{"title":"在拔牙模型中，巨噬细胞通过肿瘤坏死因子α调节间充质干细胞的功能。","authors":"Aung Ye Mun, Kentaro Akiyama, Ziyi Wang, Jiewen Zhang, Wakana Kitagawa, Teisaku Kohno, Ryuji Tagashira, Kei Ishibashi, Naoya Matsunaga, Tingling Zou, Mitsuaki Ono, Takuo Kuboki","doi":"10.1093/jbmrpl/ziae085","DOIUrl":null,"url":null,"abstract":"<p><p>Mesenchymal stem cells (MSCs) and macrophages collaboratively contribute to bone regeneration after injury. However, detailed mechanisms underlying the interaction between MSCs and inflammatory macrophages (M1) remain unclear. A macrophage-depleted tooth extraction model was generated in 5-wk-old female C57BL/6J mice using clodronate liposome (12.5 mg/kg/mouse, intraperitoneally) or saline injection (control) before maxillary first molar extraction. Mice were sacrificed on days 1, 3, 5, 7, and 10 after tooth extraction (<i>n</i> = 4). Regenerated bone volume evaluation of tooth extraction socket (TES) and histochemical analysis of CD80<sup>+</sup>M1, CD206<sup>+</sup>M2 (anti-inflammatory macrophages), PDGFRα<sup>+</sup>MSC, and TNF-α<sup>+</sup> cells were performed. In vitro, isolated MSCs with or without TNF-α stimulation (10 ng/mL, 24 h, <i>n</i> = 3) were bulk RNA-sequenced (RNA-Seq) to identify TNF-α stimulation-specific MSC transcriptomes. Day 7 micro-CT and HE staining revealed significantly lower mean bone volume (clodronate vs control: 0.01 mm<sup>3</sup> vs 0.02 mm<sup>3</sup>, <i>p</i><.0001) and mean percentage of regenerated bone area per total TES in clodronate group (41.97% vs 54.03%, <i>p</i><.0001). Clodronate group showed significant reduction in mean number of CD80<sup>+</sup>, TNF-α<sup>+</sup>, PDGFRα<sup>+</sup>, and CD80<sup>+</sup>TNF-α<sup>+</sup> cells on day 5 (306.5 vs 558.8, <i>p</i><.0001; 280.5 vs 543.8, <i>p</i><.0001; 365.0 vs 633.0, <i>p</i><.0001, 29.0 vs 42.5, <i>p</i><.0001), while these cells recovered significantly on day 7 (493.3 vs 396.0, <i>p</i>=.0004; 479.3 vs 384.5, <i>p</i>=.0008; 593.0 vs 473.0, <i>p</i>=.0010, 41.0 vs 32.5, <i>p</i>=.0003). RNA-Seq analysis showed that 15 genes (|log2FC| > 5.0, log2TPM > 5) after TNF-α stimulation were candidates for regulating MSC's immunomodulatory capacity. In vivo, <i>Clec4e</i> and <i>Gbp6</i> are involved in inflammation and bone formation. <i>Clec4e</i>, <i>Gbp6</i>, and <i>Cxcl10</i> knockdown increased osteogenic differentiation of MSCs in vitro. Temporal reduction followed by apparent recovery of TNF-α-producing M1 macrophages and MSCs after temporal macrophage depletion suggests that TNF-α activated MSCs during TES healing. In vitro mimicking the effect of TNF-α on MSCs indicated that there are 15 candidate MSC genes for regulation of immunomodulatory capacity.</p>","PeriodicalId":14611,"journal":{"name":"JBMR Plus","volume":null,"pages":null},"PeriodicalIF":3.4000,"publicationDate":"2024-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11289833/pdf/","citationCount":"0","resultStr":"{\"title\":\"Macrophages modulate mesenchymal stem cell function via tumor necrosis factor alpha in tooth extraction model.\",\"authors\":\"Aung Ye Mun, Kentaro Akiyama, Ziyi Wang, Jiewen Zhang, Wakana Kitagawa, Teisaku Kohno, Ryuji Tagashira, Kei Ishibashi, Naoya Matsunaga, Tingling Zou, Mitsuaki Ono, Takuo Kuboki\",\"doi\":\"10.1093/jbmrpl/ziae085\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Mesenchymal stem cells (MSCs) and macrophages collaboratively contribute to bone regeneration after injury. However, detailed mechanisms underlying the interaction between MSCs and inflammatory macrophages (M1) remain unclear. A macrophage-depleted tooth extraction model was generated in 5-wk-old female C57BL/6J mice using clodronate liposome (12.5 mg/kg/mouse, intraperitoneally) or saline injection (control) before maxillary first molar extraction. Mice were sacrificed on days 1, 3, 5, 7, and 10 after tooth extraction (<i>n</i> = 4). Regenerated bone volume evaluation of tooth extraction socket (TES) and histochemical analysis of CD80<sup>+</sup>M1, CD206<sup>+</sup>M2 (anti-inflammatory macrophages), PDGFRα<sup>+</sup>MSC, and TNF-α<sup>+</sup> cells were performed. In vitro, isolated MSCs with or without TNF-α stimulation (10 ng/mL, 24 h, <i>n</i> = 3) were bulk RNA-sequenced (RNA-Seq) to identify TNF-α stimulation-specific MSC transcriptomes. Day 7 micro-CT and HE staining revealed significantly lower mean bone volume (clodronate vs control: 0.01 mm<sup>3</sup> vs 0.02 mm<sup>3</sup>, <i>p</i><.0001) and mean percentage of regenerated bone area per total TES in clodronate group (41.97% vs 54.03%, <i>p</i><.0001). Clodronate group showed significant reduction in mean number of CD80<sup>+</sup>, TNF-α<sup>+</sup>, PDGFRα<sup>+</sup>, and CD80<sup>+</sup>TNF-α<sup>+</sup> cells on day 5 (306.5 vs 558.8, <i>p</i><.0001; 280.5 vs 543.8, <i>p</i><.0001; 365.0 vs 633.0, <i>p</i><.0001, 29.0 vs 42.5, <i>p</i><.0001), while these cells recovered significantly on day 7 (493.3 vs 396.0, <i>p</i>=.0004; 479.3 vs 384.5, <i>p</i>=.0008; 593.0 vs 473.0, <i>p</i>=.0010, 41.0 vs 32.5, <i>p</i>=.0003). RNA-Seq analysis showed that 15 genes (|log2FC| > 5.0, log2TPM > 5) after TNF-α stimulation were candidates for regulating MSC's immunomodulatory capacity. In vivo, <i>Clec4e</i> and <i>Gbp6</i> are involved in inflammation and bone formation. <i>Clec4e</i>, <i>Gbp6</i>, and <i>Cxcl10</i> knockdown increased osteogenic differentiation of MSCs in vitro. Temporal reduction followed by apparent recovery of TNF-α-producing M1 macrophages and MSCs after temporal macrophage depletion suggests that TNF-α activated MSCs during TES healing. In vitro mimicking the effect of TNF-α on MSCs indicated that there are 15 candidate MSC genes for regulation of immunomodulatory capacity.</p>\",\"PeriodicalId\":14611,\"journal\":{\"name\":\"JBMR Plus\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.4000,\"publicationDate\":\"2024-07-04\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11289833/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"JBMR Plus\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1093/jbmrpl/ziae085\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/8/1 0:00:00\",\"PubModel\":\"eCollection\",\"JCR\":\"Q2\",\"JCRName\":\"ENDOCRINOLOGY & METABOLISM\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"JBMR Plus","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1093/jbmrpl/ziae085","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/8/1 0:00:00","PubModel":"eCollection","JCR":"Q2","JCRName":"ENDOCRINOLOGY & METABOLISM","Score":null,"Total":0}
引用次数: 0
摘要
间充质干细胞（MSCs）和巨噬细胞共同促进损伤后的骨再生。然而，间充质干细胞与炎性巨噬细胞（M1）之间相互作用的详细机制仍不清楚。在上颌第一磨牙拔除前，使用克罗膦酸脂质体（12.5 mg/kg/只小鼠，腹腔注射）或生理盐水注射（对照组）在5周大的雌性C57BL/6J小鼠中建立了巨噬细胞缺失的拔牙模型。小鼠在拔牙后第 1、3、5、7 和 10 天处死（n = 4）。对拔牙窝（TES）的再生骨量进行评估，并对 CD80+M1、CD206+M2（抗炎巨噬细胞）、PDGFRα+间充质干细胞和 TNF-α+ 细胞进行组织化学分析。在体外，对有或没有 TNF-α 刺激（10 ng/mL，24 h，n = 3）的分离间充质干细胞进行大量 RNA 序列分析（RNA-Seq），以确定 TNF-α 刺激特异性间充质干细胞转录组。第 7 天的 micro-CT 和 HE 染色显示，第 5 天的平均骨量（氯膦酸盐 vs 对照组：0.01 mm3 vs 0.02 mm3、pp+、TNF-α+、PDGFRα+ 和 CD80+TNF-α+ 细胞）明显降低（306.5 vs 558.8，pppp=.0004；479.3 vs 384.5，p=.0008；593.0 vs 473.0，p=.0010，41.0 vs 32.5，p=.0003）。RNA-Seq分析显示，15个基因（|log2FC| > 5.0，log2TPM > 5）在TNF-α刺激后成为调节间充质干细胞免疫调节能力的候选基因。在体内，Clec4e 和 Gbp6 参与炎症和骨形成。Clec4e、Gbp6和Cxcl10的敲除增加了间充质干细胞在体外的成骨分化。在暂时性巨噬细胞耗竭后，TNF-α产生的M1巨噬细胞和间充质干细胞暂时性减少并明显恢复，这表明TNF-α在TES愈合过程中激活了间充质干细胞。体外模拟 TNF-α 对间叶干细胞的影响表明，有 15 个候选间叶干细胞基因可调节免疫调节能力。
Macrophages modulate mesenchymal stem cell function via tumor necrosis factor alpha in tooth extraction model.
Mesenchymal stem cells (MSCs) and macrophages collaboratively contribute to bone regeneration after injury. However, detailed mechanisms underlying the interaction between MSCs and inflammatory macrophages (M1) remain unclear. A macrophage-depleted tooth extraction model was generated in 5-wk-old female C57BL/6J mice using clodronate liposome (12.5 mg/kg/mouse, intraperitoneally) or saline injection (control) before maxillary first molar extraction. Mice were sacrificed on days 1, 3, 5, 7, and 10 after tooth extraction (n = 4). Regenerated bone volume evaluation of tooth extraction socket (TES) and histochemical analysis of CD80+M1, CD206+M2 (anti-inflammatory macrophages), PDGFRα+MSC, and TNF-α+ cells were performed. In vitro, isolated MSCs with or without TNF-α stimulation (10 ng/mL, 24 h, n = 3) were bulk RNA-sequenced (RNA-Seq) to identify TNF-α stimulation-specific MSC transcriptomes. Day 7 micro-CT and HE staining revealed significantly lower mean bone volume (clodronate vs control: 0.01 mm3 vs 0.02 mm3, p<.0001) and mean percentage of regenerated bone area per total TES in clodronate group (41.97% vs 54.03%, p<.0001). Clodronate group showed significant reduction in mean number of CD80+, TNF-α+, PDGFRα+, and CD80+TNF-α+ cells on day 5 (306.5 vs 558.8, p<.0001; 280.5 vs 543.8, p<.0001; 365.0 vs 633.0, p<.0001, 29.0 vs 42.5, p<.0001), while these cells recovered significantly on day 7 (493.3 vs 396.0, p=.0004; 479.3 vs 384.5, p=.0008; 593.0 vs 473.0, p=.0010, 41.0 vs 32.5, p=.0003). RNA-Seq analysis showed that 15 genes (|log2FC| > 5.0, log2TPM > 5) after TNF-α stimulation were candidates for regulating MSC's immunomodulatory capacity. In vivo, Clec4e and Gbp6 are involved in inflammation and bone formation. Clec4e, Gbp6, and Cxcl10 knockdown increased osteogenic differentiation of MSCs in vitro. Temporal reduction followed by apparent recovery of TNF-α-producing M1 macrophages and MSCs after temporal macrophage depletion suggests that TNF-α activated MSCs during TES healing. In vitro mimicking the effect of TNF-α on MSCs indicated that there are 15 candidate MSC genes for regulation of immunomodulatory capacity.
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