Impact of HSC-derived immune training on granulocyte/macrophage progenitor gene expression

Trained immunity occurs when inflammatory stimulation reprograms myeloid cells to have increased effector functions upon secondary stimulation. Interestingly, the secondary stimulation can occur well beyond the lifetime of an individual myeloid cell, thus memory of immune training must reside in the hematopoietic system. After showing that long-term hematopoietic stem cells (HSCLT) from mice treated with pristane for eight weeks generate trained macrophages, we sought to uncover mechanisms underlying the heritable trained immunity program in myeloid cells. To do this we transplanted HSCLT from pristane mice into congenic recipients, and after 18 weeks of engraftment sorted out donor derived granulocyte monocyte progenitors (GMP). We then analyzed the chromatin landscape and transcriptome using low input chromatin accessibility and transcriptomics sequencing (LiCAT-seq) which allows for the co-generation of RNA-seq and ATAC-seq libraries from the same cellular pool. With this approach we found a unique epigenetic phenotype of reduced chromatin accessibility at metabolic genes in GMP from pristane HSCLT, which resulted in reduced transcription of metabolic genes, leading to reduced metabolism in mature macrophages. Mice were treated ± pristane for eight weeks, then HSCLT were sorted out and transplanted into lethally irradiated congenic recipients. After 18 weeks of engraftment donor derived CD45.2+ GMP were sorted for LiCAT-seq analysis.

训练免疫（trained immunity）指炎症刺激对髓系细胞进行重编程，使其在二次刺激下展现出增强的效应功能。值得注意的是，二次刺激甚至可发生在单个髓系细胞的寿命周期之外，因此免疫训练的记忆必然存储于造血系统中。本研究前期已证实，经普里斯坦（pristane）处理8周的小鼠的长期造血干细胞（long-term hematopoietic stem cells, HSCLT）可分化为训练性巨噬细胞，后续我们旨在揭示髓系细胞中可遗传的训练免疫程序的潜在调控机制。为此，我们将普里斯坦处理小鼠的长期造血干细胞移植至同类系受体小鼠体内，在造血植入18周后，分选供体来源的粒细胞-单核细胞前体（granulocyte monocyte progenitors, GMP）。随后，我们采用低起始量染色质开放与转录组测序（low input chromatin accessibility and transcriptomics sequencing, LiCAT-seq）分析其染色质图谱与转录组特征——该技术可从同一细胞池中同时构建RNA测序（RNA-seq）与转座酶可及性染色质测序（ATAC-seq）文库。通过该方法，我们发现来自普里斯坦处理组长期造血干细胞的GMP，其代谢基因位点的染色质开放程度显著降低，进而导致代谢基因的转录水平下调，最终使成熟巨噬细胞的代谢能力减弱。实验中，我们将小鼠分为普里斯坦处理组与空白对照组，处理8周后分离其长期造血干细胞并移植至经致死辐照的同类系受体小鼠体内；造血植入18周后，分选供体来源的CD45.2阳性粒细胞-单核细胞前体用于LiCAT-seq分析。