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Lipopolysaccharide increases the number and activity of peripheral osteoclast progenitor cells

Objectives: Lipopolysaccharide (LPS) from gram-negative bacteria causes chronic inflammation and subsequent bone loss, and is involved in the pathogenesis of several bacterially induced bone diseases. We investigated the effects of LPS on bone metabolism and osteoclast differentiation from hematopoietic cells. Methods: C57BL/6 mice were injected during 4 weeks (10 µ g LPS/g body weight) and sacrificed at different time-points. Cells from several tissue sources (bone-marrow, homogenized bone shafts, spleen and peripheral blood) were cultured with RANKL (40 ng/mL) and M-CSF (15 ng/mL) to stimulate osteoclast (OCL) differentiation. OCL were identified as TRAP-positive multinucleated cells ?three nuclei/cell. Femoral sections (5 µ m) were stained with Goldner-trichrome and TRAP. Microtomography (µ CT) was performed using high resolution SkyScan1172. OCL progenitors were characterized by flow-cytometry as a population negative for lymphoid markers (B220, CD3, NK1.1) and positive for CD115 and CD117, within both CD11b negative/low and CD11b positive populations. Gene expression analysis of OCL differentiation genes was performed by qPCR. Results: Three weeks after LPS stimulation, the number of OCL differentiated from cells extracted from bone shafts (504.8± 74.28) was higher compared with controls (383.3± 30.48 ; p< 0.005). This was in correlation to the decrease in bone volume and trabecular thickness detected by µ CT. Femoral sections showed that LPS altered bone metabolism by inducing increased osteoresorption in bone cortex starting from the periosteal surface. This was confirmed by gene expression analysis of bone shafts, showing increased expression of OCL differentiation genes RANK and cFms. Flow-cytometry indicated that enhanced bone resorption starting at the periosteal surface may be caused by homing of peripheral OCL progenitors, since we found 2-3-fold increase of OCL progenitor cell populations in peripheral blood and spleen 10 days after LPS treatment. The presence of increased number of OCL progenitors among peripheral hematopoietic cells was supported by higher number of differentiated OCL from both blood and spleen (181± 32.16 and 299.25± 61.37 respectively) compared with control (43.8± 25.7 and 189± 54.88 respectively, p< 0.005). Conclusion: LPS administration stimulates homing of OCL progenitors to periosteal bone surface and supports osteoclast differentiation. Our further aim is to identify factors induced by LPS that mediate this osteoclastogenic effect and cause enhanced bone resorption.

Lipopolysaccharide; Osteoclasts; Resorption; Inflammation

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