Pyocyanin-enhanced neutrophil extracellular trap formation requires the NADPH oxidase.

Beyond intracellular killing, a novel neutrophil-based antimicrobial mechanism has been recently discovered: entrapment and killing by neutrophil extracellular traps (NETs). NETs consist of extruded nuclear DNA webs decorated with granule proteins. Although NET formation is an important innate immune mechanism, uncontrolled NET release damages host tissues and has been linked to several diseases including cystic fibrosis (CF). The major CF airway pathogen Pseudomonas aeruginosa establishes chronic infection. Pseudomonas imbedded within biofilms is protected against the immune system, but maintains chronic inflammation that worsens disease symptoms. Aberrant NET release from recruited neutrophils was found in CF, but the underlying mechanisms remain unclear. One of the most important Pseudomonas virulence factors is pyocyanin, a redox-active pigment that has been associated with diminished lung function in CF. Here we show that pyocyanin promotes NET formation in a time- and dose-dependent manner. Most CF Pseudomonas clinical isolates tested produce pyocyanin in vitro. Pyocyanin-derived reactive oxygen species are required for its NET release. Inhibitor experiments demonstrated involvement of Jun N-terminal Kinase (JNK) and phosphatidylinositol 3-Kinase (PI3K) in pyocyanin-induced NET formation. Pyocyanin-induced NETs also require the NADPH oxidase because NET release in chronic granulomatous disease neutrophils was greatly reduced. Comparison of neutrophils from gp91phox- and p47phox-deficient patients revealed that pyocyanin-triggered NET formation is proportional to their residual superoxide production. Our studies identify pyocyanin as the first secreted bacterial toxin that enhances NET formation. The involvement of NADPH oxidase in pyocyanin-induced NET formation represents a novel mechanism of pyocyanin toxicity.

NADPH oxidase-2 derived ROS dictates murine DC cytokine-mediated cell fate decisions during CD4 T helper-cell commitment.

NADPH oxidase-2 (Nox2)/gp91(phox) and p47(phox) deficient mice are prone to hyper-inflammatory responses suggesting a paradoxical role for Nox2-derived reactive oxygen species (ROS) as anti-inflammatory mediators. The molecular basis for this mode of control remains unclear. Here we demonstrate that IFNγ/LPS matured p47(phox-/-)-ROS deficient mouse dendritic cells (DC) secrete more IL-12p70 than similarly treated wild type DC, and in an in vitro co-culture model IFNγ/LPS matured p47(phox-/-) DC bias more ovalbumin-specific CD4(+) T lymphocytes toward a Th1 phenotype than wild type (WT) DC through a ROS-dependent mechanism linking IL-12p70 expression to regulation of p38-MAPK activation. The Nox2-dependent ROS production in DC negatively regulates proinflammatory IL-12 expression in DC by constraining p38-MAPK activity. Increasing endogenous H(2)O(2) attenuates p38-MAPK activity in IFNγ/LPS stimulated WT and p47(phox-/-) DC, which suggests that endogenous Nox 2-derived ROS functions as a secondary messenger in the activated p38-MAPK signaling pathway during IL-12 expression. These findings indicate that ROS, generated endogenously by innate and adaptive immune cells, can function as important secondary messengers that can regulate cytokine production and immune cell cross-talk to control during the inflammatory response.

Hepatitis C virus (HCV) proteins induce NADPH oxidase 4 expression in a transforming growth factor beta-dependent manner: a new contributor to HCV-induced oxidative stress.

Viral hepatitis-induced oxidative stress accompanied by increased levels of transforming growth factor beta (TGF-beta) and hepatic fibrosis are hallmarks of hepatitis C virus (HCV) infection. The mechanisms of redox regulation in the pathogenesis of HCV-induced liver disease are not clearly understood. The results of our current studies suggest that reactive oxygen species (ROS) derived from Nox4, a member of the NADPH oxidase (Nox) family, could play a role in HCV-induced liver disease. We found that the expression of HCV (genotype 1a) cDNA constructs (full-length and subgenomic), core protein alone, viral RNA, or replicating HCV (JFH-AM2) induced Nox4 mRNA expression and ROS generation in human hepatocyte cell lines (Huh-7, Huh-7.5, HepG2, and CHL). Conversely, hepatocytes expressing Nox4 short hairpin RNA (shRNA) or an inactive dominant negative form of Nox4 showed decreased ROS production when cells were transfected with HCV. The promoters of both human and murine Nox4 were used to demonstrate transcriptional regulation of Nox4 mRNA by HCV, and a luciferase reporter tied to an approximately 2-kb promoter region of Nox4 identified HCV-responsive regulatory regions modulating the expression of Nox4. Furthermore, the human Nox4 promoter was responsive to TGF-beta1, and the HCV core-dependent induction of Nox4 was blocked by antibody against TGF-beta or the expression of dominant negative TGF-beta receptor type II. These findings identified HCV as a regulator of Nox4 gene expression and subsequent ROS production through an autocrine TGF-beta-dependent mechanism. Collectively, these data provide evidence that HCV-induced Nox4 contributes to ROS production and may be related to HCV-induced liver disease.

Human cervicovaginal lavage fluid contains an inhibitor of HIV binding to dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin.

A small percentage of women at high risk for human immunodeficiency virus (HIV) exposure remain uninfected for long periods, protected by unknown mechanisms. We hypothesized that one mechanism could be inhibition of interactions between HIV and dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) in the genital tract. In an analysis of 95 cervicovaginal lavage samples, we found that 12 (12.6%) strongly inhibited the binding of laboratory-adapted and primary HIV-1 isolates to B-THP-1/DC-SIGN cells in a dose-dependent manner, independently of the donor's risk of exposure. Three of 5 primary isolates were also blocked from binding to primary DCs. The inhibitor has a high molecular weight, is heat stable, and is resistant to trypsin. It is sensitive to pronase and periodate, indicating that it is likely a glycoprotein. Mannosidase digestion and concanavalin A adsorption indicate that the terminal residues of the carbohydrate are not mannose. Mechanistic experiments indicate that the inhibitor acts via binding to DC-SIGN. Further study of such inhibitors may help to elucidate the role played by DC-SIGN in HIV transmission.