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

微生物驱动的 IFN-I 信号传导对稳态造血的调节

• 批准号: 10678151
• 负责人: Arushana Amir Maknojia
• 金额: $ 4.77万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10678151
• 关键词: Address; Adverse effects; Agonist; Ampicillin; Anemia; Antibiotics; Biological Assay; Blood; Bone Marrow; Bone Marrow Suppression; Bromodeoxyuridine; Caspase Inhibitor; Cell Wall; Cessation of life; Clinical; Communication; Cyclic GMP; Defect; Distant; Feces; Fluorochrome; Genes; Germ-Free; Hematology; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Immune; Immune signaling; Impairment; Individual; Infection; Interferon Suppression; Interferon Type I; Interferon-beta; Interferons; Label; Laboratory Finding; Left; Leukopenia; Maintenance; Measures; Mediating; Mediator; Medical Care Costs; Methylcellulose; Metronidazole; Mitochondria; Molecular; Mus; Myelopoiesis; Neomycin; Neutropenia; Pathway interactions; Patients; Phenotype; Population; Production; Proliferating; Propidium Diiodide; Public Health; Receptor Signaling; Regulation; Reporter; Risk Factors; STAT1 gene; Sampling; Sepsis; Serum; Signal Pathway; Signal Transduction; Signaling Protein; Sorting; Stains; Sting Injury; TLR3 gene; Testing; Therapeutic Intervention; Toll-Like Receptor Pathway; Toll-like receptors; Transplantation; Tretinoin; Vancomycin; Viral; Wild Type Mouse; Work; cytopenia; granulocyte; in vivo; metabolomics; microbial; microbiome; microbiota; mortality; mouse model; new therapeutic target; prevent; progenitor; screening; stool sample; therapy development

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.

摘要/项目摘要 2020年，美国开出了超过2亿个疗程的抗生素，引起了临床的重大关注 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。中性粒细胞减少症如果不及时治疗，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 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。 我们的实验室开发了一种抗生素相关骨髓抑制的小鼠模型，结果显示 长期使用抗生素导致微生物群耗竭，导致贫血、白细胞减少和其他血细胞减少症。 然而，抗生素破坏造血作用的精确分化阶段仍不清楚。我们 进一步证明微生物组促进 I 型干扰素 (IFN-I) 信号传导的基础水平，即 以 STAT1 依赖性方式维持稳态造血所需。尽管之前的研究表明 NOD1 和 TLR-MYD88 通路的激活可以支持骨髓细胞生成，我观察到正常数量的 Nod1 和 Myd88 缺陷小鼠的基线造血祖细胞和粒细胞，表明这些 对于正常的血液生产来说，通路是可有可无的。多项独立研究表明， 微生物组可以通过 TLR-TRIF、cGAS-STING 和 RIG-I-MAVS 途径诱导强效 IFN-I 信号传导， 尽管它们对造血维持的贡献尚不清楚。在非靶向代谢组学中 通过筛选，我们鉴定出 29 种微生物代谢物，这些代谢物在非非肠道粪便和血清样本中富集。 白细胞减少症小鼠与抗生素治疗后两周白细胞减少症小鼠进行比较。这些代谢物是否 是否可以支持体内造血功能仍待探索。该提案将检验微生物组的假设 利用免疫相关信号通路，例如 TLR-TRIF、cGAS-STING 和 RIG-I-MAVS 通路 在造血干细胞 (HSC) 水平支持 IFN-I 介导的稳态造血作用。 我们之前工作的一个主要限制是 IFN-I 抑制引起的 Sca-1 表达变化可能 扭曲了抗生素治疗小鼠中 HSPC 的计数。为了解决这个问题，我们首先要进行限制 稀释移植以量化抗生素和模拟治疗小鼠中的功能性 HSC。我们也将追寻命运 通过追踪 Krt18- 中的 HSPC 和粒细胞群的增殖、分化和更新 CreERT2：Rosa26-lox-STOP-lox-TdTomato 小鼠使用或不使用抗生素治疗。阐明机制 为了研究微生物组依赖性造血作用，我们将描述 Trif-/-、Sting-/- 和 使用或不使用抗生素治疗的 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 激动剂可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物维持基础 IFN-I 产生的能力。这些研究将 定义微生物组促进 IFN-I 产生以调节造血功能的机制。