Abstract
Chitooligosaccharide (COS) is an important immune enhancer and has been proven to have a variety of biological activities. Our previous research has established an M1 polarization mode by COS in blunt snout bream (Megalobrama amblycephala) macrophages, but the mechanism of COS activation of blunt snout bream macrophages remains unclear. In this study, we further explored the internalization mechanism and signal transduction pathway of chitooligosaccharide hexamer (COS6) in blunt snout bream macrophages. The results showed that mannose receptor C-type lectin-like domain 4-8 of M. amblycephala (MaMR CTLD4-8) could recognize and bind to COS6 and mediate COS6 into macrophages by both clathrin-dependent and caveolin-dependent pathways. In the inflammatory response of macrophages activated by COS6, the gene expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and nitric oxide synthase 2 (NOS2) was significantly inhibited after MaMR CTLD4-8-specific antibody blockade. However, even if it was blocked, the expression of these inflammation-related genes was still relatively upregulated, which suggested that there are other receptors involved in immune regulation. Further studies indicated that MaMR CTLD4-8 and Toll-like receptor 4 (TLR4) cooperated to regulate the pro-inflammatory response of macrophages caused by COS6. Taken together, these results revealed that mannose receptor (MR) CTLD4-8 is indispensable in the process of recognition, binding, internalization, and immunoregulation of COS in macrophages of blunt snout bream.