Department of Biochemistry and Molecular Biology, Asan Medical Center and AMIST, University of Ulsan College of Medicine, 88, Olympic-Ro 43-Gil, Songpa-Gu, Seoul, 05505, Korea.
Stem Cell Immunomodulation Research Center, University of Ulsan College of Medicine, Seoul, 05505, Korea.
Cell Commun Signal. 2023 Aug 18;21(1):213. doi: 10.1186/s12964-023-01226-w.
Toll-like receptor 4 (TLR4) conducts a highly regulated inflammatory process by limiting the extent of inflammation to avoid toxicity and tissue damage, even in bone tissues. Thus, it is plausible that strategies for the maintenance of normal bone-immunity to prevent undesirable bone damage by TLR4 activation can exist, but direct evidence is still lacking.
Osteoclast precursors (OCPs) obtained from WT or Slit3-deficient mice were differentiated into osteoclast (OC) with macrophage colony-stimulating factor (M-CSF), RANK ligand (RANKL) and lipopolysaccharide (LPS) by determining the number of TRAP-positive multinuclear cells (TRAP MNCs). To determine the alteration of OCPs population, fluorescence-activated cell sorting (FACS) was conducted in bone marrow cells in mice after LPS injection. The severity of bone loss in LPS injected WT or Slit3-deficient mice was evaluated by micro-CT analysis.
We demonstrate that TLR4 activation by LPS inhibits OC commitment by inducing the concomitant expression of miR-218-2-3p and its host gene, Slit3, in mouse OCPs. TLR4 activation by LPS induced SLIT3 and its receptor ROBO1 in BMMs, and this SLIT3-ROBO1 axis hinders RANKL-induced OC differentiation by switching the protein levels of C/EBP-β isoforms. A deficiency of SLIT3 resulted in increased RANKL-induced OC differentiation, and the elevated expression of OC marker genes including Pu.1, Nfatc1, and Ctsk. Notably, Slit3-deficient mice showed expanded OCP populations in the bone marrow. We also found that miR-218-2 was concomitantly induced with SLIT3 expression after LPS treatment, and that this miRNA directly suppressed Tnfrsf11a (RANK) expression at both gene and protein levels, linking it to a decrease in OC differentiation. An endogenous miR-218-2 block rescued the expression of RANK and subsequent OC formation in LPS-stimulated OCPs. Aligned with these results, SLIT3-deficient mice displayed increased OC formation and reduced bone density after LPS challenge.
Our findings suggest that the TLR4-dependent concomitant induction of Slit3 and miR-218-2 targets RANK in OCPs to restrain OC commitment, thereby avoiding an uncoordinated loss of bone through inflammatory processes. These observations provide a mechanistic explanation for the role of TLR4 in controlling the commitment phase of OC differentiation. Video Abstract.
Toll 样受体 4（TLR4）通过限制炎症的程度来进行高度调控的炎症过程，从而避免毒性和组织损伤，即使在骨组织中也是如此。因此，维持正常的骨免疫以防止 TLR4 激活引起的不良骨损伤的策略是有可能存在的，但目前仍缺乏直接证据。
用巨噬细胞集落刺激因子（M-CSF）、核因子-κB 配体（RANKL）和脂多糖（LPS）将从 WT 或 Slit3 缺陷型小鼠获得的破骨细胞前体细胞（OCPs）分化为破骨细胞（OC），通过 TRAP 阳性多核细胞（TRAP MNCs）的数量来确定。为了确定 OCP 群体的改变，在 LPS 注射后通过流式细胞术（FACS）对小鼠骨髓细胞进行检测。通过微 CT 分析评估 LPS 注射 WT 或 Slit3 缺陷型小鼠的骨丢失严重程度。
我们证明 LPS 激活 TLR4 通过诱导 miR-218-2-3p 及其宿主基因 Slit3 的共表达来抑制 OC 分化。LPS 激活 TLR4 在 BMMs 中诱导 SLIT3 和其受体 ROBO1，该 SLIT3-ROBO1 轴通过切换 C/EBP-β 同工型的蛋白水平来抑制 RANKL 诱导的 OC 分化。Slit3 的缺乏导致 RANKL 诱导的 OC 分化增加，OC 标记基因包括 Pu.1、Nfatc1 和 Ctsk 的表达升高。值得注意的是，Slit3 缺陷型小鼠在骨髓中显示出扩大的 OCP 群体。我们还发现，LPS 处理后 miR-218-2 与 SLIT3 表达同时诱导，该 miRNA 直接在基因和蛋白水平上抑制 Tnfrsf11a（RANK）的表达，从而导致 OC 分化减少。内源性 miR-218-2 阻断物可挽救 LPS 刺激的 OCP 中 RANK 的表达和随后的 OC 形成。这些结果表明，TLR4 依赖性 Slit3 和 miR-218-2 的共诱导靶标 RANK 在 OCP 中抑制 OC 分化，从而避免炎症过程中骨的不协调丢失。这些观察结果为 TLR4 在控制 OC 分化的启动阶段中的作用提供了机制解释。视频摘要。
