Supplementary Material for: Mesenchymal Stromal Cells Facilitate Neutrophil Trained Immunity by Reprogramming Hematopoietic Stem Cells

Novel therapeutics are urgently needed to prevent opportunistic infections in immunocompromised individuals undergoing cancer treatments or other immune suppressive therapies. Trained immunity is a promising strategy to reduce this burden of disease. We previously demonstrated that mesenchymal stromal cells (MSCs) preconditioned with a class A CpG oligodeoxynucleotide (CpG-ODN), a Toll-like receptor 9 (TLR9) agonist, can augment emergency granulopoiesis in a murine model of neutropenic sepsis. Here, we used a chimeric mouse model to demonstrate that MSCs secrete paracrine factors that act on lineage negative c-kit+ hematopoietic stem cells (HSCs), leaving them ‘poised’ to enhance emergency granulopoiesis months after transplantation. Chimeric mice developed from HSCs exposed to conditioned media from MSCs and CpG-ODN preconditioned MSCs showed significantly higher bacterial clearance and increased neutrophil granulopoiesis following lung infection than control mice. By CUT&RUN chromatin sequencing and cytometry by time of flight (CyTOF) approaches, we identified that MSC conditioned media leaves H3K4me3 histone marks in HSCs at genes involved in myelopoiesis and in signaling persistence by the mTOR pathway. Both soluble factors and extracellular vesicles (EV) from MSCs mediated these effects on HSCs and proteomic analysis by mass spectrometry revealed soluble calreticulin as a potential mediator. In summary, this study demonstrates that trained immunity can be mediated by paracrine factors from MSCs to induce neutrophil trained immunity by reprogramming HSCs for long-lasting functional changes in neutrophil mediated anti-microbial immunity.对于正在接受癌症治疗或其他免疫抑制疗法的免疫功能低下人群，亟需开发新型治疗手段以预防机会性感染。训练免疫（trained immunity）是减轻此类疾病负担的极具前景的策略。我们此前的研究证实，经A类CpG寡脱氧核苷酸 (CpG oligodeoxynucleotide, CpG-ODN，一种Toll样受体9 (Toll-like receptor 9, TLR9)激动剂) 预处理的间充质基质细胞 (mesenchymal stromal cells, MSCs)，可在中性粒细胞减少性脓毒症的小鼠模型中增强紧急粒细胞生成。本研究通过嵌合小鼠模型证实，MSCs分泌的旁分泌因子可作用于谱系阴性c-kit阳性造血干细胞 (hematopoietic stem cells, HSCs)，使其在移植数月后处于可增强紧急粒细胞生成的“待命”状态。相较于对照组小鼠，由暴露于MSCs及经CpG-ODN预处理的MSCs条件培养基的HSCs发育而来的嵌合小鼠，在肺部感染后表现出显著更高的细菌清除率与更强的中性粒细胞生成能力。通过CUT&RUN染色质测序与飞行时间流式细胞术 (cytometry by time of flight, CyTOF) 分析，我们证实MSCs条件培养基可在造血干细胞中，于髓系生成相关基因以及mTOR通路信号持续激活相关基因位点上留下H3K4me3组蛋白修饰。MSCs分泌的可溶性因子与细胞外囊泡 (extracellular vesicles, EV) 均可介导上述对HSCs的调控作用；经质谱法的蛋白质组学分析显示，可溶性钙网蛋白是潜在的介导因子。综上，本研究表明，MSCs的旁分泌因子可介导训练免疫，通过重编程造血干细胞使其在中性粒细胞介导的抗微生物免疫中产生持久的功能改变，进而诱导中性粒细胞训练免疫。