Bone marrow suppression is an adverse effect associated with many antibiotics, especially when administered for prolonged treatment courses. Recent advances in our understanding of steady-state hematopoiesis have allowed us to explore the effects of antibiotics on hematopoietic progenitors in detail using a murine model. Antibiotic-treated mice exhibited anemia, thrombocytosis, and leukopenia, with pronounced pan-lymphopenia as demonstrated by flow cytometric analysis of peripheral blood. Bone marrow progenitor analysis revealed depletion of hematopoietic stem cells and multipotent progenitors across all subtypes. Granulocytes and B cells were also diminished in the bone marrow, whereas the number of CD8 T cells increased. Reductions in progenitor activity were not observed when cells were directly incubated with antibiotics, suggesting that these effects are indirect. Hematopoietic changes were associated with a significant contraction of the fecal microbiome and were partially rescued by fecal microbiota transfer. Further, mice raised in germ-free conditions had hematopoietic abnormalities similar to those seen in antibiotic-treated mice, and antibiotic therapy of germ-free mice caused no additional abnormalities. The effects of antibiotics were phenocopied in Stat1-deficient mice, with no additional suppression by antibiotics in these mice. We conclude that microbiome depletion as a result of broad-spectrum antibiotic treatment disrupts basal Stat1 signaling and alters T-cell homeostasis, leading to impaired progenitor maintenance and granulocyte maturation. Methods to preserve the microbiome may reduce the incidence of antibiotic-associated bone marrow suppression.

Cellular quiescence is a key component of hematopoietic stem cell (HSC) homeostasis; therefore, a reliable method to measure HSC cell division is critical in many studies. However, measuring the proliferation rate of largely quiescent and rare populations of cells can be challenging. Bromo-deoxyuridine (BrdU) incorporation into replicating DNA is a commonly used and highly reproducible method to detect cell division history. Here, we describe a protocol for BrdU incorporation analysis in hematopoietic stem and progenitor cells that can provide a sensitive measure of cell division even in rare cell populations. In combination with flow cytometry, this method can be generalized to analyze other cell populations and other tissues as identified by cell surface markers.

MicroRNA-22 (miR-22) is a highly conserved microRNA that can regulate cell proliferation, oncogenesis, and cell maturation, especially during stress. In hematopoietic stem cells (HSCs) miR-22 has been reported to be involved in the regulation of key self-renewal factors including Tet2. Recent work demonstrates that miR-22 also participates in regulation of the interferon response, and expression profiling studies suggest that it is variably expressed at different stages in erythroid differentiation. We thus hypothesized that miR-22 regulates maturation of erythroid progenitors during stress hematopoiesis through its interaction with interferon. We compared the blood and bone marrow of wild type (WT) and miR-22-deficient mice at baseline and upon infectious challenge with systemic lymphochoriomeningitis (LCMV) virus. MiR-22-deficient mice maintained platelet counts better than WT mice during infection, but they showed significantly reduced red blood cells (RBC) and hemoglobin. Analysis of bone marrow progenitors demonstrated better overall survival and improved HSC homeostasis in infected miR-22-null mice compared to WT, attributable to a blunted interferon response to LCMV challenge in the miR-22-null mice. We found that miR-22 was exclusively expressed in stage II erythroid precursors and was downregulated upon infection in WT mice. Our results indicate that miR-22 promotes the interferon response to viral infection and that it functions at baseline as a brake to slow erythroid differentiation and maintain adequate erythroid potential. Impaired regulation of erythrogenesis in the absence of miR-22 can lead to anemia during infection.