Determination of Equilibrium binding constants for LPS interaction with TLR4

Toll-like receptors (TLRs) are the best characterized Pathogen Recognition Receptors (PRRs) and are directly responsible for initiating an appropriate defense against bacterial and viral infection. Among all the TLRs known, only TLR4 is able to activate both MyD88-dependent induction of genes encoding inflammatory molecules and TRIF-dependent production of type I interferon. Therefore, in this study we report the binding of TLR4 by Lipopolysaccharide (LPS) which is the component on the cell-wall of gram-negative bacteria. Binding of LPS is a prerequisite for the activation of Toll-like receptor 4 (TLR4) by LPS which increases the expression of critical proinflammatory cytokines that organize potent immune responses. The binding of LPS to TLR4 was studied using IC21 mice macrophage cell line and fluorescently labeled LPS molecule, called FITC-LPS by flow cytometry. The series of cell staining experiments were performed, which included binding of FITC-LPS to TLR4 at different temperatures as temperature influences cellular trafficking of TLR4. Since, trafficking or internalization of LPS depends on its aggregation behavior; the molecular state of LPS under experimental conditions is detected using SDS-PAGE. Trypan blue was used to identify surface bond versus internalized FITC-LPS.