Ryan Molly Mercedes, Patel Mihir, Hogan Keenan, Lipat Ariel Joy, Scandolara Rafaela, Das Rahul, Bruker Charles, Galipeau Jacques, Chinnadurai Raghavan
Department of Medicine, University of Wisconsin Carbone Comprehensive Cancer Center, University of Wisconsin-Madison, Madison, Wisconsin.
Department of Biomedical Sciences, Mercer University School of Medicine, Savannah, Georgia.
Transplant Cell Ther. 2021 May;27(5):389.e1-389.e10. doi: 10.1016/j.jtct.2021.02.002. Epub 2021 Feb 4.
Ruxolitinib is a JAK2/JAK1 inhibitor that blocks the inflammatory JAK-STAT signaling pathway. Ruxolitinib has been demonstrated to be effective in the treatment of steroid-resistant acute graft-versus-host disease (GVHD). Ruxolitinib's effect on inflammatory cells of hematopoietic origin is known. However, its effect on nonhematopoietic cell types with immune-modulating and antigen-presenting cell competency plausibly involved in pathogenesis of GVHD has not been explored. Mesenchymal stromal cells (MSCs) are CD45 nonhematopoietic cells of the bone marrow with immune modulatory functions in vivo. MSCs' immunobiology largely depends on their responsiveness to IFNγ. We aimed to define the effect of ruxolitinib on the immunobiology of MSCs that are modulated by IFNγ. Human bone marrow derived MSCs, peripheral blood mononuclear cells (PBMCs), and primary bone marrow aspirates were analyzed for their sensitivity to ruxolitinib-mediated blocking of IFNγ-induced STAT-1 phosphorylation and downstream effector molecules, utilizing Western blot, flow cytometry, secretome analysis, and phosflow techniques. IFNγ-induced cytostatic effects on MSCs are reversed by ruxolitinib. Ruxolitinib inhibits IFNγ and secretome of activated peripheral PBMC-induced STAT-1 phosphorylation on human bone marrow derived MSCs. In addition, ruxolitinib inhibits IFNγ-induced pro-GVHD pathways on MSCs, which includes HLAABC(MHCI), HLADR(MHCII), CX3CL1, and CCL2. IFNγ-induced immunosuppressive molecules IDO and PDL-1 were also inhibited by ruxolitinib on MSCs. Comparative analysis with PBMCs has demonstrated that MSCs are as equal as to HLADR PBMC populations in responding to ruxolitinib-mediated inhibition of IFNγ-induced STAT-1 phosphorylation. Ex vivo analysis of human marrow aspirates has demonstrated that ruxolitinib blocks IFNγ-induced STAT-1 phosphorylation in CD45HLADR populations at different levels, which is depending on their sensitivity to IFNγ responsiveness. These results inform the hypothesis that ruxolitinib's immune-modulatory effects in vivo may pharmacologically involve marrow and tissue-resident MSCs. Ruxolitinib affects the immunobiology of MSCs equivalent to professional HLADR antigen presenting cells, which collectively mitigate GVHD.
鲁索替尼是一种JAK2/JAK1抑制剂，可阻断炎症性JAK-STAT信号通路。已证明鲁索替尼在治疗类固醇难治性急性移植物抗宿主病（GVHD）方面有效。鲁索替尼对造血来源炎症细胞的作用是已知的。然而，其对可能参与GVHD发病机制的具有免疫调节和抗原呈递细胞能力的非造血细胞类型的作用尚未得到探索。间充质基质细胞（MSCs）是骨髓中的CD45非造血细胞，在体内具有免疫调节功能。MSCs的免疫生物学很大程度上取决于它们对IFNγ的反应性。我们旨在确定鲁索替尼对受IFNγ调节的MSCs免疫生物学的影响。利用蛋白质免疫印迹法、流式细胞术、分泌组分析和磷酸化流式细胞术技术，分析了人骨髓来源的MSCs、外周血单个核细胞（PBMCs）和原发性骨髓抽吸物对鲁索替尼介导的阻断IFNγ诱导的STAT-1磷酸化及下游效应分子的敏感性。鲁索替尼可逆转IFNγ对MSCs的细胞生长抑制作用。鲁索替尼可抑制IFNγ以及活化外周PBMC的分泌组诱导人骨髓来源MSCs上的STAT-1磷酸化。此外，鲁索替尼可抑制IFNγ诱导的MSCs上的促GVHD途径，其中包括HLAABC（MHC I）、HLADR（MHC II）、CX3CL1和CCL2。鲁索替尼还可抑制IFNγ诱导的MSCs上的免疫抑制分子IDO和PDL-1。与PBMCs的比较分析表明，在对鲁索替尼介导的抑制IFNγ诱导的STAT-1磷酸化的反应中，MSCs与HLADR PBMC群体相当。对人骨髓抽吸物的体外分析表明，鲁索替尼可在不同水平阻断IFNγ诱导的CD45HLADR群体中的STAT-1磷酸化，这取决于它们对IFNγ反应性的敏感性。这些结果支持了这样一种假说，即鲁索替尼在体内的免疫调节作用可能在药理学上涉及骨髓和组织驻留的MSCs。鲁索替尼对MSCs免疫生物学的影响等同于专业的HLADR抗原呈递细胞，共同减轻GVHD。
