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

Hattori, Hidenori; Subramanian, Kulandayan K.; Sakai, Jiro; Jia, Yonghui; Li, Yitang; Porter, Timothy F.; Loison, Fabien; Sarraj, Bara; Kasorn, Anongnard; Jo, Hakryul; Blanchard, Catlyn; Zirkle, Dorothy; McDonald, Douglas R.; Pai, Sung-Yun; Serhan, Charles N.; Luo, Hongbo

doi:10.1073/pnas.0914351107

Cited by 136 publications

(120 citation statements)

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“…Interestingly, ROS were recently identified as being important for directionality in a study of small-molecule inhibitors of directional migration. 45 That study also showed that neutrophils from chronic granulomatous disease patients, which lack significant ROS production, also exhibit defects in chemotaxis. In addition, an intriguing finding that neutrophils are able to migrate along H 2 O 2 gradients could be explained by this mechanism.…”

Section: Discussionmentioning

confidence: 86%

Rac regulates PtdInsP3 signaling and the chemotactic compass through a redox-mediated feedback loop

Kuiper

Sun

Magalhães

et al. 2011

Blood

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IntroductionNeutrophils are important mediators of the innate immune system and are rapidly recruited from the circulation to sites of infection to eliminate pathogenic threats. Key to this recruitment is the release of chemoattractants by infected host tissue or pathogens, which subsequently form a chemical gradient that attracts neutrophils to the appropriate sites. 1 In response to a chemotactic gradient, neutrophils polarize and form a leading edge pointing toward the chemoattractant source and a posterior structure called the uropod. Actin polymerization within the leading edge drives up-gradient protrusion, whereas myosin activity in the uropod detaches the rear of the cell. The proper coordination of these polarized events is essential for directional migration and depends on the asymmetrical distribution of specific molecular determinants. A hallmark of neutrophil polarization is the asymmetrical accumulation of the phospholipid phosphatidylinositol(3,4,5)-trisphosphate [PtdIns(3,4,5)P 3 ] in the leading edge. 2,3 The formation of PtdIns(3,4,5)P 3 is catalyzed by PI3Ks and is counteracted by the activity of the lipid phosphatases phosphatase and tensin homolog (PTEN) and SH2-containing inositol phosphatase (SHIP), which generate the lipid products PtdIns(4,5)P 2 and PtdIns(3,4)P 2 , respectively. 4,5 Several studies have suggested that the establishment of a robust PtdIns(3,4,5)P 3 levels requires a positive feedback loop, and this is corroborated by the observation that the introduction of exogenous PtdIns(3,4,5)P 3 induces further accumulation of endogenous PtdIns(3,4,5)P 3 in a Rho GTPase-dependent fashion. 6,7 Rac and actin polymerization were both shown to be required for the establishment of PtdIns(3,4,5)P 3 polarization via a feedback loop mechanism. 8,9 However, the exact mechanism remains elusive. Because neutrophils produce reactive oxygen species (ROS) within the leading edge during chemotaxis, 10,11 and the activity of the lipid phosphatase PTEN is markedly reduced by ROS, 12,13 we hypothesized that local ROS production in neutrophils could regulate PtdIns(3,4,5)P 3 levels through redox regulation of PTEN. We indeed identified PTEN to be a major target of chemoattractantinduced ROS formation and demonstrate that its enzymatic activity decreases accordingly. Decreased PTEN activity led to an increase of PtdIns(3,4,5)P 3 , which could be reversed by ROS scavengers or genetic ablation of NADPH-oxidase 2 (NOX2). Consistent with this finding, reduction of ROS inhibited directional migration of neutrophils toward N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), uncapping of actin filaments, and Rac activity. Although Rac GTPases are important downstream effectors of PtdIns(3,4,5)P 3 , they are also crucial for ROS production. Rescue experiments with small GTPase Rac effector mutants revealed that Rac facilitates PtdIns(3,4,5)P 3 accumulation and that this effect depends on its ability to activate NADPH-oxidase. Methods Abs and reagentsRabbit polyclonal Ab against phosphorylated AKT (Thr308; #2...

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Section: Discussionmentioning

confidence: 86%

Rac regulates PtdInsP3 signaling and the chemotactic compass through a redox-mediated feedback loop

Kuiper

Sun

Magalhães

et al. 2011

Blood

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“…In our studies we use a concentration of 5 μM for cell labeling and we are able to successfully track labeled neutrophils in blood, bone marrow and kidneys of Candida-infected recipient mice for at least 4 hr after neutrophil transfer 6 . Besides CellTracker Green and CellTracker Orange, other dyes including CellTracker Violet, CFSE and SNARF-1 have also been used successfully to label mouse bone marrow-derived neutrophils for adoptive transfer experiments 11,19,20 . In addition, the CellTracker Green and CellTracker Orange dyes have been effectively used at a similar concentration of 5 μM for labeling mouse splenic T cells for adoptive transfer and tracking experiments 21 .…”

Section: Discussionmentioning

confidence: 99%

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Swamydas

Lionakis

2013

JoVE

194

168

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Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

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“…29 It is clear that ROS is important for migration; when nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the enzyme that generates superoxide, is blocked in neutrophils, they experience decreased migration. 30 However, excess levels of ROS are also detrimental as van Aalst et al showed that incubation of endothelial cells with oxidized lowdensity lipoprotein both increased ROS levels and decreased cell migration compared to cells cultured in a normal medium. 31 Prolonged exposure to ROS does not necessarily decrease CXCR4 expression as prior studies have shown that only very high levels of AGEs (500 lg/mL) decreased receptor expression.…”

Section: Discussionmentioning

confidence: 99%

Soluble Receptor for Advanced Glycation End Products Improves Stromal Cell–Derived Factor-1 Activity in Model Diabetic Environments

Olekson

Faulknor

Hsia

et al. 2016

Advances in Wound Care

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Objective: In diabetes, hyperglycemia causes the accumulation of advanced glycation end products (AGEs) that trigger reactive oxygen species (ROS) generation through binding the receptor for AGEs (RAGE). Because exogenous growth factors have had little success in enhancing chronic wound healing, we investigated whether hyperglycemia-induced AGEs interfere with cellular responses to extracellular signals. We used stromal cell-derived factor-1 (SDF-1), an angiogenic chemokine also known to promote stem cell recruitment in skin wounds. Approach: Human leukemia-60 (HL-60) cells and mouse peripheral blood mononuclear cells (PBMCs), which express the SDF-1 receptor CXCR-4, were incubated for 24 h in medium supplemented with 25 mM d-glucose. Soluble RAGE (sRAGE) was used to block RAGE activation. Response to SDF-1 was measured in cellular migration and ROS assays. A diabetic murine excisional wound model measured SDF-1 liposome and sRAGE activity in vivo. Results: Hyperglycemia led to significant accumulation of AGEs, decreased SDF-1-directed migration, and elevated baseline ROS levels; it suppressed the ROS spike normally triggered by SDF-1. sRAGE decreased the ROS baseline and restored both the SDF-1-mediated spike and cell migration. Topically applied sRAGE alone promoted healing and enhanced the effect of exogenous SDF-1 on diabetic murine wounds. Innovation: While there is interest in using growth factors to improve wound healing, this strategy is largely ineffective in diabetic wounds. We show that sRAGE may restore signaling, thus potentiating the effect of exogenously applied growth factors. Conclusion: Blocking RAGE with sRAGE restores SDF-1-mediated cellular responses in hyperglycemic environments and may potentiate the effectiveness of SDF-1 applied in vivo.

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

Cited by 136 publications

References 27 publications

Rac regulates PtdInsP3 signaling and the chemotactic compass through a redox-mediated feedback loop

Rac regulates PtdInsP3 signaling and the chemotactic compass through a redox-mediated feedback loop

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Soluble Receptor for Advanced Glycation End Products Improves Stromal Cell–Derived Factor-1 Activity in Model Diabetic Environments

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