Inflammation after Chemotherapy Regulates Hematopoietic Stem Cell Lineage Fate

Chemotherapy is one of the most common therapies used to treat different types of malignancies. Besides eradicating malignant cells, many healthy cells are also affected. Common chemotherapy-associated side effects include neutropenia, anemia and thrombocytopenia. Hematopoietic stem cells (HSCs) are responsible for lifelong production of all blood cells and are essential for recovery after myeloablative insult. Despite this, the activation and regulation of HSCs in response to chemotherapy is poorly understood. Herein, we sought to characterize the activation and lineage output of HSCs upon chemotherapy. Specifically, we investigate the role of inflammation and caspase-1 on HSC lineage fate decisions, to gain a better understanding of what drives myelosuppression and recovery after chemotherapy.

To follow the recovery dynamics of mature hematopoietic cells after chemotherapy treatment, we treated mice with the chemotherapy drug 5-fluououracil (5-FU) and analyzed peripheral blood at seven timepoints post chemotherapy treatment. We show that mice at day 6 following chemotherapy are pancytopenic, with low numbers of leukocytes, platelets, and erythrocytes. Between day 6 and day 12 there was an extensive expansion of the leukocytes and platelets, but not of erythrocytes. Only after day 12 when leukocyte and platelet numbers had recovered to steady state levels or surpassed them, erythrocyte numbers increase.

Next, we aimed to determine the role of HSCs in the recovery kinetics of the mature lineages. Markers for identifying HSCs are not reliable in this setting since hematopoietic stem and progenitor cells transiently lose cKit cell surface expression post treatment. By using alternative cell surface markers, Lin^-, CD48^-, CD244^-, CD150⁺, EPCR⁺, we could reliably identify HSC after chemotherapy treatment. To determine if the production kinetics of different hematopoietic lineages are established at the HSC level, we transplanted sorted HSCs two days post chemotherapy to lethally irradiated recipients and found a significantly lower output of donor-derived RBCs (p= 0.0059).

To assess what transcriptional changes might contribute to the differences in mature lineage output, we performed RNA-seq on HSCs sorted 2 days post chemotherapy treatment. Gene set enrichment analysis showed pathways related to inflammation, myeloid differentiation and long-term HSC maintenance being positively enriched, while pathways related to short-term HSCs and GATA1 targets and protein translation were negatively enriched. Examining specific transcription factors, we found upregulation of HSC specific transcription factors as well as those driving myeloid differentiation, whereas those promoting erythroid and megakaryocytic differentiation were downregulated. This indicates a transcriptional priming of HSCs towards myeloid cell production only 2 days after chemotherapy, at the expense of erythroid cells. Furthermore, we observed significant upregulation of genes linked to inflammation. Upon measuring pro-inflammatory cytokine levels in the bone marrow 24 hours after chemotherapy, we noted a multi-fold increase of several cytokines, one of which was IL-1β.

To discern the role of inflammation in lineage fate decisions we utilized a caspase-1 knockout mouse model, as caspase-1 is required to produce mature IL-1β. The cytokine IL-1β has previously been found to influence lineage fate in HSCs (Pietras et al. Nature cell Biology, 2016). Treating caspase-1 KO mice with chemotherapy lead to a diminished output of leukocytes (p= 0.0021) and higher erythroid output (p= <0.0001) compared to WT 5-FU-treated mice. These findings indicate that the lineage bias towards myeloid cells over erythrocytes following chemotherapy treatment, is rescued by the loss of caspase-1 and its role in producing mature IL-1β. In a clinical setting, patients undergoing chemotherapy treatment often experience anemia, something our data suggests could be mitigated by caspase-1 / IL-1β inhibition. Anemia is also a symptom of many inflammatory conditions and diseases such as myelodysplastic syndrome (MDS). Investigating caspase-1 and IL-1 β inhibitors in the context of mitigating anemia in patients with such conditions could pose as a viable treatment option.

No relevant conflicts of interest to declare.