Summary The regulation of actin dynamics is pivotal for cellular processes such as cell adhesion, migration, and phagocytosis, and thus is crucial for neutrophils to fulfill their roles in innate immunity. Many factors have been implicated in signal-induced actin polymerization, however the essential nature of the potential negative modulators are still poorly understood. Here we report that NADPH oxidase-dependent physiologically generated reactive oxygen species (ROS) negatively regulate actin polymerization in stimulated neutrophils via driving reversible actin glutathionylation. Disruption of glutaredoxin 1 (Grx1), an enzyme that catalyzes actin deglutathionylation, increased actin glutathionylation, attenuated actin polymerization, and consequently impaired neutrophil polarization, chemotaxis, adhesion, and phagocytosis. Consistently, Grx1-deficient murine neutrophils showed impaired in vivo recruitment to sites of inflammation and reduced bactericidal capability. Together, these results present a physiological role for glutaredoxin and ROS- induced reversible actin glutathionylation in regulation of actin dynamics in neutrophils.

The recruitment and activation of neutrophils at infected tissues is essential for host defense against invading microorganisms. However, excessive neutrophil recruitment or activation can also damage the surrounding tissues and cause unwanted inflammation. Hence, the responsiveness of neutrophils needs to be tightly regulated. In this study, we have investigated the functional role of tumor suppressor PTEN in neutrophils by using a mouse line in which PTEN is disrupted only in myeloid-derived cells. Chemoattractant-stimulated PTEN ؊/؊ neutrophils displayed significantly higher Akt phosphorylation and actin polymerization. A larger fraction of these neutrophils displayed membrane ruffles in response to chemoattractant stimulation. In addition, chemoattractant-induced transwell migration and superoxide production were also augmented. Single-cell chemotaxis assays showed that PTEN ؊/؊ neutrophils have a small (yet statistically significant) defect in directionality. However, these neutrophils also showed an increase in cell speed. As a result, overall chemotaxis, which depends on speed and directionality, was not affected. Consistent with the increased responsivenessof PTEN ؊/؊ neutrophils, the in vivo recruitment of these cells to the inflamed peritoneal cavity was significantly enhanced. Thus, as a physiologic-negative regulator, PTEN should be a promising therapeutic target for modulating neutrophil functions in various infectious and inflammatory dis- IntroductionNeutrophils are the most abundant cell type among circulating white blood cells. The recruitment and activation of neutrophils are important components of the innate immune system. In response to inflammatory stimuli, neutrophils migrate from the blood to infected tissues, where they protect their host by engulfing, killing, and digesting invading bacterial and fungal pathogens. Conversely, excessive neutrophil accumulation or hyperresponsiveness of neutrophils can also be detrimental to the system. The toxic reactive oxygen species and granule enzymes (eg, proteases) released by neutrophils can damage surrounding tissues and cause unwanted and exaggerated tissue inflammation. Hence, the response of neutrophils to inflammatory stimuli needs to be well controlled.Neutrophils get recruited to the site of infection by responding to a variety of chemokines, leukotrienes, complement peptides, and some chemicals released by bacteria directly, such as peptides bearing the N-formyl group (formyl-peptides). 1-3 All these responses are mediated by heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins)-coupled receptors (GPCRs). One essential downstream target of GPCRs is PtdIns(3,4,5)P3, an inositol phospholipid which has been implicated in a variety of neutrophil functions such as polarization, chemotaxis, and superoxide generation. [4][5][6] PtdIns(3,4,5)P3 exerts its function by mediating protein translocation via pleckstrin homology (PH) domains on the protein. 7,8 This membrane translocation is crucial for these proteins to fulfill the...

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.