title: HMGB in mollusk Crassostrea ariakensis Gould: structure, pro-inflammatory cytokine function characterization and anti-infection role of its antibody.
abstract: BACKGROUND
  Crassostrea ariakensis Gould is a representative bivalve species and an economically important oyster in China, but suffers severe mortalities in recent years that are caused by rickettsia-like organism (RLO). Prevention and control of this disease is a priority for the development of oyster aquaculture. It has been proven that mammalian HMGB (high mobility group box) can be released extracellularly and acts as an important pro-inflammatory cytokine and late mediator of inflammatory reactions. In vertebrates, HMGB's antibody (anti-HMGB) has been shown to confer significant protection against certain local and systemic inflammatory diseases. Therefore, we investigated the functions of Ca-HMGB (oyster HMGB) and anti-CaHMGB (Ca-HMGB's antibody) in oyster RLO/LPS (RLO or LPS)-induced disease or inflammation.
  
  
  RESULTS
  Sequencing analysis revealed Ca-HMGB shares conserved structures with mammalians. Tissue-specific expression indicates that Ca-HMGB has higher relative expression in hemocytes. Significant continuous up-regulation of Ca-HMGB was detected when the hemocytes were stimulated with RLO/LPS. Recombinant Ca-HMGB protein significantly up-regulated the expression levels of some cytokines. Indirect immunofluorescence study revealed that Ca-HMGB localized both in the hemocyte nucleus and cytoplasm before RLO challenge, but mainly in the cytoplasm 12 h after challenge. Western blot analysis demonstrated Ca-HMGB was released extracellularly 4-12 h after RLO challenge. Anti-CaHMGB was added to the RLO/LPS-challenged hemocyte monolayer and real-time RT-PCR showed that administration of anti-CaHMGB dramatically reduced the rate of RLO/LPS-induced up-regulation of LITAF at 4-12 h after treatment. Flow cytometry analysis indicated that administration of anti-CaHMGB reduced RLO/LPS-induced hemocyte apoptosis and necrosis rates.
  
  
  CONCLUSIONS
  Ca-HMGB can be released extracellularly and its subcellular localization varies when stimulated with RLO. Ca-HMGB is involved in oyster immune reactions and functions as a pro-inflammatory cytokine. Anti-CaHMGB can significantly suppress RLO/LPS-induced inflammatory responses and hemocyte necrosis and apoptosis, suggesting that Ca-HMGB is a potential target to prevent and control RLO/LPS-induced disease or inflammation.
authors.affiliation: Laboratory of Marine Life Science and Technology, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Xu
authors.forename: Shoubao
authors.initials: S
authors.lastname: Yang
authors.forename: Jiasong
authors.initials: J
authors.lastname: Xie
authors.forename: Shigen
authors.initials: S
authors.lastname: Ye
authors.forename: Ming
authors.initials: M
authors.lastname: Luo
authors.forename: Zewen
authors.initials: Z
authors.lastname: Zhu
authors.forename: Xinzhong
authors.initials: X
authors.lastname: Wu
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D015153:Blotting, Western; D049896:Crassostrea / Q000276:immunology* / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism; D005434:Flow Cytometry; D024002:HMGB Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010802:Phylogeny; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D000906:Antibodies; D016207:Cytokines; D024002:HMGB Proteins