From Crosstalk between Immune and Bone Cells to Bone Erosion in Infection

Abstract

Bone infection and inflammation leads to the infiltration of immune cells at the site of infection, where they modulate the differentiation and function of osteoclasts and osteoblasts by the secretion of various cytokines and signal mediators. In recent years, there has been a tremendous effort to understand the cells involved in these interactions and the complex pathways of signal transduction and their ultimate effect on bone metabolism. These crosstalk mechanisms between the bone and immune system finally emerged, forming a new field of research called osteoimmunology. Diseases falling into the category of osteoimmunology, such as osteoporosis, periodontitis, and bone infections are considered to have a significant implication in mortality and morbidity of patients, along with affecting their quality of life. There is a much-needed research focus in this new field, as the reported data on the immunomodulation of immune cells and their signaling pathways seems to have promising therapeutic benefits for patients.

Keywords: T cells; bone erosion; bone infection; bone remodeling; osteoclasts; signaling crosstalk.

Figure 1

Differentiation of blood monocytes into osteoclast. Peripheral blood mononuclear cells (PBMCs) were isolated from blood of healthy donors by ficoll separation and monocytes were then purified from PBMCs. Purified monocytes were cultured for 15 days in the presence of macrophage-colony-stimulating factor (M-CSF) with or without receptor activator of NF-κB ligand (RANKL). Finally cells were stained for tartrate-resistant acid phosphatase (TRAP), an enzymatic marker for osteoclast identification. Cells were visualized using a Zeiss light microscope at 10 × resolution. Addition of RANKL to the culture medium led to the differentiation of monocytes into multinucleated large sized pink colored osteoclasts. Black arrows indicate osteoclasts.

Figure 2

Human T cells inhibit osteoclast formation in vitro. Blood monocytes were differentiated into osteoclasts in the presence of macrophage-colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) for 15 days. T cells derived from human bone tissue, by cutting bones into fines pieces and separating cells by centrifugation, were separated into CD4 and CD8 T cells by flow cytometer. Addition of these isolated CD4 or CD8 T cells separately to the differentiating blood monocytes led to a decrease in the number of osteoclasts being formed in vitro cellular coculture model when observed after 15 days post culture. A comparative method of colorimetric assay was used to determine the number of osteoclasts formed by measuring the TRAP activity through absorbance, which is a prominent osteoclast enzyme. The data is from three independent experiments. Student t tests were used to determine statistical significance (* p < 0.05).

Figure 3

Interaction and crosstalk between immune cells, osteoblasts, and osteoclasts mediated via different cytokines regulate the extent of bone erosion during infection. Osteoclasts (OC) are the cells of myeloid origin that degrade the bone matrix, whereas osteoblasts (OB) are the bone forming cells that have mesenchymal origin. Bacterial entry into the host initiates a complex crosstalk between immune cells, mainly, T and B cells with osteoclasts. The invading pathogen is phagocytized and presented by macrophages (mac) and dendritic cells (DC) to activate T cells. These activated T cells further get differentiated into T helper (Th) 1, Th2, and Th17 subsets. Th17 is the prominent osteoclastogenic T cell subset which expresses receptor activator of NF-κB ligand (RANKL) and induces the formation of osteoclasts by binding to RANK on pre-osteoclasts. It also secretes IL-17 that induces the synovial fibroblasts as well as osteoblasts to express RANKL further leading to osteoclastogenesis. Contrarily, Th1 and Th2 subset of T cells inhibits osteoclastogenesis by secreting cytokines INF-γ, IL-4 and IL-10 respectively. B cells, being activated by innate and adaptive immune cells, secrete OPG, which acts as an inhibitor to osteoclast formation process. Osteoblasts also secrete macrophage-colony-stimulating factor (M-CSF) and RANKL that aids in the process of osteoclastogenesis. Stimulation; Inhibition.

Figure 4

Flow cytometric dot plot images showing staining patterns of T cell populations in human blood, non-infected bone and infected bone. Peripheral blood mononuclear cells (PBMCs) were separated from blood by ficoll and bone cells were isolated by cutting bone samples into fine pieces, vortexing, filtering, and centrifugation. Cells were then labeled with various monoclonal antibodies, both extracellular as well as intracellular, and analyzed by flow cytometer and doing sequential analysis. Cells were first gated on CD45 and then CD3+ population were gated on these CD45+ cells. Finally, CD4+ and CD8+ T cell populations were identified by gating on CD3+ cells. Expressions of other markers were studied on these CD4 and CD8 T cell populations. CD4+ cells that were positive for CD25 and Foxp3 double markers were considered to be Tregs. Bone tissues that were without any infection were considered non-infected, whereas those with reported bacterial infection were considered to be infected.