Center of Excellence in Hip, Scottish Rite for Children, Dallas, TX 75219, USA.
Department of Orthopaedic Surgery, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Cells. 2024 Jul 20;13(14):1227. doi: 10.3390/cells13141227.
Osteonecrosis (ON) of the femoral head (ONFH) is a devastating bone disease affecting over 20 million people worldwide. ONFH is caused by a disruption of the blood supply, leading to necrotic cell death and increased inflammation. Macrophages are the key cells mediating the inflammatory responses in ON. It is unclear what the dynamic phenotypes of macrophages are and what mechanisms may affect macrophage polarization and, therefore, the healing process. In our preliminary study, we found that there is an invasion of macrophages into the repair tissue during ON healing. Interestingly, in both ONFH patients and a mouse ON model, fat was co-labeled within macrophages using immunofluorescence staining, indicating the phagocytosis of fat by macrophages. To study the effects of fat phagocytosis on the macrophage phenotype, we set up an in vitro macrophage and fat co-culture system. We found that fat phagocytosis significantly decreased M1 marker expression, such as IL1β and iNOS, in macrophages, whereas the expression of the M2 marker Arg1 was significantly increased with fat phagocytosis. To investigate whether the polarization change is indeed mediated by phagocytosis, we treated the cells with Latrunculin A (LA, which inhibits actin polymerization and phagocytosis). LA supplementation significantly reversed the polarization marker gene changes induced by fat phagocytosis. To provide an unbiased transcriptional gene analysis, we submitted the RNA for bulk RNA sequencing. Differential gene expression (DGE) analysis revealed that the top upregulated genes were related to anti-inflammatory responses, while proinflammatory genes were significantly downregulated. Additionally, using pathway enrichment and network analyses (Metascape), we confirmed that gene-enriched categories related to proinflammatory responses were significantly downregulated in macrophages with fat phagocytosis. Finally, we validated the similar macrophage phenotype changes in vivo. To summarize, we discovered that fat phagocytosis occurs in both ONFH patients and an ON mouse model, which inhibits proinflammatory responses with increased anabolic gene expression in macrophages. This fat-phagocytosis-induced macrophage phenotype is consistent with the in vivo changes shown in the ON mouse model. Our study reveals a novel phagocytosis-mediated macrophage polarization mechanism in ON, which fills in our knowledge gaps of macrophage functions and provides new concepts in macrophage immunomodulation as a promising treatment for ON.
股骨头坏死（ON）是一种破坏性的骨骼疾病，影响全球超过 2000 万人。ON 是由血液供应中断引起的，导致坏死细胞死亡和炎症增加。巨噬细胞是介导 ON 炎症反应的关键细胞。目前尚不清楚巨噬细胞的动态表型是什么，以及哪些机制可能影响巨噬细胞极化，从而影响愈合过程。在我们的初步研究中，我们发现 ON 愈合过程中巨噬细胞会侵入修复组织。有趣的是，在 ONFH 患者和小鼠 ON 模型中，免疫荧光染色显示巨噬细胞内有脂肪共同标记，表明巨噬细胞吞噬脂肪。为了研究脂肪吞噬对巨噬细胞表型的影响，我们建立了体外巨噬细胞和脂肪共培养系统。我们发现，脂肪吞噬显著降低了巨噬细胞中 M1 标志物的表达，如 IL1β 和 iNOS，而 Arg1 的 M2 标志物的表达则随着脂肪吞噬而显著增加。为了研究这种极化变化是否确实是由吞噬作用介导的，我们用拉曲库铵 A（Latrunculin A，LA，抑制肌动蛋白聚合和吞噬作用）处理细胞。LA 补充显著逆转了脂肪吞噬作用诱导的极化标志物基因变化。为了提供无偏倚的转录基因分析，我们将 RNA 提交进行批量 RNA 测序。差异基因表达（DGE）分析显示，上调基因与抗炎反应有关，而促炎基因则显著下调。此外，使用通路富集和网络分析（Metascape），我们证实了在具有脂肪吞噬作用的巨噬细胞中，与促炎反应相关的基因富集类别显著下调。最后，我们在体内验证了类似的巨噬细胞表型变化。总之，我们发现脂肪吞噬作用不仅发生在 ONFH 患者中，也发生在 ON 小鼠模型中，它抑制了促炎反应，同时增加了巨噬细胞中合成代谢基因的表达。这种脂肪吞噬作用诱导的巨噬细胞表型与 ON 小鼠模型中的体内变化一致。我们的研究揭示了 ON 中一种新的吞噬作用介导的巨噬细胞极化机制，填补了我们对巨噬细胞功能的知识空白，并为巨噬细胞免疫调节提供了新概念，作为治疗 ON 的一种有前途的方法。
