Propionibacterium acnes-induced iNOS and COX-2 protein expression via ROS-dependent NF-κB and AP-1 activation in macrophages

Background: Propionibacterium acnes (P. acnes), a gram-positive anaerobic bacterium, plays a critical role in the development of inflammatory lesion as a result of cytokines production by keratinocytes and macrophages activation. However, effect of P. acnes on iNOS/NO and COX-2/PGE(2) production in macrophages is still uninvestigated. Objective: This study aimed at determining the reactive oxygen species (ROS), inducible nitric oxide (NO) synthase (iNOS)/nitric oxide (NO), and cyclooxygenase (COX)-2/prostaglandin (PG)E-2 produced by macrophages upon P. acnes infection, and dissecting the mechanism of P. acnes-stimulated multiplicity of infection (MOI)-dependent increases in iNOS and COX-2 protein expressions in accordance with the elevation of NO and PGE(2) production by RAW264.7 macrophages. Methods: Using an in vitro cell culture system, the effects of P. acnes on iNOS/NO, COX-2/PGE(2), ROS production, ERK/JNK, and AP-1/NF-kappa B activation were examined via Western blotting, a flow cytometric analysis, and luciferase assay. In pharmacological studies, the ROS scavenger, N-acetyl cysteine (NAC), the NADPH oxidase inhibitor, diphenylene iodide (DPI), and mitogen-activated protein kinase (MAPK) inhibitors (U0126 and SP600125) were applied to investigate the mechanism. Results: We found that P. acnes exposures increased iNOS/NO and COX-2/PGE(2) expression in RAW264.7, J774A.1, and peritoneal macrophages via a MOI-dependent manner. Increased ROS production, ERK/JNK protein phosphorylation, and elevated AP-1/NF-kappa B luciferase activity are identified in P. acnes-induced iNOS/NO and COX-2/PGE(2) production. Additionally, hispolon but not its analogs, hispolon methylether or dehydroxyhispolon, showed significant inhibition of P. acnes-induced iNOS/NO and COX-2/PGE(2) production, indicating an important role of OH at C5 for hispolon's inhibition of P. acnes-induced inflammatory events in macrophages. Conclusion: ROS-dependent stimulation of ERK, JNK, NF-kappa B, and AP-1 activation contributes to P. acnes-induced iNOS/NO and COX-2/PGE(2) in macrophages, and chemicals such as hispolon possessing ability to block iNOS/NO and COX-2/PGE(2) production reserve potential to be further developed for treatment of the early phase of inflammation elicited by P. acnes. (c) 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.