NADPH oxidase: regulator of host defense and inflammation Brahm Segal, MD Roswell Park Cancer Institute
Innate Immunity against Aspergillus Segal, BH, N Engl J Med. 2009 Apr 30;360(18):1870-84
NADPH oxidase Activation Cytoplasm MPO HOCI H2O2 Activation of primary granular proteases SOD O2 O2– Activation e– gp91phox gp91phox p22phox p22phox HEME HEME HEME HEME rac Cytoplasm p47phox GTP p67phox p40phox FAD FAD e– p47phox p7phox p67phox NADPH NADP+ + H+ rac GDP RhoGDI p40phox
Infectious complications in CGD patients
Inflammatory complications of CGD
Invasive aspergillosis in a mouse model of chronic granulomatous disease
NADPH oxidase Activation Cytoplasm MPO HOCI H2O2 Activation of primary granular proteases SOD O2 O2– Activation e– gp91phox gp91phox p22phox p22phox HEME HEME HEME HEME rac Cytoplasm p47phox GTP p67phox p40phox FAD FAD e– p47phox p7phox p67phox NADPH NADP+ + H+ rac GDP RhoGDI p40phox
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
Hypothesis Double KO Neutrophil elastase (NE) and cathepsin G (CG) mice will be as susceptible to aspergillosis as NADPH oxidase-deficient mice
Survival after Aspergillus challenge (1.25 x 104 CFU/mouse)
Survival after Aspergillus challenge (1.25 x 107 CFU/mouse)
WT and NE-/- x CG-/- have similar lung fungal burden after Aspergillus challenge (day 3, 1.25 x 107 CFU/mouse)
Invasive aspergillosis in a mouse model of chronic granulomatous disease 1.25 x 104 CFU/mouse
Lung histology in WT mouse WT mouse, day 3, 1.25 x 107 CFU/mouse
Lung histology in NE-/- x CG-/-mouse NE-/- x CG-/- mouse, day 3, 1.25 x 107 CFU/mouse
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)
Wildtype X-CGD 17
IL-23 promotes the development of an IL-17-producing CD4+ helper T cell subset IL-23 promotes the development of an IL-17–producing CD4+ helper T cell subset. IL-23 induces the differentiation of naive CD4+ T cells into IL-17–producing helper T cells (Th17/ThIL-17) via mechanisms that are distinct from the Th1 and Th2 differentiation pathways. The transcriptional factors critical for the development of Th1 (STAT1, STAT4, and T-bet) and Th2 (STAT6) cells are not required for the induction of Th17/ThIL-17 cells. The transcriptional factor(s) essential for the development of Th17/ThIL-17 cells remain unknown. IFN- and IL-4 antagonize each other in the differentiation of Th1 and Th2 cells and the promotion of their function. IFN- also suppresses the differentiation of Th17/ThIL-17 cells by reducing IL-23R expression on CD4+ T cells. IL-4 also inhibits the development of Th17/ThIL-17 cells. It is not known, however, whether Th17/ThIL-17 cells inhibit the development of Th1 and Th2 cells. Tregs, an immune-modulating subset of CD4+ T cells, suppress the differentiation and effector function of Th1 and Th2 cells. Recent studies suggest that Treg-derived TGF-ß induces the differentiation of Th17/ThIL-17 cells from naive CD4+ T cells in the presence of IL-6 in vitro (26). However, the precise effect(s) of Tregs on Th17/ThIL-17 cells are as yet unknown. Iwakura, Y. et al. J. Clin. Invest. 2006;116:1218-1222 Copyright ©2006 American Society for Clinical Investigation 18
Tryptophan metabolism
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 21
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BALF IL-17 concentration after i.t. zymosan *
Lung lymphocytes from CGD mice administered zymosan are enriched in IL-17+ cells
Lung lymphocytes from CGD mice administered zymosan have diminished Tregs versus wildtypes
Neutrophil NADPH oxidase activity after bone marrow transplantation Unstimulated PMA-stimulated
NADPH oxidase in hematopoietic cells govern the inflammatory phenotype
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
Innate Adaptive Fungal b-glucans Tn-17 Dectin-1 Tregs NADPH oxidase IDO activation Tregs NADPH oxidase activation Th-2 Activation of neutrophil proteases
NADPH oxidase as a regulator of transcription factors
Lung NF-kB activation after i.t. zymosan * * * *, p<0.01
NF-kB activation in immortalized BM-derived Macrophages after zymosan stimulation 34
CGD mice develop increased inflammation after i.t. LPS Wildtype CGD
Lung NF-kB activation after i.t. LPS
NF-kB activation in immortalized BM-derived Macrophages after LPS stimulation
? ROIs IKK complex NF-kB Pro-inflammatory cytokines and chemokines HEME ROIs IKK complex ? FAD Pro-inflammatory cytokines and chemokines NF-kB NF-kB binding motif
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 39
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)
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?
Nrf-2 activation is impaired in CGD macrophages A B C D NQO1 b-actin
A) Zymosan+ vehicle B) Zymosan+ CDDO-Im C D * * * * F E
Effect of CDDO-Im started 2 days after i.t. zymosan 44
Nrf2-/- develop self-limited increased zymosan-induced lung inflammation Day 3
PBMCs from CGD patients have increased zymosan-induced NF-kB activation vs. WT donor PBMCs Normals (n=8), CGD patients (n=5), *=p<0.05 compared to WT PBMCs
PBMCs from CGD patients have defective Nrf2 activation Normals (n=8), CGD patients (n=5), * p<0.05 comparing WT with CGD PBMCs
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.
? ROIs IKK complex Treg Tn-17 Keap1 NF-kB Nrf2 Pro-inflammatory HEME Tn-17 ROIs IKK complex ? FAD Cys Keap1 Pro-inflammatory cytokines and chemokines Antioxidant and anti-inflammatory proteins NF-kB Nrf2 NF-kB binding ARE motif sequence
Funding: CGD Research Trust, NIH Università degli Studi di Perugia Luigina Romani Paolo Puccetti Brahm Segal Robert Swamidoss Melissa Grimm Carly Dennis Jen Bushey Joy Feminella Steve Holland Don Vinh Thomas Walsh Tim Blackwell Mike Freeman Wei Han Funding: CGD Research Trust, NIH