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

## TL;DR

A new hybrid protein called hMIKO-1 was developed to reduce inflammation by blocking the overactivation of immune cells called macrophages.

## Contribution

The novel hybrid protein hMIKO-1 is introduced as a potential therapeutic for inflammatory diseases by suppressing cytokine storms.

## Key findings

- hMIKO-1 suppresses proinflammatory cytokine mRNA expression in macrophages even after lipopolysaccharide stimulation.
- hMIKO-1 forms complexes with β-actin and nuclear factor-kappa B to negatively regulate inflammatory signaling.
- The binding of hMIKO-1 to macrophages involves cell surface sugar chains, as shown by reduced binding after sialidase treatment.

## Abstract

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.

In this article, we have developed a hybrid protein, tentatively named human MIKO‐1 (hMIKO‐1). The messenger RNA expression of each proinflammatory cytokine was significantly suppressed by hMIKO‐1 despite a subsequent stimulation with lipopolysaccharide. hMIKO‐1 is a functional molecule that significantly attenuates the cytokine storm.