Behavioural immune landscapes of inflammation.

Transcriptional and proteomic profiling of individual cells have revolutionized interpretation of biological phenomena by providing cellular landscapes of healthy and diseased tissues1,2. These approaches, however, do not describe dynamic scenarios in which cells continuously change their biochemical properties and downstream 'behavioural' outputs3-5. Here we used 4D live imaging to record tens to hundreds of morpho-kinetic parameters describing the dynamics of individual leukocytes at sites of active inflammation. By analysing more than 100,000 reconstructions of cell shapes and tracks over time, we obtained behavioural descriptors of individual cells and used these high-dimensional datasets to build behavioural landscapes. These landscapes recognized leukocyte identities in the inflamed skin and trachea, and uncovered a continuum of neutrophil states inside blood vessels, including a large, sessile state that was embraced by the underlying endothelium and associated with pathogenic inflammation. Behavioural screening in 24 mouse mutants identified the kinase Fgr as a driver of this pathogenic state, and interference with Fgr protected mice from inflammatory injury. Thus, behavioural landscapes report distinct properties of dynamic environments at high cellular resolution.

Functions and mechanisms of the GPCR adaptor protein Norbin.

Norbin (Neurochondrin, NCDN) is a highly conserved 79 kDa adaptor protein that was first identified more than a quarter of a century ago as a gene up-regulated in rat hippocampus upon induction of long-term potentiation. Most research has focussed on the role of Norbin in the nervous system, where the protein is highly expressed. Norbin regulates neuronal morphology and synaptic plasticity, and is essential for normal brain development and homeostasis. Dysregulation of Norbin is linked to a variety of neurological conditions. Recently, Norbin was shown to be expressed in myeloid cells as well as neurons. Myeloid-cell specific deletion revealed an important role of Norbin as a suppressor of neutrophil-derived innate immunity. Norbin limits the ability of neutrophils to clear bacterial infections by curbing the responsiveness of these cells to inflammatory and infectious stimuli. Mechanistically, Norbin regulates cell responses through binding to its interactors, in particular to a wide range of G protein-coupled receptors (GPCRs). Norbin association with GPCRs controls GPCR trafficking and signalling. Other important Norbin interactors are the Rac guanine-nucleotide exchange factor P-Rex1 and protein kinase A. Downstream signalling pathways regulated by Norbin include ERK, Ca2+ and the small GTPase Rac. Here, we review the current understanding of Norbin structure, expression and its roles in health and disease. We also explore Norbin signalling through its interactors, with a particular focus on GPCR trafficking and signalling. Finally, we discuss avenues that could be pursued in the future to increase our understanding of Norbin biology.

PtdIns(3,4,5)P3-dependent Rac exchanger 1 (P-Rex1) promotes mammary tumor initiation and metastasis.

The Rac-GEF, P-Rex1, activates Rac1 signaling downstream of G protein-coupled receptors and PI3K. Increased P-Rex1 expression promotes melanoma progression; however, its role in breast cancer is complex, with differing reports of the effect of its expression on disease outcome. To address this we analyzed human databases, undertook gene array expression analysis, and generated unique murine models of P-Rex1 gain or loss of function. Analysis of PREX1 mRNA expression in breast cancer cDNA arrays and a METABRIC cohort revealed that higher PREX1 mRNA in ER+ve/luminal tumors was associated with poor outcome in luminal B cancers. Prex1 deletion in MMTV-neu or MMTV-PyMT mice reduced Rac1 activation in vivo and improved survival. High level MMTV-driven transgenic PREX1 expression resulted in apicobasal polarity defects and increased mammary epithelial cell proliferation associated with hyperplasia and development of de novo mammary tumors. MMTV-PREX1 expression in MMTV-neu mice increased tumor initiation and enhanced metastasis in vivo, but had no effect on primary tumor growth. Pharmacological inhibition of Rac1 or MEK1/2 reduced P-Rex1-driven tumoroid formation and cell invasion. Therefore, P-Rex1 can act as an oncogene and cooperate with HER2/neu to enhance breast cancer initiation and metastasis, despite having no effect on primary tumor growth.

Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma.

PREX2 (phosphatidylinositol-3,4,5-triphosphate-dependent Rac-exchange factor 2) is a PTEN (phosphatase and tensin homolog deleted on chromosome 10) binding protein that is significantly mutated in cutaneous melanoma and pancreatic ductal adenocarcinoma. Here, genetic and biochemical analyses were conducted to elucidate the nature and mechanistic basis of PREX2 mutation in melanoma development. By generating an inducible transgenic mouse model we showed an oncogenic role for a truncating PREX2 mutation (PREX2(E824)*) in vivo in the context of mutant NRAS. Using integrative cross-species gene expression analysis, we identified deregulated cell cycle and cytoskeleton organization as significantly perturbed biological pathways in PREX2 mutant tumors. Mechanistically, truncation of PREX2 activated its Rac1 guanine nucleotide exchange factor activity, abolished binding to PTEN and activated the PI3K (phosphatidyl inositol 3 kinase)/Akt signaling pathway. We further showed that PREX2 truncating mutations or PTEN deletion induces down-regulation of the tumor suppressor and cell cycle regulator CDKN1C (also known as p57(KIP2)). This down-regulation occurs, at least partially, through DNA hypomethylation of a differentially methylated region in chromosome 11 that is a known regulatory region for expression of the CDKN1C gene. Together, these findings identify PREX2 as a mediator of NRAS-mutant melanoma development that acts through the PI3K/PTEN/Akt pathway to regulate gene expression of a cell cycle regulator.

Rac-GTPases and Rac-GEFs in neutrophil adhesion, migration and recruitment.

Rac-GTPases and their Rac-GEF activators play important roles in the recruitment and host defence functions of neutrophils. These proteins control the activation of adhesion molecules and the cytoskeletal dynamics that enable the adhesion, migration and tissue recruitment of neutrophils. They also regulate the effector functions that allow neutrophils to kill bacterial and fungal pathogens, and to clear debris. This review focuses on the roles of Rac-GTPases and Rac-GEFs in neutrophil adhesion, migration and recruitment.

Platelets in neutrophil recruitment to sites of inflammation.

PURPOSE OF REVIEW: This review describes the essential roles of platelets in neutrophil recruitment from the bloodstream into inflamed and infected tissues, with a focus on recent findings. RECENT FINDINGS: Platelets are required for the recruitment of neutrophils to sites of inflammation and infection. They fulfil this role largely by enabling contacts of circulating neutrophils with the inflamed blood vessel wall prior to extravasation. Platelets promote both early stages of neutrophil recruitment (tethering, rolling, arrest, firm adhesion) and - as recent work has demonstrated - later stages (intravascular crawling and diapedesis). Recent studies have also begun to identify platelet-signaling pathways that can elicit the underlying interactions between platelets, neutrophils and vascular endothelial cells without stimulating concomitant platelet aggregation and thrombus formation. These pathways include Rho-guanine-nucleotide binding proteins and Rho-guanine-nucleotide exchange factors. SUMMARY: Recent findings have contributed to our burgeoning understanding of the platelet-dependent mechanisms that control neutrophil recruitment to sites of inflammation and have opened up new avenues of research aimed at increasing our knowledge of these mechanisms further. These insights might lead to the development of novel anti-inflammatory drugs that will be useful in a wide range of inflammatory diseases without causing immunodeficiency.

Norbin Stimulates the Catalytic Activity and Plasma Membrane Localization of the Guanine-Nucleotide Exchange Factor P-Rex1.

P-Rex1 is a guanine-nucleotide exchange factor (GEF) that activates the small G protein (GTPase) Rac1 to control Rac1-dependent cytoskeletal dynamics, and thus cell morphology. Three mechanisms of P-Rex1 regulation are currently known: (i) binding of the phosphoinositide second messenger PIP3, (ii) binding of the Gßγ subunits of heterotrimeric G proteins, and (iii) phosphorylation of various serine residues. Using recombinant P-Rex1 protein to search for new binding partners, we isolated the G-protein-coupled receptor (GPCR)-adaptor protein Norbin (Neurochondrin, NCDN) from mouse brain fractions. Coimmunoprecipitation confirmed the interaction between overexpressed P-Rex1 and Norbin in COS-7 cells, as well as between endogenous P-Rex1 and Norbin in HEK-293 cells. Binding assays with purified recombinant proteins showed that their interaction is direct, and mutational analysis revealed that the pleckstrin homology domain of P-Rex1 is required. Rac-GEF activity assays with purified recombinant proteins showed that direct interaction with Norbin increases the basal, PIP3- and Gßγ-stimulated Rac-GEF activity of P-Rex1. Pak-CRIB pulldown assays demonstrated that Norbin promotes the P-Rex1-mediated activation of endogenous Rac1 upon stimulation of HEK-293 cells with lysophosphatidic acid. Finally, immunofluorescence microscopy and subcellular fractionation showed that coexpression of P-Rex1 and Norbin induces a robust translocation of both proteins from the cytosol to the plasma membrane, as well as promoting cell spreading, lamellipodia formation, and membrane ruffling, cell morphologies generated by active Rac1. In summary, we have identified a novel mechanism of P-Rex1 regulation through the GPCR-adaptor protein Norbin, a direct P-Rex1 interacting protein that promotes the Rac-GEF activity and membrane localization of P-Rex1.

P-Rex and Vav Rac-GEFs in platelets control leukocyte recruitment to sites of inflammation.

The small GTPase Rac is required for neutrophil recruitment during inflammation, but its guanine-nucleotide exchange factor (GEF) activators seem dispensable for this process, which led us to investigate the possibility of cooperation between Rac-GEF families. Thioglycollate-induced neutrophil recruitment into the peritoneum was more severely impaired in P-Rex1(-/-) Vav1(-/-) (P1V1) or P-Rex1(-/-) Vav3(-/-) (P1V3) mice than in P-Rex null or Vav null mice, suggesting cooperation between P-Rex and Vav Rac-GEFs in this process. Neutrophil transmigration and airway infiltration were all but lost in P1V1 and P1V3 mice during lipopolysaccharide (LPS)-induced pulmonary inflammation, with altered intercellular adhesion molecule 1-dependent slow neutrophil rolling and strongly reduced L- and E-selectin-dependent adhesion in airway postcapillary venules. Analysis of adhesion molecule expression, neutrophil adhesion, spreading, and migration suggested that these defects were only partially neutrophil-intrinsic and were not obviously involving vascular endothelial cells. Instead, P1V1 and P1V3 platelets recapitulated the impairment of LPS-induced intravascular neutrophil adhesion and recruitment, showing P-Rex and Vav expression in platelets to be crucial. Similarly, during ovalbumin-induced allergic inflammation, pulmonary recruitment of P1V1 and P1V3 eosinophils, monocytes, and lymphocytes was compromised in a platelet-dependent manner, and airway inflammation was essentially abolished, resulting in improved airway responsiveness. Therefore, platelet P-Rex and Vav family Rac-GEFs play important proinflammatory roles in leukocyte recruitment.

Small GTPases and their guanine-nucleotide exchange factors and GTPase-activating proteins in neutrophil recruitment.

PURPOSE OF REVIEW: The review describes the roles of Rho- and Rap-guanosine triphosphatases (GTPases) and of their activators, guanine-nucleotide exchange factors (GEFs), and inhibitors, GTPase activating proteins (GAPs), in neutrophil recruitment from the blood stream into inflamed tissues, with a focus on recently identified roles in neutrophils, endothelial cells, and platelets. RECENT FINDINGS: Recent studies have identified important roles of Rho- and Rap-GTPases, and of their GEFs and GAPs, in the neutrophil recruitment cascade. These proteins control the upregulation and/or activation of adhesion molecules on the surface of neutrophils, endothelial cells, and platelets, and they alter cell/cell adhesion in the vascular endothelium. This enables the capture of neutrophils from the blood stream, their migration along and through the vessel wall, and their passage into the inflamed tissue. In particular, it has recently become clear that P-Rex and Vav family Rac-GEFs in platelets are crucial for neutrophil recruitment. SUMMARY: These recent findings have contributed greatly to our understanding of the signalling pathways that control neutrophil recruitment to sites of inflammation and have opened up new avenues of research in this field.

P-Rex1 directly activates RhoG to regulate GPCR-driven Rac signalling and actin polarity in neutrophils.

G-protein-coupled receptors (GPCRs) regulate the organisation of the actin cytoskeleton by activating the Rac subfamily of small GTPases. The guanine-nucleotide-exchange factor (GEF) P-Rex1 is engaged downstream of GPCRs and phosphoinositide 3-kinase (PI3K) in many cell types, and promotes tumorigenic signalling and metastasis in breast cancer and melanoma, respectively. Although P-Rex1-dependent functions have been attributed to its GEF activity towards Rac1, we show that P-Rex1 also acts as a GEF for the Rac-related GTPase RhoG, both in vitro and in GPCR-stimulated primary mouse neutrophils. Furthermore, loss of either P-Rex1 or RhoG caused equivalent reductions in GPCR-driven Rac activation and Rac-dependent NADPH oxidase activity, suggesting they both function upstream of Rac in this system. Loss of RhoG also impaired GPCR-driven recruitment of the Rac GEF DOCK2, and F-actin, to the leading edge of migrating neutrophils. Taken together, our results reveal a new signalling hierarchy in which P-Rex1, acting as a GEF for RhoG, regulates Rac-dependent functions indirectly through RhoG-dependent recruitment of DOCK2. These findings thus have broad implications for our understanding of GPCR signalling to Rho GTPases and the actin cytoskeleton.