Identification of a Hematopoietic Stem Cell Fraction That Retains Normal Function Following Chronic Inflammation

Chronic inflammation has been suggested to impair hematopoietic stem cell (HSC) function, resulting in decreased self-renewal and myeloid overproduction at the expense of other lineages. We previously identified the pro-inflammatory cytokine interleukin (IL)-1 as a key activator of precocious HSC differentiation that simultaneously impairs HSC self-renewal. However, recent identification of multiple HSC subsets with distinct functional and molecular characteristics calls into question whether the HSC compartment is uniformly affected by chronic inflammatory signals. Moreover, a growing body of work suggests that traditional markers in the mouse that define HSC potential under homeostatic conditions, such as Sca-1 and CD34, undergo expression changes that limit their utility in prospective identification of functional HSCs during non-homeostatic conditions such as inflammation. Here we use injection of IL-1β to model chronic inflammation in the mouse, and demonstrate that the rare EPCR⁺CD34^- fraction contained within the phenotypic Lin^-cKit⁺Sca-1⁺Flk2^-CD48^-CD150⁺ (SLAM) HSC compartment is uniquely able to maintain its function despite chronic IL-1 exposure.

To assess the impact of chronic inflammation on HSC compartment dynamics, we injected wild-type (WT) C57BL/6 mice with recombinant murine IL-1β for 20 days, followed by analysis of HSC populations using multi-parametric flow-cytometry. Strikingly, t-Stochastic Neighbor Embedding (tSNE) analysis of our flow cytometry data identified changes in the relative abundance of several novel HSC sub-populations following IL-1 exposure, including a sharp decrease in the number of CD34^- cells also expressing the long-term HSC (LT-HSC) marker EPCR (CD201). Consistent with the characteristics of a long-term HSC, CD34^-EPCR⁺ HSCs exhibited slow growth and differentiation kinetics in liquid culture, and elevated colony forming potential in serial re-plating. Moreover, molecular analyses demonstrated EPCR⁺CD34^- HSCs are uniquely enriched for expression of LT-HSC genes including Hoxb5, Fgd5 and Cpt1a . Nonetheless, EPCR⁺CD34^- cells express Il1r1 and underwent accelerated myeloid differentiation in the presence of IL-1, suggesting they are directly responsive to this cytokine.

To determine the impact of sustained IL-1 exposure on the blood-forming capacity of EPCR⁺CD34^- HSCs in vivo, we assessed their growth and potential ex vivo following treatment of mice with PBS or IL-1β for 20 days. Notably, prior exposure to IL-1 did not significantly alter the growth or short-term potential of EPCR⁺CD34^- HSCs based on in vitro assays. Strikingly, ongoing transplantation experiments of IL-1-exposed EPCR⁺CD34^- HSCs versus total SLAM HSCs into irradiated recipient mice have revealed that while the total SLAM HSC compartment exhibits immediate impairment of reconstitution capacity, EPCR⁺CD34^- HSCs exhibit no functional impairment, suggesting IL-1 signaling may not directly impact their self-renewal capacity. We are currently examining the molecular and epigenetic features of these cells to understand how they retain their self-renewal capacity in the face of inflammation, and assessing whether this compartment retains its functionality in a range of inflammatory disease contexts.

Overall, our findings have uncovered a distinct fraction within the phenotypic SLAM HSC that retains essentially normal function despite exposure to chronic inflammation. This supports the notion that HSCs respond to inflammation in a heterogeneous manner and that a functional LT-HSC compartment is present even during chronic inflammatory contexts. These investigations stand to provide critical insights that will increase our understanding of the impact of inflammation on long-term blood system function in the context of chronic inflammatory disease, bone marrow transplant and hematological malignancy.