Neutrophil DREAM promotes neutrophil recruitment in vascular inflammation

2021 ◽  
Vol 219 (1) ◽  
Author(s):  
Jing Li ◽  
Tripti Kumari ◽  
Andrew Barazia ◽  
Vishwanath Jha ◽  
Si-Yeon Jeong ◽  
...  
Keyword(s):  
Therapeutic Strategy ◽  
Inflammatory Diseases ◽  
Vascular Inflammation ◽  
Binding Activity ◽  
Neutrophil Recruitment ◽  
Negative Regulator ◽  
Iκb Kinase ◽  
Tnf Α ◽  
Inflammatory Molecules ◽  
Ligand Binding Activity

The interaction between neutrophils and endothelial cells is critical for the pathogenesis of vascular inflammation. However, the regulation of neutrophil adhesive function remains not fully understood. Intravital microscopy demonstrates that neutrophil DREAM promotes neutrophil recruitment to sites of inflammation induced by TNF-α but not MIP-2 or fMLP. We observe that neutrophil DREAM represses expression of A20, a negative regulator of NF-κB activity, and enhances expression of pro-inflammatory molecules and phosphorylation of IκB kinase (IKK) after TNF-α stimulation. Studies using genetic and pharmacologic approaches reveal that DREAM deficiency and IKKβ inhibition significantly diminish the ligand-binding activity of β2 integrins in TNF-α–stimulated neutrophils or neutrophil-like HL-60 cells. Neutrophil DREAM promotes degranulation through IKKβ-mediated SNAP-23 phosphorylation. Using sickle cell disease mice lacking DREAM, we show that hematopoietic DREAM promotes vaso-occlusive events in microvessels following TNF-α challenge. Our study provides evidence that targeting DREAM might be a novel therapeutic strategy to reduce excessive neutrophil recruitment in inflammatory diseases.

scholarly journals Neutrophil DREAM Promotes Neutrophil Recruitment in Vascular Inflammation Via Nuclear Factor Kappa B-Dependent and Independent Mechanisms

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 435-435
Author(s):  
Tripti Kumari ◽  
Jing Li ◽  
Andrew Barazia, ◽  
Vishwanath Jha ◽  
Amber Hansch ◽  
...  
Keyword(s):  
Vascular Inflammation ◽  
Term Treatment ◽  
Neutrophil Recruitment ◽  
Neutrophil Adhesion ◽  
Short Term ◽  
Short Term Treatment ◽  
Tnf Α ◽  
Inflammatory Molecules ◽  
Β2 Integrins

Abstract The interaction between neutrophils and endothelial cells (ECs) is critical for the pathogenesis of vascular inflammation. Neutrophil recruitment to inflamed tissues is initiated by rolling on activated ECs through the interactions between P-/E-selectins and their ligands. Subsequently, activated integrins (mainly αLβ2 and αMβ2) and chemokine receptors bind to their ligands on ECs and mediate slow-rolling, adhesion, crawling, and transmigration of neutrophils. Although many neutrophil adhesion receptors have been identified, the regulation of their ligand-binding function remains not fully understood. Using real-time intravital microscopy with mice lacking downstream regulatory element antagonist modulator (DREAM) and their bone marrow chimeric mice, we demonstrated that hematopoietic cell DREAM contributes to neutrophil recruitment to sites of vascular inflammation induced by TNF-α- but not a G protein-coupled receptor ligand, MIP-2 or fMLP. Our studies using adoptive neutrophil transfers and flow chamber assays revealed that neutrophil DREAM positively regulates the neutrophil recruitment processes under TNF-α-induced inflammatory conditions. Using RNA-seq and biochemical and cell biological studies, we found that neutrophil DREAM upregulates numerous pro-inflammatory molecules and down-regulates anti-inflammatory molecules after TNF-α treatment. In particular, neutrophil DREAM repressed expression of A20, a negative regulator of NF-κB signaling, and enhanced phosphorylation of IκB kinase (IKK) in response to TNF-α, suggesting the role of neutrophil DREAM in NF-κB activity. Furthermore, we observed that DREAM deletion and IKK inhibition significantly diminishes the ligand-binding activity of β2 integrins in neutrophils after short-term treatment with TNF-α and that deletion of neutrophil DREAM does not affect the expression of other neutrophil adhesion receptors, such as PSGL-1, L-selectin, CD44, CXCR2, and CXCR4. As assessed by flow cytometry using conformation-specific reporter antibodies, knockdown of DREAM in neutrophil-like HL-60 cells decreased TNF-α-induced activation of β2 integrins. Neutrophil DREAM promoted degranulation through IKK-mediated SNAP-23 phosphorylation after short-term treatment with TNF-α, implying the role of neutrophil DREAM-IKK signaling in NF-κB-independent signaling. Using intravital microscopy with Berkeley mice (a mouse model of sickle cell disease) deficient in hematopoietic or nonhematopoietic DREAM, we demonstrated that hematopoietic cell DREAM is crucial for inducing intravascular cell-cell aggregation and vaso-occlusive events in microvessels following the TNF-α challenge. Furthermore, infusion of DREAM KO neutrophils, compared with WT neutrophils, significantly reduced neutrophil recruitment and vaso-occlusive events in TNF-α-challenged SCD mice. These results demonstrate that neutrophil DREAM positively regulates β2 integrin function and promotes neutrophil recruitment during sterile inflammation via NF-κB-dependent and independent mechanisms. Our study provides evidence that targeting DREAM might be a novel therapeutic strategy to reduce excessive neutrophil recruitment in inflammatory diseases. Disclosures No relevant conflicts of interest to declare.

scholarly journals Extracellular protein disulfide isomerase regulates ligand-binding activity of αMβ2 integrin and neutrophil recruitment during vascular inflammation

Blood ◽  
2013 ◽  
Vol 121 (19) ◽  
pp. 3789-3800 ◽  
Author(s):  
Eunsil Hahm ◽  
Jing Li ◽  
Kyungho Kim ◽  
Sungjin Huh ◽  
Snezna Rogelj ◽  
...  
Keyword(s):  
Protein Disulfide Isomerase ◽  
Vascular Inflammation ◽  
Binding Activity ◽  
Neutrophil Recruitment ◽  
Disulfide Isomerase ◽  
Isomerase Activity ◽  
Key Points ◽  
Protein Disulfide ◽  
Ligand Binding Activity ◽  
Neutrophil Surface

Key Points This work is the first identification of a neutrophil surface thiol isomerase regulating adhesive function of αMβ2 integrin. PDI is required for neutrophil recruitment during vascular inflammation and its isomerase activity is critical for the regulatory effect.

scholarly journals Negative Regulation of NF-κB Signaling by PIAS1

2005 ◽  
Vol 25 (3) ◽  
pp. 1113-1123 ◽  
Author(s):  
Bin Liu ◽  
Randy Yang ◽  
Kelly A. Wong ◽  
Crescent Getman ◽  
Natalie Stein ◽  
...  
Keyword(s):  
Inflammatory Diseases ◽  
Gene Activation ◽  
Binding Activity ◽  
Negative Regulator ◽  
Necrosis Factor Alpha ◽  
Dna Binding Activity ◽  
Important Negative Regulator ◽  
Cytokine Stimulation

ABSTRACT The NF-κB family of transcription factors is activated by a wide variety of signals to regulate a spectrum of cellular processes. The proper regulation of NF-κB activity is critical, since abnormal NF-κB signaling is associated with a number of human illnesses, such as chronic inflammatory diseases and cancer. We report here that PIAS1 (protein inhibitor of activated STAT1) is an important negative regulator of NF-κB. Upon cytokine stimulation, the p65 subunit of NF-κB translocates into the nucleus, where it interacts with PIAS1. The binding of PIAS1 to p65 inhibits cytokine-induced NF-κB-dependent gene activation. PIAS1 blocks the DNA binding activity of p65 both in vitro and in vivo. Consistently, chromatin immunoprecipitation assays indicate that the binding of p65 to the promoters of NF-κB-regulated genes is significantly enhanced in Pias1 −/− cells. Microarray analysis indicates that the removal of PIAS1 results in an increased expression of a subset of NF-κB-mediated genes in response to tumor necrosis factor alpha and lipopolysaccharide. Consistently, Pias1 null mice showed elevated proinflammatory cytokines. Our results identify PIAS1 as a novel negative regulator of NF-κB.

Abstract 13405: Genetic Deletion of Stimulator of Interferon Genes Attenuates Atherogenesis in Apolipoprotein E-deficient Mice

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Phuong T Pham ◽  
Daiju Fukuda ◽  
Masataka Sata
Keyword(s):  
Innate Immunity ◽  
Apolipoprotein E ◽  
Aortic Arch ◽  
Inflammatory Responses ◽  
Inflammatory Diseases ◽  
Vascular Inflammation ◽  
Atherosclerotic Plaques ◽  
Atherosclerotic Lesions ◽  
Gene And Protein Expression ◽  
Inflammatory Molecules

Introduction: Recent studies show the contribution of innate immunity system to the pathogenesis of inflammatory diseases, including atherosclerosis. Stimulation of interferon genes (STING), originally known as a cytosolic DNA sensor, recognizes cytosolic DNA fragments, activating innate immunity. Here, we investigated whether STING contributes to the development of vascular inflammation and atherogenesis in apolipoprotein E-deficient (Apoe –/– ) mice. Methods and Results: The expression of STING increased in the atherosclerotic aorta in both gene and protein expression levels. STING-deficient Apoe –/– (STING –/– Apoe –/– ) mice reduced atherosclerotic lesions in the aortic arch ( P <0.05), along with the reduction of lipid and macrophage accumulation in atherosclerotic plaques ( P <0.05, respectively), and inflammatory molecule expression in the aorta compared with those in Apoe –/– mice after 20 weeks on a western-type diet. Also, pharmacologic blockade of STING in Apoe –/– mice for 12 weeks treatment attenuated atherogenesis in the aortic arch ( P <0.05), reduced the accumulation of lipid in atherosclerotic plaques ( P <0.05) with no alteration of metabolic parameters. Restoration of STING in bone marrow in STING –/– Apoe –/– mice promoted atherogenesis ( P <0.05), lipid deposition ( P <0.05), and vascular inflammation. cGAMP, a specific STING agonist, or mitochondrial DNA extracted from macrophages promoted expression of inflammatory molecules more effectively in Apoe -/- macrophages than in STING –/– Apoe –/– macrophages, while C-176, a specific STING inhibitor, attenuated these inflammatory responses. The results of western blotting showed the involvement of NF-κB and IRF-3 signaling in STING-associated vascular inflammation and macrophage activation. Furthermore, in humans, STING expression was confirmed in atherosclerotic lesions in the carotid artery. Conclusion: STING signaling activates macrophages, promotes vascular inflammation and atherosclerosis in Apoe -/- mice. Our results suggest that STING may serve as a potential therapeutic target for atherosclerosis.

scholarly journals Sulfasalazine and BAY 11-7082 Interfere with the Nuclear Factor-κB and IκB Kinase Pathway to Regulate the Release of Proinflammatory Cytokines from Human Adipose Tissue and Skeletal Muscle in Vitro

Endocrinology ◽  
2005 ◽  
Vol 146 (3) ◽  
pp. 1491-1497 ◽  
Author(s):  
Martha Lappas ◽  
Kirin Yee ◽  
Michael Permezel ◽  
Gregory E. Rice
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Adipose Tissue ◽  
Human Adipose Tissue ◽  
Nuclear Factor Κb ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Iκb Kinase ◽  
Β Protein ◽  
Tnf Α

There is much evidence to indicate a role for adipocytokines in insulin resistance and/or type 2 diabetes mellitus. In experimental models, oral salicylates, through their ability to interfere with the nuclear factor-κB (NF-κB) transcription pathway, have been demonstrated to reverse insulin resistance. The aim of this study was to investigate whether NF-κB regulates the release of adipocytokines in human adipose tissue and skeletal muscle. Human sc adipose tissue and skeletal muscle (obtained from normal pregnant women) were incubated in the absence (control) or presence of two NF-κB inhibitors sulfasalazine (1.25, 2.5, and 5 mm) and BAY 11-7082 (25, 50, and 100 μm). After an 18-h incubation, the tissues were collected, and NF-κB p65 DNA-binding activity and IκB kinase (IKK-β) and insulin receptor-β protein expression were assessed by ELISA and Western blotting, respectively. The incubation medium was collected, and the release of TNF-α, IL-6, IL-8, resistin, adiponectin, and leptin was quantified by ELISA. Treatment of adipose tissue and skeletal muscle with sulfasalazine and BAY 11-7082 significantly inhibited the release of IL-6, IL-8, and TNF-α; NF-κB p65 DNA-binding activity; and IKK-β protein expression (P &lt; 0.05, by Newman-Keuls test). There was no effect of sulfasalazine and BAY 11-7082 on resistin, adiponectin, or leptin release. Both sulfasalazine and BAY 11-7082 increased the adipose tissue and skeletal muscle expression of insulin receptor-β. The data presented in this study demonstrate that the IKK-β/NF-κB transcription pathway is a key regulator of IL-6, IL-8, and TNF-α release from adipose tissue and skeletal muscle. Control of the IKK-β/NF-κB pathway may therefore provide an alternative therapeutic strategy for regulating aberrant cytokine release and thereby alleviating insulin resistance in type 2 diabetes mellitus.

Neutrophil DREAM Is a Novel Regulator for Beta2 Integrin Function through NF-Kappab Signaling and Modulates Neutrophil Recruitment during Vascular Inflammation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 888-888 ◽  
Author(s):  
Andrew Barazia ◽  
Jing Li ◽  
Kyungho Kim ◽  
Chinnaswamy Tiruppathi ◽  
Jaehyung Cho
Keyword(s):  
Target Genes ◽  
Conflicts Of Interest ◽  
Regulatory Element ◽  
Vascular Inflammation ◽  
Surface Expression ◽  
Neutrophil Recruitment ◽  
Β2 Integrin ◽  
Inflammatory Conditions ◽  
Tnf Α

Abstract The interaction between neutrophils and activated endothelial cells (ECs) is critical for the pathogenesis of vascular inflammation. However, it remains poorly understood how the cell-cell interactions are regulated under inflammatory conditions. Using intravital microscopy in mice lacking DREAM (downstream regulatory element antagonist modulator), a member of the neuronal Ca2+ sensors and a transcriptional repressor, we have found that DREAM plays an important role in neutrophil rolling and adhesion to the TNF-α-inflamed cremaster muscle venules. Studies with DREAM bone marrow chimeras revealed that both hematopoietic and EC DREAM are important for neutrophil recruitment. We found that neutrophils lacking DREAM exhibit reduced αMβ2 surface expression and decrease fibrinogen binding following stimulation with TNF-α, but not fMLF, implying the role for neutrophil DREAM in regulating β2 integrin function through a specific signaling pathway. Since recent studies demonstrated the important role of endothelial cell DREAM in NF-κB signaling, we further examined whether neutrophil DREAM regulates NF-κB signaling. Indeed, the protein expression of A20 (an inhibitor of NF-κB signaling) and p65 (a key subunit of the NF-κB complex) was significantly up- and down-regulated, respectively, in DREAM-deficient neutrophils, compared to WT neutrophils. Moreover, DREAM deletion impaired the phosphorylation of IKKa/β following TNF-a-stimulation, suggesting the role of neutrophil DREAM in NF-κB signaling. Consistently, we found that the transcription of the NF-κB target genes, such as IL-1β, TNF-α, and IL-6, was inhibited by DREAM deletion. Taken together, our results provide evidence that neutrophil DREAM is a novel regulator for β2 integrin function through NF-κB signaling and could be a therapeutic target for treatment of inflammatory disease. Disclosures No relevant conflicts of interest to declare.

scholarly journals Raf Kinase Inhibitor Protein Interacts with NF-κB-Inducing Kinase and TAK1 and Inhibits NF-κB Activation

2001 ◽  
Vol 21 (21) ◽  
pp. 7207-7217 ◽  
Author(s):  
Kam C. Yeung ◽  
David W. Rose ◽  
Amardeep S. Dhillon ◽  
Diane Yaros ◽  
Marcus Gustafsson ◽  
...  
Keyword(s):  
Transforming Growth Factor ◽  
Kinase Inhibitor ◽  
Mapk Pathway ◽  
Ectopic Expression ◽  
Negative Regulator ◽  
Inhibitor Protein ◽  
Raf Kinase ◽  
Raf Kinase Inhibitor Protein ◽  
Iκb Kinase ◽  
Tnf Α

ABSTRACT The Raf kinase inhibitor protein (RKIP) acts as a negative regulator of the mitogen-activated protein (MAP) kinase (MAPK) cascade initiated by Raf-1. RKIP inhibits the phosphorylation of MAP/extracellular signal-regulated kinase 1 (MEK1) by Raf-1 by disrupting the interaction between these two kinases. We show here that RKIP also antagonizes the signal transduction pathways that mediate the activation of the transcription factor nuclear factor kappa B (NF-κB) in response to stimulation with tumor necrosis factor alpha (TNF-α) or interleukin 1 beta. Modulation of RKIP expression levels affected NF-κB signaling independent of the MAPK pathway. Genetic epistasis analysis involving the ectopic expression of kinases acting in the NF-κB pathway indicated that RKIP acts upstream of the kinase complex that mediates the phosphorylation and inactivation of the inhibitor of NF-κB (IκB). In vitro kinase assays showed that RKIP antagonizes the activation of the IκB kinase (IKK) activity elicited by TNF-α. RKIP physically interacted with four kinases of the NF-κB activation pathway, NF-κB-inducing kinase, transforming growth factor beta-activated kinase 1, IKKα, and IKKβ. This mode of action bears striking similarities to the interactions of RKIP with Raf-1 and MEK1 in the MAPK pathway. Emerging data from diverse organisms suggest that RKIP and RKIP-related proteins represent a new and evolutionarily highly conserved family of protein kinase regulators. Since the MAPK and NF-κB pathways have physiologically distinct roles, the function of RKIP may be, in part, to coordinate the regulation of these pathways.

Fas-associated death-domain protein inhibits TNF-α mediated NF-κB activation in cardiomyocytes

2005 ◽  
Vol 289 (5) ◽  
pp. H2073-H2080 ◽  
Author(s):  
Wei Chao ◽  
Yan Shen ◽  
Ling Li ◽  
Huailong Zhao ◽  
Steffen E. Meiler ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Dna Binding ◽  
Nuclear Translocation ◽  
Binding Activity ◽  
Death Domain ◽  
Dna Binding Activity ◽  
Iκb Kinase ◽  
Tnf Α ◽  
Domain Protein

Fas-associated death-domain protein (FADD) is an adaptor molecule that links death receptors to caspase-8 in many cell types including cardiomyocytes (CMs). Although FADD has previously been reported to play an important role in CM apoptosis, the effect of FADD on CM NF-κB signaling, which is a proinflammatory pathway, has not been delineated. To investigate the role of FADD in CM NF-κB activation, we utilized adenoviral gene transfer of wild-type FADD and a truncation mutant that lacks the death-effector domain (FADD-DED) in rat CMs in vitro TNF-α activated NF-κB in CMs as demonstrated by phosphorylation and degradation of inhibitory-κB (IκB)-α-enhanced nuclear p65 and NF-κB DNA-binding activity as well as increased mRNA for the NF-κB-dependent adhesion molecule VCAM-1 (19 ± 4.1-fold) as measured by quantitative RT-PCR. Gene transfer of FADD inhibited TNF-α-induced IκB-α phosphorylation, decreased p65 nuclear translocation and NF-κB DNA-binding activity, and reduced VCAM-1 transcript levels by 53–65%. Interestingly, FADD-DED exhibited a similar but weaker inhibitory effect on NF-κB activation. The effects of FADD on NF-κB were cell-type specific. FADD expression also inhibited TNF-α-mediated NF-κB activation in human endothelial cells but not in rat pulmonary artery smooth muscle cells. In contrast, FADD expression actually activated NF-κB in human embryonic kidney (HEK)-293 cells. In CMs, FADD inhibited NF-κB activation as well as phosphorylation of IκB-α and IκB kinase (IKK)-β in response to cytokine stimulation or expression of the upstream kinases NF-κB-inducing kinase and IKK-β. These data demonstrate that FADD inhibits NF-κB activation in CMs, and this inhibition likely occurs at the level of phosphorylation and activation of IKK-β.

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