Inhibition of Notch Signaling Stimulates Osteoclastogenesis From the Common Trilineage Progenitor Under Inflammatory Conditions

Abstract

Osteoclasts, macrophages and dendritic cells (DCs) can be derived from a common trilineage myeloid progenitor of hematopoietic origin. Progenitor commitment is susceptible to regulation through Notch signaling. Our aim was to determine the effects of Notch modulation on trilineage progenitor commitment and functional properties of differentiated cells under inflammatory conditions. We used the conditional inducible CX3CR1CreERT2 mouse strain to achieve overexpression of the Notch 1 intracellular domain (NICD1) or to inhibit Notch signaling via deletion of the transcription factor RBP-J in a bone marrow population, used as a source of the trilineage progenitor (CD45+Ly6G−CD3−B220−NK1.1−CD11b–/loCD115+). Cre-recombinase, under the control of the CX3CR1 promoter, expressed in the monocyte/macrophage lineage, was induced in vitro by 4-hydroxytamoxifen. Differentiation of osteoclasts was induced by M-CSF/RANKL; macrophages by M-CSF; DCs by IL-4/GM-CSF, and inflammation by LPS. Functionally, DCs were tested for the ability to process and present antigen, macrophages to phagocytose E. coli particles, and osteoclasts to resorb bone and express tartrate-resistant acid phosphatase (TRAP). We found that Notch 1 signal activation suppressed osteoclast formation, whereas disruption of the Notch canonical pathway enhanced osteoclastogenesis, resulting in a higher number and size of osteoclasts. RANK protein and Ctsk gene expression were upregulated in osteoclastogenic cultures from RBP-J+ mice, with the opposing results in NICD1+ mice. Notch modulation did not affect the number of in vitro differentiated macrophages and DCs. However, RBP-J deletion stimulated Il12b and Cd86 expression in macrophages and DCs, respectively. Functional assays under inflammatory conditions confirmed that Notch silencing amplifies TRAP expression by osteoclasts, whereas the enhanced phagocytosis by macrophages was observed in both NICD1+ and RBP-J+ strains. Finally, antigen presentation by LPS-stimulated DCs was significantly downregulated with NICD1 overexpression. This experimental setting allowed us to define a cell-autonomous response to Notch signaling at the trilineage progenitor stage. Although Notch signaling modulation affected the activity of all three lineages, the major effect was observed in osteoclasts, resulting in enhanced differentiation and function with inhibition of canonical Notch signaling. Our results indicate that Notch signaling participates as the negative regulator of osteoclast activity during inflammation, which may be relevant in immune and bone diseases.

Transcriptome profiling of osteoclast subsets associated with arthritis: A pathogenic role of CCR2hi osteoclast progenitors

“…CIA was induced in male B6 mice by a modified protocol as previously described ( 9 ), and periarticular bone marrow of distal tibia was collected for flow cytometric analysis and cell sorting 33-35 days following immunization ( Figure 1A ). To identify osteoclastogenic cell subsets in the periarticular area, gating strategy was performed as previously described ( 9 , 32 ). Bone marrow OCPs were dissected by delineation of live bone marrow single-cells that were further gated for hematopoietic (CD45 + ) agranulocytes (Ly6G − ), followed by gating of non-lymphoid (CD3 − B220 − NK1.1 − ) cells with low expression of CD11b (CD11b −/lo ) ( Supplementary Figure 1A ).…”

“…Moreover, proportion of OCPs expressing protein level of Kit/CD117 was higher in CCR2 lo than in CCR2 hi subset. CD115 + CD117 + double-positive cells, lacking expression of other mature hematopoietic markers, were shown to exhibit characteristics of monocyte progenitors with the multilineage potency to differentiate into osteoclasts, dendritic cells and macrophages ( 8 , 32 ). Further dissection of the bone marrow using CD115 and CD117 markers showed that, as OCPs mature, they downregulate CD117 but remain CD115 + ( 6 ), which is in line with our observation of lower Kit expression in OCPs stimulated by RANKL and M-CSF in vitro compared to pre-cultured OCPs.…”

“…Sorting of CCR2 hi OCPs (CD45 + Ly6G − CD3 − B220 − NK1.1 − CD11b –/lo CD115 + CCR2 hi ) and CCR2 lo OCPs (CD45 + Ly6G − CD3 − B220 − NK1.1 − CD11b –/lo CD115 + CCR2 lo ) from periarticular bone marrow was performed on the BD FACSAria II instrument, using gating strategies as described previously ( 9 , 32 ). Gates were set according to unstained, fluorescence minus one and/or isotype controls.…”