title: Identification and characterization of a novel NOD-like receptor family CARD domain containing 3 gene in response to extracellular ATP stimulation and its role in regulating LPS-induced innate immune response in Japanese flounder (Paralichthys olivaceus) head kidney macrophages.
abstract: Nucleotide oligomerization domain (NOD)-like receptor (NLR) family with a caspase activation and recruitment domain (CARD) containing 3 (NLRC3) protein is an important cytosolic pattern recognition receptor that negatively regulates innate immune response in mammals. Hitherto, the immunological significance of NLRC3 protein in fish remains largely uncharacterized. Here we identified and characterized a novel NLRC3 gene (named poNLRC3) implicated in regulation of fish innate immunity from Japanese flounder Paralichthys olivaceus. The poNLRC3 protein is a cytoplasmic protein with an undefined N-terminal domain, a NACHT domain, a fish-specific NACHT associated domain, six LRR motifs, and a C-terminal fish-specific PYR/SPYR (B30.2) domain but only shares less than 40% sequence identities with the known Japanese flounder NLRC proteins. poNLRC3 gene is ubiquitously expressed in all tested tissues and is dominantly expressed in the Japanese flounder head kidney macrophages (HKMs). We for the first time showed that poNLRC3 expression was significantly modulated by the stimulation of extracellular ATP, an important danger/damage-associated molecular pattern in activating innate immunity in P. olivaceus. Importantly, we revealed that poNLRC3 plays an important role in positively regulating ATP-induced IL-1beta and IL-6 gene expression, suggesting the involvement of poNLRC3 in extracellular ATP-mediated immune signaling. In addition, we showed that poNLRC3 mRNA expression was up-regulated in response to LPS and Edwardsiella tarda immune challenges. Finally, we showed that down-regulating the endogenous poNLRC3 expression with small interfering RNA significantly reduced LPS-induced proinflammatory cytokine gene expression in the Japanese flounder HKM cells. Altogether, we have identified a novel inducible fish NLR member, poNLRC3, which is involved in extracellular ATP-mediated immune signaling and may positively regulate the LPS-induced innate immune response in the Japanese flounder HKM cells.
authors.affiliation: Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Sciences, Tianjin Normal University, 393 West Binshui Road, Xiqing District, Tianjin 300387, China. Electronic address: shuo76@yahoo.com.
authors.forename: Shuo
authors.initials: S
authors.lastname: Li
authors.affiliation: Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Sciences, Tianjin Normal University, 393 West Binshui Road, Xiqing District, Tianjin 300387, China.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Chen
authors.affiliation: Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Sciences, Tianjin Normal University, 393 West Binshui Road, Xiqing District, Tianjin 300387, China.
authors.forename: Gaixiang
authors.initials: G
authors.lastname: Hao
authors.affiliation: Tianjin Center for Control and Prevention of Aquatic Animal Infectious Disease, 442 South Jiefang Road, Hexi District, Tianjin 300221, China.
authors.forename: Xuyun
authors.initials: X
authors.lastname: Geng
authors.affiliation: Laboratory of Pathology and Immunology of Aquatic Animals, LMMEC, Ocean University of China, Qingdao 266003, China.
authors.forename: Wenbin
authors.initials: W
authors.lastname: Zhan
authors.affiliation: Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Sciences, Tianjin Normal University, 393 West Binshui Road, Xiqing District, Tianjin 300387, China. Electronic address: jssun1965@aliyun.com.
authors.forename: Jinsheng
authors.initials: J
authors.lastname: Sun
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016207:Cytokines / Q000378:metabolism; D020609:Edwardsiella tarda / Q000502:physiology; D004756:Enterobacteriaceae Infections / Q000235:genetics / Q000276:immunology / Q000382:microbiology / Q000662:veterinary*; D005393:Fish Diseases / Q000235:genetics / Q000276:immunology* / Q000382:microbiology; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005412:Flatfishes; D005786:Gene Expression Regulation; D007113:Immunity, Innate / Q000187:drug effects; D008070:Lipopolysaccharides / Q000494:pharmacology; D000070576:NLR Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D017433:Protein Structure, Secondary; D016415:Sequence Alignment / Q000662:veterinary
chemical_list: D016207:Cytokines; D029941:Fish Proteins; D008070:Lipopolysaccharides; D000070576:NLR Proteins; D000255:Adenosine Triphosphate
keywords: Extracellular ATP; Lipopolysaccharide-induced innate immunity; NLR; NLRC3; Paralichthys olivaceus