Role of NADPH oxidase versus neutrophil proteases in antimicrobial host defense

Abstract

NADPH oxidase is a crucial enzyme in mediating antimicrobial host defense and in regulating inflammation. Patients with chronic granulomatous disease, an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates (ROIs), suffer from life-threatening bacterial and fungal infections. The mechanisms by which NADPH oxidase mediate host defense are unclear. In addition to ROI generation, neutrophil NADPH oxidase activation is linked to the release of sequestered proteases that are posited to be critical effectors of host defense. To definitively determine the contribution of NADPH oxidase versus neutrophil serine proteases, we evaluated susceptibility to fungal and bacterial infection in mice with engineered disruptions of these pathways. NADPH oxidase-deficient mice (p47(phox-/-)) were highly susceptible to pulmonary infection with Aspergillus fumigatus. In contrast, double knockout neutrophil elastase (NE)(-/-)×cathepsin G (CG)(-/-) mice and lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI)-deficient mice that are defective in neutrophil serine protease activation demonstrated no impairment in antifungal host defense. In separate studies of systemic Burkholderia cepacia infection, uniform fatality occurred in p47(phox-/-) mice, whereas NE(-/-)×CG(-/-) mice cleared infection. Together, these results show a critical role for NADPH oxidase in antimicrobial host defense against A. fumigatus and B. cepacia, whereas the proteases we evaluated were dispensable. Our results indicate that NADPH oxidase dependent pathways separate from neutrophil serine protease activation are required for host defense against specific pathogens.

Figure 1. Kaplan-Meier survival curves of WT, p47^phox

^−/− , and NE^−/−×CG^−/− mice after administration of A. fumigatus . Mice were administered A) 1.25×10⁴ conidia or B) 1.25×10⁷ conidia by oropharyngeal aspiration. n = 5 mice per genotype per treatment. Log-rank analysis, p<0.0001 comparing WT with p47^{phox −/−} mice and NE^−/−×CG^−/− mice with p47^{phox −/−} mice.

Figure 2. Lung histology and airway inflammation in WT and NE^−/−×CG^−/− mice after A. fumigatus administration.

Mice were administered A. fumigatus (1.25×10⁷ conidia per mouse) by oropharyngeal aspiration and sacrificed on day 3. A) BALF leukocyte recovery and B) percent lung inflammation were similar in WT and NE^−/−×CG^−/− mice. Representative lung histology from WT (C and D) and NE^−/−×CG^−/− mice (E and F). Predominantly peribronchovascular neutrophilic and lymphohistiocytic inflammation occurred in both genotypes (C and E; H&E, 40×). GMS staining (400×) of lung sections from WT (D) and NE^−/−×CG^−/− (F) mice showed what appeared to be degenerated hyphal fragments, but no evidence of intact invasive hyphae. Results are representative of 15 WT and 10 NE^−/−×CG^−/− mice. By comparison, p47^phox−/− mice administered A. fumigatus at 0.1% of this inoculum (1.25×10⁴ conidia per mouse) and sacrificed on day 3 had evidence of fungal pneumonia characterized by G) multiple foci of neutrophilic consolidation (H&E, 40×), and H) hyphal parenchymal invasion (arrow) (GMS, 400×).

Figure 3. Fungal burden in WT and NE^−/−×CG^−/− mice after A. fumigatus administration.

Mice were administered A. fumigatus (1.25×10⁷ conidia per mouse) by oropharyngeal aspiration and sacrificed on day 3. A) Quantitative fungal cultures in lung homogenates, B) serum galactomannan, C) BALF galactomannan. n = 10 mice per genotype subjected to infection (A. fum), and n = 1 mouse per genotype subjected to sham-infection. No significant differences occurred in quantitative lung fungal cultures, serum galactomannan, and BALF galactomannan between Aspergillus-infected WT mice and NE^−/−×CG^−/− mice.

Figure 4. Lung histology in WT and DPPI^−/− mice on day 3 after oropharyngeal A. fumigatus (1.25×10⁷ conidia per mouse) administration.

In both WT (A) and DPPI^−/− (B) mice, mild predominantly peribronchovascular inflammation occurred (H&E, 100×). No evidence of invasive hyphae was present with GMS staining (400×) in either WT (C) or DPPI^−/− (D) mice. n = 5 mice per genotype.

Figure 5. WT mice and NE^−/−×CG^−/− mice were resistant to Burkholderia cepacia infection, whereas p47^phox−/− mice were highly susceptible.

A) Kaplan-Meier survival curves in WT, p47^{phox −/−} and NE^−/−×CG^−/− mice administered intraperitoneal B. cepacia (4×10⁷ CFUs/mouse). Log-rank analysis, p<0.0002 comparing WT with p47^{phox −/−} mice and p<0.0002 comparing NE^−/−×CG^−/− mice with p47^{phox −/−} mice. n = 10 mice per genotype. B) In separate experiments, mice (n = 5 per genotype) were administered the same inoculum of B. cepacia, and quantitative cultures were performed at 24 h. WT and NE^−/−×CG^−/− mice cleared infection, whereas bacterial infection persisted in the peritoneum and spleens of p47^{phox −/−} mice. Circles, no growth. *, p<0.03; **, p<0.01.