Department of Physiology and Immunology, University of Zagreb School of Medicine, Zagreb, Croatia.
Laboratory for Molecular Immunology, Croatian Institute for Brain Research, University of Zagreb School of Medicine, Zagreb, Croatia.
Front Immunol. 2022 Jul 5;13:902947. doi: 10.3389/fimmu.2022.902947. eCollection 2022.
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 deletion of the transcription factor RBP-J in a bone marrow population, used as a source of the trilineage progenitor (CD45Ly6GCD3B220NK1.1CD11bCD115). Cre-recombinase, under the control of the CX3CR1 promoter, expressed in the monocyte/macrophage lineage, was induced 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 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 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 differentiated macrophages and DCs. However, RBP-J deletion stimulated and 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.
破骨细胞、巨噬细胞和树突状细胞 (DC) 可由造血来源的共同三系髓样祖细胞衍生而来。祖细胞的定向是易受 Notch 信号调节的。我们的目的是确定 Notch 调节对炎症条件下三系祖细胞定向和分化细胞功能特性的影响。我们使用条件性诱导的 CX3CR1CreERT2 小鼠品系，通过过表达 Notch1 胞内结构域（NICD1）或在骨髓群体中抑制 Notch 信号转导（转录因子 RBP-J 的缺失）来实现，作为三系祖细胞（CD45Ly6GCD3B220NK1.1CD11bCD115）的来源。在单核细胞/巨噬细胞谱系中由 CX3CR1 启动子控制的 Cre 重组酶，通过 4-羟基他莫昔芬诱导表达。诱导 DC 的分化由 IL-4/GM-CSF 完成，由 LPS 诱导炎症。在功能上，通过处理和呈递抗原来测试 DC 的能力，通过吞噬颗粒来测试巨噬细胞的能力，通过吸收骨和表达抗酒石酸酸性磷酸酶 (TRAP) 来测试破骨细胞的能力。我们发现 Notch1 信号激活抑制破骨细胞形成，而 Notch 经典途径的破坏增强破骨细胞生成，导致破骨细胞数量和大小增加。在 RBP-J 小鼠的破骨细胞生成培养物中，RANK 蛋白和基因表达上调，而在 NICD1 小鼠中则出现相反的结果。Notch 调节不影响分化的巨噬细胞和 DC 的数量。然而，RBP-J 缺失分别刺激巨噬细胞和 DC 中的 和 表达。在炎症条件下的功能测定证实，Notch 沉默增强破骨细胞中 TRAP 的表达，而在 NICD1 和 RBP-J 品系中均观察到巨噬细胞吞噬作用增强。最后，LPS 刺激的 DC 的抗原呈递通过 NICD1 过表达显著下调。这种实验设置允许我们在三系祖细胞阶段定义对 Notch 信号的细胞自主反应。尽管 Notch 信号调节影响所有三个谱系的活性，但主要影响发生在破骨细胞中，通过抑制经典 Notch 信号转导来增强分化和功能。我们的结果表明，Notch 信号在炎症期间作为破骨细胞活性的负调节剂参与，这在免疫和骨骼疾病中可能相关。
