A-246 Desialylation Profiles of Monocytes and Macrophages Upon LPS Stimulation

Abstract Background Cell surface expresses a dense layer of glycans terminated with sialic acids (Sias), known as sialylation, which often represent different cellular statuses. Meanwhile, the removal of Sias from the glycans catalyzed by sialidase (desialylation) is also involved in many biological processes. Previous studies have established that lipopolysaccharide (LPS) can induce the expression of endogenous sialidases in monocytes and macrophages, leading to desialylation of the LPS receptor TLR4. This desialylation of TLR4 has been shown to impact the LPS/TLR4 signaling pathway. In this study, we conduct a profiling of desialylation in THP-1 monocytes and THP-1 macrophages in response to LPS stimulation. This will provide insights on the intricate connection between sialylation, desialylation, and LPS-induced signaling. Methods The activity of sialidases was assessed by utilizing 4-methylumbelliferyl-N-acetylneuraminic acid (4-MU-NANA) and ganglioside GM3 as substrates. The total sialic acid in the THP1 was quantified using liquid chromatography tandem mass spectrometry (LC-MS/MS). Flow cytometry was used to analyze the exposure of galactose on THP-1 monocytes and macrophages after LPS stimulation (100 ng/mL LPS for 24 h), using PNA-FITC staining. The expression level of Neu1 and Neu3 sialidases in cell lysates of THP-1 monocytes and macrophages were assessed by western blot technique after LPS stimulation. ELISA was used to measure the levels of sialidases in the cell supernatant of THP-1 monocytes and macrophages upon LPS stimulation. Result We found a significant reduction in total sialic acid levels in both THP-1 monocytes and macrophages upon LPS stimulation. This was further confirmed by the significant increase in total sialidase activities which measured by both 4-MU-Neu5Ac and GM3 substrates in THP-1 monocytes and macrophages upon LPS stimulation. Surprisingly, we also found that sialidase activities significantly increased in the cell supernatant of both THP-1 monocytes and macrophages, indicting sialidases secretion upon LPS stimulation. Western blot analysis further confirmed the high expression and secretion of both Neu1 and Neu3 sialidases from THP-1 monocytes and macrophages after LPS stimulation. Conclusion The present study conducted the first systematic profiling of desialylation and sialidase expression in monocytes and macrophages upon LPS stimulation. This results demonstrate that endogenous sialidase expression and secretion play a role in the LPS/TLR4 signaling pathway and subsequent cellular processes and functions in the context of inflammation and immune response.