NADPH Oxidase Limits Innate Immune Responses in the Lungs in Mice

Background: Chronic granulomatous disease (CGD), an inherited disorder of the NADPH oxidase in which phagocytes are defective in generating superoxide anion and downstream reactive oxidant intermediates (ROIs), is characterized by recurrent bacterial and fungal infections and by excessive inflammation (e.g., inflammatory bowel disease). The mechanisms by which NADPH oxidase regulates inflammation are not well understood. Methodology/Principal Findings: We found that NADPH oxidase restrains inflammation by modulating redox-sensitive innate immune pathways. When challenged with either intratracheal zymosan or LPS, NADPH oxidase-deficient p47phox-/- mice and gp91phox-deficient mice developed exaggerated and progressive lung inflammation, augmented NF-kB activation, and elevated downstream pro-inflammatory cytokines (TNF-α, IL-17, and G-CSF) compared to wildtype mice. Replacement of functional NADPH oxidase in bone marrow-derived cells restored the normal lung inflammatory response. Studies in vivo and in isolated macrophages demonstrated that in the absence of functional NADPH oxidase, zymosan failed to activate Nrf2, a key redox-sensitive anti-inflammatory regulator. The triterpenoid, CDDO-Im, activated Nrf2 independently of NADPH oxidase and reduced zymosan-induced lung inflammation in CGD mice. Consistent with these findings, zymosan-treated peripheral blood mononuclear cells from X-linked CGD patients showed impaired Nrf2 activity and increased NF-kB activation. Conclusions/Significance: These studies support a model in which NADPH oxidase-dependent, redox-mediated signaling is critical for termination of lung inflammation and suggest new potential therapeutic targets for CGD

NADPH oxidase limits innate immune responses in the lungs in mice.

BackgroundChronic granulomatous disease (CGD), an inherited disorder of the NADPH oxidase in which phagocytes are defective in generating superoxide anion and downstream reactive oxidant intermediates (ROIs), is characterized by recurrent bacterial and fungal infections and by excessive inflammation (e.g., inflammatory bowel disease). The mechanisms by which NADPH oxidase regulates inflammation are not well understood.Methodology/principal findingsWe found that NADPH oxidase restrains inflammation by modulating redox-sensitive innate immune pathways. When challenged with either intratracheal zymosan or LPS, NADPH oxidase-deficient p47(phox-/-) mice and gp91(phox)-deficient mice developed exaggerated and progressive lung inflammation, augmented NF-kappaB activation, and elevated downstream pro-inflammatory cytokines (TNF-alpha, IL-17, and G-CSF) compared to wildtype mice. Replacement of functional NADPH oxidase in bone marrow-derived cells restored the normal lung inflammatory response. Studies in vivo and in isolated macrophages demonstrated that in the absence of functional NADPH oxidase, zymosan failed to activate Nrf2, a key redox-sensitive anti-inflammatory regulator. The triterpenoid, CDDO-Im, activated Nrf2 independently of NADPH oxidase and reduced zymosan-induced lung inflammation in CGD mice. Consistent with these findings, zymosan-treated peripheral blood mononuclear cells from X-linked CGD patients showed impaired Nrf2 activity and increased NF-kappaB activation.Conclusions/significanceThese studies support a model in which NADPH oxidase-dependent, redox-mediated signaling is critical for termination of lung inflammation and suggest new potential therapeutic targets for CGD

p47<i>^phox−/−</i> mice develop increased zymosan-induced lung inflammation compared to wildtype (WT) mice.

<p>Representative lung histology of WT (A) and p47<i>^phox−/−</i> (CGD) mice (B) at days 3, 7, 14, 25 after i.t. zymosan. All slides are H&E stained, 20x, and are representative of at least 3 mice per genotype per time point. C) Percent of lung parenchyma with consolidation or granulomatous inflammation. Bronchoalveolar lavage fluid (BALF) neutrophil (D) and macrophage (E) concentrations. 2-way ANOVA showed a significant difference between genotypes in % lung inflammation (p<0.0001) and BALF neutrophil concentration (p<0.0001), with significant differences at the indicated time points by Boneferroni post-test. *, p<0.01; **, p<0.001).</p

Intratracheal zymosan caused increased lung inflammation and NF-κB activation, in gp91<i>^phox-</i>deficient (X-linked CGD) versus wildtype mice.

<p>A) Representative lung section of gp91<i>^phox-</i>deficient mouse 6 days after i.t. zymosan showing extensive inflammation (H&E, 100x). Similarly treated wildtype (WT) mice had no lung inflammation (not shown). B) On day 6 after i.t. zymosan, both NADPH oxidase deficient genotypes (p47<i>^phox−/−</i> and gp91<i>^phox−/</i>) had similar BALF neutrophilic leukocytosis, whereas monocytes predominated in WT BALF. C) gp91<i>^phox-</i>deficient/HLL mice had increased whole lung NF-κB activation compared to WT/HLL mice.</p

Intratracheal zymosan treatment results in higher levels of pro-inflammatory cytokines and NF-κB activation in lungs of p47<i>^phox−/−</i> mice.

<p>A) BALF levels of TNF-α, IL-17, and G-CSF in wild type (WT) and p47<i>^phox−/−</i> (CGD) mice administered i.t. zymosan. Note that the Y-axes in the TNF-α and IL-17 graphs are in log-scale. The interaction of genotype (p47<i>^phox−/−</i> vs. WT) and time was assessed by 2-way ANOVA and was significant for each of the 3 cytokines (p<0.001). Bonferroni post-test was used to test for significance at each time point (*, p<0.05). B) Whole lung NF-κB activation measured by bioluminescence imaging over the chest after i.v. luciferin in NF-κB reporter mice (p47<i>^phox−/−</i>/HLL and WT/HLL). C) NF-κB dependent luciferase activity in bone marrow-derived macrophages (BMDMs) from p47<i>^phox−/−</i>/HLL and WT/HLL mice after <i>in vitro</i> stimulation with zymosan (20 µg/ml). For (B) and (C), 2-way ANOVA indicated p<0.0001 between genotypes with significant differences at the indicated time points by Bonferroni post-test. *, p<0.05; **, p<0.01; ***, p<0.001).</p

Zymosan-induced Nrf2 nuclear translocation is NADPH oxidase-dependent in isolated macrophages and <i>in vivo</i>.

<p>Wild type (WT) and p47<i>^phox−/−</i> (CGD) bone marrow-derived macrophages (BMDMs) cultured in DMEM media with 10% serum were stimulated with zymosan (20 µ/ml). A) Western blot for Nrf2 and Tata box binding protein (TBP) in nuclear fractions of BMDMs from p47<i>^phox−/−</i> and WT mice at baseline, 1, 4, and 24 hours after zymosan. B) Densitometry from 3 separate experiments. p<.05 by ANOVA using Tukey post-test. C) Western blot showing expression of NQO1 in cytoplasmic extracts. D) Western blot for nuclear Nrf2 from gp91<i>^phox−/</i> and p47<i>^phox−/−</i> macrophages treated with vehicle, zymosan (20 µg/ml), or the Nrf2 agonist electrophiles, CDDO-Im (1.0 µM) or sulforaphane (50 µM), for 4 hours. E) Nrf2 DNA binding activity of lung nuclear extracts in unstimulated (baseline) mice and 6 days after i.t. zymosan. n = 3−6 mice per group. Lung Nrf2 binding activity in zymosan-stimulated WT mice was significantly greater than that in unstimulated WT mice (student's t-test, p<0.05), whereas zymosan treatment had no effect on lung Nrf2 activation in CGD mice.</p

Bone marrow chimera experiments demonstrate that NADPH oxidase in hematopoietic cells, but not lung stromal cells, is required to restrain zymosan-induced lung inflammation.

<p>Wild type (WT) and p47<i>^phox−/−</i> (CGD) mice were administered myeloablative total body irradiation, and rescued with marrow-derived WT or p47<i>^phox−/−</i> donor cells. The transplants were as follows: WT donor/CGD recipient (n = 3); CGD donor/WT recipient (n = 3); WT donor/WT recipient (n = 2); CGD donor/CGD recipient (n = 2); the latter two transplants were performed as specificity controls for artifacts introduced by transplantation. Recipient mice were administered i.t. zymosan at 31 days after transplant. Seven days later, peripheral blood and lungs were harvested. A) NADPH oxidase activity in peripheral neutrophils of transplanted mice was evaluated using the fluorescent probe, dihydrorhodamine 123 (DHR). In a representative experiment, neutrophils were unstimulated (top row) or stimulated with PMA (100 ng/ml) (bottom row) to activate NADPH oxidase. Hydrogen peroxide, a metabolite of NADPH oxidase activation, activates DHR fluorescence. In all transplants, the donor genotype determined NADPH oxidase competence. B) Representative lung histology (H&E, 40x) and C) percent of lung parenchyma with consolidation or granulomatous inflammation in transplanted mice at day 7 after i.t. zymosan administration. Percent lung inflammation was significantly greater in transplanted mice with CGD compared to wildtype donors (abbreviated, “D” in the figure) (unpaired t-test, p<0.0001), whereas recipient (abbreviated, “R” in the figure) genotype had no effect on the inflammatory response.</p

NADPH oxidase augments Nrf2 activation and restrains NF-κB activation in human PBMCs.

<p>PBMCs from normal donors (n = 8) and X-linked CGD patients (n = 5) were stimulated with zymosan (20 µg/ml). A) Nrf2 activation. B) NF-κB activation. *, p<0.05 comparing normal donor and CGD PBMCs.</p

The triterpenoid, CDDO-Im, a Nrf2 inducer, reduces zymosan-induced lung inflammation and pro-inflammatory BALF cytokines in p47<i>^phox−/−</i> mice.

<p>CDDO-Im (0.2 mg/mouse by i.p. injection) or vehicle (control) was administered daily to p47<i>^phox−/−</i> mice from day −1 to +2 in relation to i.t. zymosan, and BALF and lungs were harvested on day +3. Representative H&E stained lung sections of p47<i>^phox−/−</i> mice administered zymosan plus vehicle (A) or zymosan plus CDDO-Im (B). Neutrophil (C) and cytokine (D) concentrations were assessed in BALF obtained at day 3 after zymosan treatment. Significant differences were observed for neutrophils (p = 0.03), IL-23 (p = 0.008), IL-17 (p = 0.02), TNF-α (p = 0.02), and LIX (p = 0.03) (Mann-Whitney two-tailed test). E) Lung NF-κB activation, measured by bioluminescence, was similar in p47<i>^phox−/−</i> /HLL mice administered zymosan plus CDDO-Im versus zymosan plus vehicle (Two-way ANOVA, p = NS). F) Representative Western blot of lung homogenates for NQO1 and (G) densitometry (normalized to β-actin) (G) for 3 mice per genotype per treatment (p<.05 by ANOVA using Tukey post-test). Untreated  =  no experimental manipulation; zymosan  =  i.t. zymosan plus i.p. vehicle; zymosan + CDDO-Im  =  i.t. zymosan plus i.p. CDDO-Im. H) Measurement of Nrf2 activity by TransAM™ ELISA from whole lung nuclear protein extracts from p47<i>^phox−/−</i> mice treated with zymosan plus vehicle or zymosan plus CDDO-Im. Results are presented as increase over background O.D. measurement in lung nuclear protein samples from Nrf2^−/− mice (p<.05 using unpaired t-test).</p

CGD mice develop increased lipopolysaccharide-induced lung inflammation and NF-κB activation compared to wildtype (WT) mice.

<p>Wildtype (WT)/HLL and p47<i>^phox−/−</i> /HLL (CGD) mice were administered intratracheal (i.t.) LPS (3 µg/g per mouse). Representative lung histology of a WT/HLL (A) and p47<i>^phox−/−</i>/HLL (B) mouse 6 days after i.t. LPS (H&E, 100x). Minimal to no lung inflammation was present in WT/HLL mice, whereas mixed neutrophilic and lymphohistiocytic infiltrates involving ∼10% of the lung and interstitial edema was present in p47<i>^phox−/−</i>/HLL mouse lungs. Whole lung NF-κB activation (C) was augmented in p47<i>^phox−/−</i>/HLL mice administered i.t. LPS compared to similarly treated WT/HLL mice (2-way ANOVA, p<0.0001, with Bonferroni post-test showing significant differences between genotypes at the indicated time points). D) Isolated p47<i>^phox−/−</i>/HLL bone marrow-derived macrophages had augmented NF-κB activation in response to LPS compared to similarly treated WT/HLL macrophages (2-way ANOVA, p<0.0001). *, p<0.05; **, p<0.01; ***, p<0.001.</p