Role of DREAM in Neutrophil Function and Platelet-Neutrophil Interactions in Thrombo-Inflammatory Disease

The interaction between neutrophils and activated endothelial cells (ECs) is critical for the pathogenesis of vascular inflammation. However, it remains poorly understood how neutrophil-EC interactions are regulated. Using intravital microscopy with mice lacking downstream regulatory element antagonist modulator (DREAM), a transcriptional repressor, we demonstrated that intravascular cell DREAM was important for neutrophil adhesion to TNF-α-activated ECs during vascular inflammation. Studies with bone marrow chimeras revealed that both hematopoietic and EC DREAM were required for neutrophil-EC interactions. DREAM-null neutrophils showed reduced membrane translocation and ligand-binding activity of αMβ2 integrin and β2-talin1 binding following stimulation with TNF-α, but not fMLF. Deletion of neutrophil DREAM resulted in upregulated expression of A20, a negative regulator of NF-κB signaling and downregulated expression of p65, a critical subunit of the NF-κB complex. Further, we found that neutrophil DREAM was required for gene transcription of TNFα, IL-1β, and IL-6, and phosphorylation of IκB kinase (IKK) following TNF- stimulation. Inhibition of IKK activity reduced the membrane translocation of αMβ2 integrin and degranulation in TNF-α-stimulated WT, but not DREAM-null, neutrophils. Thus, our results suggest that neutrophil DREAM plays an important role in both NF-κB-dependent gene transcription of pro-inflammatory cytokines and NF-κB-independent IKK activation during vascular inflammation. Since αMβ2 integrin and neutrophil-derived cytokines are important for the interaction of neutrophils with platelets, we further determined whether DREAM regulates platelet-neutrophil interaction during vascular inflammation. Our fluorescence intravital microscopy demonstrated that both hematopoietic and EC DREAM are crucial for regulating the interaction of platelets with adherent neutrophils in TNFα-inflamed venules in live mice. Additionally, using an in vitro heterotypic cell-cell aggregation assay, we found that both neutrophil and platelet DREAM are important in regulation neutrophil-platelet interactions under stirring conditions mimicking venous shear. Compared with WT platelets, DREAM KO platelets exhibited a significant reduction in P-selectin exposure on activated platelets which is required for platelet-neutrophil interaction. Taken together, these results provide important evidence that intravascular cell DREAM could be a novel therapeutic target for treatment of inflammatory diseases

ARQ 092, an orally-available, selective AKT inhibitor, attenuates neutrophil-platelet interactions in sickle cell disease

Previous studies identified the Ser/Thr protein kinase, AKT, as a therapeutic target in thrombo-inflammatory diseases. Here we report that specific inhibition of AKT with ARQ 092, an orally-available AKT inhibitor currently in phase Ib clinical trials as an anti-cancer drug, attenuates the adhesive function of neutrophils and platelets from sickle cell disease patients in vitro and cell-cell interactions in a mouse model of sickle cell disease. Studies using neutrophils and platelets isolated from sickle cell disease patients revealed that treatment with 50–500 nM ARQ 092 significantly blocks αMβ2 integrin function in neutrophils and reduces P-selectin exposure and glycoprotein Ib/IX/V-mediated agglutination in platelets. Treatment of isolated platelets and neutrophils with ARQ 092 inhibited heterotypic cell-cell aggregation under shear conditions. Intravital microscopic studies demonstrated that short-term oral administration of ARQ 092 or hydroxyurea, a major therapy for sickle cell disease, diminishes heterotypic cell-cell interactions in venules of sickle cell disease mice challenged with tumor necrosis factor-α. Co-administration of hydroxyurea and ARQ 092 further reduced the adhesive function of neutrophils in venules and neutrophil transmigration into alveoli, inhibited expression of E-selectin and intercellular adhesion molecule-1 in cremaster vessels, and improved survival in these mice. Ex vivo studies in sickle cell disease mice suggested that co-administration of hydroxyurea and ARQ 092 efficiently blocks neutrophil and platelet activation and that the beneficial effect of hydroxyurea results from nitric oxide production. Our results provide important evidence that ARQ 092 could be a novel drug for the prevention and treatment of acute vaso-occlusive complications in patients with sickle cell disease

Neutrophil DREAM promotes neutrophil recruitment in vascular inflammation

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

Hydroxyurea with AKT2 inhibition decreases vaso-occlusive events in sickle cell disease mice

Heterotypic cell-cell adhesion and aggregation mediate vaso-occlusive events in patients with sickle cell disease (SCD). Although hydroxyurea (HU), an inducer of fetal hemoglobin, is the main therapy for treatment of SCD, it is unclear whether it has immediate benefits in acute vaso-occlusive events in SCD patients. Using real-time fluorescence intravital microscopy, we demonstrated that short-term coadministration of HU and Akti XII, an AKT2 inhibitor, efficiently reduced neutrophil adhesion and platelet-neutrophil aggregation in venules of Berkeley (SCD) mice challenged with tumor necrosis factor α (TNF-α) or hypoxia/reoxygenation. Importantly, compared with HU or Akti XII treatment alone, short-term treatment with both agents significantly improved survival in those mice. We found that the level of plasma nitric oxide species was elevated by HU but not Akti XII, AKT2 phosphorylation levels in activated neutrophils and platelets were reduced by Akti XII but not HU, and the expression of endothelial E-selectin and intercellular adhesion molecule 1 was decreased by either agent. Our results suggest that short-term coadministration of HU and Akti XII has immediate benefits for acute vaso-occlusive events and survival in SCD mice exceeding those seen for single therapy