Hematopoietic Stem Cells Respond Heterogeneously to Inflammatory Signals in an Age-Dependent Manner

Hematopoietic stem cells (HSCs) have the unique responsibility to produce balanced immune cell output throughout an organism's life. Importantly, they must do so robustly despite a plethora of external stress, including frequent inflammatory challenge. With age, the accumulation of these stresses leads to impaired HSC function and myeloid-biased output. Aged HSCs are also more prone to pathological hematopoiesis, such as myeloproliferative disorder, leukemia and autoimmune diseases. However, little is known about the subcellular mechanisms that govern the inflammatory response of HSCs with age, which in turn might contribute to pathologic transformation.

We show that young hematopoietic stem and progenitor cells (HSPCs) demonstrate a robust transcriptional response to toll-like receptor (TLR) ligands. Interestingly, this response is similar to that of mature immune cell types such as dendritic cells. Using single-cell proteomic assays, we found that young HSPCs secrete a diverse array of myeloid and lymphoid cytokines. However, when challenged with TLR ligands in vivo, young mice acutely increase myeloid-biased output but return rapidly to baseline hematopoietic output of both lymphoid and myeloid cells. Moreover, inflammatory challenge of young long-term HSCs in vitro did not perturb the function and output of these cells in bone marrow reconstitution experiments.

In contrast to their counterparts from young mice, we found HSPCs obtained from aged mice have a diminished ability to secrete cytokines in response to TLR ligands. Furthermore, they secrete a homogenous subset of myeloid-biased cytokines. When challenged with TLR ligands in vivo, aged mice acutely increased myeloid output and maintain elevated myeloid output for several months implying memory of the inflammatory challenge. Consistent with this, we also found that pre-stimulation of aged HSCs prior to bone marrow transplant results in a sustained increase in myeloid output compared to unstimulated aged HSCs.

To elucidate the differential heterogeneity between young and aged HSPCs in response to TLR signaling, we next performed single-cell RNA sequencing (RNA-seq) experiments. We found that the sustained myeloid output in aged mice after TLR stimulation is largely due to expansion of a myeloid-biased HSC subset in the aged HSC pool. By characterizing the gene expression networks that define these myeloid-biased HSCs under stimulation conditions, we were then able to identify a myeloid-biased HSC subset in both the unperturbed young and aged HSC pools. Moreover, we found that these cells are more abundant in aged mice at steady-state, and that these HSCs demonstrate a unique response to inflammatory challenge. We further identify putative transcriptional regulators, including Klf4, Klf5, Ikzf1 and Stat3, among others, that define gene expression in these myeloid-biased HSCs. We further show that loss of function of these factors can differentially alter myeloid output in young and aged mice both in vitro and in vivo.

Our results demonstrate that there is a differential response of young and aged HSCs to inflammatory signals. Using single-cell RNA-seq and protein secretion studies, we elucidate the molecular heterogeneity of the HSC pool at steady state and with TLR stimulation. By resolving heterogeneous subsets of cells in both the young and aged HSC pool, and by uncovering the transcriptional regulators that influence their function, we thus propose a new model of inflammatory hematopoiesis that may have implications to understanding age-related defects in immune development.