Regulation Of Grass Carp IL-6 On Expression Of Inflammation-related Cytokines In Head Kidney Leukocytes

In mammals,interleukin-6(IL-6)is a multifunctional cytokine that plays a significant role in cell differentiation,proliferation,migration and apoptosis.When the host invaded by extracellular pathogens or inflammation occurs in tissues and organs,IL-6 can rapidly induce the production of inflammation-related factors and enhance the inflammatory response to eliminate pathogens and necrotic cells.The immuno-regulatory functions of IL-6 are largely unknown in teleosts.Teleosts have different immune organs compared to mammals and four isoforms of STAT3.This indicates different functions and mechanisms of IL-6 in regulating immune responses in fish species.In the present study,grass carp was used as the animal model,and grass carp recombinant IL-6 protein was used as a tool to explore the immune effects and functional mechanisms of fish IL-6 in head kidney of teleost with mammals.Firstly,the recombinant grass carp IL-6 protein was expressed using a prokaryotic expression system and purified.The biological activity of this protein was verified through measuring the expression of its downstream target gene suppressor of cytokine signaling 3(SOCS3)in grass carp head kidney leukocyte(HKL)and kidney cell line(CIK)cells.In addition,the mRNA levels of the four STAT3 variants(STAT3?1,STAT3?2,STAT3?1 and STAT3?2)stimulated by IL-6 were also detected.This not only further confirmed the activity of IL-6,but also demonstrated that the four splice isoforms of STAT3 play different roles in signal transduction of grass carp IL-6.Secondly,grass carp IL-6 could significantly induce the mRNA levels of several proinflammatory cytokines(IL-1?,IL-8 and IL-6)and STAT3 is involved in these responses.Moreover,ERK was also involved in the regulation of IL-6 on IL-1? mRNA expression.Further studies showed that grass carp IL-6 could also regulate the gene expression of IL-10 in HKL.Interestingly,results showed that IL-6 stimulated IL-10 mRNA expression after 3 hours' treatment and inhibited its expression after 24 hours' treatment.STAT3 were involved in both of these reverse effects.Subsequently,we found that grass carp IL-6 did not affect the half-life of IL-10 mRNA in HKL using a RNA synthesis inhibitor(Actinomycin D).Luciferase reporter assays demonstrated that grass carp IL-6 could increase the activity of IL-10 promoter,suggesting that it can directly regulate IL-10 mRNA levels on the promoter level.Further studies found that sustained activation of STAT3?2 enhanced the activity of IL-10 promoter,whereas the sustained activation of STAT3?1 inhibited the activity of IL-10 promoter.It implies that the different regulations of IL-10 mRNA expression after different treatment duration by IL-6 might be led by activation of different STAT3 isoforms.In conclusion,grass carp IL-6 not only affects the expression of pro-inflammatory cytokines in HKL,but also reversely regulates the expression of an anti-inflammatory cytokine,IL-10 with short-and long-time treatment in the same cell model.It reveals the multifunctional effects of IL-6 in the regulation of teleost inflammation,providing a clue to compare immune regulations in different species and new knowledge about the regulatory mechanisms of IL-6 in fish immune cells.