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.