NADPH oxidase-regulator of host defense and inflammation (CGD) PowerPoint Presentation

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NADPH oxidase: regulator of host defense and inflammation Brahm Segal, MD Roswell Park Cancer Institute

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Innate Immunity against Aspergillus Segal, BH, N Engl J Med. 2009 Apr 30;360(18):1870-84

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HEME HOCI H2O2 SOD NADP+ + H+ NADPH MPO O2 O2– p22phox gp91phox e– FAD e– rac GTP HEME HEME p22phox gp91phox FAD HEME rac p7phox RhoGDI GDP p67phox p47phox p40phox p67phox p47phox p40phox NADPH oxidase Activation Cytoplasm Activation of primary granular proteases

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Infectious complications in CGD patients

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Inflammatory complications of CGD

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Invasive aspergillosis in a mouse model of chronic granulomatous disease

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HEME HOCI H2O2 SOD NADP+ + H+ NADPH MPO O2 O2– p22phox gp91phox e– FAD e– rac GTP HEME HEME p22phox gp91phox FAD HEME rac p7phox RhoGDI GDP p67phox p47phox p40phox p67phox p47phox p40phox NADPH oxidase Activation Cytoplasm Activation of primary granular proteases

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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

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Hypothesis Double KO Neutrophil elastase (NE) and cathepsin G (CG) mice will be as susceptible to aspergillosis as NADPH oxidase-deficient mice

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Survival after Aspergillus challenge (1.25 x 104 CFU/mouse)

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Survival after Aspergillus challenge (1.25 x 107 CFU/mouse)

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WT and NE-/- x CG-/- have similar lung fungal burden after Aspergillus challenge (day 3, 1.25 x 107 CFU/mouse)

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Invasive aspergillosis in a mouse model of chronic granulomatous disease 1.25 x 104 CFU/mouse

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Lung histology in WT mouse WT mouse, day 3, 1.25 x 107 CFU/mouse

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Lung histology in NE-/- x CG-/-mouse NE-/- x CG-/- mouse, day 3, 1.25 x 107 CFU/mouse

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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)

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Wildtype X-CGD

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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

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Tryptophan metabolism

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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

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BALF IL-17 concentration after i.t. zymosan *

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Lung lymphocytes from CGD mice administered zymosan are enriched in IL-17+ cells

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Lung lymphocytes from CGD mice administered zymosan have diminished Tregs versus wildtypes

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Unstimulated PMA-stimulated Neutrophil NADPH oxidase activity after bone marrow transplantation

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NADPH oxidase in hematopoietic cells govern the inflammatory phenotype

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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

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Fungal b-glucans Dectin-1 NADPH oxidase activation Activation of neutrophil proteases Tregs IDO activation Innate Adaptive Tn-17 Th-2

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NADPH oxidase as a regulator of transcription factors

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Lung NF-kB activation after i.t. zymosan * * * *, p<0.01

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NF-kB activation in immortalized BM-derived Macrophages after zymosan stimulation

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CGD mice develop increased inflammation after i.t. LPS Wildtype CGD

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Lung NF-kB activation after i.t. LPS

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NF-kB activation in immortalized BM-derived Macrophages after LPS stimulation

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IKK complex NF-kB NF-kB binding motif HEME FAD ROIs Pro-inflammatory cytokines and chemokines ?

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Nrf-2 Transcriptional factor: binds to antioxidant response element (ARE) Nrf2-regulated genes encode almost all of the relevant antioxidant proteins HO-1, g-glutamyl cysteine synthase, and several members of the GST family Under basal conditions, Nrf2 undergoes rapid ubiquitination by CUL3 (a ubiquitin ligase), with subsequent proteasome-dependent degradation Keap1 is an oxidative stress sensor that functions as an adaptor for CUL3 Oxidation or adduction of critical Keap1 cysteine residues induces a conformational change in Keap1 that inhibits its ability to bind to CUL3, thereby abrogating Nrf2 ubiquitination

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Phenotype of Nrf2-/- mice Increased inflammation in multiple experimental models (e.g., colitis, diesel particles, LPS-induced shock, smoking-induced lung disease) Increased sensitivity to multiple experimental tumor models (e.g., chemically-induced gastric and bladder cancer)

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NADPH oxidase and Nrf2 Is NADPH oxidase required for Nrf2 activation during lung inflammation? Can excessive inflammation in CGD mice be restrained by pharmacological Nrf2 activation?

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NQO1 b-actin A B C D Nrf-2 activation is impaired in CGD macrophages

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C D E F * * * * B) Zymosan+ CDDO-Im A) Zymosan+ vehicle

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Effect of CDDO-Im started 2 days after i.t. zymosan

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Nrf2-/- develop self-limited increased zymosan-induced lung inflammation Day 3 D

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Normals (n=8), CGD patients (n=5), *=p<0.05 compared to WT PBMCs PBMCs from CGD patients have increased zymosan-induced NF-kB activation vs. WT donor PBMCs

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Normals (n=8), CGD patients (n=5), * p<0.05 comparing WT with CGD PBMCs PBMCs from CGD patients have defective Nrf2 activation

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Summary NADPH oxidase downregulates LPS- and zymosan-stimulated inflammation and NF-kB activation. CDDO-Im, an Nrf2 activator, significantly reduced zymosan-induced inflammation and BALF TNF-a and IL-17 levels in CGD mice CGD and Nrf2-/- mice have distinct inflammatory phenotypes Nrf2 is likely to be an important, but not the sole, mechanisms by which NADPH oxidase restrains inflammation Consistent with mouse data, studies of PBMCs from X-linked CGD patients demonstrate NADPH oxidase as a Nrf2 activator, while restraining NF-kB activation.

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Nrf2 Keap1 Antioxidant and anti-inflammatory proteins IKK complex NF-kB NF-kB binding ARE motif sequence HEME FAD Cys ? ROIs Pro-inflammatory cytokines and chemokines Tn-17 Treg

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Luigina Romani Paolo Puccetti Università degli Studi di Perugia Steve Holland Don Vinh Thomas Walsh Brahm Segal Robert Swamidoss Melissa Grimm Carly Dennis Jen Bushey Joy Feminella Tim Blackwell Mike Freeman Wei Han Funding: CGD Research Trust, NIH