Staphylococcus aureus induces expression of receptor activator of NF-kappaB ligand and prostaglandin E2 in infected murine osteoblasts

Abstract

Osteomyelitis is an inflammatory disease of the bone that is characterized by the presence of necrotic bone tissue and increased osteoclast activity. Staphylococcus aureus is responsible for approximately 80% of all cases of human osteomyelitis. While the disease is especially difficult to treat, the pathogenesis of S. aureus-induced osteomyelitis is poorly understood. Elucidating the molecular mechanisms by which S. aureus induces osteomyelitis could lead to a better understanding of the disease and its progression and development of new treatments. Osteoblasts can produce several soluble factors that serve to modulate the activity or formation of osteoclasts. Receptor activator of NF-kappaB ligand (RANK-L) and prostaglandin E(2) (PGE(2)) are two such molecules which can promote osteoclastogenesis and stimulate bone resorption. In addition, previous studies in our laboratory have shown that osteoblasts produce inflammatory cytokines, such as interleukin 6, following infection with S. aureus, which could induce COX-2 and in turn PGE(2), further modulating osteoclast recruitment and differentiation. Therefore, we hypothesized that following infection with S. aureus, osteoblasts will express increased levels of RANK-L and PGE(2). The results presented in this study provide evidence for the first time that RANK-L mRNA and protein and PGE(2) expression are upregulated in S. aureus-infected primary osteoblasts. In addition, through the use of the specific COX-2 inhibitor NS 398, we show that when PGE(2) production is inhibited, RANK-L production is decreased. These data suggest a mechanism whereby osteoblasts regulate the production of RANK-L during infection.

FIG. 1.

Expression of RANK-L mRNA by osteoblasts. Primary mouse osteoblasts either were not infected (MOI = 0) or were infected with S. aureus strain UAMS-1 (MOI = 25:1 or 75:1). Following a 45-min incubation with or without bacteria (time zero hours), total RNA was extracted at various time points and subjected to quantitative real-time reverse transcription-PCR analysis for RANK-L mRNA. All values were normalized to G3PDH and are expressed as n-fold change over baseline levels (0 h; MOI = 0). Data are expressed as the means ± standard errors for three separate osteoblast cultures. *, P < 0.05 versus results for MOI of 75:1 at 0 min; #, P < 0.05 versus results for MOI of 0 at time zero min; $, P < 0.05 versus results for MOI of 25:1 at 4 h.

FIG. 2.

Expression of RANK-L protein by osteoblasts. Primary mouse osteoblasts were either uninfected (MOI 0) or infected with either S. aureus strain UAMS-1 (MOI = 25:1 or 75:1), UV-killed strain UAMS-1 (MOI = 25:1), S. carnosus (MOI = 25:1), or S. aureus nasopharyngeal isolate N1, N2, N3, N4, or N5 (MOI = 25:1). Following a 45-min incubation with or without bacteria and a 1-h treatment with 25 μg/ml gentamicin sulfate, whole-cell extracts were taken at time zero hours, 6 h, and 24 h. RANK-L concentrations were determined using an ELISA, and the results are displayed in pg/ml. Data are expressed as the means ± standard errors for three separate osteoblast cultures. *, P < 0.05 versus results with MOI of 0 at all time points; #, P < 0.05 versus results with MOI of 75:1 0 h and 6 h; $, P < 0.05 versus results with MOI of 25:1 at 6 h or 24 h.

FIG. 3.

Expression of PGE₂ by osteoblasts. Primary mouse osteoblasts were either uninfected (MOI = 0) or infected with either S. aureus strain UAMS-1 (MOI = 25:1 or 75:1), UV-killed strain UAMS-1 (MOI = 25:1), S. carnosus (MOI = 25:1,) or S. aureus nasopharyngeal isolate N1, N2, N3, N4, or N5 (MOI = 25:1). Following a 45-min incubation with or without bacteria and a 1-h treatment with 25 μg/ml gentamicin sulfate, supernatants were collected at time zero, 6 h, and 24 h. PGE₂ concentrations were determined by an EIA and are expressed in pg/ml. Data are expressed as the means ± standard errors for three separate osteoblast cultures. *, P < 0.05 versus results with an MOI of 0 at 0, 6 h, or 24 h.

FIG. 4.

Expression of PGE₂ and the effects of NS-398. Primary mouse osteoblasts were cultured in the presence of 0.025% DMSO or 20 μM NS-398 in DMSO 48 h prior to infection. Osteoblasts were either uninfected (MOI = 0) or infected with S. aureus strain UAMS-1 (MOI = 25:1 or 75:1). Following a 45-min incubation with or without bacteria and a 1-h treatment with 25 μg/ml gentamicin sulfate, supernatants were collected at time zero, 6 h, and 24 h. PGE₂ concentrations were determined by an EIA and are expressed in ng/ml. Data are expressed as the means ± standard errors for three separate osteoblast cultures. *, P < 0.05 versus results with MOI of 0 and treatment with DMSO; #, P < 0.05 versus results for all groups treated with NS-398 at all time points.

FIG. 5.

Expression of RANK-L and the effects of NS-398. Primary mouse osteoblasts were cultured in the presence of 0.025% DMSO or 20 μM NS-398 in DMSO 48 h prior to infection. Osteoblasts were either uninfected (MOI = 0) or infected with S. aureus strain UAMS-1 (MOI = 25:1 or 75:1). Following a 45-min incubation with or without bacteria and a 1-h treatment with 25 μg/ml gentamicin sulfate, whole-cell extracts were collected at time zero, 6 h, and 24 h. RANK-L concentrations were determined by an ELISA and are expressed in pg/ml. Data are expressed as the means ± standard errors for three separate osteoblast cultures. *, P < 0.05 versus results with MOI of 0 and treatment with DMSO at 0 h, 6 h, or 24 h; #, P < 0.05 versus results for all groups treated with NS-398 at all time points; $, P < 0.05 versus MOI of 25:1 at 24 h.