Force Characteristics of Yersinia pestis Lipopolysaccharide Interaction with TLR4 and CD14 Receptors on J774 Macrophages: Atomic Force Microscopy

Abstract

One of the main stages of the infectious process, which largely determines the course and outcome of the disease, is the primary contact of the pathogen with the host cells. A key role in this interaction of gram-negative bacteria with immunocompetent cells of the macroorganism is played by lipopolysaccharide of the outer membrane, which initiates the launch and development of immune reactions by interacting with a number of specific receptors, primarily CD14 and TLR4. The aim of this study was to quantify by atomic force microscopy the force characteristics of the interaction of Yersinia pestis lipopolysaccharide of the EV vaccine strain with CD14 and TLR4 receptors on the surface of murine J774 macrophages. The lipopolysaccharide was isolated from Y. pestis cells of the EV vaccine strain grown at 27°C. Fluorescence and confocal microscopy were used to evaluate the expression of receptors on the cell surface. Using monoclonal antibodies to CD14 and TLR4 receptors, the force characteristics of the interaction of lipopolysaccharide on the surface of the cantilever probe (tip) with J774 macrophages were evaluated by force spectroscopy. The conditions of immobilization of J774 macrophages on glass made it possible to scan their surface and assess the force of adhesion to the cells of target antigens by atomic force microscopy. Incubation of immobilized macrophages in solutions with monoclonal antibodies to CD14 and TLR4 receptors caused a decrease in the main force characteristics of interaction in the J774 macrophage–Y. pestis lipopolysaccharide system compared with intact, untreated cells. A similar effect was observed after pretreatment of cells with a solution of the same lipopolysaccharide without monoclonal antibodies. The results obtained indicate the ability of the lipopolysaccharide chemically bound to the probe to interact with CD14 and TLR4 receptors on the surface of macrophages.