Introduction
Interleukin-6 (IL-6) is a key cytokine that orchestrates immune responses and is widely recognized for its roles in promoting inflammation, regulating cell survival and apoptosis, and providing protection against bacterial and viral infections. Previous research using il-6 knockout mice has highlighted its importance in bacterial defence; these mice exhibited lower survival rates and impaired bacterial clearance. While the immunological functions of IL-6 have been well characterized in mammals, its roles in teleost remain unclear. In this study, we utilized zebrafish (Danio rerio) to investigate the significance of IL-6 in antibacterial defence against Edwardsiella piscicida (E. piscicida). E. piscicida, a gram-negative bacterium of the Enterobacteriaceae family, is known to pose major threats by causing organ damage, ulceration, and eye abnormalities, often resulting in high mortality of various freshwater and marine organisms. We employed CRISPR/Cas9 gene editing technology for comparative analyses between wild-type (WT) and il-6 knockout (il-6 -/-) zebrafish.
Materials and methods
Utilizing the CRISPR/Cas9 technology, we constructed an il-6 -/- zebrafish line. Both WT and il-6 -/- fish were challenged with a low (1x107 CFU/ml) and high (1x108 CFU/ml) concentration of E. piscicida though immersion. To assess the host’s susceptibility to the pathogen, we monitored cumulative mortality up to 11 days post-infection (dpi) and measured the amount of E. piscicida gDNA within the fish. Lastly, expression patterns of immune related genes such as pro-inflammatory cytokines (il-1β, tnf-α), signalling molecules (stat3, socs3), and antimicrobial peptides (mpx, mpeg) were observed through RT-qPCR.
Results
A 7 base pair deletion was formed in the il-6 sequence, resulting in a knockout of the gene which yielded a non-functional IL-6 protein. The genetic alteration led to dysregulated expression of immune related genes. These fish showed significantly elevated expression levels of proinflammatory cytokines, which coincided with a downregulation of the negative immune regulator socs3. Meanwhile, antimicrobial peptides were suppressed. The E. piscicida challenge assay revealed an increase in susceptibility to E. piscicida infection in knockout fish. These fish showed significantly higher mortality rates with earliest deaths occurring at 3 dpi. This heightened vulnerability corresponded with higher bacterial loads, indicating a flawed pathogen clearance. After the bacterial challenge, immune related gene expression in il-6-/- fish mostly peaked at 24 hours post-infection, although il-1β and mpx showed delayed expression peaks at 48 hours. Overall, the altered expression profiles of key immune genes indicate a disrupted antibacterial signalling network in the absence of IL-6. These findings underscore the critical role of IL-6 in initiating and coordinating immune responses following bacterial infections in zebrafish.