STING-dependent interferon signatures restrict osteoclast differentiation and bone loss in mice

Stimulator of Interferon Genes (STING) is a key mediator of type-I interferon (IFN-I) signaling in response to a variety of stimuli, but the contribution of STING to homeostatic processes is not fully characterized. Previous studies showed ligand activation of STING limits osteoclast differentiation in vitro through the induction of IFN and IFN-I Interferon Stimulated Genes (ISGs). In a disease model (SAVI) driven by the V154M gain-of-function mutation in STING, fewer osteoclasts form from SAVI precursors in response to RANKL in an IFN-I-dependent manner. Due to the described role of STING-mediated regulation of osteoclastogenesis in activation settings, we sought to determine whether basal STING signaling contributes to bone homeostasis, an unexplored area. Using whole-body and myeloid-specific deficiency, we show that STING signaling prevents trabecular bone loss in mice over time and that myeloid-restricted STING activity is sufficient for this effect. STING-deficient osteoclast precursors differentiate with greater efficiency than wildtypes. RNA sequencing of wildtype and STING-deficient osteoclast precursor cells and differentiating osteoclasts reveals novel clusters of ISGs including a previously undescribed ISG set expressed in RANKL naïve precursors (tonic expression) and downregulated during differentiation. We identify a 50 gene tonic ISG signature that is STING-dependent and shapes osteoclast differentiation. From this list, we identify Interferon Stimulated Gene 15 (ISG15) as a tonic STING-regulated ISG that limits osteoclast formation. Thus, STING is an important upstream regulator of tonic IFN-I signatures shaping the commitment to osteoclast fates, providing evidence for a nuanced and novel role for this pathway in bone homeostasis. Bulk RNA sequencing of murine bone marrow derived osteoclast precursors at baseline (d0) and following RANKL stimulation for 2, 3 and 4 days differentiated in vitro. Samples are from either WT or STING-deficient bone marrow. Samples were isolated from three mice per genotype (triplicates).

干扰素基因刺激因子（Stimulator of Interferon Genes, STING）是应对多种刺激时I型干扰素（type-I interferon, IFN-I）信号通路的关键介导因子，但STING在机体稳态生理过程中的调控作用尚未得到充分阐释。既往研究证实，STING经配体激活后，可通过诱导干扰素β（IFN-β）及I型干扰素刺激基因（Interferon Stimulated Genes, ISGs）的表达，在体外抑制破骨细胞的分化。在由STING的V154M功能获得性突变驱动的SAVI疾病模型中，SAVI来源的前体细胞经核因子κB受体活化因子配体（Receptor Activator of Nuclear Factor κB Ligand, RANKL）刺激后生成的破骨细胞数量更少，且该效应依赖IFN-I信号通路。鉴于已有研究揭示了STING介导的破骨细胞生成调控在激活状态下的作用，我们旨在探究基础STING信号是否参与骨稳态调控——这一此前未被探索的研究方向。通过构建全身敲除及髓系特异性敲除小鼠模型，我们证实STING信号可随时间推移防止小鼠小梁骨丢失，且髓系限制性STING活性足以介导该保护效应。STING缺陷型破骨细胞前体细胞的分化效率显著高于野生型细胞。对野生型及STING缺陷型破骨细胞前体细胞与分化中的破骨细胞进行RNA测序，我们发现了一类新的干扰素刺激基因簇，其中包含一组此前未被报道的ISG：该基因集在未受RANKL刺激的前体细胞中呈基础表达，且在细胞分化过程中被下调。我们鉴定出一个包含50个基因的STING依赖性基础表达ISG特征谱，该特征谱可调控破骨细胞的分化。从该基因列表中，我们进一步鉴定出干扰素刺激基因15（Interferon Stimulated Gene 15, ISG15）作为受STING基础调控的ISG，其可抑制破骨细胞的生成。综上，STING是调控基础IFN-I特征谱的重要上游调节因子，该特征谱可影响破骨细胞的命运决定，为该通路在骨稳态中发挥精细且全新的调控作用提供了实验证据。本数据集包含小鼠骨髓来源破骨细胞前体细胞在基线状态（第0天），以及体外经RANKL刺激2、3、4天后的分化阶段中的批量RNA测序数据。样本分别来自野生型（WT）及STING缺陷型小鼠的骨髓，每种基因型的样本均分离自3只小鼠，设置生物学重复三次。