Myeloperoxidase is required for neutrophil extracellular trap formation: implications for innate immunity

Abstract

IntroductionMyeloperoxidase (MPO) is one of the most abundant proteins in neutrophils, accounting for 5% of the dry weight of the cell. 1 Stored in the azurophilic granules and released when neutrophils are stimulated, MPO catalyzes the oxidation of chloride and other halide ions in the presence of hydrogen peroxide 2,3 to generate hypochlorous acid and other highly reactive products that mediate efficient antimicrobial action. 4,5 Several inherited mutations and deletions in the gene encoding MPO result in decreased enzyme production and activity. 6,7 Using automated hematological devices, clinicians can distinguish between partial and complete MPO deficiencies. 8 MPO deficiency is reported to have an incidence of 1 in 2000-4000 in the United States and Europe and 1 in 55 000 in Japan. 9-13 Candida infections are common in MPO-deficient patients, especially in those that also develop diabetes. 9,14-18 Occasionally, serious infectious or inflammatory complications have been observed in completely MPOdeficient patients as well. 8 Consistently, MPO knockout mice are susceptible to particular bacterial and fungal infections. 19 Neutrophil extracellular traps (NETs) are part of the neutrophil response to microbes. Activated neutrophils die and release these structures composed of decondensed chromatin and antimicrobial proteins 20,21 that trap and inhibit a broad range of microbes. 22 Little is known about the molecular mechanism that regulates NET formation, making the antimicrobial role of NETs in vivo difficult to assess.Interestingly, neutrophils from chronic granulomatous disease (CGD) patients fail to make NETs. 20 CGD is caused by mutations that disrupt the ability of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to generate superoxide, which dismutates to hydrogen peroxide, the substrate of MPO. CGD patients are prone to recurrent and severe infections, as well as to persistent inflammation that can occur independently of infection. [23][24][25] NET formation by CGD neutrophils is restored by the addition of exogenous hydrogen peroxide, indicating that reactive oxygen species are required for NET formation. 20 Here we show that MPO is necessary for making NETs and suggest that defective NET formation may undermine host defense in patients lacking MPO. Methods Donor consentAll donors gave consent to blood drawing in accordance with the Declaration of Helsinki and to functional and genetic analysis. Samples were collected with approval from the ethical committees at each institution. Neutrophil isolationNeutrophils were isolated by centrifuging heparinized venous blood over Histopaque 1119 (Sigma-Aldrich) and subsequently over a discontinuous Percoll (Amersham Biosciences) gradient as described previously. 20 Cells were stored in Hank buffered salt solution (-) or Dulbecco phosphatebuffered saline (-), without calcium or magnesium, before experiments. NET formation and visualizationNeutrophils (5 ϫ 10 4 ) were seeded per well in 24-well tissue culture plates, in Hanks buffered salt solution (...

α-Enolase of Streptococcus pneumoniae Induces Formation of Neutrophil Extracellular Traps

“…NET formation is dependent on hydrogen peroxide generated by NADPH oxidase and further metabolized by myeloperoxidase. Thus, chronic granulomatous disease neutrophils and myeloperoxidase-deficient neutrophils do not form NETs (11,29).…”

Priming of neutrophils toward NETosis promotes tumor growth

“…Interestingly, it has been shown that neutrophils from chronic granulomatous disease patients, which harbor NADPH oxidase mutations, and myeloperoxidase (MPO)-deficient neutrophils fail to suppress lymphocyte function 33 and also do not make NETs. [34][35][36] Thus, NETs likely play a role in several aspects of tumor biology.…”

Defective Stromal Remodeling and Neutrophil Extracellular Traps in Lymphoid Tissues Favor the Transition from Autoimmunity to Lymphoma

“…29 ). In addition, NETosis requires myeloperoxidase (MPO) expression ( 30 ), which was increased in the spleens of lpr/lpr/ Sparc −/− mice with NET-like extracellular structures ( Fig. 3C ).…”