Enhanced dual function of osteoclast precursors following calvarial Porphyromonas gingivalis infection

Abstract

Background and objective:Excessive osteoclast activity is a major characteristic of pathogenic bone loss in inflammatory bone diseases including periodontitis.However, beyond the knowledge that osteoclasts are differentiated from the monocyte/macrophage lineage and share common ancestry with macrophages and DC, the nature and function of osteoclast precursors are not completely understood. Furthermore, little is known about how osteoclast precursors respond to bacterial infection in vivo. We have previously demonstrated in vitro that the periodontal pathogen Porphyromonas gingivalis (Pg) plays a biphasic role on the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation. In this study, we investigated the in vivo effect of Pg infection on the regulation of osteoclast precursors, using a mouse calvarial infection model. Methods and results: C57BL/6 wild-type and the myeloid differentiation factor 88 knockout (MyD88 −/− ) mice were infected with Pg by calvarial injection. Local and systemic bone loss, and the number and function of CD11b + c-fms + cells from bone marrow and spleen were analyzed. Our results show that Pg infection induces localized inflammatory infiltration and osteoclastogenesis, as well as increased number and osteoclastogenic potential of CD11b + c-fms + osteoclast precursors in the bone marrow and periphery. We also show that CD11b + c-fms + RANK + and CD11b + c-fms + RANK − are precursors with similar osteoclastogenic and pro-inflammatory potentials. In addition, CD11b + c-fms + cells exhibit an antigen-specific T-cell immune-suppressive activity, which are increased with Pg infection. Moreover, we demonstrate that MyD88 is involved in the regulation of osteoclast precursors upon Pg infection. Conclusions:In this study, we demonstrate an enhanced dual function of osteoclast precursors following calvarial Pg infection. Based on our findings, we propose the following model: Pg infection increases a pool of precursor cells that can be shunted toward osteoclast formation at the infection/inflammation sites, while at the same

Frontline Science: Characterization and regulation of osteoclast precursors following chronic Porphyromonas gingivalis infection

“…Pg ATCC 33277 was cultured and maintained on enriched trypticase soy agar plates containing 1% yeast extract, 5% defibrinated sheep blood, 5 μg/ml hemin, and 1 μg/ml menadione, at 37°C in an anaerobic atmosphere of 10% H 2 , 5% CO 2 , and 85% N 2 32 . To prepare Pg for infection, the bacteria were grown in trypsin soy broth (BD Biosciences, San Jose, CA, USA) containing 1% yeast extract, 5 μg/ml heme, and 2.5 μg/ml menadione.…”

“…To prepare Pg for infection, the bacteria were grown in trypsin soy broth (BD Biosciences, San Jose, CA, USA) containing 1% yeast extract, 5 μg/ml heme, and 2.5 μg/ml menadione. The bacteria were collected by centrifugation and washed in PBS, and the number of bacteria (colony‐forming units/ml) was determined by measuring the optical density at 600 nm and extrapolating using a standard curve, with a culture of OD 600 of 1 equals 10 9 CFU/ml 32 …”

“…Single‐cell suspensions were prepared from BM, SPL, or PB, as previously described 31,32,35,36 . Briefly, femur and tibiae were isolated and both ends of the bones were cut off and BM was flushed with PBS using a 25‐gauge needle.…”

“…Cells were stained for tartrate‐resistant acid phosphatase (TRAP) activity using a leukocyte acid phosphatase kit (Sigma, St. Louis, MO, USA). TRAP + MNCs (≥3 nuclei) were considered as OCs 32 . To evaluate F‐actin ring formation of OCs, differentiated cells were fixed, permeabilized, and stained with Rhodamine Phalloidin (Invitrogen, Waltham, MA, USA) 32 .…”

Multitasking by the OC Lineage during Bone Infection: Bone Resorption, Immune Modulation, and Microbial Niche

“…P. gingivalis increases the osteoclastogenic potential of OC precursors both locally and systemically [ 49 ]. In addition to LPS-mediated TLR activation, P. gingivalis releases gingipains, cysteine proteases that can induce osteoclastogenesis of RANKL-treated OCs and enhance OC activity through an increase in integrin αvβ3 and degradation of OPG in co-culture with OBs [ 50 , 51 ].…”