The P-Rex1 Regulator Norbin Suppressess Neutrophil-Dependent Antibacterial Immunity

P-Rex1 is a guanine-nucleotide exchange factor (GEF) that activates the small G protein Rac, thus regulating a wide range of physiological and pathophysiological responses. Neutrophils are leukocytes of the innate immune system in which P-Rex1 regulates several Rac-dependent responses, especially those elicited by GPCR signalling. The aim of my project was to assess the functional importance of a new interactor of P-Rex1, the GPCR adaptor protein Norbin, in neutrophils. Norbin is an essential neuronal protein that binds directly to GPCRs, regulating GPCR signalling and trafficking, through unknown mechanisms. Our laboratory recently identified that Norbin stimulates P-Rex1 Rac-GEF activity and promotes P-Rex1 membrane localisation. Furthermore, we showed that Norbin is expressed in myeloid cells. To investigate, we generated mouse strains with conditional deletion of Norbin in myeloid cells and with combined Norbin/Prex1 deficiency. Unexpectedly, I found increased adhesion, spreading, ROS production and degranulation responses in isolated Norbin-deficient neutrophils. Moreover, Norbin-deficient neutrophils had an increased ROS-dependent capacity to kill Staphylococcus aureus. In vivo, Norbin deficiency provided increased immunity against pulmonary infection with Streptococcus pneumoniae, even in immune-deficient Prex‒/‒ mice. Neutrophil depletion showed that Norbin deficiency renders neutrophils important for combatting this infection. Mostly, Norbin deficiency overrode the functional impairments caused by Prex1 deficiency, although some neutrophil responses remained P-Rex1-dependent. Mechanistically, the Norbin deficiency caused constitutive upregulation of some GPCRs onto the neutrophil surface and promoted the GPCR-dependent activities of Rac and Erk, whereas several other signalling pathways and the surface levels of adhesion molecules were not obviously affected. Together, my data indicate that the GPCR adaptor and P-Rex1 regulator Norbin plays an important role in suppressing the host defence functions of neutrophils. A subset of Norbin functions are P-Rex1 dependent, whereas others are likely mediated through other regulators of Rac and of Erk, as well as through the control of GPCR trafficking.BBRSC DT

Dock2 generates characteristic spatiotemporal patterns of Rac activity to regulate neutrophil polarisation, migration and phagocytosis

IntroductionRac-GTPases and their Rac-GEF activators play important roles in neutrophil-mediated host defence. These proteins control the adhesion molecules and cytoskeletal dynamics required for neutrophil recruitment to inflamed and infected organs, and the neutrophil effector responses that kill pathogens.MethodsHere, we used live cell TIRF-FRET imaging in neutrophils from Rac-FRET reporter mice with deficiencies in the Rac-GEFs Dock2, Tiam1 or Prex1/Vav1 to evaluate if these proteins activate spatiotemporally distinct pools of Rac, and to correlate patterns of Rac activity with the neutrophil responses they control.ResultsAll the GEFs were required for neutrophil adhesion, and Prex1/Vav1 were important during spreading and for the velocity of migration during chemotaxis. However, Dock2 emerged as the prominent regulator of neutrophil responses, as this GEF was required for neutrophil polarisation and random migration, for migration velocity during chemokinesis, for the likelihood to migrate and for the speed of migration and of turning during chemotaxis, as well as for rapid particle engulfment during phagocytosis. We identified characteristic spatiotemporal patterns of Rac activity generated by Dock2 which correlate with the importance of the Rac-GEF in these neutrophil responses. We also demonstrate a requirement for Dock2 in neutrophil recruitment during aseptic peritonitis.DiscussionCollectively, our data provide a first direct comparison of the pools of Rac activity generated by different types of Rac-GEFs, and identify Dock2 as a key regulator of polarisation, migration and phagocytosis in primary neutrophils

The Rac-GEF Tiam1 controls integrin-dependent neutrophil responses

Rac GTPases are required for neutrophil adhesion and migration, and for the neutrophil effector responses that kill pathogens. These Rac-dependent functions are impaired when neutrophils lack the activators of Rac, Rac-GEFs from the Prex, Vav, and Dock families. In this study, we demonstrate that Tiam1 is also expressed in neutrophils, governing focal complexes, actin cytoskeletal dynamics, polarisation, and migration, in a manner depending on the integrin ligand to which the cells adhere. Tiam1 is dispensable for the generation of reactive oxygen species but mediates degranulation and NETs release in adherent neutrophils, as well as the killing of bacteria. In vivo, Tiam1 is required for neutrophil recruitment during aseptic peritonitis and for the clearance of Streptococcus pneumoniae during pulmonary infection. However, Tiam1 functions differently to other Rac-GEFs. Instead of promoting neutrophil adhesion to ICAM1 and stimulating β2 integrin activity as could be expected, Tiam1 restricts these processes. In accordance with these paradoxical inhibitory roles, Tiam1 limits the fMLP-stimulated activation of Rac1 and Rac2 in adherent neutrophils, rather than activating Rac as expected. Tiam1 promotes the expression of several regulators of small GTPases and cytoskeletal dynamics, including αPix, Psd4, Rasa3, and Tiam2. It also controls the association of Rasa3, and potentially αPix, Git2, Psd4, and 14-3-3ζ/δ, with Rac. We propose these latter roles of Tiam1 underlie its effects on Rac and β2 integrin activity and on cell responses. Hence, Tiam1 is a novel regulator of Rac-dependent neutrophil responses that functions differently to other known neutrophil Rac-GEFs

The GPCR adaptor protein Norbin regulates S1PR1 trafficking and the morphology, cell cycle and survival of PC12 cells

Abstract Norbin is an adaptor protein that binds numerous G protein-coupled receptors (GPCRs), is highly expressed in neurons, and is essential for a functioning nervous system in rodent models. Yet, beyond its control of neurite outgrowth and synaptic plasticity, few cellular roles of Norbin have been investigated to date. Furthermore, while Norbin is known to regulate the steady-state cell surface levels of several GPCRs, only in one case has the protein been shown to control the agonist-induced receptor internalisation which serves to attenuate GPCR signalling. Here, we generated a Norbin-deficient PC12 cell line which enabled us to study both the cellular functions of Norbin and its roles in GPCR trafficking and signalling. We show that Norbin limits cell size and spreading, and is required for the growth, viability and cell cycle progression of PC12 cells. We also found that Norbin regulates both the steady-state surface level and agonist-induced internalisation of the GPCR sphingosine-1-phosphate receptor 1 (S1PR1) in these cells, suggesting that its role in agonist-dependent GPCR trafficking is more widespread than previously appreciated. Finally, we show that Norbin limits the S1P-stimulated activation of Akt and p38 Mapk, and is required for the activation of Erk in PC12 cells. Together, our findings provide a better understanding of the cellular functions of Norbin and its control of GPCR trafficking

The GPCR adaptor protein norbin suppresses the neutrophil-mediated immunity of mice to pneumococcal infection

Streptococcal pneumonia is a worldwide health problem that kills 2 million people each year, particularly young children, the elderly, and immunosuppressed individuals. Alveolar macrophages and neutrophils provide the early innate immune response to clear pneumococcus from infected lungs. However, the level of neutrophil involvement is context dependent, both in humans and in mouse models of the disease, influenced by factors such as bacterial load, age, and coinfections. Here, we show that the G protein coupled receptor (GPCR) adaptor protein norbin (neurochondrin, NCDN), which was hitherto known as a regulator of neuronal function, is a suppressor of neutrophilmediated innate immunity. Myeloid norbin deficiency improved the immunity of mice to pneumococcal infection by increasing the involvement of neutrophils in clearing the bacteria, without affecting neutrophil recruitment or causing autoinflammation. It also improved immunity during Escherichia coli induced septic peritonitis. It increased the responsiveness of neutrophils to a range of stimuli, promoting their ability to kill bacteria in a reactive oxygen species dependent manner, enhancing degranulation, phagocytosis, and the production of reactive oxygen species and neutrophil extracellular traps, raising the cell surface levels of selected GPCRs, and increasing GPCR-dependent Rac and Erk signaling. The Rac guanine-nucleotide exchange factor Prex1, a known effector of norbin, was dispensable for most of these effects, which suggested that norbin controls additional downstream targets. We identified the Rac guanine-nucleotide exchange factor Vav as one of these effectors. In summary, our study presents the GPCR adaptor protein norbin as an immune suppressor that limits the ability of neutrophils to clear bacterial infections

Memory of stochastic single-cell apoptotic signaling promotes chemoresistance in neuroblastoma

Gene expression noise is known to promote stochastic drug resistance through the elevated expression of individual genes in rare cancer cells. However, we now demonstrate that chemoresistant neuroblastoma cells emerge at a much higher frequency when the influence of noise is integrated across multiple components of an apoptotic signaling network. Using a JNK activity biosensor with longitudinal high-content and in vivo intravital imaging, we identify a population of stochastic, JNK-impaired, chemoresistant cells that exist because of noise within this signaling network. Furthermore, we reveal that the memory of this initially random state is retained following chemotherapy treatment across a series of in vitro, in vivo, and patient models. Using matched PDX models established at diagnosis and relapse from individual patients, we show that HDAC inhibitor priming cannot erase the memory of this resistant state within relapsed neuroblastomas but improves response in the first-line setting by restoring drug-induced JNK activity within the chemoresistant population of treatment-naive tumors