Neuropeptide F interacts with its receptor and regulates the immune response of Sepiella japonica

In mammals, neuropeptide Y (NPY) has been recognized for its role in modulating the immune response of host. However, invertebrate neuropeptide F (NPF), as a homologous gene of NPY, has been minimally explored immunomodulatory function. In this study, NPF and NPF receptor (NPFR) mRNAs were significantly up-regulated in sick Sepiella japonica, and in juvenile S. japonica after bath infection of Vibrio harveyi. Subsequently, hemocytes of S. japonica were isolated, and Sj_NPF and Sj_NPFR mRNAs were notably induced in the hemocyte of S. japonica after Lipopolysaccharide (LPS) stimulation. Flow cytometry and pull-down assay confirmed the interaction between Sj_NPF and Sj_NPFR. Afterwards, knockdown of NPF notably decreased the mRNA expression of NPFR, Nitric oxide synthase (NOS), Toll-like receptor (TLR) and Interleukin-17-7 (IL-17-7) in the white body of S. japonica. Furthermore, after in vivo injection of NPF mature peptide, NPFR, NOS, TLR, TNF and IL-17-7 mRNAs were significantly up-regulated in the white body of S. japonica, with TNF and IL-17-7 mRNAs also showing significant induction in the liver of S. japonica. Interestingly, NPFR, TLR, MyD88 and IL-17-7 mRNAs were significantly decreased in the gill of S. japonica after in vivo injection of NPF mature peptide. Finally, the colocalization of Sj_NPF and Sj_NOS mRNAs was observed in the dorsal area of juvenile cuttlefish using whole-mount in situ hybridization (WISH), and NO content was increased at 24 h and 48 h in the liver after in vivo injection of NPF mature peptide. Collectively, these results uncover that the Sj_NPF may interact with its receptor and mediate immune response to eliminate invasive pathogens.