The cytokine network is an important homeostatic system in an organism’s body. It is actively contributing in immune surveillance, development, growth, and repair processes. The interleukin-1b (IL-1b) is considered a key pro-inflammatory cytokine, especially in host –defense in response to inflammation, injury and pathogenic invasion. IL-1β is produced and secreted by various types of cells including monocytes and macrophages. IL-1β is produced as a precursor called as pro-IL-1β in response to receptor binding of pathogen associated molecular patterns (PAMP’s). The pro-IL-1β is cleaved by the action of caspase-1. After the maturation mature IL-1β is secreted from cells.
In the current experiment, homologues sequence of IL-1β-like was identified from constructed cDNA library of rock fish. in-silico analysis of the respective nucleotide and amino acid sequences was carried out using appropriate bioinformatics tools and software. Tissue specific expression of the IL-1β-like gene was an
Tissue specific expression of IL-1β-like gene was analyzed by quantitative real time PCR (qPCR) using cDNA synthesized from twelve different tissues of fish. Healthy Rock fish discerned into four groups were individually immune challenged with Streptococcus iniae, Poly I: C, lipopolysaccharide (LPS) and phosphate buffered saline (PBS) as control. In different time points, selected tissues of the immunized fish were dissected, mRNA was extracted and cDNA was synthesized and used in qPCR to determine the expression of IL-1β-like gene under different immune challenges.
In- silico analysis revealed that the complete ORF of the IL-1β-like of rock fish consisted of 792 base pairs encoding 263 amino acids with a predicted molecular weight of 29.19 kDa. The multiple sequence alignment show that the IL-1 family signature is highly conserved through the evolution from fish to human. Similarly, amino acids which comprised in receptor binding sites are much conserved. 3D modeling of IL-1β-like indicate that IL-1 superfamily domain composed with β sheets and other parts are composed with α helixes.
Tissue specific mRNA expression examination indicate that spleen tissues give the highest expression of IL-1β-like gene. Second highest expression was observed in head kidney tissues. Both of the spleen and head kidney are very important immune tissues in fish immune system. In the immune challenge experiment, IL-1β-like gene has highly up regulated by LPS and poly I:C at the early time points of challenge and gradually reduced with time. The live Streptococcus iniae challenge cause in up regulation of the gene at the later hours of the challenge. This might due to increase the bacterial number with time which provide more stimulants to up regulate inflammatory cytokines. Collectively, another homolog of IL-1β gene from black rock fish was identified and characterized. The results indicate that the IL-1β-like homolog also plays an active role in defense mechanism in black rock fish.