Chugh Rishi Man, Bhanja Payel, Zitter Ryan, Gunewardena Sumedha, Badkul Rajeev, Saha Subhrajit
Department of Radiation Oncology, University of Kansas Medical Center, Kansas City, KS, 66160, USA.
Department of Cell Biology and Physiology, University of Kansas Medical Center, Kansas City, KS, 66160, USA.
Cell Commun Signal. 2025 Feb 11;23(1):78. doi: 10.1186/s12964-025-02065-7.
Macrophages are the major source of WNT ligands. However, the regulation of WNT expression in macrophages has not been studied. In the present study, we have discovered that activation of canonical β-Catenin signaling suppresses WNT expression in macrophages. EVs from these pre-conditioned macrophages promoted intestinal stem cell regeneration and mitigated intestinal injury.
ChIP-seq analysis and validation studies using recombinant DNA construct expressing Luciferase reporter under WNT promoter (e.g. WNT5a and WNT9b) were conducted to demonstrate the involvement of β-Catenin in the transcriptional regulation of WNT expression. The regulatory role of β-Catenin in WNT expression in macrophages was examined by treating these cells with a Tankyrase inhibitor. In addition, the gene expressing β-Catenin was deleted in macrophages using Csf1r.iCre; Ctnnb1 mice model. Both pharmacological and genetically modulated macrophages were examined for WNT expression and activity by qPCR and TCF/LEF luciferase assay respectively. Additionally, Csf1r.iCre; Ctnnb1 mice were exposed to irradiation to compare the radiosensitivity with their wildtype littermate. Extracellular vesicles (EVs) were isolated from pre-conditioned WNT-enriched macrophages and infused in irradiated C57BL/6 and Lgr5/eGFP-IRES-Cre-ERT2; R26-ACTB-tdTomato-EGFP mice to determine the regenerative response of intestinal stem cell (ISC) and epithelial repair. Regenerative effects of EVs were also examined in mice model DSS induced colitis.
ChIP-seq analysis and subsequent validation study suggested physical association of β-Catenin with WNT promoters to suppress WNT expression. Macrophage specific deletion of gene expressing β-Catenin or pharmacological inhibition of Tankyrase improves the WNT expression in macrophages several folds compared to control. Transfusion of these preconditioned macrophages or EVs from these cells delivers optimum level of morphogenic WNT to injured epithelium, activates ISC regeneration and mitigated radiation induced intestinal injury. Intestinal epithelium in Csf1r.iCre; Ctnnb1 mice also showed radioresistance compared to wild type littermate. Moreover, EVs derived from WNT enriched macrophages can mitigate intestinal injury in mice model of DSS induced acute colitis.
The study provides substantial evidence that macrophage-targeted modulation of canonical WNT signaling induces WNT expression in macrophages. Treatment with preconditioned macrophage derived WNT-enriched EVs can be a promising therapeutic approach against intestinal injury.
巨噬细胞是WNT配体的主要来源。然而，巨噬细胞中WNT表达的调控尚未得到研究。在本研究中，我们发现经典β-连环蛋白信号通路的激活会抑制巨噬细胞中WNT的表达。来自这些预处理巨噬细胞的细胞外囊泡（EVs）促进了肠道干细胞再生并减轻了肠道损伤。
进行染色质免疫沉淀测序（ChIP-seq）分析以及使用在WNT启动子（如WNT5a和WNT9b）下表达荧光素酶报告基因的重组DNA构建体的验证研究，以证明β-连环蛋白参与WNT表达的转录调控。通过用端锚聚合酶抑制剂处理这些细胞来检测β-连环蛋白在巨噬细胞WNT表达中的调节作用。此外，使用Csf1r.iCre; Ctnnb1小鼠模型在巨噬细胞中删除表达β-连环蛋白的基因。分别通过定量聚合酶链反应（qPCR）和TCF/LEF荧光素酶测定法检测经药物和基因调节的巨噬细胞的WNT表达和活性。另外，将Csf1r.iCre; Ctnnb1小鼠暴露于辐射下，以与其野生型同窝小鼠比较放射敏感性。从预处理的富含WNT的巨噬细胞中分离细胞外囊泡，并注入经辐射的C57BL/6和Lgr5/eGFP-IRES-Cre-ERT2; R26-ACTB-tdTomato-EGFP小鼠中，以确定肠道干细胞（ISC）的再生反应和上皮修复。还在葡聚糖硫酸钠（DSS）诱导的结肠炎小鼠模型中检测了细胞外囊泡的再生作用。
ChIP-seq分析及随后的验证研究表明β-连环蛋白与WNT启动子存在物理关联以抑制WNT表达。与对照相比，巨噬细胞特异性删除表达β-连环蛋白的基因或对端锚聚合酶进行药物抑制可使巨噬细胞中的WNT表达提高数倍。输注这些预处理的巨噬细胞或来自这些细胞 的细胞外囊泡可将最佳水平的形态发生性WNT传递至受损上皮，激活肠道干细胞再生并减轻辐射诱导的肠道损伤。与野生型同窝小鼠相比，Csf1r.iCre; Ctnnb1小鼠的肠道上皮也表现出放射抗性。此外，源自富含WNT的巨噬细胞的细胞外囊泡可减轻DSS诱导的急性结肠炎小鼠模型中的肠道损伤。
该研究提供了大量证据表明巨噬细胞靶向调节经典WNT信号通路可诱导巨噬细胞中WNT的表达。用预处理的巨噬细胞衍生的富含WNT的细胞外囊泡进行治疗可能是一种有前景的治疗肠道损伤的方法。
