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Proinflammatory M1 Macrophages Inhibit RANKL-Induced Osteoclastogenesis.

ABSTRACT: In response to a defined panel of stimuli, immature macrophages can be classified into two major phenotypes: proinflammatory (M1) and anti-inflammatory (M2). Although both phenotypes have been implicated in several chronic inflammatory diseases, their direct role in bone resorption remains unclear. The present study investigated the possible effects of M1 and M2 macrophages on RANKL-induced osteoclastogenesis. In osteoclastogenesis assays using RAW264.7 cells or bone marrow cells as osteoclast precursors, addition of M1 macrophages significantly suppressed RANKL-induced osteoclastogenesis compared to nonstimulated conditions (M0), addition of M2 macrophages, or no macrophage addition (P < 0.05), suggesting that M1 macrophages can downregulate osteoclastogenesis. This effect was maintained when direct contact between M1 and osteoclast precursors was interrupted by cell culture insertion, indicating engagement of soluble factors released from M1. M1 macrophages developed from interferon gamma (IFN-?) knockout (IFN-?-KO) mice lost the ability to downregulate osteoclastogenesis. Antibody-based neutralization of interleukin-12 (IL-12), but not IL-10, produced by M1 macrophages also abrogated M1-mediated downregulation of osteoclastogenesis. Real-time PCR analyses showed that IFN-? suppressed gene expression of NFATc1, a master regulator of osteoclastogenesis, whereas IL-12 increased the apoptosis of osteoclasts, suggesting molecular mechanisms underlying the possible roles of IFN-? or IL-12 in M1-mediated inhibition of osteoclastogenesis. These findings were confirmed in an in vivo ligature-induced mouse periodontitis model in which adoptive transfer of M1 macrophages showed a significantly lower level of bone loss and less tartrate-resistant acid phosphatase (TRAP)-positive cell induction than M0 or M2 macrophage transfer. In conclusion, by its secretion of IFN-? and IL-12, M1, but not M0 or M2, was demonstrated to inhibit osteoclastogenesis.

SUBMITTER: Yamaguchi T

PROVIDER: S-EPMC5038061 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Proinflammatory M1 Macrophages Inhibit RANKL-Induced Osteoclastogenesis.

Yamaguchi Tsuguno T Movila Alexandru A Kataoka Shinsuke S Wisitrasameewong Wichaya W Ruiz Torruella Montserrat M Murakoshi Michiaki M Murakami Shinya S Kawai Toshihisa T

Infection and immunity 20160919 10

In response to a defined panel of stimuli, immature macrophages can be classified into two major phenotypes: proinflammatory (M1) and anti-inflammatory (M2). Although both phenotypes have been implicated in several chronic inflammatory diseases, their direct role in bone resorption remains unclear. The present study investigated the possible effects of M1 and M2 macrophages on RANKL-induced osteoclastogenesis. In osteoclastogenesis assays using RAW264.7 cells or bone marrow cells as osteoclast p ...[more]

PMID: 27456834

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Project description:Osteoclasts are large multinucleated cells responsible for bone resorption. Excessive inflammatory activation of osteoclasts leads to bony erosions, which are the hallmark of several diseases such as rheumatoid arthritis (RA). Salt-inducible kinases (SIK) constitute a subfamily of kinases comprising three members (SIK1, -2, and -3). Inhibition of SIK kinase activity induces an anti-inflammatory phenotype in macrophages. Since osteoclasts originate from precursors of macrophage origin, we hypothesized a role of SIK in osteoclastogenesis. We analyzed SIK1, -2 and -3 expression and function in osteoclast differentiation using the mouse macrophage cell line RAW264.7 and bone marrow-derived macrophages (BMM). We show that all three SIK are expressed in fully differentiated osteoclasts and that in BMM-derived osteoclasts there is an increased expression of SIK1 and SIK3 proteins. Interestingly, the pan-SIK inhibitor HG-9-91-01 significantly inhibited osteoclastogenesis by dose dependently reducing osteoclast differentiation markers (i.e. CathepsinK, MMP-9 and TRAP) and bone resorbing activity. Analysis of the signaling pathways activated by RANKL in RAW cells showed that SIK inhibitors did not affect RANKL-induced ERK1/2, JNK, p38 or NF-?B activation, but induced a significant downregulation in c-Fos and NFATc1 protein levels, the two main transcription factors involved in the regulation of osteoclast-specific genes. Moreover, SIK inhibition partially increased the proteasome-mediated degradation of c-Fos. SIK2 and SIK3 knockout RAW cells were generated by the CRISPR/Cas9 approach. SIK2 KO and, to a lesser extent, SIK3 KO recapitulated the effect of SIK small molecule inhibitor, thus confirming the specificity of the effect of SIK inhibition on the reduction of osteoclastogenesis. Overall, our results support the notion that the SIK signaling pathway plays a significant role among the check-points controlling osteoclastogenesis. SIK kinase inhibitors could thus represent a potential novel therapy to prevent bone erosions.

Dataset Information

Proinflammatory M1 Macrophages Inhibit RANKL-Induced Osteoclastogenesis.

Publications

Proinflammatory M1 Macrophages Inhibit RANKL-Induced Osteoclastogenesis.

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