Gut Microbiota-Driven Tonic Type I IFN Signaling Supports Steady-State Hematopoiesis

Prior studies have shown that up to 20% of patients treated with antibiotics (Abx) for 2 weeks or longer develop hematological adverse effects such neutropenia and other cytopenias, leading to increased medical costs and mortality. Yet, the mechanisms underlying antibiotic-associated blood and bone marrow suppression remain poorly understood. Our lab has shown that microbiome depletion on prolonged antibiotics impairs steady-state hematopoiesis. Furthermore, the gut microbiota induces a basal level of type I interferon (IFN-I) signaling to support steady-state hematopoiesis in a STAT1-dependent manner. The goal of this work is 1) to identify the receptors that sense bacterial components to trigger IFN-I production, and 2) to identify key bacterial species and metabolites that contribute to microbiota-dependent hematopoiesis.

Bacterial cues are known to trigger IFN-I production via TLR-MYD88, NOD1, NOD2, cGAS-STING and RIG-I-MAVS pathways. However, the contribution of these pathways in supporting IFN-I-mediated hematopoiesis remain unknown. Using global knockout mouse models, we showed for the first time that microbial metabolites can activate cytosolic pattern recognition receptor RIG-I in the hematopoietic compartment to induce a basal level of type I IFN (IFN-I) production. Deletion of MAVS, a downstream adaptor protein in the RIG-I pathway, resulted in profound depletion of hematopoietic progenitors (lineage-, cKit+, Sca+; LSK), hemoglobin, and platelets which phenocopied Abx-treated mice, suggesting that the RIG-I MAVS pathway is indispensable for hematopoietic maintenance. In contrast, LSK numbers were normal in mice containing deletion mutations in Myd88, Nod1, Nod2, and Sting. Activation of RIG-I signaling by intraperitoneal administration of dsRNA in Abx-treated mice rescued both HSPC and granulocytic defects in an IFN-I dependent manner. We performed multiplex serum cytokine analysis and single-cell RNA sequencing on bone marrow samples obtained from Abx-treated and control mice which confirmed that microbiome depletion is associated with suppression of tonic IFN-I signaling in hematopoietic cells.

To identify the specific bacterial species and metabolites that can contribute to normal hematopoiesis, we conducted a multicenter, non-interventional IRB-approved study in pediatric patients that did or did not develop neutropenia after prolonged (≥ 2 weeks) antibiotic therapy. Fifty one patients completed the study, of whom ten developed absolute neutrophil count < 1500 /uL. Clinically, length of intravenously administered antibiotics and intensive care unit stay were correlated with neutropenia, whereas no association was found between type of infection or type of antibiotics and neutropenia. We performed liquid chromatography-mass spectrometry-based metabolomic profiling and 16S sequencing on stool samples collected at the beginning and cessation of therapy. A similar approach was used to screen metabolites in serum and stool samples from leukopenic and nonleukopenic mice after Abx treatment. Notably, several metabolites or classes of metabolites were shared in common between the human and murine studies. We tested the ability of several metabolites that were differentially abundant in neutropenic mice and human post-treatment to induce IFN-I via RIG-I signaling in vitro. We found that only uridine diphosphate glucose, a central intermediate in bacterial carbohydrate metabolism, was able to induce IFN-I production in a RAW-Lucia ISG reporter cell line. Our 16S rRNA sequencing data showed reduced alpha diversity and depletion of Lachnospiraceae in patients with neutropenia compared to non-neutropenic controls. Members of the Lachnospiraceae family such as Blautia faecis have been reported to be associated with engraftment after hematopoietic stem cell transplant, lending credence to the idea that these bacteria may play a role in normal granulopoiesis. Studies are currently ongoing to assess whether UDP-glucose or Lachnospiraceae can rescue Abx-associated hematopoietic defects in the murine model.

Overall, our work sheds light on molecular crosstalk between the microbiome and hematopoietic progenitors to sustain steady-state hematopoiesis. Our work will advance translational approaches, particularly the development of next-generation pre- and probiotics and metabolite supplements that can support a healthy immune system.

Maknojia:Danone North America: Research Funding.