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Molecular and immune response characterizations of IL-6 in large yellow croaker (Larimichthys crocea)
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Abstracts / Fish & Shellfish Immunology 53 (2016) 58e93
O-057. Molecular and immune response characterizations of IL-6 in large yellow croaker (Larimichthys crocea) Qian Zhu y, Chan Li y, Zhen-Xing Yu, Peng-Fei Zou, Qing-Xiang Meng, Cui-Luan Yao* Fisheries College/ Fujian Provincial Key Laboratory of Marine Fishery Resources and Eco-environment, Jimei University, Xiamen 361021, PR China Abstract Interleukin-6 (IL-6) is a multifunctional inflammatory cytokine which exists in multiple tissues and cell lines. In the present study, the full-length cDNA and the genomic sequence of IL-6 (LcIL-6) were cloned from large yellow croaker, Larimichthys crocea. The full-length cDNA of LcIL-6 was 1066 base pairs (bp), containing an open reading frame (ORF) of 678 bp encoding for 225 amino acids, a 5’ untranslated region (UTR) of 71 bp and a 3’ UTR of 317 bp. The predicted LcIL-6 protein included a 24 amino acids (aa) signal peptide and a conserved IL-6 domain. However, the polypeptide sequence identities between LcIL-6 and its counterparts in mammals and other fish are from 12% to 45%. The genome sequence of LcIL-6 gene was composed of 2126 bp, including five exons and four introns. Phylogenetic analysis revealed that LcIL-6 showed a close relationship with the IL-6 from other bony fish. Quantitative real-time PCR (qRT-PCR) analysis revealed that LcIL-6 mRNA was expressed in most examined tissues, with the most predominant expression in stomach, followed by blood and very weak expression in other tissues. The expression levels of LcIL-6 after challenged with LPS, poly I:C and Vibrio parahaemolyticus were investigated in spleen, head-kidney and liver. LcIL-6 transcripts were induced significantly after immune challenge, with the peak-value of 33.5 times as much as the control in the head-kidney at 3 h after LPS injection (p < 0.05). Overexpression of LcIL-6 enhanced tumor necrosis factor (TNF)-a transcripts significantly (p < 0.05) in L. crocea kidney (LCK) cells. Additionally, recombinant LcIL-6 mature peptide was obtained in the supernatant of Escherichia coli BL21 (DE3). The purified recombinant LcIL-6 fusion protein was also demonstrated to improve the transcriptional expression levels of TNF-a significantly in LCK cells (p < 0.05). However, no significant changes of Mx (myxovirus resistant protein), IL-1b, janus kinase (JAK)2, signal transducers and activators of transcription (STAT)3 and STAT5 in LCK cells was detected after LcIL-6 overexpression or recombinant LcIL-6 protein stimulation. Our results indicated that LcIL-6 might be important in large yellow croaker immune response and improve the inflammatory response by through activation TNF-a expression. Keywords: Interleukin-6; gene structure; large yellow croaker; immune challenge; overexpression; recombinant expression; inflammatory response (This research was supported by NSFC (31101882; 41276178) and 973 Program (2011CB111604) to C.L.Y) * Corresponding author. E-mail address: [email protected] (C.-L. Yao). y Both authors have the same contribution.
O-058. Identification and functional characterization of two granulocyte colony-stimulating factors of common carp Fumihiko Katakura§, Erika Hino, Kohei Nishiya, Jiro Miyamae, Tadaaki Moritomo Department of Veterinary Medicine, Nihon University, Fujisawa, Kanagawa, Japan Abstract Granulocyte colony-stimulating factor (GCSF) is a cytokine that regulates the proliferation, differentiation, survival and immunological activation of myeloid cells such as neutrophils and monocytes, and their progenitors in mammals. GCSF is used as a recombinant therapeutics against mammalian
neutropenia to prevent infectious diseases. However, functions of GCSF are yet to be deeply characterized in lower vertebrates. Analysis of carp genome showed that carp have two paralogous GCSF molecules, GCSFa and GCSFb, similar to other teleosts, presumably resulting from a whole genome duplication event in teleosts. These molecules were predicted to possess functional domains and structures, although there are quite low sequence identities between vertebrate species. Expression analysis of carp gcsfa and gcsfb revealed the highest gcsfa mRNA levels in spleen, while the highest gcsfb mRNA levels in heart. Both GCSFa and GCSFb appear to be involved in immunity based on their up-regulation in kidney leukocytes following in vitro stimulation of lipopolysaccharide and a combination of Concanavalin A and Phorbol myristate acetate. Additionally, recombinant carp GCSFa and GCSFb stimulated the proliferation and the colony-formation of carp kidney leukocytes in a dose dependent manner, suggesting that both ligands play a role as a hematopoietic growth factor. Furthermore, recombinant GCSFb induced the development of morphologically mature neutrophils and up-regulated mRNA expression of GCSF receptor in the kidney leukocytes. Our results indicate that carp GCSFs are involved in the immune system and at least GCSFb has parallel functions to mammalian GCSF as a regulator of neutrophilic myelopoiesis. Keywords: Granulocyte colony-stimulating factor; Common carp; Hematopoiesis; Neutrophils; In vitro x Corresponding author. Tel.: þ81 (0)466 84 3443; Fax: þ81 (0)466 84 3374. E-mail address: [email protected] (F. Katakura).
O-059. RNA-Seq analysis in gills of atlantic salmon (Salmo salar) infected with newly identified ISAV strains in the atlantic Canada region Francis LeBlanc 1, Steven Leadbeater 2, Mark Laflamme 1, Nellie Gagn e 1,* 1 Fisheries & Oceans Canada, Gulf Fisheries Center, Moncton, NB, Canada 2 Fisheries & Oceans Canada, St Andrews Biological Station, St Andrews, NB, Canada
The Infectious salmon anemia virus (ISAV) is an important viral disease of farmed Atlantic salmon (Salmo salar) which has caused severe financial losses for salmon farmers around the world, including Atlantic Canada. It is listed as an OIE notifiable disease and to this day eradication of infected cages remains the current practice in many countries to mitigate the spread of the virus. All strains known to display any level of virulence are characterised by deletions in the highly polymorphic region (HPR) of the hemagglutinin-esterase (HE) protein compared to ISAV-HPR0 strains and are designated ISAV-HPRD. In contrast, HPR0 strains are non-virulent and not detected by viral culture. In Atlantic Canada, improved management and husbandry practices resulted in no outbreaks caused by ISAV-HPRD strains from 2007 to 2012, with however ISAV-HPR0 strains (European genotype) still being detected. Starting in 2012, new ISAV-HPRD strains were responsible for disease outbreaks in fish farms in Nova Scotia, Newfoundland and New-Brunswick. This study was thus designed to characterize virulence associated to three of the newly identified ISAVHPRD strains and investigate gene expression responses in gills of infected fish using RNA-Seq. All three ISAV-HPRD strains studied through the use of in vivo challenges resulted in cumulative mortalities below 40 percent and caused the differential expression of up to 1000 genes at the peak of infection compared to non-infected fish. Many genes linked to innate immunity were over-expressed; similar to what had been observed in the head-kidney through previous work. The use of RNA-Seq also enabled us to look at ISAV transcript abundance and showed that segment 8 had the highest abundance of all 8 segments of the genome. This information has given us great insight on how ISAV interacts with his host during the course of an infection. Keywords: Atlantic salmon, ISAV, RNA-Seq, Transcriptome, ISAV transcript abundance
Abstracts / Fish & Shellfish Immunology 53 (2016) 58e93
O-057. Molecular and immune response characterizations of IL-6 in large yellow croaker (Larimichthys crocea) Qian Zhu y, Chan Li y, Zhen-Xing Yu, Peng-Fei Zou, Qing-Xiang Meng, Cui-Luan Yao* Fisheries College/ Fujian Provincial Key Laboratory of Marine Fishery Resources and Eco-environment, Jimei University, Xiamen 361021, PR China Abstract Interleukin-6 (IL-6) is a multifunctional inflammatory cytokine which exists in multiple tissues and cell lines. In the present study, the full-length cDNA and the genomic sequence of IL-6 (LcIL-6) were cloned from large yellow croaker, Larimichthys crocea. The full-length cDNA of LcIL-6 was 1066 base pairs (bp), containing an open reading frame (ORF) of 678 bp encoding for 225 amino acids, a 5’ untranslated region (UTR) of 71 bp and a 3’ UTR of 317 bp. The predicted LcIL-6 protein included a 24 amino acids (aa) signal peptide and a conserved IL-6 domain. However, the polypeptide sequence identities between LcIL-6 and its counterparts in mammals and other fish are from 12% to 45%. The genome sequence of LcIL-6 gene was composed of 2126 bp, including five exons and four introns. Phylogenetic analysis revealed that LcIL-6 showed a close relationship with the IL-6 from other bony fish. Quantitative real-time PCR (qRT-PCR) analysis revealed that LcIL-6 mRNA was expressed in most examined tissues, with the most predominant expression in stomach, followed by blood and very weak expression in other tissues. The expression levels of LcIL-6 after challenged with LPS, poly I:C and Vibrio parahaemolyticus were investigated in spleen, head-kidney and liver. LcIL-6 transcripts were induced significantly after immune challenge, with the peak-value of 33.5 times as much as the control in the head-kidney at 3 h after LPS injection (p < 0.05). Overexpression of LcIL-6 enhanced tumor necrosis factor (TNF)-a transcripts significantly (p < 0.05) in L. crocea kidney (LCK) cells. Additionally, recombinant LcIL-6 mature peptide was obtained in the supernatant of Escherichia coli BL21 (DE3). The purified recombinant LcIL-6 fusion protein was also demonstrated to improve the transcriptional expression levels of TNF-a significantly in LCK cells (p < 0.05). However, no significant changes of Mx (myxovirus resistant protein), IL-1b, janus kinase (JAK)2, signal transducers and activators of transcription (STAT)3 and STAT5 in LCK cells was detected after LcIL-6 overexpression or recombinant LcIL-6 protein stimulation. Our results indicated that LcIL-6 might be important in large yellow croaker immune response and improve the inflammatory response by through activation TNF-a expression. Keywords: Interleukin-6; gene structure; large yellow croaker; immune challenge; overexpression; recombinant expression; inflammatory response (This research was supported by NSFC (31101882; 41276178) and 973 Program (2011CB111604) to C.L.Y) * Corresponding author. E-mail address: [email protected] (C.-L. Yao). y Both authors have the same contribution.
O-058. Identification and functional characterization of two granulocyte colony-stimulating factors of common carp Fumihiko Katakura§, Erika Hino, Kohei Nishiya, Jiro Miyamae, Tadaaki Moritomo Department of Veterinary Medicine, Nihon University, Fujisawa, Kanagawa, Japan Abstract Granulocyte colony-stimulating factor (GCSF) is a cytokine that regulates the proliferation, differentiation, survival and immunological activation of myeloid cells such as neutrophils and monocytes, and their progenitors in mammals. GCSF is used as a recombinant therapeutics against mammalian
neutropenia to prevent infectious diseases. However, functions of GCSF are yet to be deeply characterized in lower vertebrates. Analysis of carp genome showed that carp have two paralogous GCSF molecules, GCSFa and GCSFb, similar to other teleosts, presumably resulting from a whole genome duplication event in teleosts. These molecules were predicted to possess functional domains and structures, although there are quite low sequence identities between vertebrate species. Expression analysis of carp gcsfa and gcsfb revealed the highest gcsfa mRNA levels in spleen, while the highest gcsfb mRNA levels in heart. Both GCSFa and GCSFb appear to be involved in immunity based on their up-regulation in kidney leukocytes following in vitro stimulation of lipopolysaccharide and a combination of Concanavalin A and Phorbol myristate acetate. Additionally, recombinant carp GCSFa and GCSFb stimulated the proliferation and the colony-formation of carp kidney leukocytes in a dose dependent manner, suggesting that both ligands play a role as a hematopoietic growth factor. Furthermore, recombinant GCSFb induced the development of morphologically mature neutrophils and up-regulated mRNA expression of GCSF receptor in the kidney leukocytes. Our results indicate that carp GCSFs are involved in the immune system and at least GCSFb has parallel functions to mammalian GCSF as a regulator of neutrophilic myelopoiesis. Keywords: Granulocyte colony-stimulating factor; Common carp; Hematopoiesis; Neutrophils; In vitro x Corresponding author. Tel.: þ81 (0)466 84 3443; Fax: þ81 (0)466 84 3374. E-mail address: [email protected] (F. Katakura).
O-059. RNA-Seq analysis in gills of atlantic salmon (Salmo salar) infected with newly identified ISAV strains in the atlantic Canada region Francis LeBlanc 1, Steven Leadbeater 2, Mark Laflamme 1, Nellie Gagn e 1,* 1 Fisheries & Oceans Canada, Gulf Fisheries Center, Moncton, NB, Canada 2 Fisheries & Oceans Canada, St Andrews Biological Station, St Andrews, NB, Canada
The Infectious salmon anemia virus (ISAV) is an important viral disease of farmed Atlantic salmon (Salmo salar) which has caused severe financial losses for salmon farmers around the world, including Atlantic Canada. It is listed as an OIE notifiable disease and to this day eradication of infected cages remains the current practice in many countries to mitigate the spread of the virus. All strains known to display any level of virulence are characterised by deletions in the highly polymorphic region (HPR) of the hemagglutinin-esterase (HE) protein compared to ISAV-HPR0 strains and are designated ISAV-HPRD. In contrast, HPR0 strains are non-virulent and not detected by viral culture. In Atlantic Canada, improved management and husbandry practices resulted in no outbreaks caused by ISAV-HPRD strains from 2007 to 2012, with however ISAV-HPR0 strains (European genotype) still being detected. Starting in 2012, new ISAV-HPRD strains were responsible for disease outbreaks in fish farms in Nova Scotia, Newfoundland and New-Brunswick. This study was thus designed to characterize virulence associated to three of the newly identified ISAVHPRD strains and investigate gene expression responses in gills of infected fish using RNA-Seq. All three ISAV-HPRD strains studied through the use of in vivo challenges resulted in cumulative mortalities below 40 percent and caused the differential expression of up to 1000 genes at the peak of infection compared to non-infected fish. Many genes linked to innate immunity were over-expressed; similar to what had been observed in the head-kidney through previous work. The use of RNA-Seq also enabled us to look at ISAV transcript abundance and showed that segment 8 had the highest abundance of all 8 segments of the genome. This information has given us great insight on how ISAV interacts with his host during the course of an infection. Keywords: Atlantic salmon, ISAV, RNA-Seq, Transcriptome, ISAV transcript abundance