Lipopolysaccharide-induced osteoclastogenesis from mononuclear precursors: a mechanism for osteolysis in chronic otitis

Abstract

Osteoclasts are the only cells capable of carrying out bone resorption and therefore are responsible for the osteolysis seen in infectious diseases such as chronic otitis media and infected cholesteatoma. Pseudomonas aeruginosa is the most common organism isolated from these infectious middle ear diseases. In this study, we examined the mechanisms by which P. aeruginosa lipopolysaccharide (LPS) stimulates osteoclastogenesis directly from mononuclear osteoclast precursor cells. Osteoclast precursors demonstrated robust, bone-resorbing osteoclast formation when stimulated by P. aeruginosa LPS only if previously primed with permissive, sub-osteoclastogenic doses of receptor activator of NF-kappaB ligand (RANKL), suggesting that LPS is osteoclastogenic only during a specific developmental window. Numerous LPS-elicited cytokines were found to be released by osteoclast precursors undergoing P. aeruginosa LPS-mediated osteoclast formation. Two lines of evidence suggest that several cytokines promote Oc formation in an autocrine/paracrine manner. First, inhibition of several cytokine pathways including TNF-alpha, IL-1, and IL-6 block the osteoclastogenesis induced by LPS. Secondly, increased expression of the receptors for TNF-alpha and IL-1 was demonstrated by real-time quantitative polymerase chain reaction. Such a mechanism has not previously been established and demonstrates the ability of osteoclast precursors to autonomously facilitate bone destruction.

FIG. 1

Time course study of duration of RANKL priming on P. aeruginosa LPS-mediated Oc differentiation of primary pOcs. pOcs derived from bone marrow cells of C57BL/6 mice were influenced to undergo LPS-mediated Oc formation with media containing LPS and M-CSF with/without RANKL. LPS-mediated Oc formation refers to RANKL (10 ng/ml) priming of pOcs for specified durations prior to P. aeruginosa LPS exposure. A Increases in Oc number were observed with increased duration of RANKL priming. Wells shown here received media containing LPS, M-CSF, and permissive RANKL after priming. B Dose-dependent increases in Oc number were observed with increased duration of RANKL priming in both treatment groups (^ap < 0.01, black and white bars). Furthermore, in groups undergoing 24, 48, and 72 h of RANKL priming, Oc formation was always significantly higher in those groups where RANKL was continuously present throughout the experiment (black versus white bars, ^bp < 0.01). C Time course study of duration of RANKL priming on P. aeruginosa LPS-mediated Oc differentiation of RAW 264.7 cells. RAW cells were influenced to undergo LPS-mediated Oc formation (as above) with media containing LPS with/without RANKL. Dose-dependent increases in Oc area fraction were observed with increased duration of RANKL priming in both treatment groups (^ap < 0.01). D Representative photographs of resorption areas formed by primary pOcs on dentin (upper) and hydroxyapatite (lower) during P. aeruginosa LPS-mediated Oc formation. All data are expressed as the mean ± SEM of at least three experiments performed in triplicate wells.

FIG. 2

Cytokine profile of pOcs undergoing RANKL priming and P. aeruginosa LPS-mediated osteoclastogenesis. A Cell culture supernatants were probed for the cytokines shown in the cytokine antibody array shown. B–D Array images represent one of two independent experiments that found similar patterns of expression. Untreated RAW cells (B), RAW cells primed with RANKL for 72 h (C), and RAW cells undergoing P. aeruginosa LPS-mediated Oc formation (D) were compared. In this case, P. aeruginosa LPS-mediated Oc formation refers to RAW cells primed for 72 h with RANKL (10 ng/ml) followed by P. aeruginosa LPS stimulation for 48 additional hours. D Cytokine ratios were calculated between the P. aeruginosa LPS-mediated osteoclastogenesis and 72 h RANKL prime groups. Untreated RAW cell culture supernatants were used as the baseline control. Results presented are those cytokines whose mean cytokine concentrations were threefold or greater in pOcs undergoing LPS-mediated Oc formation compared to those receiving only RANKL priming (highlighted in black boxes).

FIG. 3

RANKL-mediated osteoclastogenesis in wild-type and various knockout pOcs. Wild-type pOcs were compared to pOcs with targeted deletions of either IL-6 (IL-6^−/−), TNF receptors (TNFR1^−/−, TNFR2^−/−, TNFR1/2^−/−), or IL-1 receptor 1 (IL-1R1^−/−) in their ability to form Ocs by exposing them to RANKL at 100 ng/ml for 6 days in the presence of M-CSF (10 ng/ml). There were no differences in the abilities of any of these cell types to form Ocs under these conditions (overall p = 0.105 by one-way ANOVA with post hoc Bonferroni test). All data are expressed as the mean ± SD of at least three experiments performed in triplicate wells.

FIG. 4

Autocrine IL-6, TNF-α, and IL-1 involvement in LPS-mediated osteoclastogenesis. A–C pOcs derived from mice incapable of generating IL-6 (IL-6^−/−), mice lacking one or both TNF receptors (TNFR1^−/−, TNFR2^−/−, or TNFR1/2^−/−), or mice incapable of responding to IL-1 (IL-1R1^−/−) were influenced to undergo P. aeruginosa LPS-mediated Oc formation. In this case, P. aeruginosa LPS-mediated Oc formation refers to 72 h of RANKL (10 ng/ml) priming followed by P. aeruginosa LPS stimulation for an additional 96 h. Clearly, IL-6^−/− pOcs (A), all TNF receptor knockout subtype pOcs (B), and IL-1R1^−/− pOcs (C) were impaired in their ability to undergo LPS-mediated Oc formation when compared to C57BL/6 wild-type control pOcs (^ap < 0.01). Furthermore, TNFR1/2^−/− double knockout pOcs demonstrated significantly more impairment than either TNFR1^−/− or TNFR2^−/− pOcs (overall p < 0.001, ^bmain p < 0.005 by one-way ANOVA with post hoc Tukey test). D. C57BL/6 wild-type pOcs undergoing P. aeruginosa LPS-mediated Oc formation were concomitantly exposed to IL-1ra (0, 1, 10, 20 μg/ml). Clearly, decreased Oc formation was seen with increasing IL-1ra dose and achieved statistical significance at 10 and 20 μg/ml (^ap < 0.01). All data are expressed as the mean ± SEM of at least three experiments performed in triplicate wells.

FIG. 5

Quantitative PCR analysis of TNFR1, TNFR2, IL-1R1, and NFATc1 expression by pOcs undergoing P. aeruginosa LPS-mediated Oc formation. Here, P. aeruginosa LPS-mediated Oc formation refers to 72 h of RANKL (10 ng/ml) priming followed by P. aeruginosa LPS stimulation for an additional 96 h. RNA was harvested for PCR analysis at the following time points: (1) after 72 h of RANKL priming; (2) 24 h after P. aeruginosa LPS addition; and (3) 96 h after P. aeruginosa LPS addition. RAW 264.7 pOcs undergoing P. aeruginosa LPS-mediated Oc formation clearly show significantly increased expression of IL-1R1, TNFR1, TNFR2, and NFATc1 as maturation into Ocs progresses (^ap < 0.01). The data are expressed as the mean ± SD of at least two experiments performed in triplicate.