title: SjCa8, a calcium-binding protein from Schistosoma japonicum, inhibits cell migration and suppresses nitric oxide release of RAW264.7 macrophages.
abstract: BACKGROUND
  Schistosomiasis is considered second only to malaria as the most devastating parasitic disease in tropical countries. Schistosome cercariae invade the host by penetrating the skin and migrate though the lungs and portal circulation to their final destination in the hepatic portal system and eventually the mesenteric veins. Previous studies have shown that the cytotoxic pathways that target schistosomulum in the lung-stage involve nitric oxide (NO) produced by macrophages. By contrast, skin-stage schistosomulas can evade clearance, indicating that they might be freed from macrophage NO-mediated cytotoxicity to achieve immune evasion; however, the critical molecules and mechanisms involved remain unknown.
  
  
  METHODS
  Recombinant SjCa8 (rSjCa8), an 8-kDa calcium-binding protein that is stage-specifically expressed in cercaria and early skin-stage schistosomulas of Schistosoma japonicum, was incubated with mouse RAW264.7 macrophages. Effects on macrophage proliferation were determined using Cell Counting Kit-8. Next, transwell assay was carried out to further investigate the role of rSjCa8 in macrophage migration. The effects of rSjCa8 on macrophage apoptosis were evaluated using confocal microscopy and flow cytometry. Additional impacts of rSjCa8 on NO release by lipopolysaccharide (LPS)-stimulated macrophages as well as the underlying mechanisms were explored using fluorescent probe, nitric oxide signaling pathway microarray, quantitative real-time PCR, mutagenesis, and neutralizing antibody approaches.
  
  
  RESULTS
  rSjCa8 exhibited a striking inhibitory effect on macrophage migration, but did not markedly increase cell proliferation or apoptosis. Additionally, rSjCa8 potently inhibited NO release by LPS-stimulated macrophages in a dose- and time-dependent manner, and the inhibitory mechanism was closely associated with intracellular Ca(2+) levels, the up-regulation of catalase expression, and the down-regulation of the expression of 47 genes, including Myc, Gadd45a, Txnip, Fas, Sod2, Nos2, and Hmgb1. Vaccination with rSjCa8 increased NO concentration in the challenging skin area of infected mice and reduced the number of migrated schistosomula after skin penetration by cercariae.
  
  
  CONCLUSIONS
  Our findings indicate that SjCa8 might be a novel molecule that plays a critical role in immune evasion by S. japonicum cercaria during the process of skin penetration. The inhibitory impacts of rSjCa8 on macrophage migration and [Ca(2+)]i-dependent NO release suggest it might represent a novel vaccine candidate and chemotherapeutic target for the prevention and treatment of schistosomiasis.
authors.affiliation: Zhongshan School of Medicine, Sun Yat-sen University, 74 2nd Zhongshan Road, Guangzhou, 510080, China. 1224989703@qq.com.|Key Laboratory for Tropical Diseases Control of Ministry of Education, Sun Yat-sen University, Guangzhou, 510080, China. 1224989703@qq.com.
authors.forename: Ji
authors.initials: J
authors.lastname: Liu
authors.affiliation: Zhongshan School of Medicine, Sun Yat-sen University, 74 2nd Zhongshan Road, Guangzhou, 510080, China. 19679932453@qq.com.|Key Laboratory for Tropical Diseases Control of Ministry of Education, Sun Yat-sen University, Guangzhou, 510080, China. 19679932453@qq.com.
authors.forename: Tong
authors.initials: T
authors.lastname: Pan
authors.affiliation: Zhongshan School of Medicine, Sun Yat-sen University, 74 2nd Zhongshan Road, Guangzhou, 510080, China. 45976103@qq.com.|Key Laboratory for Tropical Diseases Control of Ministry of Education, Sun Yat-sen University, Guangzhou, 510080, China. 45976103@qq.com.
authors.forename: Xu
authors.initials: X
authors.lastname: You
authors.affiliation: Zhongshan School of Medicine, Sun Yat-sen University, 74 2nd Zhongshan Road, Guangzhou, 510080, China. 723385673@qq.com.|Key Laboratory for Tropical Diseases Control of Ministry of Education, Sun Yat-sen University, Guangzhou, 510080, China. 723385673@qq.com.
authors.forename: Yiyue
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Zhongshan School of Medicine, Sun Yat-sen University, 74 2nd Zhongshan Road, Guangzhou, 510080, China. 1291157375@qq.com.|Key Laboratory for Tropical Diseases Control of Ministry of Education, Sun Yat-sen University, Guangzhou, 510080, China. 1291157375@qq.com.
authors.forename: Jinyi
authors.initials: J
authors.lastname: Liang
authors.affiliation: Department of Social and Environmental Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand. yanin.lim@mahidol.ac.th.
authors.forename: Yanin
authors.initials: Y
authors.lastname: Limpanont
authors.affiliation: Zhongshan School of Medicine, Sun Yat-sen University, 74 2nd Zhongshan Road, Guangzhou, 510080, China. shenlan_362096394@qq.com.|Key Laboratory for Tropical Diseases Control of Ministry of Education, Sun Yat-sen University, Guangzhou, 510080, China. shenlan_362096394@qq.com.
authors.forename: Xi
authors.initials: X
authors.lastname: Sun
authors.affiliation: Department of Social and Environmental Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand. kamolnetr.oka@mahidol.ac.th.
authors.forename: Kamolnetr
authors.initials: K
authors.lastname: Okanurak
authors.affiliation: Zhongshan School of Medicine, Sun Yat-sen University, 74 2nd Zhongshan Road, Guangzhou, 510080, China. zhenghq@mail.sysu.edu.cn.|Key Laboratory for Tropical Diseases Control of Ministry of Education, Sun Yat-sen University, Guangzhou, 510080, China. zhenghq@mail.sysu.edu.cn.
authors.forename: Huanqin
authors.initials: H
authors.lastname: Zheng
authors.affiliation: Zhongshan School of Medicine, Sun Yat-sen University, 74 2nd Zhongshan Road, Guangzhou, 510080, China. wuzhd@mail.sysu.edu.cn.|Key Laboratory for Tropical Diseases Control of Ministry of Education, Sun Yat-sen University, Guangzhou, 510080, China. wuzhd@mail.sysu.edu.cn.
authors.forename: Zhongdao
authors.initials: Z
authors.lastname: Wu
authors.affiliation: Zhongshan School of Medicine, Sun Yat-sen University, 74 2nd Zhongshan Road, Guangzhou, 510080, China. lvzhiyue@mail.sysu.edu.cn.|Key Laboratory for Tropical Diseases Control of Ministry of Education, Sun Yat-sen University, Guangzhou, 510080, China. lvzhiyue@mail.sysu.edu.cn.
authors.forename: Zhiyue
authors.initials: Z
authors.lastname: Lv
mesh_terms: D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002465:Cell Movement; D049109:Cell Proliferation; D058487:Cercaria; D015536:Down-Regulation; D015801:Helminth Proteins / Q000235:genetics / Q000378:metabolism; D008099:Liver / Q000469:parasitology; D008264:Macrophages / Q000187:drug effects; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism*; D011994:Recombinant Proteins; D012549:Schistosoma japonicum / Q000235:genetics* / Q000378:metabolism; D012554:Schistosomiasis japonica / Q000469:parasitology*; D015854:Up-Regulation
chemical_list: D002135:Calcium-Binding Proteins; D015801:Helminth Proteins; D011994:Recombinant Proteins; D009569:Nitric Oxide