillai, Shiv, Ley, Klaus, Hedrick, Catherine C., and McNamara, Coleen A.
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- 2023
- Full Text
- View/download PDF
25. Altered Gut Microbiota and Host Metabolite Profiles in Women With Human Immunodeficiency Virus
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Wang, Zheng, Usyk, Mykhaylo, Sollecito, Christopher C, Qiu, Yunping, Williams-Nguyen, Jessica, Hua, Simin, Gradissimo, Ana, Wang, Tao, Xue, Xiaonan, Kurland, Irwin J, Ley, Klaus, Landay, Alan L, Anastos, Kathryn, Knight, Rob, Kaplan, Robert C, Burk, Robert D, and Qi, Qibin
- Subjects
Medical Biochemistry and Metabolomics ,Medical Microbiology ,Biomedical and Clinical Sciences ,Infectious Diseases ,HIV/AIDS ,2.1 Biological and endogenous factors ,Aetiology ,Infection ,Female ,Gastrointestinal Microbiome ,HIV ,HIV Infections ,Humans ,Metabolomics ,RNA ,Ribosomal ,16S ,HIV infection ,gut microbiota ,metabolomics ,integrative analysis ,Biological Sciences ,Medical and Health Sciences ,Microbiology ,Clinical sciences - Abstract
BackgroundAlterations in gut microbiota (GMB) and host metabolites have been noted in individuals with HIV. However, it remains unclear whether alterations in GMB and related functional groups contribute to disrupted host metabolite profiles in these individuals.MethodsThis study included 185 women (128 with longstanding HIV infection, 88% under antiretroviral therapy; and 57 women without HIV from the same geographic location with comparable characteristics). Stool samples were analyzed by 16S rRNA V4 region sequencing, and GMB function was inferred by PICRUSt. Plasma metabolomic profiling was performed using liquid chromatography-tandem mass spectrometry, and 133 metabolites (amino acids, biogenic amines, acylcarnitines, and lipids) were analyzed.ResultsFour predominant bacterial genera were identified as associated with HIV infection, with higher abundances of Ruminococcus and Oscillospira and lower abundances of Bifidobacterium and Collinsella in women with HIV than in those without. Women with HIV showed a distinct plasma metabolite profile, which featured elevated glycerophospholipid levels compared with those without HIV. Functional analyses also indicated that GMB lipid metabolism was enriched in women with HIV. Ruminococcus and Oscillospira were among the top bacterial genera contributing to the GMB glycerophospholipid metabolism pathway and showed positive correlations with host plasma glycerophospholipid levels. One bacterial functional capacity in the acetate and propionate biosynthesis pathway was identified to be mainly contributed by Bifidobacterium; this functional capacity was lower in women with HIV than in women without HIV.ConclusionsOur integrative analyses identified altered GMB with related functional capacities that might be associated with disrupted plasma metabolite profiles in women with HIV.
- Published
- 2020
26. Frontline Science: A flexible kink in the transmembrane domain impairs β2 integrin extension and cell arrest from rolling
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Sun, Hao, Fan, Zhichao, Gingras, Alexandre R, Lopez-Ramirez, Miguel A, Ginsberg, Mark H, and Ley, Klaus
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Biomedical and Clinical Sciences ,Immunology ,CD18 Antigens ,Cell Cycle Checkpoints ,HL-60 Cells ,Humans ,Intercellular Adhesion Molecule-1 ,Interleukin-8 ,Leukocyte Rolling ,Mutation ,P-Selectin ,Proline ,Protein Conformation ,Protein Domains ,talin ,activation ,affinity ,adhesion ,spreading ,Biochemistry and Cell Biology - Abstract
β2 integrins are the main adhesion molecules in neutrophils and other leukocytes and are rapidly activated by inside-out signaling, which results in conformational changes that are transmitted through the transmembrane domain (TMD). Here, we investigated the biologic effect of introducing a proline mutation in the β2 integrin TMD to create a flexible kink that uncouples the topology of the inner half of the TMD from the outer half and impairs integrin activation. The β2 integrin alpha chains, αL, αM, αX, and αD, all contain an inserted (I) domain with homology to von Willebrand factor A domain. β2 activation was monitored in a homogenous binding assay of 2 reporter monoclonal antibodies: KIM127 reporting extension (E+ ) and mAb24 reporting the high-affinity (H+ ) conformation of the β2 I-like domain. The proline mutation partially diminished chemokine-induced extension, but not the high-affinity conformation. The proline mutation in the TMD of β2 completely inhibited arrest of rolling HL-60 cells in response to the chemokine IL-8. TMD mutant HL-60 cells rolling on P-selectin and ICAM-1 were unable to reduce their rolling velocity in response to IL-8. Quantitative dynamic footprinting live-cell imaging showed that blocking TMD topology transmission impaired the chemokine-induced activation of β2, limiting the appearance of extended high-affinity (E+ H+ ) β2. This also resulted in a defect in early spreading (3 min after arrest), which could be overcome by forced integrin activation using Mn2+ . We conclude that the TMD proline mutation severely impairs β2 integrin extension, cell arrest, and early spreading.
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- 2020
27. Deleting interleukin-10 from myeloid cells exacerbates atherosclerosis in Apoe−/− mice
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Orecchioni, Marco, Wolf, Dennis, Suryawanshi, Vasantika, Winkels, Holger, Kobiyama, Kouji, Makings, Jeffrey, Kiosses, William B., and Ley, Klaus
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- 2023
- Full Text
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28. Single cell transcriptomics and TCR reconstruction reveal CD4 T cell response to MHC-II-restricted APOB epitope in human cardiovascular disease
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Saigusa, Ryosuke, Roy, Payel, Freuchet, Antoine, Gulati, Rishab, Ghosheh, Yanal, Armstrong Suthahar, Sujit Silas, Durant, Christopher P., Hanna, David B., Kiosses, William B., Orecchioni, Marco, Wen, Lai, Wu, Runpei, Kuniholm, Mark H., Landay, Alan L., Anastos, Kathryn, Tien, Phyllis C., Gange, Stephen J., Kassaye, Seble, Vallejo, Jenifer, Hedrick, Catherine C., Kwok, William W., Sette, Alessandro, Hodis, Howard N., Kaplan, Robert C., and Ley, Klaus
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- 2022
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29. How the immune system shapes atherosclerosis: roles of innate and adaptive immunity
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Roy, Payel, Orecchioni, Marco, and Ley, Klaus
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- 2022
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30. Rap1 binding and a lipid-dependent helix in talin F1 domain promote integrin activation in tandem
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Gingras, Alexandre R, Lagarrigue, Frederic, Cuevas, Monica N, Valadez, Andrew J, Zorovich, Marcus, McLaughlin, Wilma, Lopez-Ramirez, Miguel Alejandro, Seban, Nicolas, Ley, Klaus, Kiosses, William B, and Ginsberg, Mark H
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1.1 Normal biological development and functioning ,Underpinning research ,Animals ,Binding Sites ,CHO Cells ,Cricetinae ,Cricetulus ,Humans ,Integrins ,Lipids ,Mutation ,Protein Binding ,Protein Conformation ,Protein Domains ,Shelterin Complex ,Talin ,Telomere-Binding Proteins ,Biological Sciences ,Medical and Health Sciences ,Developmental Biology - Abstract
Rap1 GTPases bind effectors, such as RIAM, to enable talin1 to induce integrin activation. In addition, Rap1 binds directly to the talin1 F0 domain (F0); however, this interaction makes a limited contribution to integrin activation in CHO cells or platelets. Here, we show that talin1 F1 domain (F1) contains a previously undetected Rap1-binding site of similar affinity to that in F0. A structure-guided point mutant (R118E) in F1, which blocks Rap1 binding, abolishes the capacity of Rap1 to potentiate talin1-induced integrin activation. The capacity of F1 to mediate Rap1-dependent integrin activation depends on a unique loop in F1 that has a propensity to form a helix upon binding to membrane lipids. Basic membrane-facing residues of this helix are critical, as charge-reversal mutations led to dramatic suppression of talin1-dependent activation. Thus, a novel Rap1-binding site and a transient lipid-dependent helix in F1 work in tandem to enable a direct Rap1-talin1 interaction to cause integrin activation.
- Published
- 2019
31. CX3CL1-Fc treatment prevents atherosclerosis in Ldlr KO mice.
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Riopel, Matthew, Vassallo, Melanie, Ehinger, Erik, Pattison, Jennifer, Bowden, Karen, Winkels, Holger, Wilson, Maria, de Jong, Ron, Patel, Sanjay, Balakrishna, Deepika, Bilakovics, James, Fanjul, Andrea, Plonowski, Artur, Larson, Christopher J, Ley, Klaus, Cabrales, Pedro, Witztum, Joseph L, Olefsky, Jerrold M, and Lee, Yun Sok
- Subjects
Aorta ,Cells ,Cultured ,Animals ,Mice ,Inbred C57BL ,Mice ,Receptors ,LDL ,Recombinant Proteins ,Immunoglobulin Fc Fragments ,Male ,Atherosclerosis ,Chemokine CX3CL1 ,Plaque ,Atherosclerotic ,CX3CR1 ,Fractalkine ,Inflammation ,Ldlr KO ,Monocyte adhesion ,Cells ,Cultured ,Inbred C57BL ,Receptors ,LDL ,Plaque ,Atherosclerotic ,Biochemistry and Cell Biology ,Physiology - Abstract
OBJECTIVE:Atherosclerosis is a major cause of cardiovascular disease. Monocyte-endothelial cell interactions are partly mediated by expression of monocyte CX3CR1 and endothelial cell fractalkine (CX3CL1). Interrupting the interaction between this ligand-receptor pair should reduce monocyte binding to the endothelial wall and reduce atherosclerosis. We sought to reduce atherosclerosis by preventing monocyte-endothelial cell interactions through use of a long-acting CX3CR1 agonist. METHODS:In this study, the chemokine domain of CX3CL1 was fused to the mouse Fc region to generate a long-acting soluble form of CX3CL1 suitable for chronic studies. CX3CL1-Fc or saline was injected twice a week (30 mg/kg) for 4 months into Ldlr knockout (KO) mice on an atherogenic western diet. RESULTS:CX3CL1-Fc-treated Ldlr KO mice showed decreased en face aortic lesion surface area and reduced aortic root lesion size with decreased necrotic core area. Flow cytometry analyses of CX3CL1-Fc-treated aortic wall cell digests revealed a decrease in M1-like polarized macrophages and T cells. Moreover, CX3CL1-Fc administration reduced diet-induced atherosclerosis after switching from an atherogenic to a normal chow diet. In vitro monocyte adhesion studies revealed that CX3CL1-Fc treatment caused fewer monocytes to adhere to a human umbilical vein endothelial cell monolayer. Furthermore, a dorsal window chamber model demonstrated that CX3CL1-Fc treatment decreased in vivo leukocyte adhesion and rolling in live capillaries after short-term ischemia-reperfusion. CONCLUSION:These results indicate that CX3CL1-Fc can inhibit monocyte/endothelial cell adhesion as well as reduce atherosclerosis.
- Published
- 2019
32. High-Affinity Bent β2-Integrin Molecules in Arresting Neutrophils Face Each Other through Binding to ICAMs In cis
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Fan, Zhichao, Kiosses, William Bill, Sun, Hao, Orecchioni, Marco, Ghosheh, Yanal, Zajonc, Dirk M, Arnaout, M Amin, Gutierrez, Edgar, Groisman, Alex, Ginsberg, Mark H, and Ley, Klaus
- Subjects
Biochemistry and Cell Biology ,Biological Sciences ,5.1 Pharmaceuticals ,Development of treatments and therapeutic interventions ,CD18 Antigens ,Cell Adhesion Molecules ,Humans ,Neutrophils ,Protein Binding ,STORM ,TIRF ,human ,integrin ,integrin activation ,molecular modeling ,neutrophil ,superresolution ,Medical Physiology ,Biological sciences - Abstract
Leukocyte adhesion requires β2-integrin activation. Resting integrins exist in a bent-closed conformation-i.e., not extended (E-) and not high affinity (H-)-unable to bind ligand. Fully activated E+H+ integrin binds intercellular adhesion molecules (ICAMs) expressed on the opposing cell in trans. E-H- transitions to E+H+ through E+H- or through E-H+, which binds to ICAMs on the same cell in cis. Spatial patterning of activated integrins is thought to be required for effective arrest, but no high-resolution cell surface localization maps of activated integrins exist. Here, we developed Super-STORM by combining super-resolution microscopy with molecular modeling to precisely localize activated integrin molecules and identify the molecular patterns of activated integrins on primary human neutrophils. At the time of neutrophil arrest, E-H+ integrins face each other to form oriented (non-random) nanoclusters. To address the mechanism causing this pattern, we blocked integrin binding to ICAMs in cis, which significantly relieved the face-to-face orientation.
- Published
- 2019
33. De-stressing plaques attenuates atherosclerosis progression
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Nettersheim, Felix Sebastian and Ley, Klaus
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- 2022
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34. Molecular mechanisms of leukocyte β2 integrin activation
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Wen, Lai, Moser, Markus, and Ley, Klaus
- Published
- 2022
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35. Titration of 124 antibodies using CITE-Seq on human PBMCs
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Nettersheim, Felix Sebastian, Armstrong, Sujit Silas, Durant, Christopher, Blanco-Dominguez, Rafael, Roy, Payel, Orecchioni, Marco, Suryawanshi, Vasantika, and Ley, Klaus
- Published
- 2022
- Full Text
- View/download PDF
36. Deconvolution of pro- and antiviral genomic responses in Zika virus-infected and bystander macrophages
- Author
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Carlin, Aaron F, Vizcarra, Edward A, Branche, Emilie, Viramontes, Karla M, Suarez-Amaran, Lester, Ley, Klaus, Heinz, Sven, Benner, Christopher, Shresta, Sujan, and Glass, Christopher K
- Subjects
Infectious Diseases ,Vaccine Related ,Prevention ,Human Genome ,Biodefense ,Genetics ,2.1 Biological and endogenous factors ,Aetiology ,2.2 Factors relating to the physical environment ,Infection ,Good Health and Well Being ,Bystander Effect ,Female ,Humans ,Immunity ,Innate ,Interferon-beta ,Macrophages ,Male ,Proteolysis ,RNA Polymerase II ,STAT2 Transcription Factor ,Zika Virus ,Zika Virus Infection ,Zika virus ,macrophage ,immune evasion ,genomics ,transcription - Abstract
Genome-wide investigations of host-pathogen interactions are often limited by analyses of mixed populations of infected and uninfected cells, which lower sensitivity and accuracy. To overcome these obstacles and identify key mechanisms by which Zika virus (ZIKV) manipulates host responses, we developed a system that enables simultaneous characterization of genome-wide transcriptional and epigenetic changes in ZIKV-infected and neighboring uninfected primary human macrophages. We demonstrate that transcriptional responses in ZIKV-infected macrophages differed radically from those in uninfected neighbors and that studying the cell population as a whole produces misleading results. Notably, the uninfected population of macrophages exhibits the most rapid and extensive changes in gene expression, related to type I IFN signaling. In contrast, infected macrophages exhibit a delayed and attenuated transcriptional response distinguished by preferential expression of IFNB1 at late time points. Biochemical and genomic studies of infected macrophages indicate that ZIKV infection causes both a targeted defect in the type I IFN response due to degradation of STAT2 and reduces RNA polymerase II protein levels and DNA occupancy, particularly at genes required for macrophage identity. Simultaneous evaluation of transcriptomic and epigenetic features of infected and uninfected macrophages thereby reveals the coincident evolution of dominant proviral or antiviral mechanisms, respectively, that determine the outcome of ZIKV exposure.
- Published
- 2018
37. Regulatory CD4+ T Cells Recognize Major Histocompatibility Complex Class II Molecule–Restricted Peptide Epitopes of Apolipoprotein B
- Author
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Kimura, Takayuki, Kobiyama, Kouji, Winkels, Holger, Tse, Kevin, Miller, Jacqueline, Vassallo, Melanie, Wolf, Dennis, Ryden, Christian, Orecchioni, Marco, Dileepan, Thamotharampillai, Jenkins, Marc K, James, Eddie A, Kwok, William W, Hanna, David B, Kaplan, Robert C, Strickler, Howard D, Durkin, Helen G, Kassaye, Seble G, Karim, Roksana, Tien, Phyllis C, Landay, Alan L, Gange, Stephen J, Sidney, John, Sette, Alessandro, and Ley, Klaus
- Subjects
Biomedical and Clinical Sciences ,Immunology ,Immunization ,Cardiovascular ,Prevention ,Vaccine Related ,Atherosclerosis ,Heart Disease ,2.1 Biological and endogenous factors ,Aetiology ,Good Health and Well Being ,Adjuvants ,Immunologic ,Animals ,Aorta ,Aortic Diseases ,Apolipoprotein B-100 ,Apolipoproteins B ,Disease Models ,Animal ,Epitope Mapping ,Epitopes ,T-Lymphocyte ,Female ,Freund's Adjuvant ,Histocompatibility Antigens Class II ,Humans ,Lymphocyte Activation ,Male ,Mice ,Mice ,Inbred C57BL ,Mice ,Knockout ,ApoE ,Peptide Fragments ,Plaque ,Atherosclerotic ,T-Lymphocytes ,Regulatory ,Vaccination ,antigen specificity ,apoB-100 ,atherosclerosis ,regulatory T cells ,vaccination ,Cardiorespiratory Medicine and Haematology ,Clinical Sciences ,Public Health and Health Services ,Cardiovascular System & Hematology ,Cardiovascular medicine and haematology ,Clinical sciences ,Sports science and exercise - Abstract
BackgroundCD4+ T cells play an important role in atherosclerosis, but their antigen specificity is poorly understood. Immunization with apolipoprotein B (ApoB, core protein of low density lipoprotein) is known to be atheroprotective in animal models. Here, we report on a human APOB peptide, p18, that is sequence-identical in mouse ApoB and binds to both mouse and human major histocompatibility complex class II molecules.MethodsWe constructed p18 tetramers to detect human and mouse APOB-specific T cells and assayed their phenotype by flow cytometry including CD4 lineage transcription factors, intracellular cytokines, and T cell receptor activation. Apolipoprotein E-deficient ( Apoe-/-) mice were vaccinated with p18 peptide or adjuvants alone, and atherosclerotic burden in the aorta was determined.ResultsIn human peripheral blood mononuclear cells from donors without cardiovascular disease, p18 specific CD4+ T cells detected by a new human leukocyte antigen-antigen D related-p18 tetramers were mostly Foxp3+ regulatory T cells (Tregs). Donors with subclinical cardiovascular disease as detected by carotid artery ultrasound had Tregs coexpressing retinoic acid-related orphan receptor gamma t or T-bet, which were both almost absent in donors without cardiovascular disease. In Apoe-/- mice, immunization with p18 induced Tregs and reduced atherosclerotic lesions. After peptide restimulation, responding CD4+ T cells identified by Nur77-GFP (green fluorescent protein) were highly enriched in Tregs. A new mouse I-Ab-p18 tetramer identified the expansion of p18-specific CD4+ T cells on vaccination, which were enriched for interleukin-10-producing Tregs.ConclusionsThese findings show that APOB p18-specific CD4+ T cells are mainly Tregs in healthy donors, but coexpress other CD4 lineage transcription factors in donors with subclinical cardiovascular disease. This study identifies ApoB peptide 18 as the first Treg epitope in human and mouse atherosclerosis.
- Published
- 2018
38. Oxidized phospholipids are proinflammatory and proatherogenic in hypercholesterolaemic mice.
- Author
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Que, Xuchu, Hung, Ming-Yow, Yeang, Calvin, Gonen, Ayelet, Prohaska, Thomas A, Sun, Xiaoli, Diehl, Cody, Määttä, Antti, Gaddis, Dalia E, Bowden, Karen, Pattison, Jennifer, MacDonald, Jeffrey G, Ylä-Herttuala, Seppo, Mellon, Pamela L, Hedrick, Catherine C, Ley, Klaus, Miller, Yury I, Glass, Christopher K, Peterson, Kirk L, Binder, Christoph J, Tsimikas, Sotirios, and Witztum, Joseph L
- Subjects
Macrophages ,Peritoneal ,Animals ,Mice ,Inbred C57BL ,Mice ,Transgenic ,Mice ,Fatty Liver ,Aortic Valve Stenosis ,Hypercholesterolemia ,Disease Progression ,Inflammation ,Phosphorylcholine ,Cholesterol ,Lipoproteins ,LDL ,Phospholipids ,Immunoglobulin M ,Receptors ,LDL ,Apoptosis ,Oxidation-Reduction ,Female ,Atherosclerosis ,Single-Chain Antibodies ,Macrophages ,Peritoneal ,Inbred C57BL ,Transgenic ,Lipoproteins ,LDL ,Receptors ,General Science & Technology - Abstract
Oxidized phospholipids (OxPL) are ubiquitous, are formed in many inflammatory tissues, including atherosclerotic lesions, and frequently mediate proinflammatory changes 1 . Because OxPL are mostly the products of non-enzymatic lipid peroxidation, mechanisms to specifically neutralize them are unavailable and their roles in vivo are largely unknown. We previously cloned the IgM natural antibody E06, which binds to the phosphocholine headgroup of OxPL, and blocks the uptake of oxidized low-density lipoprotein (OxLDL) by macrophages and inhibits the proinflammatory properties of OxPL2-4. Here, to determine the role of OxPL in vivo in the context of atherogenesis, we generated transgenic mice in the Ldlr-/- background that expressed a single-chain variable fragment of E06 (E06-scFv) using the Apoe promoter. E06-scFv was secreted into the plasma from the liver and macrophages, and achieved sufficient plasma levels to inhibit in vivo macrophage uptake of OxLDL and to prevent OxPL-induced inflammatory signalling. Compared to Ldlr-/- mice, Ldlr -/- E06-scFv mice had 57-28% less atherosclerosis after 4, 7 and even 12 months of 1% high-cholesterol diet. Echocardiographic and histologic evaluation of the aortic valves demonstrated that E06-scFv ameliorated the development of aortic valve gradients and decreased aortic valve calcification. Both cholesterol accumulation and in vivo uptake of OxLDL were decreased in peritoneal macrophages, and both peritoneal and aortic macrophages had a decreased inflammatory phenotype. Serum amyloid A was decreased by 32%, indicating decreased systemic inflammation, and hepatic steatosis and inflammation were also decreased. Finally, the E06-scFv prolonged life as measured over 15 months. Because the E06-scFv lacks the functional effects of an intact antibody other than the ability to bind OxPL and inhibit OxLDL uptake in macrophages, these data support a major proatherogenic role of OxLDL and demonstrate that OxPL are proinflammatory and proatherogenic, which E06 counteracts in vivo. These studies suggest that therapies inactivating OxPL may be beneficial for reducing generalized inflammation, including the progression of atherosclerosis, aortic stenosis and hepatic steatosis.
- Published
- 2018
39. Transmission of integrin β7 transmembrane domain topology enables gut lymphoid tissue development
- Author
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Sun, Hao, Lagarrigue, Frederic, Gingras, Alexandre R, Fan, Zhichao, Ley, Klaus, and Ginsberg, Mark H
- Subjects
2.1 Biological and endogenous factors ,Aetiology ,1.1 Normal biological development and functioning ,Underpinning research ,Animals ,Cell Adhesion ,Female ,Gastrointestinal Tract ,HEK293 Cells ,Humans ,Integrin beta Chains ,Integrins ,Jurkat Cells ,Leukocyte Rolling ,Lymphocyte Activation ,Lymphocytes ,Lymphoid Tissue ,Male ,Mice ,Inbred C57BL ,Mice ,Transgenic ,Mutation ,Protein Binding ,Protein Interaction Domains and Motifs ,Signal Transduction ,Structure-Activity Relationship ,Talin ,Biological Sciences ,Medical and Health Sciences ,Developmental Biology - Abstract
Integrin activation regulates adhesion, extracellular matrix assembly, and cell migration, thereby playing an indispensable role in development and in many pathological processes. A proline mutation in the central integrin β3 transmembrane domain (TMD) creates a flexible kink that uncouples the topology of the inner half of the TMD from the outer half. In this study, using leukocyte integrin α4β7, which enables development of gut-associated lymphoid tissue (GALT), we examined the biological effect of such a proline mutation and report that it impairs agonist-induced talin-mediated activation of integrin α4β7, thereby inhibiting rolling lymphocyte arrest, a key step in transmigration. Furthermore, the α4β7(L721P) mutation blocks lymphocyte homing to and development of the GALT. These studies show that impairing the ability of an integrin β TMD to transmit talin-induced TMD topology inhibits agonist-induced physiological integrin activation and biological function in development.
- Published
- 2018
40. Scavenger Receptor CD36 Directs Nonclassical Monocyte Patrolling Along the Endothelium During Early Atherogenesis
- Author
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Marcovecchio, Paola M, Thomas, Graham D, Mikulski, Zbigniew, Ehinger, Erik, Mueller, Karin AL, Blatchley, Amy, Wu, Runpei, Miller, Yury I, Nguyen, Anh Tram, Taylor, Angela M, McNamara, Coleen A, Ley, Klaus, and Hedrick, Catherine C
- Subjects
Biomedical and Clinical Sciences ,Cardiovascular Medicine and Haematology ,Clinical Sciences ,Cardiovascular ,Atherosclerosis ,Aetiology ,2.1 Biological and endogenous factors ,Actin Cytoskeleton ,Adaptor Proteins ,Signal Transducing ,Animals ,Apolipoproteins E ,CD36 Antigens ,Diet ,Western ,Disease Models ,Animal ,Endothelial Cells ,Endothelium ,Vascular ,Femoral Artery ,Genetic Predisposition to Disease ,Humans ,Intravital Microscopy ,Leukocyte Rolling ,Lipoproteins ,LDL ,Mice ,Inbred C57BL ,Mice ,Knockout ,Monocytes ,Phenotype ,Signal Transduction ,Time Factors ,src-Family Kinases ,atherosclerosis ,humans ,mice ,monocytes ,phosphorylation ,Cardiorespiratory Medicine and Haematology ,Cardiovascular System & Hematology ,Cardiovascular medicine and haematology ,Clinical sciences - Abstract
ObjectiveNonclassical monocytes (NCM) function to maintain vascular homeostasis by crawling or patrolling along the vessel wall. This subset of monocytes responds to viruses, tumor cells, and other pathogens to aid in protection of the host. In this study, we wished to determine how early atherogenesis impacts NCM patrolling in the vasculature.Approach and resultsTo study the role of NCM in early atherogenesis, we quantified the patrolling behaviors of NCM in ApoE-/- (apolipoprotein E) and C57BL/6J mice fed a Western diet. Using intravital imaging, we found that NCM from Western diet-fed mice display a 4-fold increase in patrolling activity within large peripheral blood vessels. Both human and mouse NCM preferentially engulfed OxLDL (oxidized low-density lipoprotein) in the vasculature, and we observed that OxLDL selectively induced NCM patrolling in vivo. Induction of patrolling during early atherogenesis required scavenger receptor CD36, as CD36-/- mice revealed a significant reduction in patrolling activity along the femoral vasculature. Mechanistically, we found that CD36-regulated patrolling was mediated by a SFK (src family kinase) through DAP12 (DNAX activating protein of 12KDa) adaptor protein.ConclusionsOur studies show a novel pathway for induction of NCM patrolling along the vascular wall during early atherogenesis. Mice fed a Western diet showed increased NCM patrolling activity with a concurrent increase in SFK phosphorylation. This patrolling activity was lost in the absence of either CD36 or DAP12. These data suggest that NCM function in an atheroprotective manner through sensing and responding to oxidized lipoprotein moieties via scavenger receptor engagement during early atherogenesis.
- Published
- 2017
41. Flow Cytometry and Mass Cytometry for Measuring the Immune Cell Infiltrate in Atherosclerotic Arteries
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Orecchioni, Marco, primary, Meyer, Melissa A., additional, Hedrick, Catherine C., additional, and Ley, Klaus, additional
- Published
- 2022
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42. Single-Cell Antibody Sequencing in Atherosclerosis Research
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Saigusa, Ryosuke, primary, Durant, Christopher P., additional, Suryawanshi, Vasantika, additional, and Ley, Klaus, additional
- Published
- 2022
- Full Text
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43. CITE-seq analysis reveals human cytomegalovirus and diabetes-associated adaptive NK cell alterations in cardiovascular disease.
- Author
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Armstrong, Sujit Silas, primary, Chen, Daniel G., additional, Kumar, Sunil, additional, Heath, James R., additional, Feinstein, Matthew J., additional, Greenland, John R., additional, Calabrese, Daniel R., additional, Lanier, Lewis L., additional, Ley, Klaus, additional, and Shemesh, Avishai, additional
- Published
- 2024
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44. Single Cell High Dimensional Analysis of Human Peripheral Blood Mononuclear Cells Reveals Unique Intermediate Monocyte Subsets Associated with Sex Differences in Coronary Artery Disease
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Chatterjee, Nandini, primary, Komaravolu, Ravi K., additional, Durant, Christopher P., additional, Wu, Runpei, additional, McSkimming, Chantel, additional, Drago, Fabrizio, additional, Kumar, Sunil, additional, Valentin-Guillama, Gabriel, additional, Miller, Yury I., additional, McNamara, Coleen A., additional, Ley, Klaus, additional, Taylor, Angela, additional, Alimadadi, Ahmad, additional, and Hedrick, Catherine C., additional
- Published
- 2024
- Full Text
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45. Myeloid cell-specific Irf5 deficiency stabilizes atherosclerotic plaques in Apoe–/– mice
- Author
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Leipner, Julia, Dederichs, Tsai-Sang, von Ehr, Alexander, Rauterberg, Simon, Ehlert, Carolin, Merz, Julian, Dufner, Bianca, Hoppe, Natalie, Krebs, Katja, Heidt, Timo, von zur Muehlen, Constantin, Stachon, Peter, Ley, Klaus, Wolf, Dennis, Zirlik, Andreas, Bode, Christoph, Hilgendorf, Ingo, and Härdtner, Carmen
- Published
- 2021
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46. Single-cell profiling of CD11c+ B cells in atherosclerosis
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Pattarabanjird, Tanyaporn, primary, Srikakulapu, Prasad, additional, Ransegnola, Brett, additional, Marshall, Melissa A., additional, Ghosheh, Yanal, additional, Gulati, Rishab, additional, Durant, Chistopher, additional, Drago, Fabrizio, additional, Taylor, Angela M., additional, Ley, Klaus, additional, and McNamara, Coleen A., additional
- Published
- 2024
- Full Text
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47. Kindlin-3 recruitment to the plasma membrane precedes high-affinity β2-integrin and neutrophil arrest from rolling
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Wen, Lai, Marki, Alex, Roy, Payel, McArdle, Sara, Sun, Hao, Fan, Zhichao, Gingras, Alexandre R., Ginsberg, Mark H., and Ley, Klaus
- Published
- 2021
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48. Identification of apolipoprotein B-reactive CDR3 motifs allows tracking of atherosclerosisrelated memory CD4+T cells in multiple donors.
- Author
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Roy, Payel, Suthahar, Sujit Silas Armstrong, Makings, Jeffrey, and Ley, Klaus
- Subjects
APOLIPOPROTEIN B ,MEMORY ,BLOOD cells ,T cells ,ODDS ratio - Abstract
Introduction: Atherosclerosis is a major pathological condition that underlies many cardiovascular diseases (CVDs). Its etiology involves breach of tolerance to self, leading to clonal expansion of autoreactive apolipoprotein B (APOB)-reactive CD4
+ T cells that correlates with clinical CVD. The T-cell receptor (TCR) sequences that mediate activation of APOB-specific CD4+ T cells are unknown. Methods: In a previous study, we had profiled the hypervariable complementarity determining region 3 (CDR3) of CD4+ T cells that respond to six immunodominant APOB epitopes in most donors. Here, we comprehensively analyze this dataset of 149,065 APOB-reactive and 199,211 non-reactive control CDR3s from six human leukocyte antigen-typed donors. Results: We identified 672 highly expanded (frequency threshold > 1.39E-03) clones that were significantly enriched in the APOB-reactive group as compared to the controls (log10 odds ratio =1, Fisher's test p < 0.01). Analysis of 114,755 naïve, 91,001 central memory (TCM) and 29,839 effector memory (TEM) CDR3 sequences from the same donors revealed that APOB+ clones can be traced to the complex repertoire of unenriched blood T cells. The fraction of APOB+ clones that overlapped with memory CDR3s ranged from 2.2% to 46% (average 16.4%). This was significantly higher than their overlap with the naïve pool, which ranged from 0.7% to 2% (average 1.36%). CDR3 motif analysis with the machine learning-based in-silico tool, GLIPHs (grouping of lymphocyte interactions by paratope hotspots), identified 532 APOB+ motifs. Analysis of naïve and memory CDR3 sequences with GLIPH revealed that ~40% (209 of 532) of these APOB+motifs were enriched in the memory pool. Network analysis with Cytoscape revealed extensive sharing of the memory-affiliated APOB+ motifs across multiple donors. We identified six motifs that were present in TCM and TEM CDR3 sequences from >80% of the donors and were highly enriched in the APOB-reactive TCR repertoire. Discussion: The identified APOB-reactive expanded CD4+ T cell clones and conserved motifs can be used to annotate and track human atherosclerosisrelated autoreactive CD4+ T cells and measure their clonal expansion. [ABSTRACT FROM AUTHOR]- Published
- 2024
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49. Spiking Pandemic Potential: Structural and Immunological Aspects of SARS-CoV-2
- Author
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Wang, Ying-Ting, Landeras-Bueno, Sara, Hsieh, Li-En, Terada, Yutaka, Kim, Kenneth, Ley, Klaus, Shresta, Sujan, Saphire, Erica Ollmann, and Regla-Nava, Jose Angel
- Published
- 2020
- Full Text
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50. Neutrophil recruitment limited by high-affinity bent β2 integrin binding ligand in cis.
- Author
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Fan, Zhichao, McArdle, Sara, Marki, Alex, Mikulski, Zbigniew, Gutierrez, Edgar, Engelhardt, Britta, Deutsch, Urban, Ginsberg, Mark, Groisman, Alex, and Ley, Klaus
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Neutrophils ,Animals ,Mice ,Inbred C57BL ,Transplantation Chimera ,Mice ,Knockout ,Humans ,Mice ,Inflammation ,Cell Adhesion Molecules ,Recombinant Proteins ,Ligands ,Imaging ,Three-Dimensional ,Bone Marrow Transplantation ,Neutrophil Infiltration ,Protein Conformation ,Protein Binding ,Stereoisomerism ,Male ,Molecular Imaging ,Healthy Volunteers ,Intravital Microscopy ,CD18 Antigens ,Imaging ,Three-Dimensional ,Inbred C57BL ,Knockout - Abstract
Neutrophils are essential for innate immunity and inflammation and many neutrophil functions are β2 integrin-dependent. Integrins can extend (E(+)) and acquire a high-affinity conformation with an 'open' headpiece (H(+)). The canonical switchblade model of integrin activation proposes that the E(+) conformation precedes H(+), and the two are believed to be structurally linked. Here we show, using high-resolution quantitative dynamic footprinting (qDF) microscopy combined with a homogenous conformation-reporter binding assay in a microfluidic device, that a substantial fraction of β2 integrins on human neutrophils acquire an unexpected E(-)H(+) conformation. E(-)H(+) β2 integrins bind intercellular adhesion molecules (ICAMs) in cis, which inhibits leukocyte adhesion in vitro and in vivo. This endogenous anti-inflammatory mechanism inhibits neutrophil aggregation, accumulation and inflammation.
- Published
- 2016
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