PROFILING AND MODULATING DESIALYLATION OF LPS/TLR4 SIGNALING PATHWAY

Abstract

The cell surface is coated with glycans with terminal sialic acids (Sias), a process known as sialylation that plays important roles in a variety of biological processes. Removal of Sias (desialylation) by sialidase, on the other hand, is often involved in a number of pathological pathways. Neu1, Neu2, Neu3, and Neu4 are the four isozymes of mammalian sialidase, which differ in subcellular localization and substrate specificity. Lipopolysaccharide (LPS) induces endogenous sialidase expression in macrophages, which could result in desialylation of the TLR4, thereby initiating the LPS/TLR4 signaling pathway. In this dissertation study, a variety of analytical methods have been applied to investigate desialylation and localization of sialidases such as Neu1 and Neu3. Higher expression of sialidases was confirmed on THP-1 monocytes and macrophages upon LPS stimulation. Furthermore, lectin blot analysis and flow cytometry showed desialylation of total proteins of LPS-stimulated THP-1 macrophages. Next, we investigated the impact of sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA) on the regulation of LPS/TLR4 signaling pathways. As a result, DANA effectively mitigates the inflammatory responses induced by LPS in THP1 macrophages. Our findings demonstrate that the presence of LPS and the sialidase inhibitor DANA reduces both NF-κB phosphorylation and cytokine release. Finally, we investigated the secretion of Neu1 and Neu3 in the cell culture media of THP1 monocyte and macrophage upon stimulation with LPS. Surprisingly, sialidase levels significantly increased in the cell culture media, indicating sialidases secretion from THP-1 macrophages and monocytes. Furthermore, we investigated the secretion of exosomes from THP-1 macrophages to assess the presence of Neu1 and Neu3 in response to LPS stimulation. The exosome release from THP-1 macrophages is significantly increased by LPS treatment and Vacuolin-1 since it is inhibiting lysosomal exocytosis. Despite this, we were unable to detect Neu1 and Neu3 on the exosomes in either lysed or non-lysed conditions. Although their presence on exosomes was not observed, the substantial release of sialidases into the cell culture media suggests their involvement in extracellular signaling mechanisms. Overall, this indicates that Neu1 and Neu3 expression and secretion are involved in the LPS/TLR4 signaling pathway and sialidase inhibitors could serve as anti- inflammatory agents.