D and E: Nuclei stained with DAPI showing alveoli (white arrows) or occlusion in the bronchiole (defined by the white collection). and survive even in the presence of NETs by generating endonucleases (which digest NETs) or catalase (which metabolizes hydrogen peroxide).36,38 NETs formation in viral infections is virtually unknown, except for a recent study that exhibited modulation of NETs formation in feline leukemia virus infections.39 Here, we exploited macrophage- or neutrophil-depleted animals to unravel the association of these cells with ALI in influenza pneumonia. Our results show that macrophage-depleted animals suffered complications of ARDS when challenged with sublethal influenza computer SR-3029 virus contamination, in contrast to neutrophil-depleted animals that developed only mild lung injury. For the first time, we exhibited the induction of NETs during influenza computer virus contamination both and studies revealed the participation of NETs in endothelial damage, and that the regulation of NETs formation was dependent on activation of redox enzymes. SR-3029 Materials and Methods Virus, Animals, and Ethics Approval Influenza A computer virus A/Puerto Rico/8/34 H1N1 (PR8) obtained from the American Type Culture Collection (Manassas, VA) was propagated in embryonated eggs at 37C for 72 hours, and the allantoic fluid was harvested. Computer virus titers were determined by the plaque assay via contamination of Madin-Darby canine kidney (MDCK) cells. Female 4- to 6-week-old BALB/c mice were housed in microisolator cages in an animal BSL-2 laboratory facility. All animal experiments were approved by the Institutional Animal Care and Use Committee, National University SR-3029 of Singapore (protocol number 035/07), and were performed in strict accordance with the recommendations. Macrophage and Neutrophil Depletion and Infection Macrophage depletion in lungs was achieved using the liposome-mediated macrophage suicide technique. Liposome-encapsulated dichloromethylene diphosphonate (clodronate-liposome, L-Cl2MDP) is taken up by macrophages by phagocytosis and induces apoptosis.40 Clodronate was a gift from Roche Diagnostics (Mannheim, Germany), and was encapsulated in liposomes as described earlier.41 L-Cl2MDP was administered intranasally (50 L per dose per mouse) at days ?4 and ?1 before virus challenge. For depletion of neutrophils, purified rat anti-Ly6G monoclonal antibody 1A842 was administered intraperitoneally (at a dose of 200 g per mouse) 1 day before infection and every 48 hours thereafter. Animals were divided into five groups, ie, uninfected allantoic fluidCinoculated control (Con), clodronate-liposomeCtreated (CL), infected (INF), clodronate-liposome-treated and infected (CL-I), and 1A8-treated and infected (1A8-I). Two separate sets of mice (each consisting of five animals) were tested, one set for bronchoalveolar lavage fluid (BALF) SR-3029 and virus titer analyses, and the other for histopathology. The group of animals treated with 1A8 alone did not show any pathological changes in their lungs, and were thus excluded from further study. Within the infected groups, each animal was challenged with a sublethal dose of 100 plaque-forming units (PFU) of PR8 virus through intranasal inoculation. Animals were monitored daily for clinical signs of infection and body weight loss. In separate experiments involving lethal virus challenge, each animal was infected with 500 PFU of virus. Lung Histopathology and BALF Analysis The lungs of one group of animals were harvested for BALF and lung homogenate, whereas lungs for histopathology were obtained from a separate group of mice. After BALF collection, the corresponding lungs were not used for any further experiments. For histopathological analyses, lungs were fixed in neutral-buffered formalin and embedded in paraffin. The severity of ALI was evaluated by a semiquantitative histology score in a blinded manner as described previously.43 Animals were anesthetized with intraperitoneal injections of ketamine (75 mg/kg) and medetomidine (1 mg/kg), and bronchoalveolar lavage was performed immediately after SR-3029 sacrifice as described previously.43 The left lung was tied with nylon thread, and the right lung was lavaged twice with a total volume of 800 L of sterile phosphate-buffered saline (PBS) inserted through an endotracheal tube. The recovery of BALF was more than 90% for all animals. For differential cell counts, the cells were processed onto microscopic slides using a CytoFuge 2 cytocentrifuge (StatSpin, Westwood, MA), and subjected to modified Giemsa staining. Cells Rabbit polyclonal to ZMYND19 (500 per animal) were counted at a magnification of 400. The supernatants were stored at ?70C for measurement of protein content and enzyme activities. Virus titers were determined, and BALF protein content was measured by the DC Protein Assay Kit (Bio-Rad, Hercules, CA). To evaluate secondary bacterial infection, 40 L of each BALF sample was plated onto.