Inhibition of mCD14 inhibits TNFα secretion and NO production in RAW264.7 cells stimulated by Brucella melitensis infection

• Published in: Veterinary microbiology 2012-12, Vol.160 (3-4), p.362-368
• Main Authors: Lei, Ming, Du, Li, Jiao, Hanwei, Cheng, Ying, Zhang, Donglin, Hao, Yongchang, Li, Gangshan, Qiu, Wei, Fan, Quanshui, Li, Chengyao, Chen, Chuanfu, Wang, Fengyang
• Format: Article
• Language: English
• Subjects: Animals; Bacteriology; Biological and medical sciences; Brucella melitensis; Brucella melitensis - immunology; Brucellosis - immunology; Brucellosis - metabolism; Cell Line; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation; Gene Knockdown Techniques; HEK293 Cells; Humans; Inflammation - prevention & control; Lipopolysaccharide Receptors - genetics; Lipopolysaccharide Receptors - metabolism; mCD14; Mice; Microbiology; Miscellaneous; Nitric Oxide - biosynthesis; Nitric Oxide Synthase - metabolism; Nitric Oxide Synthase Type II - metabolism; RNA Interference; TNFα; Tumor Necrosis Factor-alpha - genetics; Tumor Necrosis Factor-alpha - secretion
• ISSN: 0378-1135; 1873-2542
• DOI: 10.1016/j.vetmic.2012.05.039

Brucellosis, caused by Brucella spp., is an important disease affecting not only human health, but also a number of animal species around the world. A receptor for LPS of Brucella and important innate immune molecule, CD14, has been implicated in the initiation of the inflammatory response to sepsis. Evidence indicates that upstream inhibition of the LPS initiated inflammatory pathway is an effective therapeutic approach for attenuating damaging immune activation. The aim of this study was to explore the possibility of using RNA interference (RNAi) targeting mCD14 as a strategy for inhibiting the secretion of tumor necrosis factor alpha (TNFα) and the production of nitric oxide (NO) from Brucella-stimulated RAW264.7 cells and attenuating damaging immune activation. The current study stably incorporated mCD14-shRNA-224 into the RAW264.7 cell line by lentiviral gene transfer to successfully knockdown mCD14, and was then challenged with Brucella melitensis M5-90. The secretion of TNFα, interleukin (IL)-12, CXCL1/KC, and inducible nitric oxide synthase (iNOS) protein expression, and NO production were evaluated. The mCD14-shRNA-224 knockdown was shown to effectively inhibit B. melitensis M5-90-stimulated TNFα release, iNOS protein expression, and NO production, but no significant differences were observed for IL-12 and CXCL1/KC. These findings provide the basis for the development of RNAi-based prophylaxis and therapy for B. melitensis induced inflammatory disease.