RESUMO
Unmethylated CpG motifs in DNA are recognised by vertebrate immune cells as pathogen signatures. Consequently, oligodeoxynucleotides containing CpG motifs (CpG ODNs) are able to enhance and direct immune responses. Recent studies have demonstrated that CpG ODNs activate antiviral immune responses in Atlantic salmon (Salmo salar L.) leukocytes, and are therefore promising agents as vaccine adjuvants or immunostimulants in fish. In this work, we report synergy of CpG ODN and cationic proteins in the stimulation of type I IFN activity in Atlantic salmon leukocytes. Different fractions of cationic histone proteins derived from cod milt or poly-l-arginine and poly-l-lysine were screened for their ability to enhance CpG ODN induced type I IFN activity in Atlantic salmon leukocytes. Optimal ratio of histones to CpG ODN was identified, and effects on transcription of type I IFN and antiviral Mx genes were studied. Delivery of CpG ODN with cationic proteins enhanced the production of type I IFN and succeeding Mx transcripts after two and five days of stimulation at substimulatory concentrations of CpG ODN. These results indicate that co-delivery of CpG ODN and cationic proteins enhance antiviral mechanisms in Atlantic salmon leukocytes as compared to CpG ODN alone.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/farmacologia , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Leucócitos/imunologia , Oligonucleotídeos/farmacologia , Salmo salar/imunologia , Animais , Sequência de Bases , Ilhas de CpG/genética , Primers do DNA , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica/imunologia , Interferon Tipo I/genética , Luciferases , Proteínas de Resistência a Myxovirus , Oligonucleotídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
This study was carried out to reveal some characteristics of cationic proteins from Atlantic cod (Gadus morhua) milt chromatin and to investigate their ability to activate Atlantic salmon (Salmo salar) macrophages. Cationic proteins extracted from cod milt chromatin were fractionated on a cation exchange chromatography column. SDS-PAGE and amino acid analyses of the resulting fractions indicated that these proteins are similar to calf thymus histones. Two cationic protein fractions were used to stimulate leucocytes from Atlantic salmon in vitro and in vivo. Increased production of superoxide, measured as reduction of nitroblue tetrazolium (NBT), was used as indication of macrophage activation. Both fractions induced elevated superoxide anion production in the macrophages after 3 and 6 days of in vitro stimulation. Intraperitoneal injection of the cationic protein fractions in Atlantic salmon (100 mg kg(-1)) four days prior to slaughtering stimulated superoxide production when assayed after one and two days of cell cultivation. In macrophages from fish slaughtered two days after injection, activation could first be seen after two days of cell cultivation.