Single-cell RNA analysis of chemokine expression in heterogeneous CD14+ monocytes with lipopolysaccharide-induced bone resorption

Lipopolysaccharide (LPS)-induced bone resorption has normally been found in inflammatory bone diseases, but the underlying mechanism is currently unclear. Since LPS binds to CD14 and activates Toll-like receptor 4 (TLR4) in monocytes, the present study focused on CD14+ monocytes and observed their responses after LPS treatment during the progression of local bone destruction. CD14+ monocytes were obtained from human peripheral blood mononuclear cells (PBMCs) by magnetic cell separation (MACS), and their classification was confirmed by fluorescence-activated cell sorting (FACS). Single-cell RNA sequencing (scRNA-seq) was further utilized to analyze their subpopulations, and the results showed that physiological CD14+ monocytes were heterogeneous and divided into 6 subsets, that could be easily agitated. After priming with a suitable concentration of LPS, heterogeneous CD14+ monocytes became pathological and expressed a large number of chemokines as a “cascade effect”. Some of these chemokines have been validated in an animal model of mouse calvarial bone invasion. Taken together, our research has linked enhanced chemokine expression with stimulation of heterogeneous CD14+ monocytes, and indicated that inflammatory responses caused by microbiome infection are responsible for the recruitment and mobilization of CD14+ monocytes into bone resorption sites, which may explain the pathogenesis of LPS-associated bone diseases.