Olfm4 deletion enhances defense against Staphylococcus aureus in chronic granulomatous disease

Abstract

Chronic granulomatous disease (CGD) patients have recurrent life-threatening bacterial and fungal infections. Olfactomedin 4 (OLFM4) is a neutrophil granule protein that negatively regulates host defense against bacterial infection. The goal of this study was to evaluate the impact of Olfm4 deletion on host defense against Staphylococcus aureus and Aspergillus fumigatus in a murine X-linked gp91phox-deficiency CGD model. We found that intracellular killing and in vivo clearance of S. aureus, as well as resistance to S. aureus sepsis, were significantly increased in gp91phox and Olfm4 double-deficient mice compared with CGD mice. The activities of cathepsin C and its downstream proteases (neutrophil elastase and cathepsin G) and serum levels of IL-1β, IL-6, IL-12p40, CXCL2, G-CSF, and GM-CSF in Olfm4-deficient as well as gp91phox and Olfm4 double-deficient mice were significantly higher than those in WT and CGD mice after challenge with S. aureus. We did not observe enhanced defense against A. fumigatus in Olfm4-deficient mice using a lung infection model. These results show that Olfm4 deletion can successfully enhance immune defense against S. aureus, but not A. fumigatus, in CGD mice. These data suggest that OLFM4 may be an important target in CGD patients for the augmentation of host defense against bacterial infection.

Figure 1. Intracellular killing and in vivo peritoneal clearance of S. aureus in Olfm4- and gp91phox-deficient mice.

(A) NBT assays and (B) superoxide burst assays were performed to confirm the loss of NAPDH oxidase activity in different genotypes of mice. Original magnification, ×200 in NBT assays. (C) Neutrophils derived from the bone marrow of Olfm4- and gp91phox-deficient mice were incubated with preopsonized S. aureus (Rosenbach or USA300). The number of viable bacteria (CFU) after the cells were treated with gentamicin (50 μg/ml) was determined on plates. Data are expressed as the mean ± SD (n = 5). (D) Mice were challenged i.p. with S. aureus (Rosenbach or USA300). After 6 hours, the peritoneal cavity was lavaged, and the number of viable bacteria (CFU) was determined on plates. Data are expressed as the mean ± SD (n = 5). *P < 0.05 when compared with WT (gp91phox^+/+Olfm4^+/+) mice or as indicated.

Figure 2. Susceptibility of Olfm4- and gp91phox-deficient mice to S. aureus or A. fumigatus infection.

(A) Survival probability plots (Kaplan-Meier) of experimental groups infected i.p. with S. aureus (Rosenbach or USA300) (2 × 10⁸ CFU per mouse; n = 10). Survival was monitored every 6 hours. (B) Survival probability plots (Kaplan-Meier) of experimental groups infected with oropharyngeal aspiration of A. fumigatus (5 × 10⁵ CFU per mouse; n = 5). Survival was monitored twice a day.

Figure 3. Cathepsin C and serine protease activities and cytokine/chemokine serum levels in Olfm4- and gp91phox-deficient mice.

(A–C) Neutrophils (5 × 10⁶) derived from the bone marrow of mice with different genotypes 6 hours after i.p. infection with S. aureus (5 × 10⁷ CFU per mouse) were lysed, and an equal amount of lysate was used for assays of cathepsin C (Cat C) (A), neutrophil elastase (B), and cathepsin G (Cat G) (C) activity using the corresponding AMC-labeled substrate. *P < 0.05 when compared with WT (gp91phox^+/+Olfm4^+/+) mice. Data are expressed as the mean ± SD (n = 5). RFU, relative fluorescence unit. (D) Cytokine and chemokine levels in the serum of mice with different genotypes 6 hours after i.p. infection with S. aureus (5 × 10⁷ CFU per mouse) were determined by high-throughput immunoassay. Data are expressed as the mean ± SD for each experimental group (n = 5). *P < 0.05 versus WT (gp91phox^+/+Olfm4^+/+) mice.