S. aureus and E. coli
infections are still major threats to human health, especially because of emerging antimicrobial resistance (21
). Neutrophils play essential roles in killing invading bacteria. Therefore, it is important to understand the molecular mechanisms underlying neutrophil activity to better combat bacteria infection. In this study, we demonstrate that OLFM4 is a critical regulator of neutrophil’s ability to kill S. aureus
and E. coli.
This function is associated with restriction of cathepsin C and downstream serine proteases, but not with NADPH-mediated oxidative burst.
We demonstrate that OLFM4 physically associates with cathepsin C in neutrophils. We show that OLFM4 is localized in multiple granule compartments with most abundance in specific granules, but also in azurophilic granules where cathepsin C and serine proteases are mostly localized (15
). As previously described, there is a functional interplay between different granule subsets (22
). For example, neutrophil gelatinase-associated lipocalin that is normally found in the specific granules is able to complex with gelatinase (23
). Proteases from azurophil granules may activate the cathelicidin present in specific granules (22
). Therefore, OLFM4 abundantly present in specific granules may also interact with cathepsin C in azurophil granules, especially when azurophil granules and specific granules are fused with phagosomes to form phagolysosomes after bacteria phagocytosis by neutrophils.
Recent studies showed that cathepsin C is involved in multiple pathological processes, particularly in neutrophils where it plays an important role in bacterial clearance (20
) and the inflammatory response (24
). Thus, cathepsin C represents a potential therapeutic target for the treatment of inflammatory disease. Many small peptides have been selected to be potent cathepsin C inhibitors, but they have poor stability and/or pharmacokinetics (25
). Cathepsin C is also inhibited by protein inhibitors, such as rat stefin A and chicken cystatin F, two important inhibitors of cysteine peptidases from the cystatin superfamily (26
). Cystatin F is expressed primarily in CD8+
T cells and in CD56+
NK cells, and has been shown to complex with cathepsin C in human monocytic and NK cells (27
). Our study showed that OLFM4, a granule protein, was associated with cathepsin C in neutrophils, and strongly and specifically inhibited cathepsin C both in vitro and in vivo. Moreover, the data from the OLFM4−/−
mouse model demonstrated that OLFM4 also affects multiple serine protease activities indirectly. Thus, OLFM4 represents a novel protein inhibitor of cathepsin C, specifically in neutrophils.
Our results show that enhanced bacteria killing activity in OLFM4-deficient neutrophils is associated with increased cathepsin C and downstream serine protease activities. It has been reported that bacterial clearance is impaired in cathepsin C−/−
mice using a cecal ligation and puncture model of septic peritonitis (20
), demonstrating that cathepsin C is an important regulator for bacteria control and clearance. Although a regulatory role of cathepsin C on specific bacteria such as S. aureus
and E. coli
used in this study has not been reported, mice clearance of E. coli
and S. aureus
are impaired in absence of active neutrophil elastase and cathepsin G, which requires activation from cathepsin C (1
). The mode of action of OLFM4 through cathepsin C is further confirmed by experiments in OLFM4 and cathepsin C double-deficient mice. The enhanced bactericidal abilities in the neutrophils of OLFM4−/−
mice were compromised, but not eliminated, by further deletion of cathepsin C gene, confirming the involvement of cathepsin C in the OLFM4-mediated effect. This suggests that the increased bactericidal activity in OLFM4−/−
neutrophils is, at least in part, due to the enhanced cathepsin C activity. However, these data do not rule out possible involvement of other mechanisms in the observed phenotypes. For example, it was previously reported that OLFM4 negatively regulates the nucleotide-binding oligomerization domain (Nod) 1 and Nod2 (12
). Nod1 has a broad influence on neutrophil function, activating both oxidative and nonoxidative mechanisms of killing (28
). Therefore, the increased bactericidal activity in OLFM4-deficient neutrophils may also be partially caused by subsequent enhanced Nod1 activity in neutrophils.
OLFM4 and other olfactomedin-related proteins have a high m.w. multimer structure with intramolecular and intermolecular disulfide bonds (13
). It has been previously reported that OLFM4 complexes with cadherin (13
) and Nods (12
) proteins. These observations together with this study suggest a possibility that OLFM4 may be a component of multiple protein complexes as an adaptor or scaffold protein. The notion that olfactomedin class of proteins may be scaffolds for proteases and their substrates was also recently suggested by Inomata and coworkers (29
) based on their finding that a Xenopus
secreted olfactomedin 1 recruits the Tolloid proteases to their substrate chordin and is required for normal chordin degradation.
In summary, we demonstrate that OLFM4 is a neutrophil granule protein, which proved to be an important negative regulator of bacteria killing as evidenced by that neutrophils from OLFM4−/−
mice have increased capability to kill S. aureus
and E. coli
, and OLFM4−/−
mice are more resistant to systemic sepsis. Taken together with the previous study (12
), which demonstrated that OLFM4 downregulates host innate immunity against H. pylori
, OLFM4 appears to be an important regulator of host innate immunity against a broad array of bacterial infections. OLFM4 may prove to be a potential target for therapeutic augmentation of host innate immunity in genetic immune-deficient patients such as those with chronic granulomatous disease.