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

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.