Unveiling the anti-inflammatory potential of RF16, an interleukin 8-derived therapeutic peptide in macrophages

Interleukin 8 (IL-8, also known as CXCL-8) regulates inflammation and breast cancer formation by activating its cognate receptor CXCR1/2. In our previous study, a RF16 peptide, derived from the IL-8 binding region of CXCR1/2, was synthesized to inhibit IL-8-stimulated and LPS-induced adhesion and transmigration of monocytes. However, the anti-inflammatory effects of RF16 on THP-1 cells remain unknown.

THP-1 cells were differentiated with PMA overnight, followed by an one hour pretreatment of the IL-8 antagonist peptide RF16. IL-8 or TNF-α was then added for further incubation. The supernatant, mRNA, and protein of the cells were harvested. The secretion of TNF-α, IL-1β, and IL-6 was measured, along with assessment of ROS production and oxLDL uptake. Protein expression of different signaling pathways was analyzed. RF16 was also treated in a mouse model of acute sepsis to evaluate the white blood cell (WBC) count and serum IL-6 levels.

Our results indicate that RF16 was able to reduce mRNA and protein expression of inflammatory cytokines including TNF-α, IL-8, IL-6, and IL-1β levels in TNF-α/IL-8-induced THP-1 cells, with some results showing a dose-dependent trend. We found that RF16 was able to inhibit TNF-α/IL-8-induced ROS generation, thereby suppressing the inflammatory response. Moreover, a higher concentration (1 uM) of RF16 can alleviate oxLDL uptake induced by TNF-α and IL-8. Further investigation revealed that NF-κB, PI3K, and MAPK signaling pathways were the main mechanism responsible for the inhibition of inflammation. In addition, using the LPS-induced sepsis animal model, RF16 was able to reduce the number of white blood cells in peripheral blood, and subsequently reduce IL-6 levels.

Our results demonstrated that RF16 has the ability to suppress the expressions and secretions of TNF-α, IL-8, and LPS-induced pro-inflammatory cytokines in both in vitro and in vivo.