Abstract
As a potent pleiotropic cytokine, tumor necrosis factor-alpha (TNF-α) plays an important role in innate immune responses. The cDNA sequence and genomic structure of the TNF-α gene (Aj TNF-α) in the Japanese eel (Anguilla japonica) were identified and characterized. The full-length AjTNF-α cDNA was 1 546 bp, including a 5′-untranslated region (UTR) of 13 bp, a 3′-UTR of 879 bp and an open reading frame of 654 bp encoding a protein of 218 amino acids. The full-length genomic sequence of AjTNF-α was 2 392 bp and included four exons and three introns. The putative AjTNF-α protein contained TNF family signature motifs, including a protease cleavage site, a transmembrane domain and two conserved cysteine residues. Quantitative real-time reverse transcription PCR analysis revealed AjTNF-α expression in a wide range of tissues, with predominant expression in blood and liver. Lower levels of expression were seen in spleen, gills, kidney, intestine, heart, and skin, with very low levels in muscle. The modulation of AjTNF-α expression after injection of eels with lipopolysaccharide (LPS), the viral mimic, poly I:C, or Aeromonas hydrophila was assessed in blood, liver, and kidney. In blood, TNF-α mRNA levels increased rapidly and then rapidly decreased after stimulation with LPS, poly I:C or A. hydrophila. However, the response to LPS and A. hydrophila peaked at 6 h while for poly I:C the peak was at 12 h. In liver, after injection with A. hydrophila, an up- and down-regulation of AjTNF-α expression occurred twice, peaking at 6 h and 24 h, respectively. No remarkable increase of AjTNF-α expression appeared in liver until 72 h after LPS or poly I:C treatment. In kidney, AjTNF-α expression increased significantly only at 72 h post-stimulation with LPS or A. hydrophila. Our results suggest that AjTNF-α plays an important role in fish in the defense against viral and bacterial infection.
Similar content being viewed by others
References
Aggarwal B B, Gupta S C, Kim J H. 2012. Historical perspectives on tumor necrosis factor and its superfamily: 25 years later, a golden journey. Blood, 119: 651–665.
Basu M, Swain B, Sahoo B, Maiti N, Samanta M. 2012. Induction of toll-like receptor (TLR)2, and MyD88-dependent TLR-signaling in response to ligand stimulation and bacterial infections in the Indian major carp, mrigal (Cirrhinus mrigala). Mol. Biol. Rep., 39: 6 015–6 028.
Branch D R, Guilbert L J. 1996. Differential expression of tumor necrosis factor-α isoforms from lipopolysaccharide and cytokine-stimulated mouse macrophages. Int. J. Biochem. Cell Biol., 28: 949–955.
Brook M, Sully G, Clark A R, Saklatvala J. 2000. Regulation of tumour necrosis factor α mRNA stability by the mitogen-activated protein kinase p38 signalling cascade. FEBS Lett., 483: 57–61.
Caput D, Beutler B, Hartog K, Thayer R, Brown-Shimer S, Cerami A. 1986. Identification of a common nucleotide sequence in the 3′-untranslated region of mRNA molecules specifying inflammatory mediators. Proc. Natl. Acad. Sci., 83: 1 670–1 674.
Covello J M, Bird S, Morrison R N, Battaglene S C, Secombes C J, Nowak B F. 2009. Cloning and expression analysis of three striped trumpeter (Latris lineate) pro-inflamatory cytokines, TNF-α, IL-1β and IL-8, in response to infection by the ectoparasitic, Chondracanthus goldsmidi. Fish Shellfish Immunol., 26: 773–786.
Garcia-Castillo J, Chaves-Pozo E, Olivares P, Pelegin P, Meseguer J, Mulero V. 2004. The tumor necrosis factor a of the bony fish seabream exhibits the in vivo proinflammatory and proliferative activities of its mammalian counterparts, yet it functions in a speciesspecific manner. Cell. Mol. Life Sci., 61: 1 331–1 340.
Garcia-Castillo J, Pelegrin P, Mulero V, Meseguer J. 2002. Molecular cloning and expression analysis of tumor necrosis factor a from a marine fish reveal its constitutive expression and ubiquitous nature. Immunogenetics, 54: 200–207.
Garlanda C, Bottazzi B, Bastone A, Mantovani A. 2005. Pentraxins at the crossroads between innate immunity, inflammation, matrix deposition, and female fertility. Annu. Rev. Immunol., 23: 337–366.
Gerlier D, Lyles D S. 2011. Interplay between innate immunity and negative-strand RNA viruses: towards a rational model. Mol. Biol. Rev., 75: 468–490.
Goetz F W, Planas J V, MacKenzie S. 2004. Tumor necrosis factors. Dev. Comp. Immunol., 28: 487–497.
Grayfer L, Walsh J G, Belosevic M. 2008. Characterization and functional analysis of goldfish (Carassius auratus L.) tumor necrosis factor-alpha. Dev. Comp. Immunol., 32: 532–543.
Guo S L, Feng J J, Xiong J, Guan R Z. 2011. Immunohistochemistry distribution and pathological characteristics of Aeromonas hydrophila in several organs of European eel (Anguilla anguilla) after artificial infection. J. Huazhong Agric. Univ., 30: 494–499. (in Chinese with English abstract)
Guo S L, Feng J J, Yang Q H, Guan R Z, Wang Y, Lu P P. 2012. Immune effects of bathing European eels in live pathogenic bacteria, Aeromonas hydrophila. Aquacult. Res., http://dx.doi.org/10.1111/are.12035.
Hirono I, Nam B H, Kurobe T, Aoki T. 2000. Molecular cloning, characterization, and expression of TNF cDNA and gene from Japanese flounder Paralychthys olivaceus. J. Immunol., 165: 4 423–4 427.
Iliev D B, Roach J C, Mackenzie S, Planas J V, Goetz F W. 2005. Endotoxin recognition: in fish or not in fish? FEBS Lett., 579: 6 519–6 528.
Kadowaki T, Harada H, Sawada Y, Kohchi C, Soma G I, Takahashi Y, Inagawa H. 2009. Two types of tumor necrosis factor-α in bluefin tuna (Thunnus orientalis) genes: molecular cloning and expression profile in response to several immunological stimulants. Fish Shellfish Immunol., 27: 585–594.
Kisich K O, Higgins M, Diamond G, Heifets L. 2002. Tumor necrosis factor alpha stimulates killing of Mycobacterium tuberculosis by human neutrophils. Infect. Immun., 70: 4 591–4 599.
Kumar H, Kawai T, Akira S. 2011. Pathogen recognition by the innate immune system. Int. Rev. Immunol., 30: 16–34.
Laing K J, Wang T, Zou J, Holland J, Hong S, Bols N, Hirono I, Aoki T, Secombes C J. 2001. Cloning and expression analysis of rainbow trout Oncorhynchus mykiss tumour necrosis factor-α. Eur. J. Biochem., 268: 1 315–1 322.
Lam F W S, Wu S Y, Lin S J, Lin C C, Chen Y M, Wang H C, Chen T Y, Lin H T, Lin J H Y. 2011. The expression of two novel orange-spotted grouper (Epinephelus coioides) TNF genes in peripheral blood leukocytes, various organs, and fish larvae. Fish Shellfish Immunol., 30: 618–629.
Lee W C, Chen Y H, Lee Y C, Liao I C. 2003. The competitiveness of the eel aquaculture in Taiwan, Japan, and China. Aquaculture, 221: 115–124.
Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using Real-Time Quantitative PCR and the 2-ΔΔCt method. Methods, 25: 402–408.
Locksley R M, Killeen N, Lenardo M J. 2001. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell, 104: 487–501.
Medzhitov R. 2007. Recognition of microorganisms and activation of the immune response. Nature, 449: 819–826.
Mizui Y, Yamazaki K, Sagane K, Tanaka I. 1999. cDNA cloning of mouse tumor necrosis factor-α converting enzyme (TACE) and partial analysis of its promoter. Gene, 233: 67–74.
Mladineo I, Block B A. 2010. Expression of cytokines IL-1β and TNF-α in tissues and cysts surrounding Didymocystis wedli (Digenea, Didymozoidae) in the Pacific bluefin tuna (Thunnus orientalis). Fish Shellfish Immunol., 29: 487–493.
Morgan M J, Kim YS, Liu Z. 2008. TNFα and reactive oxygen species in necrotic cell death. Cell Res., 18: 343–349.
Morrison R N, Zou J, Secombes C J, Scapigliati G, Adams M B, Nowak B F. 2007. Molecular cloning and expression analysis of tumour necrosis factor-α in amoebic gill disease (AGD)-affected Atlantic salmon (Salmo salar L.). Fish Shellfish Immunol., 23: 1 015–1 031.
Nakamoto T, Yoshimura H, Honda T, Nakata K, Taniguchi Y, Yoshida A, Uenobe M, Yoshioka N, Yamaguchi T, Inagawa H. 2007. Treatments for the activating macrophages that reduces surgical stress and postoperative mortalities from bacterial infections and tumor metastases. In Vivo, 21: 357–364.
Nascimento D S, Pereira P J B, Reis M I R, do Vale A, Zou J, Silva M T, Secombes C J, dos Santos N M S. 2007. Molecular cloning and expression analysis of sea bass (Dicentrarchus labrax L.) tumor necrosis factor-α (TNF-α). Fish Shellfish Immunol., 23: 701–710.
Navarro L, Alto N M, Dixon J E. 2005. Functions of the Yersinia effector proteins in inhibiting host immune responses. Curr. Opin. Microbiol., 8: 21–27.
Ordás M C, Costa M M, Roca F J, López-Castejón G, Mulero V, Meseguer J, Figueras A, Novoa B. 2007. Turbot TNFα gene: molecular characterization and biological activity of the recombinant protein. Mol. Immunol., 44: 389–400.
Pérez-Sánchez T, Balcázar J L, Merrifield D L, Carnevali O, Gioacchini G, de Blas I, Ruiz-Zarzuela I. 2011. Expression of immune-related genes in rainbow trout (Oncorhynchus mykiss) induced by probiotic bacteria during Lactococcus garvieae infection. Fish Shellfish Immunol., 31: 196–201.
Poisa-Beiro L, Dios S, Ahmed H, Vasta G R, Martínez-López A, Estepa A, Alonso-Gutiérrez J, Figueras A, Novoa B. 2009. Nodavirus infection of sea bass (Dicentrarchus labrax) induces up-regulation of galectin-1 expression with potential anti-inflammatory activity. J. Immunol., 183: 6 600–6 611.
Praveen K, Evans D L, Jaso-Friedmann L. 2006. Constitutive expression of tumor necrosis factor alpha in cytotoxic cells of teleost fishs and its role in regulation of cellmediated cytotoxicity. Mol. Immunol., 43: 279–291.
Raida M K, Buchmann K. 2008. Bath vaccination of rainbow trout (Oncorhynchus mykiss Walbaum) against Yersinia ruckeri: Effects of temperature on protection and gene expression. Vaccine, 26: 1 050–1 062.
Roca F J, Mulero I, López-Muñoz A, Sepulcre M P, Renshaw S A, Meseguer J, Mulero V. 2008. Evolution of the inflammatory response in vertebrates: Fish TNF-α is a powerful activator of endothelial cells but hardly activates phagocytes. J. Immunol., 181: 5 071–5 081.
Saeij J P J, Stet R J M, de Vries B J, van Muiswinkel W B, Wiegertjes G F. 2003. Molecular and functional characterization of carp TNF: a link between TNF polymorphism and trypanotolerance? Dev. Comp. Immunol., 27: 29–41.
Savan R, Kono T, Igawa D, Sakai M. 2005. A novel tumor necrosis factor (TNF) gene present in tandem with the TNF-α gene on the same chromosome in teleost fishs. Immunogenetics, 57: 140–150.
Wang W L, Hong J R, Lin G H, Liu W, Gong H Y, Lu M W, Lin C C, Wu J L. 2011. Stage-specific expression of TNFα regulates bad/bid-mediated apoptosis and RIP1/ROSmediated secondary necrosis in birnavirus-infected fish cells. PLoS One, 6: 1–11.
Watts T H. 2005. TNF/TNFR family members in costimulation of T cell responses. Annu. Rev. Immunol., 23: 23–68.
Xiao J, Zhou Z C, Chen C, Huo W L, Yin Z X, Weng S P, Chan S M, Yu X Q, He J G. 2007. Tumor necrosis factor-alpha gene from mandarin fish, Siniperca chuatsi. Molecular cloning, cytotoxicity analysis and expression profile. Mol. Immunol., 44: 3 615–3 622.
Xie F, Zhang Z, Lin P, Wang S, Zou Z, Wang Y. 2008. Cloning and infection response of tumour necrosis factor alpha in large yellow croaker Pseudosciaena crocea (Richardson). J. Fish Biol., 73: 1 149–1 160.
Yamamoto T, Batts W, Arakawa C, Winton J. 1990. Multiplication of infectious hematopoietic necrosis virus in rainbow trout following immersion infection: Wholebody assay and immunohistochemistry. J. Aquat. Anim. Health, 2: 271–280.
Zhang A, Chen D, Wei H, Du L, Zhao T, Wang X, Zhou H. 2012. Functional characterization of TNF-α in grass carp head kidney leukocytes: Induction and involvement in the regulation of NF-κB signaling. Fish Shellfish Immunol., 33: 1 123–1 132.
Zhang Z, Swain T, Bøgwald J, Dalmo R A, Kumari J. 2009. Bath immunostimulation of rainbow trout (Oncorhynchus mykiss) fry induces enhancement of inflammatory cytokine transcripts, while repeated bath induce no changes. Fish Shellfish Immunol., 26: 677–684.
Zhu L Y, Nie L, Zhu G, Xiang L X, Shao J Z. 2013. Advances in research of fish immune-relevant genes: A comparative overview of innate and adaptive immunity in teleost fishs. Dev. Comp. Immunol., 39: 39–62.
Zou J, Peddie S, Scapigliati G, Zhang Y, Bols N C, Ellis A E, Secombes C J. 2003a. Functional characterisation of the recombinant tumor necrosis factors in rainbow trout, Oncorhynchus mykiss. Dev. Comp. Immunol., 27: 813–822.
Zou J, Secombes C J, Long S, Miller N, Clem L W, Chinchar V G. 2003b. Molecular identification and expression analysis of tumor necrosis factor in channel catfish (Ictalurus punctatus). Dev. Comp. Immunol., 27: 845–858.
Zou J, Wang T, Hirono I, Aoki T, Inagawa H, Honda T, Soma G I, Ototake M, Nakanishi T, Ellis A E, Secombes C J. 2002. Differential expression of two tumor necrosis factor genes in rainbow trout, Oncorhynchus mykiss. Dev. Comp. Immunol., 26: 161–172.
Author information
Authors and Affiliations
Corresponding author
Additional information
Supported by the Ocean and Fishery Bureau of Fujian Province (No. 201212140006), the Scientific Research Foundation of Jimei University, China (No. ZQ2008013), the National Natural Science Foundation of China (No. 31272685), and the Program of the Education Department of Fujian Province (No. JA11150)
Rights and permissions
About this article
Cite this article
Feng, J., Guan, R., Guo, S. et al. Molecular cloning of Japanese eel Anguilla japonica TNF-α and characterization of its expression in response to LPS, poly I:C and Aeromonas hydrophila infection. Chin. J. Ocean. Limnol. 32, 1046–1059 (2014). https://doi.org/10.1007/s00343-014-3231-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00343-014-3231-2