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

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

3. 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

4. Infectious complications in CGD patients

5. Inflammatory complications of CGD

6. Invasive aspergillosis in a mouse model of chronic granulomatous disease

7. 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

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

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

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

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

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

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

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

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

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)

17. Wildtype X-CGD 17

18. 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:
Copyright ©2006 American Society for Clinical Investigation 18

19. Tryptophan metabolism

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 21

22. 22

23. 23

24. 24

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

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

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

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

29. NADPH oxidase in hematopoietic cells govern the inflammatory phenotype

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

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

32. NADPH oxidase as a regulator of transcription factors

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

34. NF-kB activation in immortalized BM-derived Macrophages
after zymosan stimulation 34

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

36. Lung NF-kB activation after i.t. LPS

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

38. ? 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

39. 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

40. 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)

41. 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?

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

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

44. Effect of CDDO-Im started 2 days after i.t. zymosan44

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

46. 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

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

48. 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.

49. ? 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

50. 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