<i>P. gingivalis</i> induces osteoclastogenesis in RANKL-primed RAW-D cells in the absence of TNF-α.

<p>Analysis of TNF-α mRNA expression (A) or production of TNF-α protein (B) by <i>P. gingivalis</i> infected RANKL-primed RAW-D cells and unprimed cells. (C) Effect of neutralizing antibody against mouse TNF-α on osteoclast formation in RANKL-primed RAW-D cells induced by TNF-α or live <i>P. gingivalis</i>. RAW-D cells were primed with RANKL (50 ng/ml) for 22 h and then retreated with TNF-α or live <i>P. gingivalis</i> in the presence or absence of neutralizing antibody against mouse TNF-α or control IgG. After 24 h, RNA was extracted, and TNF-α mRNA expression was assessed by real-time PCR. After 48 h, cell supernatants were collected and analyzed for TNF-α by ELISA. After 48 h, the culture was stained for TRAP, and the number of TRAP-positive MNCs was counted. Data are expressed as mean ± S.D. of four independent cultures. Statistical significance was determined with Student’s <i>t</i> test. **P<0.01, *P<0.05 compared with unprimed infected RAW-D or RANKL-primed uninfected control (A), unprimed control (B), or control IgG1 (C).</p

Infection of RANKL-primed RAW-D macrophages with <i>P. gingivalis</i> induces osteoclastogenesis.

<p>(A) (B) <i>P. gingivalis</i> infection of RANKL-primed RAW-D cells induces the formation of TRAP-positive MNCs. (C) <i>P. gingivalis</i> infection of RANKL-primed RAW-D cells induces mRNA expression of the osteoclast-specific gene, cathepsin K. Total RNA was isolated, and cathepsin K expression was assessed by real-time PCR. Expression levels were normalized to GAPDH. Effect of pretreatment (D), or OPG (E) on osteoclastogenesis induced by infection with <i>P. gingivalis</i>. RAW-D cells were primed with RANKL (50 ng/ml) or TNF-α (10 ng/ml) for 22 h, then infected with <i>P. gingivalis</i>, and cultured for 24–48 h. After 24 h, RNA was extracted and analyzed for gene expression. After 48 h, the culture was stained for TRAP, and TRAP-positive MNCs were counted. Data are expressed as mean ± S.D. of four independent cultures. Statistical significance was determined with Student’s <i>t</i> test. **P<0.01, *P<0.05 compared to uninfected control (B, C) or untreated control (D).</p

Infection of RANKL-primed BMM with <i>P. gingivalis</i> induces osteoclastogenesis in the absence of TNF-α.

<p>(A) Infection of RANKL-primed BMM with <i>P. gingivalis</i> induces osteoclastogenesis. Representative photographs are shown. (B) Effect of neutralizing antibody against mouse TNF-α on osteoclastogenesis in RANKL-primed BMM induced by TNF-α or live <i>P. gingivalis</i>. BMM were stimulated with RANKL (50 ng/ml) for 22 h and then re-stimulated by TNF-α or live <i>P. gingivalis</i> (m.o.i.  = 10) in the presence or absence of neutralizing antibody against mouse TNF-α or control IgG. At the end of culture, the culture was stained for TRAP, and TRAP-positive MNCs were counted. Data are expressed as mean ± S.D. of four independent cultures. Statistical significance was determined with Student’s <i>t</i> test. **P<0.01, compared with control IgG1.</p

Possible role of TNF-α in osteoclastogenesis in the presence or absence of <i>P. gingivalis</i>.

<p>Macrophages respond to infection with <i>P. gingivalis</i> by producing TNF-α, which stimulates osteoclastogenesis in osteoclast precursor cells in the absence of <i>P. gingivalis</i> (A). However, osteoclast precursor cells primed with RANKL do not produce TNF-α and respond differentially to various stimuli. (B) Cells that are not stimulated do not differentiate into osteoclasts. (C) Cells that are continuously re-stimulated with RANKL differentiate into osteoclasts in an NFATc1- and NF-κB-dependent manner in the presence of <i>P. gingivalis</i>. (D) Cells that are infected with <i>P. gingivalis</i> differentiate into osteoclasts in an NFATc1-dependent and NF-κB-independent manner. TNF-α does not stimulate osteoclastogenesis in osteoclast precursor cells in the presence of <i>P. gingivalis</i>, whereas RANKL stimulates osteoclastogenesis in the presence or absence of <i>P. gingivalis</i>.</p

Expression of osteoclast signaling proteins in osteoclastogenesis in RANKL-primed RAW-D cells induced by infection.

<p>RAW-D cells were stimulated with or without RANKL (50 ng/ml) or <i>P. gingivalis</i> for 22 h. RANKL-primed RAW-D cells were then retreated with or without RANKL (50 ng/ml) or <i>P. gingivalis</i> for 24 h. Total RNA was prepared, cDNA was synthesized, and real-time PCR analysis was performed using NFATc1 (A), c-fos (B), or IFNβ (C) Taqman probes. Statistical significance was determined with Student’s <i>t</i> test. **P<0.01, *P<0.05 compared with unprimed control or RANKL-primed control.</p

RANKL but not TNF-α promotes osteoclastogenesis in RANKL-primed RAW-D cells in the presence of <i>P. gingivalis</i>.

<p>RAW-D cells were stimulated with RANKL (50 ng/ml) for 22 h and then re-stimulated with RANKL or TNF-α in the absence (A) or presence (B) of live <i>P. gingivalis.</i> The culture was stained for TRAP activity after 48 h of retreatment, and TRAP-positive MNCs were counted. Data are expressed as mean ± S.D. of four independent cultures. Statistical significance was determined with Student’s <i>t</i> test. **P<0.01, compared to cultures without RANKL or TNF-α. (C) Expression of mRNAs for p55 and p75 TNF receptors, TLR2, and TLR4 in RAW-D cells treated with or without <i>P. gingivalis</i> for 22 h, or RANKL-primed RAW-D cells retreated with or without RANKL or <i>P. gingivalis</i> for 24 h. Total RNA was isolated, and mRNA expression was assessed by semi-quantitative RT-PCR using specific primers as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038500#s4" target="_blank">Materials and Methods</a></i>.</p

TLR4 is not involved in osteoclastogenesis in RANKL-primed RAW-D cells induced by infection with <i>P. gingivalis</i>.

<p>Effect of <i>E.coli</i> LPS or Pam3CSK4 (A) or <i>P. gingivalis</i> LPS (B) on osteoclastogenesis in RANKL-primed RAW-D cells. (C) Effect of heat treatment of <i>P. gingivalis</i> on osteoclastogenesis in RANKL-primed RAW-D cells. (D) Effect of polymyxin B on osteoclast formation in RANKL-primed RAW-D cells induced by <i>E. coli</i> LPS, Pam3CSK4, live <i>P. gingivalis</i>, or <i>P. gingivalis</i> LPS. RAW-D cells were primed with RANKL (50 ng/ml) for 22 h and then treated with <i>E. coli</i> LPS (100 ng/ml), Pam3CSK4 (100 ng/ml), <i>P. gingivalis</i> LPS (10 µg/ml) or live <i>P. gingivalis</i> (m.o.i.  = 10) in the presence of various concentrations of polymyxin B. After 48 h, the culture was stained for TRAP, and the number of TRAP-positive MNCs was counted. Data are expressed as mean ± S.D. of four independent cultures. Statistical significance was determined with Student’s <i>t</i> test. **P<0.01 compared to untreated controls (A, B, and C) or controls without polymyxin B (D).</p

Analysis of expression of genes encoding cell surface proteins in BMM, preosteoclasts, and MNCs.

<p>(A) Relative mRNA expression levels of <i>emr1</i> (F4/80), <i>itgam</i> (CD11b), <i>CCR1, CCR2,</i> and <i>CCR5</i> in cultures of BMM, preosteoclasts (pre-OC) and MNCs. (B) Immunofluorescence images of preosteoclasts stained for Kat1 antigen (red) and CD11b/c (green). (C) Percentages of Kat1<b>⁻</b>CD11b/c⁺, Kat1⁺CD11b/c⁺ or Kat1⁺CD11b/c<b>⁻</b> cells among cells induced by M-CSF in combination with TNF-α and/or TGF-β. NABMCs were cultured for 3 days in the presence of various combinations of TNF-α, TGF-β, and M-CSF to induce formation of BMM or preosteoclasts. After formation of preosteoclasts, RANKL was added to induce formation of MNCs. Total RNA from cells was prepared and analyzed by real-time RT-PCR using specific primers as in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047930#s2" target="_blank">Materials and Methods</a></i>. Expression levels were normalized with <i>gapdh</i> (A). The cells were stained for Kat1 and CD11b and analyzed by confocal microscopy using LSM5Pascal software. The photographs show typical cultures. (B). Each value represents the mean ± SEM (n = 3, A) or (cell counts from ten-microscope fields, C). Statistical significance was determined by Student’s <i>t</i> test; *P<0.05, **P<0.01 compared with BMM (A) or TGF-β alone (C). Bar = 20 µm. The experiments were performed three times, and similar results were obtained.</p

TNF-α and TGF-β specifically increase subpopulations of Kat1⁺c-fms⁺ and Kat1⁺c-fms⁻ preosteoclasts.

<p>(A) Confocal microscopic analysis of Kat1⁺, c-fms⁺, or Kat1⁺c-fms⁺ cells in cultures induced by M-CSF and various concentrations of TNF-α, and TGF-β. Bar = 50 µm. (B) Percentages of Kat1<b>⁻</b>c-fms⁺, Kat1⁺c-fms⁺ or Kat1⁺c-fms<b>⁻</b> cells among cells induced by M-CSF and various concentrations of TNF-α and TGF-β. NABMCs were cultured for 2.5 days. Cells were stained for Kat1 and c-fms, and analyzed by confocal microscopy with LSM5Pascal software. The photographs show typical cultures. Each value represents the mean ± SEM of ten fields in a typical culture. Statistical significance was determined by Student’s <i>t</i> test; **P<0.01 compared with TNF-α or TGF-β alone. We have repeated these experiments and similar results were obtained.</p

Kat1⁺ cells represent a late differentiation stage of preosteoclasts.

<p>(A) Photographs of cells that were Kat1<b>⁻</b>c-fms⁺, Kat1⁺c-fms⁺, or Kat1⁺c-fms<b>⁻</b> in time-course cultures. Cells were stained for Kat1 and c-fms, and analyzed by confocal microscopy with LSM5Pascal software. Bar = 50 µm. (B) Distribution of cells from cultures shown in (A). Each value represents the mean ± SEM of ten fields in a typical culture. Statistical significance was determined by Student’s <i>t</i> test; *P<0.05, **P<0.01 compared with 48 hours. (C) Formation of TRAP⁺ MNCs from enriched populations of Kat1⁺ or Kat1⁻ cells in preosteoclasts cultures. NABMCs were cultured with TNF-α, TGF-β, and M-CSF for 2.5 days. Kat1⁺ cells and Kat1⁻ cells in preosteoclasts cultures were isolated by panning with mAb Kat1 as described in <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047930#s2" target="_blank">Materials and Methods</a></i>, and cultured for 2 days in the presence of RANKL (30 ng/ml) and M-CSF (3 ng/ml). Statistical significance was determined by Student’s <i>t</i> test. **P<0.01 compared with Kat-1⁻ cells. We have repeated this experiments and similar results were obtained.</p