Force Characteristics of <i>Yersinia pestis</i> Lipopolysaccharide Interaction with TLR4 and CD14 Receptors on J774 Macrophages. Atomic Force Microscopy

Abstract

One of the main stages of infectious process, which mostly determines the course and outcome of the disease, is the initial contact of the pathogen with the host cells. The lipopolysaccharide as a component of the outer membrane is crucially involved in the interaction between Gram-negative bacteria and immunocompetent host cells. It triggers immune reactions by interaction with specific receptors, mainly CD14 and TLR4. The aim of this work was to quantify the force characteristics of the interaction of Yersinia pestis EV lipopolysaccharide with CD14 and TLR4 receptors on the surface of mouse macrophages J774 by atomic force microscopy. Lipopolysaccharide was extracted from Y. pestis cells (vaccine strain EV) grown at 27°С. The expression of receptors on the cell surface was evaluated by fluorescent and confocal microscopy. Using monoclonal antibodies against CD14 and TLR4 receptors, force spectroscopy was used to estimate the force characteristics of the interaction between lipopolysaccharide on the cantilever surface and J774 macrophages immobilized on a glass substrate. The conditions for immobilization of J774 macrophages on glass were developed that allowed scanning the cell surface and estimating the adhesion force of target antigens to the cells. Incubation of macrophages in solutions with monoclonal antibodies against CD14 and TLR4 receptors caused a decrease in the major force characteristics of the interaction in the J774 macrophage – Y. pestis lipopolysaccharide system compared to the system containing untreated macrophages. A similar effect was observed after pretreatment of the cells with a solution containing the same lipopolysaccharide without monoclonal antibodies. The results show the ability of the Y. pestis lipopolysaccharide chemically bound to the cantilever to interact with CD14 and TLR4 receptors on the macrophage surface.