CXC chemokines are primarily expressed in immune organs
CXC chemokines are primarily expressed in immune organs and the central nervous system: the mouse thymus and spleen (Heesen et al., 1996), chicken bursa (Liang et al., 2001), primate (Macaca mulatta) Vanoxerine sale (Federsppiel et al., 1993) and cattle locus coeruleus, cerebellum and pons (Rimland et al., 1991; Jia and Zhang, 2009). In orange-spotted grouper, most CXCL and CXCR genes were primarily expressed in immune-relevant tissues. As shown in Fig. 4, CXCL2, CXCL12, CXCL13, CXCL14, CXCL11, CXCL19, CXCR1b, CXCR4a and CXCR3 (CXCLR3.1, CXCLR3.2 and CXCLR3.3) were found to be ubiquitously expressed, with relatively high expression levels, suggesting their basic and important roles in immunologic function. Some genes, such as CXCL12b, CXCL13 and CXCL14 in the brain, CXCL19 in the gonad and CXCR3.2 in the heart, exhibited relatively high expression levels, suggesting that these family members evolved toward specific functions in relevant tissues. CXC chemokines not only have vital roles in development and homeostasis but also participate in inflammatory responses in humans (Zlotnik and Yoshie, 2000, 2012; Raman et al., 2011). Conversely, the involvement of CXC chemokines in immune responses in fish species has only been sporadically reported (Xu et al., 2010; Jia and Zhang, 2009; Fu et al., 2017a, 2017b). Therefore, we examined the expression profiles of 9 CXC chemokine ligands and 8 receptors following SGIV infection in orange-spotted grouper, and observed notable expression patterns of the former in resistant and susceptible fish after SGIV infection. For example, CXCL8 and CXCL12a were significantly upregulated after SGIV infection in the spleen, kidney or liver of resistant fish. These results indicate that high expression of these CXC chemokines might be beneficial to the host, enhancing its immunity against the pathogen without tissue damage. As shown in Fig. 6 B and Fig. 7 B, both the spleen and kidney of resistant fish showed significantly increased levels of CXCR1b and CXCR4a after SGIV infection, whereas CXCR3.3 and CXCR4a were upregulated in the liver of these fish. In general, CXCR genes, but not all CXC chemokines, were induced after SGIV infection; as an example, CXCR1a was downregulated after infection (Fig. 8 B). A recent study showed that CXCR4 plays a role in the immune response to microbial infection (Lin et al., 2012), though the exact mechanism by which CXCR1a and CXCR1b exert their beneficial effect in teleost fish remains to be elucidated. Therefore, in general, caution must be exercised when comparing gene expression. Nonetheless, some highly differentially expressed genes in these fish with drastically different resistance phenotypes may provide insight into genes that are potentially important for disease resistance. Most chemokine receptors are promiscuous because each can bind with high affinity to multiple chemokine ligands, and there is also a high degree of redundancy in the chemokine family, as multiple chemokines bind to the same receptor (Bieche et al., 2007; Sun et al., 2011). Some chemokines bind to multiple receptors, and some receptors in turn bind multiple chemokines; conversely, certain chemokines bind to only one or two chemokine receptors. Among the CXCL8/CXCR2, CXCL8/CXCR1, CXCL12/CXCR4, CXCL12/CXCR7, CXCL11/CXCR3, CXCL10/CXCR3 and CXCL9/CXCR3 interactions, that of CXCL12/CXCR4 in humans has a critical role in immune regulation (Huskens et al., 2007; Nakase et al., 2012; Aboumrad et al., 2007; Belperio et al., 2004; Sun et al., 2011). Regardless, research on chemokine ligands and receptor binding in fish species is currently limited. In this study, we found that expression of the CXC chemokine ligand CXCL12a was significantly higher in all three organs (spleen, kidney and liver) of resistant fish compared to susceptible fish, as was expression of CXCR4a. We conclude that CXCL12a/CXCR4a binding has immune regulatory functions in orange-spotted grouper, though the exact mechanism requires future studies. In addition, CXCL8 expression in the spleen, kidney and liver was similar to that of CXCR4 after SGIV infection; therefore, these molecules may play a role in combination with each other. The newly identified CXC chemokine receptor CXCR1b was highly expressed in the spleen and kidney of resistant fish, which was consistent with expression of the ligands CXCL8 and CXCL12a. Based on the complicated mechanisms of CXC chemokine ligand receptor binding (Sun et al., 2011), we hypothesize that CXCR1b binds CXCL8 or CXCL12a ligands to exert its beneficial effect in teleost fish. However, the exact mechanisms require further analysis.