Enhancing immune responses in Japanese flounder (Paralichthys olivaceus) to Keyhole Limpet Hemocyanin by intramuscularly conjugating a new type of chemokine, JfCXCL8_L1b

Abstract

CXCL8 is a high-profile chemokine in vertebrates. In our previous study, CXCL8_L1b was first identified in Japanese flounder and triggered varying immune responses in infected fish compared with Japanese flounder CXCL8_L1a (JfCXCL8_L1a). To further understand the immune functions of Japanese flounder CXCL8_L1b (JfCXCL8_L1b), in vitro and in vivo experiments were performed using the recombinant protein rCXCL8_L1b. Our results showed that rCXCL8_L1b significantly induced all the detected immune genes except IgM and IgD in peripheral blood leukocytes (PBLs) of Japanese flounder. Intramuscular injection of Keyhole Limpet Hemocyanin (KLH) did not cause inflammation responses in fish muscle. In the spleen of injected fish, IgM was the only detected gene expression. In contrast, all detected genes in the KLH group were significantly up-regulated in the head kidney, indicating the primary immune organ response to KLH was the head kidney instead of the spleen. At the injection site, we observed significantly induced receptor gene CXCR1 in KLH + rCXCL8_L1a, KLH + rCXCL8_L1b, and KLH + rCXCL8_L1a + rCXCL8_L1b groups on day 1, while significantly induced receptor gene CXCR2 was only observed in the KLH + rCXCL8_L1b group on day 3. Conversely, gene expression of JfCXCL8_L1a and CXCR2 were down-regulated in the head kidney of the KLH + rCXCL8_L1b group compared with the KLH + rCXCL8_L1a group. Intramuscular injection of KLH significantly induced an antibody titer response to KLH at weeks 2 and 4. Compared with rCXCL8_L1a, rCXCL8_L1b, as the immunologic adjuvant of KLH, significantly enhanced the antibody titer response to KLH at weeks 2 and 4. Collectively, this study reveals that JfCXCL8_L1a and JfCXCL8_L1b have different immune pathways, and JfCXCL8_L1b plays a significant role in enhancing the adaptive immunity of T cell-dependent antigen.