Infection of RANKL-primed RAW-D macrophages with Porphyromonas gingivalis promotes osteoclastogenesis in a TNF-α-independent manner.
Infection of macrophages with bacteria induces the production of pro-inflammatory cytokines including TNF-α. TNF-α directly stimulates osteoclast differentiation from bone marrow macrophages in vitro as well as indirectly via osteoblasts. Recently, it was reported that bacterial components such as LPS inhibited RANKL-induced osteoclastogenesis in early stages, but promoted osteoclast differentiation in late stages. However, the contribution to osteoclast differentiation of TNF-α produced by infected macrophages remains unclear. We show here that Porphyromonas gingivalis, one of the major pathogens in periodontitis, directly promotes osteoclastogenesis from RANKL-primed RAW-D (subclone of RAW264) mouse macrophages, and we show that TNF-α is not involved in the stimulatory effect on osteoclastogenesis. P. gingivalis infection of RANKL-primed RAW-D macrophages markedly stimulated osteoclastogenesis in a RANKL-independent manner. In the presence of the TLR4 inhibitor, polymyxin B, infection of RANKL-primed RAW-D cells with P. gingivalis also induced osteoclastogenesis, indicating that TLR4 is not involved. Infection of RAW-D cells with P. gingivalis stimulated the production of TNF-α, whereas the production of TNF-α by similarly infected RANKL-primed RAW-D cells was markedly down-regulated. In addition, infection of RANKL-primed macrophages with P. gingivalis induced osteoclastogenesis in the presence of neutralizing antibody against TNF-α. Inhibitors of NFATc1 and p38MAPK, but not of NF-κB signaling, significantly suppressed P. gingivalis-induced osteoclastogenesis from RANKL-primed macrophages. Moreover, re-treatment of RANKL-primed macrophages with RANKL stimulated osteoclastogenesis in the presence or absence of P. gingivalis infection, whereas re-treatment of RANKL-primed macrophages with TNF-α did not enhance osteoclastogenesis in the presence of live P. gingivalis. Thus, P. gingivalis infection of RANKL-primed macrophages promoted osteoclastogenesis in a TNF-α independent manner, and RANKL but not TNF-α was effective in inducing osteoclastogenesis from RANKL-primed RAW-D cells in the presence of P. gingivalis.
Department of Microbiology, Faculty of Medicine, Saga University, Saga, Japan. kukita@cc.saga-u.ac.jp
Akiko
A
Kukita
Yuka
Y
Ichigi
Ippei
I
Takigawa
Toshiyuki
T
Watanabe
Toshio
T
Kukita
Hiroshi
H
Miyamoto
D000818:Animals; D002460:Cell Line; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects / Q000382:microbiology* / Q000502:physiology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D016966:Porphyromonas gingivalis; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D053245:RANK Ligand / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D016328:NF-kappa B; D050778:NFATC Transcription Factors; D016760:Proto-Oncogene Proteins c-fos; D053245:RANK Ligand; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4