IRF10 was reported to play important roles in immune responses to both viral and bacterial infections in teleost, which can inhibit the activation of IFN promoters and negatively regulated fish antiviral genes expression to avoid an excessive immune response [10, 11, 32]. In the present study, a non-mammalian IRF10 gene was cloned from the common carp, and the antiviral and antibacterial immune function of CcIRF10 was investigated. The predicted CcIRF10 protein contained two conserved functional domains, the N-terminus DBD and the C-terminus IAD, suggesting that the functions of IRF10 may be conserved throughout the vertebrates. The DBD contains a highly conserved five tryptophan repeat, which can bind to the IFN stimulating response element (ISRE) and IRF regulatory element (IRF-E) in target promoters [4, 33]. It is noteworthy that, the five well conserved tryptophan residues in D. rerio, C. idella, G. gallus, as well as C. carpio were all at position 10, 25, 37, 57 and 76 in the N terminus. Different from the DBD, the IAD is less conserved, which is responsible for homo/hetero-dimers interactions of the IRFs and association with other transcription factors [5]. Phylogenetic analysis of predicted IRF10 protein sequences of C. carpio and other vertebrate species supported the division of IRF10 into two branches: teleosts and bird. These results matched the established evolutionary relationships among the teleost and other vertebrate species and supported the authenticity of the nomenclature for IRF10.
IRF10 was first identified in G. gallus and highly expressed in white blood cells and splenic lymphocytes, whereas low expression levels were found in other tissues [8]. In the present study, constitutive expression of CcIRF10 gene was detectable in all the eleven tissues of C. carpio when analysed by Real-time PCR, despite differences at the level of expression. This ubiquitous tissue expression pattern supported previous studies of IRFs in teleost, including M. albus [17], G. morhua [13], D. rerio [34], O. mykiss [35], turbot (Scophthalmus maximus) [36], P. olivaceus [12, 37], rock bream (Oplegnathus fasciatu) [38], blunt snout bream (Megalobrama amblycephala) [39] and half-smooth tongue sole (Cynoglossus semilaevis) [40]. CcIRF10 was found to be most highly expressed in gonad (Fig. 3), which was different from the P. solivaceus, C. idella and M. albus. In P. solivaceus, the expression of IRF10 was very high in gills, intestine, trunk kidney, heart, stomach, head kidney and PBLs; in C. idella, IRF10 expression was high in all tested tissues with the highest expression in thymus and gills; in M. albus, the highest expression level was observed in intestine, whereas the lowest level was found in liver [12, 15, 17]. However, D. rerio IRF10 was highly expressed in testis, which is also a reproductive organ [9]. These results suggested that IRF10 may not only play an important role in the immune system, but also likely participate the regulation of reproductive system in teleost.
Previous studies in C. carpio showed that expression of IRF1, IRF3, IRF5 and IRF7 were up-regulated upon stimulation with poly I:C or viruses [25–27]. In the present study, following poly I:C injection, CcIRF10 in the foregut (27.5-fold) is much stronger than the other tissues (4.5 to 7.5-fold), revealing the important role of CcIRF10 in the mucosal immune system against poly I:C (Fig. 4). Moreover, the similar results were observed in PBLs and HKLs with poly I:C stimulation (Fig. 6). Expression of IRF10 in P. solivaceus was also up-regulated by poly I:C stimulation in PBLs [12]. In G. morhua, two isoforms of IRF10 had been identified, and expression of the long isoform reached the peak at 6 hpi at 16 °C and 24 hpi at 10 °C, whereas the highest expression of short isoform was observed at 6 hpi at both 16 °C and 10 °C after poly I:C stimulation [13, 41]. Moreover, IRF10 of P. solivaceus, C. idella, M. albus and zebrafish embryo fibroblast-like ZF4 cells can be up-regulated by virus [viral hemorrhagic septicemia virus (VHSV) or grass carp haemorrhagic virus (GCHV)] or poly I:C [9, 12]. The observed expression of IRF10 induced by various viruses and poly I:C suggested that fish IRF10 may play a crucial role in protecting host from viral infection.
A. hydrophila, a well-known fish pathogenic bacterium, is primarily found in temperate and freshwater environments and causes infections in various organisms. Fish are becoming more and more susceptible to A. hydrophila, because of the increasing intensive rearing methods used in the aquaculture [42]. Moreover, A. hydrophila breakouts have caused great economic losses around the world [43]. In order to gain insights into the immune mechanism of CcIRF10 in antibacterial response, its expression pattern in response to A. hydrophila was investigated using Real-time PCR. When challenged with A. hydrophila, the levels of CcIRF10 were up-regulated in all four tissues with the highest induction in the foregut (Fig. 4). IRF10 of P. solivaceus can also be induced by Edwardsiella tarda and Streptococcus iniae [37]. Upon Aeromonas salmonicida infection, the greatest expression of long isoform of G. morhua IRF10 was occurred at 24 hpi, whereas the highest expression of short isoform was observed at 6 hpi at 10 °C and 16 °C, suggesting the distinct roles of the two isoforms in the immune system of G. morhua [13, 41]. It should be noted that E. coioides IRF10 was responsive to both poly I:C stimulation and Vibrio parahemolyticus infection, with a higher increase to poly I:C [10]. In accordance with their results, our study showed that the fold change of CcIRF10 induced by poly I:C (4.5 to 27.5-fold) stimulation was higher than the A. hydrophila (3.0 to 13.7-fold) infection. LPS is the major component of the outer membrane of Gram-negative bacteria, but it has been reported that lower vertebrates (such as fish) may be resistant to the toxic effects of LPS [44]. Therefore, it is not strange that CcIRF10 was not up-regulated by LPS in the PBLs (Fig. 6A). PGN, a major component of the bacterial cell wall, consists of sugars and amino acids. Similar with the study in head kidney macrophages of O. mykiss, CcIRF10 was up-regulated by PGN stimulation in vitro [15]. Hence, our in vivo and in vitro findings, together with these analogous results, suggesting that CcIRF10 was more susceptible to the poly I:C compared with the A. hydrophila infection, and played a substantial role in the foregut which is a mucosal immune organ. Moreover, fish IRF10 may play an essential role in both the antiviral and antibacterial defence as reported for G. gallus IRF10.
In mammals, IFNs are natural glycoproteins produced by the cells of the innate and adaptive immune systems in most vertebrates in response to challenge by viruses, bacteria, fungi, parasites, as well as by tumour cells [45]. Besides, IFNs can also be produced by non-immune cells such as fibroblasts and epithelial cells [45]. Similarly, fish IFNs also play a crucial role in the innate immunity [46, 47]. In order to investigate the regulatory role of CcIRF10 in the IFN response, we detected the mRNA expression of type I IFN, ISGs (PKR and ISG15) and TNFα in CcIRF10-transfected EPC cells. The transcription of PKR, ISG15 and TNFα was down-regulated in EPC cells transfected with pcIRF10 plasmid. This is in line with the previous study that overexpression of D. rerio IRF10 down-regulated the expression of IFN-related genes induced by poly I:C and promoted the replication of Spring Viremia of Carp Virus (SVCV) in EPC cells [9]. Moreover, functional domain analysis of DrIRF10 showed that DBD and IAD were sufficient for inhibiting the activation of IFN induced by poly I:C and retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) [9]. However, G. gallus IRF10 can up-regulate the expression of MHC class I and GBP [8]. These results suggested the function of IRF10 between fish and bird might be different.