In normal neutrophils, microorganisms are killed by the combined action of oxygen-dependent and oxygen-independent mechanisms. CGD neutrophils possess only oxygen-independent mechanisms, and killing mainly depends on the microbicidal effect of the neutrophil granule proteins (5
). In this study, we first investigated whether deletion of Olfm4,
a negative modulator of host defense against bacterial infection, could boost X-linked CGD mouse immunity against S
, a commonly encountered pathogen in CGD patients. X-CGD mice have a null allele for gp91phox
and are similar to patients with CGD in that they manifest impaired resistance to infections with S
and Aspergillus fumigatus
). Nitroblue tetrazolium (NBT) reduction assays (Figure A) and superoxide burst assays (Figure B) confirmed the lack of NADPH oxidase activity in gp91phox
Intracellular killing and in vivo peritoneal clearance of S. aureus in Olfm4- and gp91phox-deficient mice.
We first crossed gp91phox-deficient mice with Olfm4-deficient mice to create gp91phox and Olfm4 double-deficient mice. These double-deficient mice demonstrated normal development and growth without symptoms of colitis or skin disease. We then analyzed neutrophils derived from the bone marrow of mice with different genotypes for their intracellular killing of two strains of S. aureus: Rosenbach and USA300/LAC, the latter of which is a methicillin-resistant S. aureus (MRSA). In both strains, the neutrophils from gp91phox+/+Olfm4–/– (Olfm4-deficient) mice demonstrated increased capability to kill intracellular S. aureus, while neutrophils from gp91phox–/–Olfm4+/+ (CGD) mice had decreased bacterial-killing ability compared with those from gp91phox+/+Olfm4+/+ (WT) mice (Figure C). The bacterial-killing abilities in gp91phox–/–Olfm4–/– (double-deficient) neutrophils were restored to the levels of those from WT mice (Figure C). Neutrophils from gp91phox–/–Olfm4+/– and gp91phox–/–Olfm4+/+ (CGD) mice demonstrated a similar level of bacterial killing (Figure C). We then performed in vivo peritoneal S. aureus clearance assays for the two strains among different genotypes of mice. Similarly, Olfm4–/– (Olfm4-deficient) mice had increased in vivo peritoneal S. aureus clearance, and gp91phox–/– Olfm4+/+ (CGD) mice had decreased bacterial-clearance ability. In addition, double-deficient mice had enhanced in vivo bacterial clearance abilities compared with CGD mice and demonstrated a similar level of bacterial clearance ability to that of WT mice (Figure D). These results indicate that a homozygous deletion of the Olfm4 gene in CGD mice could enhance CGD mouse S. aureus killing and in vivo clearance.
Next, we investigated whether deletion of Olfm4 in CGD mice could increase the host defense against systemic infections of S. aureus and A. fumigatus, the latter of which is another common pathogen in CGD patients. Mice of various genotypes were infected with S. aureus of Rosenbach or USA300 by i.p. injection, and their survival was monitored. All CGD mice and gp91phox–/–Olfm4+/– mice died within 2 to 5 days (Figure A). Most WT mice died gradually during a 2-week observation period. In contrast, almost all the Olfm4-deficient and gp91phox and Olfm4 double-deficient mice survived. Consistent with our earlier observations, these results suggest that a homozygous, but not a heterozygous, deletion of Olfm4 could increase host system defense against S. aureus infection and prevent S. aureus sepsis-induced mortality in CGD mice.
Susceptibility of Olfm4- and gp91phox-deficient mice to S. aureus or A. fumigatus infection.
Pulmonary aspergillus infection is the most common microbial cause of death in CGD patients, and NADPH oxidase–deficient mice are also susceptible to experimental aspergillus challenge (11
). Therefore, we wanted to determine whether OLFM4 is involved in the mouse host defense against A
using a lung infection model. Following oropharyngeal challenge with a moderate inoculum (5 × 105
conidia per mouse), CGD, double-deficient, and gp91phox–/–Olfm4+/–
mice all died within 9 days, whereas WT and Olfm4
-deficient mice all survived (Figure B). These results showed that Olfm4
deletion did not prevent pulmonary A
infection-induced death in CGD mice. We also evaluated host immune response in Olfm4–/–
mice compared with WT mice 24 hours after a high A
inoculum (1 × 107
conidia per mouse).
Bronchoalveolar fluid leukocytosis was not significantly different between the two genotypes (Supplemental Figure 1A; supplemental material available online with this article; doi:
). No significant difference between the two genotypes was observed in the quantitative fungal burden in the lungs (Supplemental Figure 1B) or in the Grocott-Gomori methenamine-silver staining in lung sections (Supplemental Figure 1C). Lung histology showed a similar level of inflammation characterized by peribronchial and perivascular inflammatory cell infiltration, including neutrophils and mononuclear cells, in Olfm4–/–
and WT mice (Supplemental Figure 1D). Taken together, these results suggest that OLFM4 may not play a role in the mouse host defense against pulmonary aspergillosis.
Neutrophils from gp91phox
-deficient mice or CGD patients have impaired fungicidal activity, and the generation of ROS by neutrophils is considered to be the major antifungal mechanism lacking in CGD (12
). Although evidence suggests that neutrophils can mediate antifungal activities via nonoxidative mechanisms, including defensins (13
), lactoferrin (4
), and pentraxins (14
), neutrophils from CGD patients do not cause substantial damage to hyphae in vitro (15
), suggesting that cellular nonoxidative mechanisms alone are insufficient to kill hyphae under physiologic conditions. It has been reported that A
was markedly resistant to neutrophil granule extract (5
). Neutrophils use oxygen-dependent mechanisms to attack hyphae germinating from conidia that escape alveolar macrophage surveillance (16
). A recent study demonstrated that neutrophil proteases and cathepsin C, which are important for bacterial innate immunity (17
), do not play a role in mouse host defense against pulmonary aspergillosis (11
). Because OLFM4 regulates bacterial killing largely through modulation of cathepsin C and neutrophil proteases in the granules (8
), this may explain why Olfm4
deletion did not alter the mice’s defense against A
, but led to enhanced host defense against S
. These results support the conclusion that NADPH oxidase and neutrophil proteases have distinct antibacterial and antifungal functions.
To explore the mechanism through which Olfm4
deletion enhanced the bactericidal activities of CGD neutrophils, we analyzed the activities of cathepsin C and its downstream proteases (neutrophil elastase and cathepsin G) in the neutrophils from mice after S
challenge. Cathepsin C activity in the neutrophils of Olfm4
-deficient mice and double-deficient mice was significantly higher than that in WT mouse neutrophils, while cathepsin C activity in CGD (gp91phox–/–Olfm4+/+
) and gp91phox–/–Olfm4+/–
mice was similar to that in WT mice (Figure A). Accordingly, the neutrophil elastase (Figure B) and cathepsin G (Figure C) activities in Olfm4
-deficient and double-deficient mice were also substantially higher than those in WT mice as well as in CGD and gp91phox–/–Olfm4+/–
mice. These results suggest that the increase in serine protease activities observed with Olfm4
deletion is NADPH independent. The compromised neutrophil bacterial killing and host innate immunity against S
in CGD mice due to oxidative mechanism deficiency could be successfully restored by the enhancement of serine protease activities in neutrophils subsequent to Olfm4
deletion. Serine proteases are stored in granules in their active form until they are released following neutrophil exposure to inflammatory stimuli. Once released, neutrophil serine proteases are potentially fully active and have broad biological effects, including intracellular microbial killing and modulation of inflammatory cell recruitment (19
Cathepsin C and serine protease activities and cytokine/chemokine serum levels in Olfm4- and gp91phox-deficient mice.
A previous study showed that OLFM4 binds NOD1 and NOD2 and inhibits NOD-mediated NF-κB signaling (9
). Here, we analyzed cytokine and chemokine levels in the serum of mice following challenge with S
. The levels of IL-1β, IL-6, IL-12p40, CXCL2, G-CSF, and GM-CSF were significantly higher in the Olfm4
-deficient mice as well as in the gp91phox
double-deficient mice than the levels in WT and CGD mice (Figure D). All of these cytokines and chemokines are known target genes of the NF-κB pathway. We consistently found that NF-κB activity in the neutrophils from Olfm4
-deficient as well as gp91phox
double-deficient mice was significantly higher than that in WT and CGD mice (Supplemental Figure 2). The enhanced cytokine levels in Olfm4
-deficient mice could be caused by the upregulated NF-κB signaling as well as by increased serine protease activities. Neutrophil serine proteases might not only regulate the activity of chemokines and cytokines by proteolysis, but might also modulate their release through cellular activation (19
). NOD2 is critical for innate recognition and antibacterial defense against S
). Therefore, the enhanced NOD-mediated defense pathways caused by Olfm4
deletion may also contribute to the enhanced host defense against S
NOD signaling works synergistically with TLR signaling to recognize S
infection and induce inflammatory responses (22
). The TLR2-MyD88 pathway has been recognized to play important roles in mouse innate immunity against S
). To determine whether the effect of Olfm4
deletion on S
immune defense is mediated through the TLR-MyD88 pathway, we investigated the host defense of MyD88
double-deficient mice against S
systemic infection. While the MyD88-
deficient mice were highly susceptible to S
infection, additional Olfm4
elimination remarkably improved the survival of MyD88-
deficient mice and enhanced the serum levels of some NF-κB–targeted pro-inflammatory cytokines and chemokines (Supplemental Figure 3). These results suggest that enhanced mouse innate immunity due to Olfm4
deletion is TLR-MyD88 signal independent and that NOD-mediated NF-κB may play an important role. The mechanism of OLFM4 regulation of host immune defense against S
is summarized in Supplemental Figure 4.
Taken together, we found that deletion of Olfm4, a critical negative regulator of neutrophil protease activities and the NOD-mediated pathway, could enhance the immune defense against S. aureus infection in CGD mice. This finding provides a rationale for enhancing CGD patient defense against bacterial infections potentially through the modulation of OLFM4 levels.