Lymphocyte‑derived microparticles stimulate osteoclastogenesis by inducing RANKL in fibroblasts of odontogenic keratocysts

Abstract

Leukocyte-derived microparticles (LMPs) include neutrophil-, lymphocyte-and monocyte-derived MPs. LMPs act as proinflammatory mediators in autoimmune diseases, infectious diseases and vascular diseases. The present study examined the hypothesis that the percentage of LMPs was increased in patients with inflamed odontogenic keratocysts (OKCs), and investigated the biological effects of Jurkat cell-derived MPs on the fibroblasts of OKCs in vitro. Cyst fluid MPs, obtained by centrifugation of samples from 20 patients with inflamed OKCs, 3 patients with uninflamed OKCs, 15 patients with radicular cysts (RCs) and 12 patients with inflamed dentigerous cysts (DCs), were analyzed by transmission electron microscopy, dynamic light scattering and immunofluorescence staining. The percentages and concentrations of cyst fluid LMPs were further determined by flow cytometry. The cytokine levels of apoptotic Jurkat cell-derived MPs and Jurkat cell supernatants were compared by cytokine antibody arrays. Fibroblasts were isolated from 3 patients with OKC and co-cultured with apoptotic Jurkat cell-derived MPs with or without interleukin (IL)-15Rα to detect the levels of matrix metallopeptidase 9 (MMP-9) and receptor activator of nuclear factor-κB ligand (RANKL) by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The supernatant from Jurkat MPs-treated fibroblasts was collected to make conditioned medium in which the osteoclastogenesis of Raw264.7 cells was determined. Antibodies against human soluble (s)RANKL were added to the conditioned medium to investigate the inhibitory effects. Mean percentages of lymphocyte-and neutrophil-derived MPs were significantly higher in inflamed OKCs than in DCs. Significant elevations in IL-15 were detected in apoptotic Jurkat cell-derived MPs compared with that in Jurkat cell supernatant. Furthermore, higher levels of MMP-9 and RANKL were detected in Jurkat cell MP-treated OKC fibroblasts, and this was partially blocked by IL-15Rα. Increased osteoclast-like cell formation was observed in the Jurkat MPs-treated fibroblast supernatant and Raw264.7 co-culture groups. The anti-human sRANKL antibody in the Jurkat MPs-treated fibroblast supernatant group decreased the osteoclastogenesis of the Raw264.7 cells. These results indicate that LMPs serve as novel communication tools that contribute toward the bone resorption of inflamed OKCs by inducing RANKL of OKC fibroblasts via IL-15.

ENO1 Binds to ApoC3 and Impairs the Proliferation of T Cells via IL-8/STAT3 Pathway in OSCC

“…Double-labelling immunofluorescence staining procedures on CAL27 cells and paraffin-embedded tissue samples were conducted as described previously [ 60 , 68 ]. The cells and the slides were incubated with the primary antibodies against ENO1 and ApoC3, respectively, at 4 °C overnight.…”

Single-Cell RNA Sequencing Reveals CXCLs Enriched Fibroblasts Within Odontogenic Keratocysts

“…The primary fibroblast culture was performed as described previously. 23 The tissue of OKC was immediately rinsed by PBS (Servicebio, Wuhan, China) and cutted into 5 × 5 mm cubes which were placed on the cell culture dishes. DMEM (Servicebio, Wuhan, China) medium supplemented with 10% FBS and 1% penicillin and streptomycin (Servicebio, Wuhan, China).…”

“…The samples for cytokines and chemokines detection were stored as previously described. 23 Supernatant of cultured fibroblasts (5 × 10 5 /well in six-well culture plates) was collected and stored in −80°C. Following the manufacturer’s protocols, detection of cytokines and chemokines was performed by Bio-Plex Pro Human Cytokine Screening 40-plex panel using Bio-PlexMAGPIX System (Bio-Rad) (Wayen Biotechnologies (Shanghai), Inc).…”

“…Immunohistochemical staining was performed as described previously. 23 Tissue microarray (TMA) of 30 cases of primary OKC was collected (Servicebio, Wuhan, China). Briefly, tissue slides were dewaxed, hydrated, washed and endogenous peroxidase neutralized.…”

Serum-derived extracellular vesicles inhibit osteoclastogenesis in active-phase patients with SAPHO syndrome

“…[11][12][13] Previous studies have shown that EVs, mainly exosomes and shed microvesicles, are involved in bone metabolism in both normal physiological and disease states 14,15 likewise in multiple inflammatory diseases. 16 In addition, EVs from monocytes, leukocyte and macrophage exert directly effects on osteoclastogenesis and osteogenesis, [17][18][19] indicating that EVs function as mediators between the immune system and bone metabolism. As SAPHO syndrome is an inflammatory-mediated disease characterized mainly by systemic osteoarticular damage, we hypothesized that EVs play an important role in bone and joint damage in SAPHO syndrome.…”