Olfactomedin 4 (OLFM4) is a glycoprotein that has been found to be up-regulated in inflammatory bowel diseases and Helicobacter pylori (H. pylori) infected patients. However, its role in biological processes such as inflammation or other immune response is not known. In this study, we generated OLFM4 knock-out (KO) mice to investigate potential role(s) of OLFM4 in gastric mucosal responses to H. pylori infection. H. pylori colonization in the gastric mucosa of OLFM4 KO mice was significantly lower compared with wild-type littermates. The reduced bacterial load was associated with enhanced infiltration of inflammatory cells in gastric mucosa. Production and expression of proinflammatory cytokines/chemokines such as IL-1β, IL-5, IL-12 p70, and MIP-1αwas increased in OLFM4 KO mice compared with infected controls. Furthermore, we found that OLFM4 is a target gene of NF-κB pathway and has a negative feedback effect on NF-κB activation induced by H.pylori infection through a direct association with nucleotide oligomerization domain-1 (NOD1) and -2 (NOD2). Together these observations indicate that OLFM4 exerts considerable influence on the host defense against H.pylori infection acting through NOD1 and NOD2 mediated NF-κB activation and subsequent cytokines and chemokines production, which in turn inhibit host immune response and contribute to persistence of H. pylori colonization. Although OLFM4 is a target gene of the NF-kB and Notch pathways and OLFM4 down-regulates innate immunity to H. pylori infection, its potential biological functions in neutrophils still remain to be defined. The goal of our second study was to determine whether OLFM4 is involved in the bactericidal activity of neutrophils using an OLFM4 deficient mouse model. We found: 1. OLFM4 expression in neutrophils is upregulated in response to Staphylocococus aureus (Gram positive) and Escherichia coli (Gram negative) bacteria. 2. Neutrophils from OLFM4 deficient mice have increased intracellular killing of S. aureus and E. coli bacteria in vitro. 3. The OLFM4 deficient mice displayed enhanced bacterial clearance in vivo when the mice were challenged with intra-peritoneal injection of S. aureus and E. coli. 4. We performed a yeast 2-hybrid screen and found that OLFM4 interacts with cathepsin C (dipeptidyl peptidase I or DPPI), a lysosomal cysteine protease that has a degraditive function as an exopeptidase and is essential for activation of neutrophil granule-associated serine proteases including neutrophil elastase, cathepsin G and proteinase 3. The direct association of OLFM4 with cathepsin C was confirmed in human primary neutrophils. 4. We demonstrated that OLFM4 is a direct substrate of DPPI and inhibits DPPI activity in transfected 293T cells. 5. The cathepsin C activity in neutrophils from OLFM4 deficient mice was significantly higher than that in neutrophils from wild-type littermate mice in the absence or presence of bacterial infection, suggesting that OLFM4 is an endogenous inhibitor of cathepsin C in neutrophils. 6. We have also demonstrated increased activities of neutrophil elastase, cathepsin G and proteinase 3 (whose processing and maturity require cathepsin C activity) in OLFM4 deficient neutrophils compared with wild-type neutrophils. 7. Activation of NADPH oxidase, myeloperoxidase (MPO) activity, and neutrophil phagocytosis were not altered in OLFM4 deficient neutrophils compared with wild-type neutrophils. Conclusion: These results suggest that OLFM4 is an important regulator of neutrophil bacterial killing activity via negative regulation of cathepsin C activity and its down stream granule-associated serine proteases.
