2047 – AGING ENHANCES IL-1β RESPONSIVENESS IN HUMAN BONE MARROW HEMATOPOIETIC STEM CELLS

Abstract

Human hematopoietic stem cells (HSCs) demonstrate age-related molecular changes, such as increased inflammatory pathway activation; however, the functional consequences on bone marrow (BM) HSC behavior remain incompletely understood. To address this question, we characterized 104,224 human BM HSCs (CD34⁺/CD38^–/CD45RA^–/CD90⁺) from 20 vertebral BM samples spanning 8–66 years of age using single-cell RNA sequencing. We identified an age-dependent increase in interleukin-1 (IL-1)–mediated signaling within the HSC compartment. We next examined the effects of IL-1β on human BM HSCs across multiple model systems. Although aging diminished overall HSC expansion in liquid culture, IL-1β preferentially enhanced expansion and promoted myeloid differentiation in old HSCs relative to young. Mechanistically, we observed age-dependent augmentation of IL-1β–induced NF-κβ activation in old HSCs. Bulk RNA sequencing of IL-1β–treated HSCs demonstrated enrichment of NF-κβ target genes in old samples, and single-cell ATAC-sequencing revealed increased accessibility of NF-κβ–associated transcription factor motifs, including REL and RELA. Consistent with these findings, confocal microscopy demonstrated increased nuclear localization of p65 following IL-1β treatment in old HSCs. To evaluate in vivo relevance, we transplanted human HSCs into NSG mice and treated engrafted recipients with intraperitoneal IL-1β for 7 days. IL-1β treatment resulted in enhanced human monocytic differentiation in mice engrafted with old HSCs compared with young, consistent with the increased age-dependent myeloid differentiation observed in vitro. Collectively, these findings demonstrate age-associated hypersensitivity of human BM HSCs to IL-1β mediated by enhanced NF-κβ responsiveness, providing a framework to understand how aging may reshape hematopoietic responses during inflammatory stress.

Human hematopoietic stem cells (HSCs) demonstrate age-related molecular changes, such as increased inflammatory pathway activation; however, the functional consequences on bone marrow (BM) HSC behavior remain incompletely understood. To address this question, we characterized 104,224 human BM HSCs (CD34⁺/CD38^–/CD45RA^–/CD90⁺) from 20 vertebral BM samples spanning 8–66 years of age using single-cell RNA sequencing. We identified an age-dependent increase in interleukin-1 (IL-1)–mediated signaling within the HSC compartment. We next examined the effects of IL-1β on human BM HSCs across multiple model systems. Although aging diminished overall HSC expansion in liquid culture, IL-1β preferentially enhanced expansion and promoted myeloid differentiation in old HSCs relative to young. Mechanistically, we observed age-dependent augmentation of IL-1β–induced NF-κβ activation in old HSCs. Bulk RNA sequencing of IL-1β–treated HSCs demonstrated enrichment of NF-κβ target genes in old samples, and single-cell ATAC-sequencing revealed increased accessibility of NF-κβ–associated transcription factor motifs, including REL and RELA. Consistent with these findings, confocal microscopy demonstrated increased nuclear localization of p65 following IL-1β treatment in old HSCs. To evaluate in vivo relevance, we transplanted human HSCs into NSG mice and treated engrafted recipients with intraperitoneal IL-1β for 7 days. IL-1β treatment resulted in enhanced human monocytic differentiation in mice engrafted with old HSCs compared with young, consistent with the increased age-dependent myeloid differentiation observed in vitro. Collectively, these findings demonstrate age-associated hypersensitivity of human BM HSCs to IL-1β mediated by enhanced NF-κβ responsiveness, providing a framework to understand how aging may reshape hematopoietic responses during inflammatory stress.