Abstract：
BACKGROUND: Acute graft-versus-host disease (aGVHD), which is mainly mediated by allogeneic T cells, is a decisive factor in the success of allogeneic hematopoietic stem cell transplantation (allo-HCT). Prophylaxis for aGVHD in clinical patients is unsatisfactory, and there is still a huge unmet need for novel approaches. Icariin (ICA) shows potent anti-inflammatory activity and suppresses T cell-mediated immune responses. Thus, ICA is a potential drug for the prevention of aGVHD. However, there is no data assessing the impact of ICA on aGVHD after allo-HCT.
OBJECTIVE: This study aimed to investigate the protective effect of ICA against aGVHD and its mechanisms. Moreover, the impact of ICA on the graft-versus-leukemia (GVL) effect and engraftment of donor hematopoietic and immune cells were assessed.
METHODS: Different murine models of allo-HCT were developed to study the influence of the ICA on GVHD and GVL effect. Flow cytometry was used to analyze the growth of leukemia cells, alterations in different immune cells, and apoptosis. Cell proliferation was determined using a CCK-8 assay. RNA sequencing and quantitative proteomic analysis were performed to elucidate the underlying mechanisms, which were further verified by polymerase chain reaction or functional experiments.
RESULTS: Different concentrations of ICA exhibited opposite effects: low-concentration ICA promoted, while high concentrations suppressed the proliferation and function of T cells. A high dose of ICA administration during days +3 to +5 post-allo-HCT can alleviate murine aGVHD but does not affect the course of chronic GVHD (cGVHD), the GVL effect against both acute myeloid and lymphoblastic leukemia, or the recovery of donor hematological and immune cells. ICA extensively represses the expansion, function, and infiltration of donor alloreactive T cells, while preserving regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC). Quantitative proteomic analysis showed that downregulation of integrin-linked kinase (ILK) and lymphocyte cytosolic protein 2 (LCP2) expression was possibly associated with ICA-mediated aGVHD protective effects. Furthermore, an inhibitor of ILK, which can alleviate murine aGVHD administered early after allo-HCT.
CONCLUSIONS: These findings suggest that the bioactivities of ICA are associated with its concentration and that ICA can effectively mitigate aGVHD without losing GVL activity or engraftment of donor hematopoietic and immune cells. Thus, ICA may be a promising drug for preventing aGVHD in clinical settings.
OBJECTIVE: This study aimed to investigate the protective effect of ICA against aGVHD and its mechanisms. Moreover, the impact of ICA on the graft-versus-leukemia (GVL) effect and engraftment of donor hematopoietic and immune cells were assessed.
METHODS: Different murine models of allo-HCT were developed to study the influence of the ICA on GVHD and GVL effect. Flow cytometry was used to analyze the growth of leukemia cells, alterations in different immune cells, and apoptosis. Cell proliferation was determined using a CCK-8 assay. RNA sequencing and quantitative proteomic analysis were performed to elucidate the underlying mechanisms, which were further verified by polymerase chain reaction or functional experiments.
RESULTS: Different concentrations of ICA exhibited opposite effects: low-concentration ICA promoted, while high concentrations suppressed the proliferation and function of T cells. A high dose of ICA administration during days +3 to +5 post-allo-HCT can alleviate murine aGVHD but does not affect the course of chronic GVHD (cGVHD), the GVL effect against both acute myeloid and lymphoblastic leukemia, or the recovery of donor hematological and immune cells. ICA extensively represses the expansion, function, and infiltration of donor alloreactive T cells, while preserving regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC). Quantitative proteomic analysis showed that downregulation of integrin-linked kinase (ILK) and lymphocyte cytosolic protein 2 (LCP2) expression was possibly associated with ICA-mediated aGVHD protective effects. Furthermore, an inhibitor of ILK, which can alleviate murine aGVHD administered early after allo-HCT.
CONCLUSIONS: These findings suggest that the bioactivities of ICA are associated with its concentration and that ICA can effectively mitigate aGVHD without losing GVL activity or engraftment of donor hematopoietic and immune cells. Thus, ICA may be a promising drug for preventing aGVHD in clinical settings.
摘要：
背景：急性移植物抗宿主病（aGVHD），主要由同种异体T细胞介导，是异基因造血干细胞移植（allo-HCT）成功的决定性因素。临床患者对aGVHD的预防不能令人满意，对新方法仍有巨大的未满足需求。淫羊藿苷（ICA）显示出有效的抗炎活性并抑制T细胞介导的免疫反应。因此，ICA是预防aGVHD的潜在药物。然而,没有数据评估allo-HCT后ICA对aGVHD的影响。
目的：本研究旨在探讨ICA对aGVHD的保护作用及其机制。此外，评估了ICA对移植物抗白血病(GVL)效应和供体造血和免疫细胞植入的影响.
方法：开发了不同的小鼠allo-HCT模型来研究ICA对GVHD和GVL效应的影响。流式细胞术用于分析白血病细胞的生长，不同免疫细胞的改变，和凋亡。使用CCK-8测定法测定细胞增殖。进行RNA测序和定量蛋白质组学分析以阐明潜在的机制，通过聚合酶链反应或功能实验进一步验证。
结果：不同浓度的ICA表现出相反的作用：低浓度的ICA促进，高浓度抑制T细胞的增殖和功能。在allo-HCT后3至5天的高剂量ICA给药可以缓解鼠aGVHD，但不影响慢性GVHD（cGVHD）的进程，GVL对急性髓系和淋巴细胞白血病的作用，或供体血液和免疫细胞的恢复。ICA广泛压制扩张，函数,和供体同种反应性T细胞的浸润，同时保留调节性T细胞（Tregs）和骨髓来源的抑制细胞（MDSC）。定量蛋白质组学分析表明，整合素连接激酶（ILK）和淋巴细胞胞浆蛋白2（LCP2）表达的下调可能与ICA介导的aGVHD保护作用有关。此外，ILK的抑制剂，这可以减轻小鼠aGVHD在allo-HCT后早期给药。
结论：这些发现表明ICA的生物活性与其浓度相关，并且ICA可以有效缓解aGVHD，而不会失去GVL活性或供体造血和免疫细胞的植入。因此，ICA可能是临床上预防aGVHD的有前途的药物。
目的：本研究旨在探讨ICA对aGVHD的保护作用及其机制。此外，评估了ICA对移植物抗白血病(GVL)效应和供体造血和免疫细胞植入的影响.
方法：开发了不同的小鼠allo-HCT模型来研究ICA对GVHD和GVL效应的影响。流式细胞术用于分析白血病细胞的生长，不同免疫细胞的改变，和凋亡。使用CCK-8测定法测定细胞增殖。进行RNA测序和定量蛋白质组学分析以阐明潜在的机制，通过聚合酶链反应或功能实验进一步验证。
结果：不同浓度的ICA表现出相反的作用：低浓度的ICA促进，高浓度抑制T细胞的增殖和功能。在allo-HCT后3至5天的高剂量ICA给药可以缓解鼠aGVHD，但不影响慢性GVHD（cGVHD）的进程，GVL对急性髓系和淋巴细胞白血病的作用，或供体血液和免疫细胞的恢复。ICA广泛压制扩张，函数,和供体同种反应性T细胞的浸润，同时保留调节性T细胞（Tregs）和骨髓来源的抑制细胞（MDSC）。定量蛋白质组学分析表明，整合素连接激酶（ILK）和淋巴细胞胞浆蛋白2（LCP2）表达的下调可能与ICA介导的aGVHD保护作用有关。此外，ILK的抑制剂，这可以减轻小鼠aGVHD在allo-HCT后早期给药。
结论：这些发现表明ICA的生物活性与其浓度相关，并且ICA可以有效缓解aGVHD，而不会失去GVL活性或供体造血和免疫细胞的植入。因此，ICA可能是临床上预防aGVHD的有前途的药物。
