Nuclear factor interleukin 3 (NFIL3) participates in regulation of the NF-κB-mediated inflammation and antioxidant system in Litopenaeus vannamei under ammonia-N stress

Nuclear factor interleukin 3 (NFIL3) is a critical upstream regulator of the NF-κB pathway. Nevertheless, the detailed molecular mechanism of NFIL3 and its function in shrimp have not been well characterized. In the present study, NFIL3 was identified and characterized from Litopenaeus vannamei. Molecular feature analysis revealed that the open reading frame (ORF) of LvNFIL3 was 2963 bp, which codes for a polypeptide of 516 amino acids with a conserved basic region leucine zipper (bZIP) domain. Sequence alignments and phylogenetic tree analysis showed that the amino acid sequence of LvNFIL3 shared 18.82%-98.07% identity with that of NFIL3 in other species, and was closely related to Penaeus monodon NFIL3. A core promoter in the 5' flanking region of LvNFIL3 was essential for regulation of transcription. LvNFIL3 mRNA was highly expressed in gills and hepatopancreas. Subcellular localization of the protein was observed almost exclusively in the nucleus. Amplification of mRNA by RT-qPCR showed that LvNFIL3 was induced in shrimp gills, hepatopancreas, and muscle after ammonia-N stress. Moreover, silencing of LvNFIL3 increased the mortality of shrimp exposed to ammonia-N. Furthermore, dual-luciferase reporter assay data suggested that LvNFIL3 was capable of activating the NF-κB pathway. Conversely, knockdown of LvNFIL3 decreased NF-κB homolog (Dorsal and Relish) and IkB homolog (Cactus) expression, as well as expression of anti-inflammatory cytokine (IL-16) and five antioxidant-related genes (HO-1, Mn-SOD, CAT, GPx, and GST), whereas NF-κB repressing factor (NKRF) and inflammation-related genes (TNFα and Spz) were upregulated. More importantly, LvNFIL3 knockdown exacerbated the pathology in hepatopancreas exposed to ammonia-N, and the total antioxidant capacity (T-AOC) and superoxide dismutase (T-SOD) were significantly decreased, resulting in a significant increased lipid peroxidation and protein carbonization. Taken together, these data suggest that LvNFIL3 was involved in ammonia-N tolerance in L. vannamei by regulating the inflammation and antioxidant system through the NF-κB pathway.