Jiro Sakai

Small-molecule screen identifies reactive oxygen species as key regulators of neutrophil chemotaxis

Neutrophil chemotaxis plays an essential role in innate immunity, but the underlying cellular mechanism is still not fully characterized. Here, using a small-molecule functional screening, we identified NADPH oxidase–dependent reactive oxygen species as key regulators of neutrophil chemotactic migration. Neutrophils with pharmacologically inhibited oxidase, or isolated from chronic granulomatous disease (CGD) patients and mice, formed more frequent multiple pseudopodia and lost their directionality as they migrated up a chemoattractant concentration gradient. Knocking down NADPH oxidase in differentiated neutrophil-like HL60 cells also led to defective chemotaxis. Consistent with the in vitro results, adoptively transferred CGD murine neutrophils showed impaired in vivo recruitment to sites of inflammation. Together, these results present a physiological role for reactive oxygen species in regulating neutrophil functions and shed light on the pathogenesis of CGD.

Inositol hexakisphosphate kinase 1 regulates neutrophil function in innate immunity by inhibiting phosphatidylinositol-(3,4,5)-trisphosphate signaling

Inositol phosphates are widely produced throughout animal and plant tissues. Diphosphoinositol pentakisphosphate (InsP7) contains an energetic pyrophosphate bond. Here we demonstrate that disruption of inositol hexakisphosphate kinase 1 (InsP6K1), one of the three mammalian inositol hexakisphosphate kinases (InsP6Ks) that convert inositol hexakisphosphate (InsP6) to InsP7, conferred enhanced phosphatidylinositol-(3,4,5)-trisphosphate (PtdIns(3,4,5)P3)-mediated membrane translocation of the pleckstrin homology domain of the kinase Akt and thus augmented downstream PtdIns(3,4,5)P3 signaling in mouse neutrophils. Consequently, these neutrophils had greater phagocytic and bactericidal ability and amplified NADPH oxidase–mediated production of superoxide. These phenotypes were replicated in human primary neutrophils with pharmacologically inhibited InsP6Ks. In contrast, an increase in intracellular InsP7 blocked chemoattractant-elicited translocation of the pleckstrin homology domain to the membrane and substantially suppressed PtdIns(3,4,5)P3-mediated cellular events in neutrophils. Our findings establish a role for InsP7 in signal transduction and provide a mechanism for modulating PtdIns(3,4,5)P3 signaling in neutrophils.

The Effects of a Shear Flow on the Uptake of LDL and Acetylated LDL by an EC Monoculture and an EC-SMC Coculture

To elucidate the mechanisms of localized genesis and development of atherosclerosis and anastomotic intimal hyperplasia in man, a coculture of bovine aortic endothelial cells (ECs) and smooth muscle cells (SMCs) was prepared, and the effects of a shear flow on the uptake of lipoproteins by the cells was studied by incubating the EC–SMC coculture as well as an EC monoculture with a culture medium containing either DiI-LDL or DiI-Ac-LDL and subjecting to a laminar shear flow. It was found that in both the presence and absence of a shear flow that imposed the ECs an area mean shear stress of 13.3 dynes/cm2, the uptake of LDL by an EC–SMC coculture was much greater than that by an EC monoculture, whereas that of Ac-LDL was almost the same. The uptake of LDL by an EC monoculture increased slightly by being exposed to a shear flow, whereas that by an EC–SMC coculture did not. In contrast to this, the uptake of Ac-LDL by both an EC monoculture and an EC–SMC coculture decreased drastically by a shear flow, suggesting that the action of a shear flow on the uptake of Ac-LDL by vascular cells is very different from that of LDL.

Phosphoinositide lipid phosphatase SHIP1 and PTEN coordinate to regulate cell migration and adhesion.

The second messenger phosphatidylinositol(3,4,5)P(3) (PtdIns(3,4,5)P(3)) is formed by stimulation of various receptors, including G protein-coupled receptors and integrins. The lipid phosphatases PTEN and SHIP1 are critical in regulating the level of PtdIns(3,4,5)P(3) during chemotaxis. Observations that loss of PTEN had minor and loss of SHIP1 resulted in a severe chemotaxis defect in neutrophils led to the belief that SHIP1 rather than PTEN acts as a predominant phospholipid phosphatase in establishing a PtdIns(3,4,5)P(3) compass. In this study, we show that SHIP1 regulates PtdIns(3,4,5)P(3) production in response to cell adhesion and plays a limited role when cells are in suspension. SHIP1((-)/(-)) neutrophils lose their polarity upon cell adhesion and are extremely adherent, which impairs chemotaxis. However, chemo-taxis can be restored by reducing adhesion. Loss of SHIP1 elevates Akt activation following cell adhesion due to increased PtdIns(3,4,5)P(3) production. From our observations, we conclude that SHIP1 prevents formation of top-down PtdIns(3,4,5)P(3) polarity to facilitate proper cell attachment and detachment during chemotaxis.

Reactive oxygen species as signaling molecules in neutrophil chemotaxis

Neutrophil chemotaxis is a critical component in innate immunity. Recently, using a small-molecule functional screening, we identified NADPH-oxidase-dependent Reactive Oxygen Species (ROS) as key regulators of neutrophil chemotactic migration. Neutrophils depleted of ROS form more frequent multiple pseudopodia and lost their directionality as they migrate up a chemoattractant concentration gradient. Here, we further studied the role of ROS in neutrophil chemotaxis and found that multiple pseudopodia formation induced by NADPH inhibitor diphenyleneiodonium chloride (DPI) was more prominent in relatively shallow chemoattractant gradient. It was reported that, in shallow chemoattractant gradients, new pseudopods are usually generated when existing ones bifurcate. Directional sensing is mediated by maintaining the most accurate existing pseudopod, and destroying pseudopods facing the wrong direction by actin depolymerization. We propose that NADPH-mediated ROS production may be critical for disruption of misoriented pseudopods in chemotaxing neutrophils. Thus, inhibition of ROS production will lead to formation of multiple pseudopodia.

Integrin-independent role of CalDAG-GEFI in neutrophil chemotaxis.

Chemotaxis and integrin activation are essential processes for neutrophil transmigration in response to injury. CalDAG‐GEFI plays a key role in the activation of β1, β2, and β3 integrins in platelets and neutrophils by exchanging a GDP for a GTP on Rap1. Here, we explored the role of CalDAG‐GEFI and Rap1b in integrin‐independent neutrophil chemotaxis. In a transwell assay, CalDAG‐GEFI−/− neutrophils had a 46% reduction in transmigration compared with WT in response to a low concentration of LTB4. Visualization of migrating neutrophils in the presence of 10 mM EDTA revealed that CalDAG‐GEFI−/− neutrophils had abnormal chemotactic behavior compared with WT neutrophils, including reduced speed and directionality. Interestingly, Rap1b−/− neutrophils had a similar phenotype in this assay, suggesting that CalDAG‐GEFI may be acting through Rap1b. We investigated whether the deficit in integrin‐independent chemotaxis in CalDAG‐GEFI−/− neutrophils could be explained by defective cytoskeleton rearrangement. Indeed, we found that CalDAG‐GEFI−/− neutrophils had reduced formation of F‐actin pseudopodia after LTB4 stimulation, suggesting that they have a defect in polarization. Overall, our studies show that CalDAG‐GEFI helps regulate neutrophil chemotaxis, independent of its established role in integrin activation, through a mechanism that involves actin cytoskeleton and cellular polarization.

Reactive oxygen species mediate shear stress-induced fluid-phase endocytosis in vascular endothelial cells

To elucidate the role of shear stress in fluid-phase endocytosis of vascular endothelial cells (EC), we used a rotating-disk shearing apparatus to investigate the effects of shear stress on the uptake of lucifer yellow (LY) by cultured bovine aortic endothelial cells (BAEC). Exposure of EC to shear stress (area-mean value of 10 dynes/cm2) caused an increase in LY uptake that was abrogated by the antioxidant, N-acetyl-l-cysteine (NAC), the NADPH oxidase inhibitor, acetovanillone, and two inhibitors of protein kinase C (PKC), calphostin C and GF109203X. These results suggest that fluid-phase endocytosis is regulated by both reactive oxygen species (ROS) and PKC. Shear stress increased both ROS production and PKC activity in EC, and the increase in ROS was unaffected by calphostin C or GF109203X, whereas the activation of PKC was reduced by NAC and acetovanillone. We conclude that shear stress-induced increase in fluid-phase endocytosis is mediated via ROS generation followed by PKC activation in EC.

G-CSF maintains controlled neutrophil mobilization during acute inflammation by negatively regulating CXCR2 signaling

Luo et al. report that CXCR2 ligands are responsible for rapid neutrophil mobilization during early-stage acute inflammation and that G-CSF suppresses this mobilization by negatively regulating CXCR2-mediated intracellular signaling.

De Novo Chemoattractants Form Supramolecular Hydrogels for Immunomodulating Neutrophils In Vivo

Most immunomodulatory materials (e.g., vaccine adjuvants such as alum) modulate adaptive immunity, and yet little effort has focused on developing materials to regulate innate immunity, which get mentioned only when inflammation affects the biocompatibility of biomaterials. Traditionally considered as short-lived effector cells from innate immunity primarily for the clearance of invading microorganisms without specificity, neutrophils exhibit a key role in launching and shaping the immune response. Here we show that the incorporation of unnatural amino acids into a well-known chemoattractant—N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLF)—offers a facile approach to create a de novo, multifunctional chemoattractant that self-assembles to form supramolecular nanofibrils and hydrogels. This de novo chemoattractant not only exhibits preserved cross-species chemoattractant activity to human and murine neutrophils, but also effectively resists proteolysis. Thus, its hydrogel, in vivo, releases the chemoattractant and attracts neutrophils to the desired location in a sustainable manner. As a novel and general approach to generate a new class of biomaterials for modulating innate immunity, this work offers a prolonged acute inflammation model for developing various new applications.

Lipopolysaccharide-induced NF-κB nuclear translocation is primarily dependent on MyD88, but TNFα expression requires TRIF and MyD88

TLR4 signalling through the MyD88 and TRIF-dependent pathways initiates translocation of the transcription factor NF-κB into the nucleus. In cell population studies using mathematical modeling and functional analyses, Cheng et al. suggested that LPS-driven activation of MyD88, in the absence of TRIF, impairs NF-κB translocation. We tested the model proposed by Cheng et al. using real-time single cell analysis in macrophages expressing EGFP-tagged p65 and a TNFα promoter-driven mCherry. Following LPS stimulation, cells lacking TRIF show a pattern of NF-κB dynamics that is unaltered from wild-type cells, but activation of the TNFα promoter is impaired. In macrophages lacking MyD88, there is minimal NF-κB translocation to the nucleus in response to LPS stimulation, and there is no activation of the TNFα promoter. These findings confirm that signalling through MyD88 is the primary driver for LPS-dependent NF-κB translocation to the nucleus. The pattern of NF-κB dynamics in TRIF-deficient cells does not, however, directly reflect the kinetics of TNFα promoter activation, supporting the concept that TRIF-dependent signalling plays an important role in the transcription of this cytokine.