Neutrophils are the first line of cellular defense by moving rapidly to sites of infection in tissue. This migration is tightly regulated; indeed, aberrat accumulation of neutrophils causes tissue injury. Despite the clinical importance, the mechanisms of neutrophil migration into tissues remain poorly understood. Our long-term goal is to identify the signaling pathways that limit neutrophil tissue infiltration and inflammation. A critical step in that process is neutrophil migration across the endothelium, known as diapedesis. Diapedesis is not solely a migration step. It is also important for neutrophil activation and subsequent functions via neutrophil-endothelial cell interactions, and for endothelial barrier integrity. Hence, it directly participates in inflammation. Interestingly, unequivocal evidence indicates that diapedesis can occur between or through endothelial cells (paracellular or transcellular, respectively). But, the mechanisms that separately control these two modes of migration are poorly understood. How distinct migration paths impact outcome of inflammation is not known. Understanding these mechanisms is important for our understanding of neutrophil biology and inflammation. More broadly, it will reveal principle that may apply to other cells, including cancer cells. It is fundamental clinically because targeting only one mode of migration could offer new ways to dampen hyperinflammation while preserving some host defense mechanism. We found that Ras proximity 1 (Rap1) isoform Rap1b limits neutrophil diapedesis and inflammation. Rap1b-/- neutrophils exhibit increased diapedesis, due to a selective increase in transcellular migration. This is caused by enhanced PIP3-Akt activity-mediated invasive protrusions. Rap1b loss enhanced inflammation, which is prevented by Akt inhibition in vivo. Hence, neutrophil activation via signaling outputs control diapedesis route and inflammation outcome. We hypothesize that the spatiotemporal organization of signaling, regulated by Rap1b, phosphatase, PIP3 and metallo-proteinases (MMPs), specifies diapedesis route via protrusions-mediated neutrophil-endothelial cell interactions - and, as a result modulates inflammatory reactions. We will pursue the following aims.
Aim1 will study the role of Rap1b, phosphatases and PIP3 in diapedesis.
Aim 2 will test the hypothesis that specific neutrophil invadopodia components, including substrates of PIP3, proteases and MMP, control neutrophil/endothelial cell interactions for transcellular migration.
Aim 3 will use intravital imaging to investigate mechanism of neutrophil diapedesis in vivo. It will examine the relationship between route choice and inflammation in vivo. The clinical impact should be significant, since the proposed aims focus on molecules (ie, AKT, CD11b, phosphatase [SHP-1, PTEN, SHIP-1/2]) that are associated with inflammatory, immune disorders, and cancer. We anticipate the study will uncover completely novel mechanisms of neutrophil diapedesis. It will pave the way to designing novel therapeutic opportunities to inflammation, and more broadly to metastatic cell extravasation into tissues, as Rap1b inactivation and subsequent targets are associated with some metastatic cancers.
Rapid and directed migration of neutrophils to sites of infection is the first line of defense employed by the body. Failure to regulate neutrophil migration leads to an inability to control infection. However, aberrant accumulation of neutrophils in tissues is also a known cause of tissue injury. Despite its clinical importance, the molecular mechanisms regulating neutrophil migration remains incompletely understood both in physiological and pathological settings. The studies proposed in this application will provide important information on the molecular mechanisms that govern neutrophil migration across blood vessels. This study should guide designing new strategies to modulate neutrophil functions that could be used in the future for novel therapeutics of inflammatory diseases.
|Kumar, Sachin; Filippi, Marie-Dominique (2016) An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis. J Vis Exp :|
|Filippi, Marie-Dominique (2016) Mechanism of Diapedesis: Importance of the Transcellular Route. Adv Immunol 129:25-53|
|Filippi, Marie-Dominique (2015) Leukocyte transcellular diapedesis: Rap1b is in control. Tissue Barriers 3:e1052185|