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

摘要/项目摘要 2020年，美国处方了超过2亿个抗生素疗程，引起了重大的临床关注 因为两周或更长时间的抗生素疗程会导致血液学并发症，其中最严重的是 中性粒细胞减少症。中性粒细胞增多症，如果不及时治疗，是后续感染，败血症和死亡的危险因素。 阐明造血相关骨髓抑制的分子机制将使我们能够开发 用于预防或治疗需要长期使用抗生素的患者的骨髓抑制的疗法。 我们的实验室已经建立了一个小鼠骨髓抑制模型， 长期使用抗生素导致微生物组的耗竭导致贫血、白细胞减少症和其他血细胞减少症。 然而，抗生素破坏造血的确切分化阶段仍然未知。我们 进一步证明了微生物组促进基础水平的I型干扰素（IFN-I）信号传导， 需要以STAT 1依赖性方式维持稳态造血。尽管之前的研究表明 NOD 1和TLR-MYD 88通路的激活可以支持骨髓生成，我观察到正常数量的 在Nod 1和Myd 88缺陷小鼠中，造血祖细胞和粒细胞在基线时，表明这些 正常的血液生成途径。一些独立的研究表明， 微生物组可以通过TLR-TRIF、cGAS-STING和RIG-I-MAVS途径诱导紧张性IFN-I信号传导， 尽管它们对造血维持的作用还不清楚。在非靶向代谢组学中， 筛选，我们确定了29种微生物代谢产物，这些代谢产物在非结核病患者的粪便和血清样本中富集， 白细胞减少的小鼠与抗生素治疗后两周白细胞减少的小鼠进行比较。这些代谢物是否 可以支持体内造血仍然是未知的。这项提案将检验微生物组 利用免疫相关信号传导途径，如TLR-TRIF、cGAS-STING和RIG-I-MAVS途径， 在造血干细胞（HSC）水平支持IFN-1介导的稳态造血。 我们先前工作的一个主要局限是，由IFN-1抑制引起的Sca-1表达的变化可能 已经使抗肿瘤治疗的小鼠中HSPC的计数偏斜。为了解决这个问题，我们将首先执行限制 稀释移植以定量抗生素和模拟治疗小鼠中的功能性HSC。我们还将追踪 HSPCs和粒细胞群体，通过跟踪它们在Krt 18- CreERT 2：用或不用抗生素处理的Rosa 26-lox-STOP-lox-TdTomato小鼠。为了阐明 微生物群依赖性造血，我们将描述Trif-/-，Sting-/-和 用或不用抗生素处理的Mavs-/-小鼠。我们将通过评估TLR的充分性来验证我们的结果， STING和RIG-I激动剂来挽救造血相关的造血缺陷。我们还将使用IfnbΔβ-luc/Δβ-luc 报告小鼠以评价微生物代谢物维持基础IFN-1产生的能力。这些研究将 定义微生物组促进IFN-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.