Regulation of Steady-State Hematopoiesis by Microbiota-Driven IFN-I Signaling

摘要/项目摘要 2020年，美国开出了超过2亿疗程的抗生素，这引起了临床上的重大担忧 因为两周或更长时间的抗生素疗程会导致血液系统并发症，其中最严重的是 中性粒细胞减少症。如果不进行治疗，中性粒细胞减少症是随后感染、败血症和死亡的危险因素。 阐明抗生素相关骨髓抑制的分子机制将使我们能够开发 预防或治疗需要长期使用抗生素的患者骨髓抑制的治疗方法。 我们的实验室已经开发出一种与抗生素相关的骨髓抑制小鼠模型，显示出 长期使用抗生素导致微生物群枯竭，导致贫血、白细胞减少和其他红细胞减少症。 然而，抗生素扰乱造血的确切分化阶段仍不清楚。我们 进一步证明，微生物组促进I型干扰素(IFN-I)信号的基础水平，这是 需要以STAT1依赖的方式维持稳定的造血。尽管之前的研究表明 激活NOD1和TLR-MYD88通路可以支持骨髓生成，我观察到正常数量的 Nod1和MyD88缺陷小鼠基线状态下的造血祖细胞和粒细胞 对于正常的血液生产来说，通路是必不可少的。几项独立研究表明， 微生物组可通过TLR-TRIF、cGAS-STING和RIG-I-MAVS途径诱导强直的干扰素-I信号转导， 尽管它们对造血维持的贡献还不是很清楚。在非靶向代谢组学中 经过筛选，我们鉴定了29种微生物代谢物，这些代谢物在粪便和血清样本中富含 白细胞减少的小鼠与抗生素后两周白细胞减少的小鼠进行比较。这些代谢物是否 是否可以支持体内的造血仍未被探索。这一提议将检验微生物群的假设 利用免疫相关信号通路，如TLR-TRIF、cGAS-STING和RIG-I-MAVS通路 在造血干细胞(HSC)水平支持干扰素-I介导的稳态造血。 我们先前工作的一个主要局限性是，由于抑制干扰素-I而导致的SCA-1表达的变化可能 在抗生素治疗的小鼠中扭曲了HSPC的计数。为了解决这个问题，我们将首先执行一个限制 稀释法移植以量化抗生素和模拟治疗小鼠中的功能性HSCs。我们还将追查到 通过跟踪其在Krt18中的增殖、分化和周转来检测HSPC和粒细胞群 CRERT2：用或不用抗生素治疗的ROSA26-LOX-STOP-LOX-Td番茄小鼠。阐明其作用机制 对于依赖微生物组的造血，我们将描述Trif-/-、Sting-/-和 MAVS-/-使用或不使用抗生素治疗的小鼠。我们将通过评估TLR的充分性来验证我们的结果， 刺痛，和RIG-I激动剂，以挽救抗生素相关的造血缺陷。我们还将使用IFNBΔβ-LUC/Δβ-LUC 报告小鼠评估微生物代谢产物维持基础干扰素-I产生的能力。这些研究将 确定微生物群促进干扰素-I产生以调节造血的机制。

Abstract/Project Summary Over 200 million courses of antibiotics were prescribed in the U.S. in 2020, raising a significant clinical concern as antibiotic courses of two weeks or longer result in hematological complications, the most serious of which is neutropenia. Neutropenia, if left untreated, is a risk factor for subsequent infections, sepsis, and death. Elucidating the molecular mechanisms of antibiotic-associated bone marrow suppression will allow us to develop therapies to prevent or treat bone marrow suppression in patients who require prolonged antibiotics. Our lab has developed a mouse model of antibiotic-associated bone marrow suppression that showed that depletion of the microbiome on prolonged antibiotics results in anemia, leukopenia, and other cytopenias. However, the precise stage in differentiation at which antibiotics disrupt hematopoiesis remains unknown. We further demonstrated that the microbiome promotes a basal level of type I interferon (IFN-I) signaling, which is required to maintain steady-state hematopoiesis in a STAT1-dependent manner. Although prior studies showed that activation of NOD1 and TLR-MYD88 pathways can support myelopoiesis, I observed normal numbers of hematopoietic progenitors and granulocytes at baseline in Nod1 and Myd88-deficient mice, suggesting that these pathways are dispensable for normal blood production. Several independent studies have shown that the microbiome can induce tonic IFN-I signaling through TLR-TRIF, cGAS-STING, and RIG-I-MAVS pathways, though their contribution to hematopoietic maintenance is not well understood. In an untargeted metabolomics screening, we identified 29 microbial metabolites that were enriched in stool and serum samples from non- leukopenic mice compared to those that were leukopenic two weeks post-antibiotics. Whether these metabolites can support hematopoiesis in vivo remains unexplored. This proposal will test the hypothesis that the microbiome utilizes immune-related signaling pathways such as TLR-TRIF, cGAS-STING, and RIG-I-MAVS pathways to support IFN-I mediated steady-state hematopoiesis at the level of the hematopoietic stem cell (HSC). A major limitation of our prior work is that a shift in Sca-1 expression caused by IFN-I suppression could have skewed the enumeration of HSPCs in antibiotic-treated mice. To address this, we will first perform a limiting dilution transplant to quantify functional HSCs in antibiotics and mock-treated mice. We will also trace the fate of HSPCs and granulocyte populations by tracking their proliferation, differentiation, and turnover in Krt18- CreERT2:Rosa26-lox-STOP-lox-TdTomato mice treated with or without antibiotics. To elucidate the mechanism of microbiome-dependent hematopoiesis, we will characterize the hematopoietic defects in Trif-/-, Sting-/-, and Mavs-/- mice treated with or without antibiotics. We will validate our results by assessing the sufficiency of TLR, STING, and RIG-I agonists to rescue antibiotic-associated hematopoietic defects. We will also use IfnbΔβ-luc/Δβ-luc reporter mice to evaluate the ability of microbial metabolites to sustain basal IFN-I production. These studies will define the mechanisms by which the microbiome promotes IFN-I production to regulate hematopoiesis.