Hemoglobin stimulate inflammatory cytokine expression in grass carp (Ctenopharyngodon idella) head-kidney macrophages

In general, disease caused by the hemorrhagic pathogen infections, results in hemolysis, in series the hemoglobin (Hb) is discharged into the tissues. Consecutively, oxidation of Hb while exposed to air provokes serious high oxidative damage levels in the body and inducing a large amount of inflammation to release additional proinflammatory mediators. Promptly, it is critical to clear the highly cytotoxic Hb. Macrophages play a key role not only under stress conditions, but also in sustaining homeostasis by promoting erythropoiesis and hemoglobin (Hb) clearance. However, the complete mechanism of the oxidized Hb in teleost fish and the effects of macrophages on it not yet clear. In this study, head-kidney macrophages and Hb of the C. idella were taken as the research model to examine the interaction between Hb and macrophages. Stimulation of the Hb during in vivo studies led to the visible deposition of Hb in the head and middle of the kidney and elevated the levels of antioxidant-related enzymes in serum, and further promoted the expression levels of inflammatory cytokine, such as TNF-α, IL-1β, and IL-10 in head kidney. Thereafter, the indirect immunofluorescence and Prussian blue staining analyses indicated that the macrophages of the head kidney exhibited intense phagocytic activity to Hb. Analysis of gene expression level by quantitative real-time PCR, macrophage markers, including CD68, CD86, CSF, and MHC-Ⅱ genes were significantly elevated through the stimulation of Hb. Likewise, the expression levels of inflammatory cytokines (TNF-α, IL-1 β, and IL-4), chemokines (CCL20 and CCL4), IFN-α and TLR4 genes were significantly up-regulated in the head kidney macrophages after 12 h of Hb stimulation. In summary, these results indicate that C. idella head kidney macrophages possess the phagocytic ability towards Hb, and in turn, Hb also activates the expression levels of macrophage-related markers and associated cytokines. Taken together, our findings explicitly improve the understanding of Hb macrophage interactions of C. idella and provided a new reference for healthy fish farming.