Functional characterization of TNF‐α in pufferfish (Takifugu obscurus) in immune response and apoptosis against Aeromonas hydrophila

Abstract

Tumour necrosis factor‐α (TNF‐α) is a multifunctional cytokine involved in immune system homeostasis, antimicrobial defence, regulation of apoptosis, cell proliferation and differentiation. Although the pro‐inflammatory property of TNF‐α has been made new progress, detailed research on host defence against bacterial infection and inducing apoptosis remains to be revealed in early vertebrates. Here, we reported the TNF‐α homologue (ToTNF‐α) from pufferfish (Takifugu obscurus). The open reading frame (ORF) of ToTNF‐α was 753 bp, encoding a protein of 250 aa contained the TNF family signature and conserved cysteine residues. The mRNA expression of ToTNF‐α had a wide range of tested tissues, with the highest expression in the skin. After Aeromonas hydrophila infection, the mRNA expression of ToTNF‐α was significantly up‐regulated both in vivo and in vitro experiments. After stimulation by recombinant protein of ToTNF‐α ((r)ToTNF‐α), the relative expressions of endogenous TNF‐α, caspase 8, caspase 3, p53, and Bax inhibitor‐1 in head kidney leucocytes were all notably up‐regulated. These results showed that ToTNF‐α might induce apoptosis depend on pro‐ and anti‐apoptotic proteins at mRNA level. Moreover, flow cytometry analysis indicated that the (r)ToTNF‐α can induce apoptosis of head kidney leucocytes. Taken together, these characteristics suggest that ToTNF‐α can participate in immune response against A. hydrophila and induce apoptosis at mRNA and cellular level, which will help to understand the mechanism of apoptosis and immune response in teleost fish.

Nuclear Factor-κB Plays a Positive Role in TNFα Expression in Golden Pompano, Trachinotus ovatus (Linnaeus 1758)

“…In the present study, ToTNFα transcripts were constitutively expressed in all tested tissues of healthy fish, which was similar to the expression of TNFα in other teleosts (Hirono et al, 2000;García-Castillo et al, 2002;Zou et al, 2003;Savan and Sakai, 2004;Hong et al, 2013;Biswas et al, 2015;Cui et al, 2020;Huang et al, 2020;Kong et al, 2021;Ko et al, 2022). The mRNA level of ToTNFα was rich in the head-kidney, gill, skin and small intestine and poor in the stomach, liver and blood.…”

“…Previous studies have declared that viruses [grass carp reovirus (GCRV)], poly I: C, and bacteria (LPS, Vibrio alginolyticus, Photobacterium damselae, Aeromonas schubertii, A. hydrophila, Nocardia seriolae) and peptidoglycan can generate antiviral and antibacterial responses in teleosts, where NF-κB and TNFα play a role (Nascimento et al, 2007;Wang et al, 2013;Huang et al, 2020;He et al, 2021;Kong et al, 2021). Nevertheless, in fish, few studies have been performed to explain the role of TNFα in response to parasite stimulation.…”

“…In fish, TNFα has been found in more than 20 species, such as in important seawater species, Japanese flounder (Paralichthys olivaceus) (Hirono et al, 2000), gilthead seabream (Sparus aurata) (García-Castillo et al, 2002), rainbow trout (Oncorhynchus mykiss) (Zou et al, 2002;Hong et al, 2013), catfish (Ictalurus punctatus) (Zou et al, 2003), turbot (Psetta maxima) (Ordás et al, 2007), sea bass (Dicentrarchus labrax) (Nascimento et al, 2007), bluefin tuna (Thunnus orientalis) (Kadowaki et al, 2009), striped trumpeter (Latris lineata) (Covello et al, 2009), orange-spotted grouper (Epinephelus coioides) (Lam et al, 2011), rock bream (Oplegnathus fasciatus) (Kim et al, 2011;Ko et al, 2022), fugu (Fugu rubripes) (Biswas et al, 2015), large yellow croaker (Larimichthys crocea) (Huang et al, 2020), and pufferfish (Takifugu obscurus) (Kong et al, 2021), and in important freshwater species, common carp (Cyprinus carpio) (Forlenza et al, 2009;Zhao et al, 2012), zebrafish (Danio rerio) (Savan et al, 2005), ayu (Plecoglossus altivelis) (Uenobe et al, 2007), goldfish (Carassius auratus) (Grayfer et al, 2008), grass carp (Ctenopharyngodon idella) (Zhang et al, 2012), and snakehead (Channa argus) (Cui et al, 2020). Furthermore, two TNFα isoforms have been characterized in Atlantic bluefin tuna Thunnus thynnus (Lepen Pleićet al, 2014), T. orientalis (Kadowaki et al, 2009), E. coioides (Lam et al, 2011), and C. auratus (Grayfer et al, 2008), three TNFα isoforms in O. mykiss (Hong et al, 2013), and even four in C. carpio (Savan and Sakai, 2004;Zhao et al, 2012).…”

Studies of effector molecules exerting autonomous and nonautonomous influence of T lymphocyte apoptosis under the conditions of in vitro “cell neighborhood” in healthy people and patients with rheumatoid arthritis

“…Существуют различные данные, выявляющие помимо основных, автономных функций TNFαмногофункционального цитокина, участвующего в гомеостазе иммунной системы, антимикробной защите, регуляции апоптоза, пролиферации и дифференцировке клеток, обеспечивать некоторые неавтономные процессы [19] Одним из возможных вариантов индукции пролиферации вместо апоптоза в стимулированных каспазой-8 лимфоцитах может быть компартментальная активация фермента, приводящая к активации NF-κB и ядерной транслокации [29]. Помимо пролиферации, которая является установленным неавтономным следствием активации каспазы, а митогенные сигналы генерируются каспаз-экспрессирующими клетками, были описаны и другие зависимые от каспаз исходы.…”

Effects on grass carp ( Ctenopharyngodon idella ) induced by waterborne lead exposure and the association with alteration of microbiota in the intestine and water

“…The total RNA from all samples was extracted with Trizol reagent (Vazyme, China) (Fu et al, 2020), and its quality and concentration were detected using electrophoresis in 1% agarose gel and NanoDrop 2000 spectrophotometer (NanoDrop Technologies, USA) (Kong et al, 2021). Next, RNA was reversely transcribed to cDNA using PrimeScript® RT reagent Kit with gDNA Eraser (Takara, Japan).…”