A hybrid protein is a functional molecule to reduce the cytokine storm caused by excessively activated macrophages.

We recently developed a hybrid protein, tentatively named human MIKO-1 (hMIKO-1), based on the amino acid sequences of human S100A8 (hS100A8) and hS100A9. Human THP-1 macrophages (THP-1m), differentiated from THP-1 cells by phorbol 12-myristate 13-acetate, were used to investigate the immune function of hMIKO-1 as a drug for inflammatory diseases. Western blotting was conducted to confirm whether hMIKO-1 binds with β-actin and nuclear factor-kappa B to form complexes in THP-1m. A polymerase chain reaction (PCR) and quantitative PCR were performed to examine changes in the messenger RNA levels of proinflammatory cytokines in THP-1m. Fluorescent immunochemical staining was used to observe the intracellular localization of hMIKO-1 and hS100A8 or hS100A9 in THP-1m. As observed microscopically, the intracellular localization of hMIKO-1 in THP-1m was consistent with that of hS100A8, suggesting the close involvement of hS100A8 in the intracellular behavior of hMIKO-1 in THP-1m. Western blotting revealed that hMIKO-1 formed complexes with intracellular proteins, such as β-actin and nuclear factor-kappa B, to negatively regulate inflammatory signal transduction in THP-1m. Flow cytometry showed that the binding of hMIKO-1 to THP-1m significantly decreased when THP-1m were preliminarily treated with a sialidase (neuraminidases) cocktail. Therefore, the present results strongly suggest that the binding of hMIKO-1 to THP-1m closely involves the sugar chains of the surface proteins of cells. The messenger RNA expression of each proinflammatory cytokine was significantly suppressed in THP-1m preliminarily treated with hMIKO-1 despite a subsequent stimulation with lipopolysaccharide. In conclusion, hMIKO-1 is a functional molecule that significantly inhibits inflammatory signal transduction in THP-1m.