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NADPH oxidase: regulator of host defense and inflammation Brahm Segal, MD Roswell Park Cancer Institute Slide 2 Innate Immunity against Aspergillus Segal, BH, N Engl J Med. 2009 Apr 30;360(18):1870-84 Slide 3 HEME HOCIH2O2H2O2 SOD NADP + + H + NADPH MPO O2O2 O2O2 p22 phox gp91 phox ee FAD ee rac GTP HEME p22 phox gp91 phox FAD HEME rac p7 phox RhoGDI GDP p67 phox p47 phox p40 phox p67 phox p47 phox p40 phox NADPH oxidase Activation Cytoplasm Activation of primary granular proteases Slide 4 Infectious complications in CGD patients Slide 5 Inflammatory complications of CGD Slide 6 Invasive aspergillosis in a mouse model of chronic granulomatous disease Slide 7 HEME HOCIH2O2H2O2 SOD NADP + + H + NADPH MPO O2O2 O2O2 p22 phox gp91 phox ee FAD ee rac GTP HEME p22 phox gp91 phox FAD HEME rac p7 phox RhoGDI GDP p67 phox p47 phox p40 phox p67 phox p47 phox p40 phox NADPH oxidase Activation Cytoplasm Activation of primary granular proteases Slide 8 Reeves et al. Killing activity of neutrophils is mediated through activation of proteases by K+ flux Nature 2002 Tkalcevik et al. Impaired immunity and enhanced resistance to endotoxin in the absence of neutrophil elastase and cathepsin G. Immunity, 2000 Slide 9 Hypothesis Double KO Neutrophil elastase (NE) and cathepsin G (CG) mice will be as susceptible to aspergillosis as NADPH oxidase-deficient mice Slide 10 Survival after Aspergillus challenge (1.25 x 10 4 CFU/mouse) Slide 11 Survival after Aspergillus challenge (1.25 x 10 7 CFU/mouse) Slide 12 WT and NE-/- x CG-/- have similar lung fungal burden after Aspergillus challenge (day 3, 1.25 x 10 7 CFU/mouse) Slide 13 Invasive aspergillosis in a mouse model of chronic granulomatous disease 1.25 x 10 4 CFU/mouse Slide 14 Lung histology in WT mouse WT mouse, day 3, 1.25 x 10 7 CFU/mouse Slide 15 Lung histology in NE-/- x CG-/- mouse NE-/- x CG-/- mouse, day 3, 1.25 x 10 7 CFU/mouse Slide 16 Conclusions NADPH oxidase is critical -- but NE and CG are dispensable -- in host defense against pulmonary aspergillosis Potentially, NADPH oxidase-mediated activation of other antimicrobial proteins compensate for loss of NE and CG Future studies: We will evaluate the requirement for NADPH oxidase vs. NE and CG in a bacterial challenge model (Burkholderia cepacia) Slide 17 Wildtype X-CGD Slide 18 Copyright 2006 American Society for Clinical Investigation Iwakura, Y. et al. J. Clin. Invest. 2006;116:1218-1222 IL-23 promotes the development of an IL-17-producing CD4+ helper T cell subset Slide 19 Tryptophan metabolism Slide 20 Slide 21 NADPH oxidase and inflammation Aim: evaluate the role of NADPH oxidase in regulating inflammatory responses following challenge with sterile intratracheal zymosan Rationale for zymosan Pro-inflammatory fungal cell wall constituent used widely in models of inflammation Avoids confounding effect of impaired host defense against aspergillosis in CGD mice Ligand of Dectin-1, TLR-2, and CR3 Activates NADPH oxidase via Dectin-1-dependent signalling Slide 22 Slide 23 Slide 24 Slide 25 BALF IL-17 concentration after i.t. zymosan * Slide 26 Lung lymphocytes from CGD mice administered zymosan are enriched in IL-17+ cells Slide 27 Lung lymphocytes from CGD mice administered zymosan have diminished Tregs versus wildtypes Slide 28 Unstimulated PMA-stimulated Neutrophil NADPH oxidase activity after bone marrow transplantation Slide 29 NADPH oxidase in hematopoietic cells govern the inflammatory phenotype Slide 30 Dectin-1-/- mice and aspergillosis Dectin-1-/- neutrophils diminished NADPH oxidase activation after exposure to A. fumigatus Diminished antifungal activity in vitro In vivo, Dectin-1-/- mice have impaired host defense following A. fumigatus challenge Impaired neutrophil recruitment to the lungs and production of IL-17 and other pro- inflammatory cytokines and chemokines Werner et al. J Immunol, 2009 Slide 31 Fungal -glucans Dectin-1 NADPH oxidase activation Activation of neutrophil proteases Tregs IDO activation Innate Adaptive Tn-17 Th-2 Slide 32 NADPH oxidase as a regulator of transcription factors Slide 33 Lung NF-kB activation after i.t. zymosan * * * *, p