Platelet Factor 4 (PF4) Regulates Hematopoietic Stem Cell Aging

Hematopoietic stem cells (HSCs), responsible for blood cell production, and their bone marrow regulatory niches undergo age-related changes, impacting immune responses and predisposing individuals to hematologic malignancies. Extensive research has elucidated cell-autonomous processes underlying HSC aging, while recent studies also highlight the impact of bone marrow niche-derived factors. Our previous studies in young mice showed that HSCs reside within specialized bone marrow niches, which encompass megakaryocytes, crucial for maintaining myeloid-biased HSC quiescence and supporting their reconstitution potential via platelet factor 4 (PF4) secretion (Nature Medicine 2014; Developmental Cell 2018).Remarkably, the plasma levels of PF4 decline in both aged mice and humans.

Here, we investigated the impact of PF4 on murine and human HSC aging. Our studies revealed significant alterations in the aged (22-month-old) mouse bone marrow megakaryocytic niche, characterized by compromised megakaryocyte maturation evidenced by smaller size and decreased ploidy levels, alongside an increase in megakaryocyte progenitors (P<0.0001). RNA sequencing analysis of young and old megakaryocytes further confirmed this immature phenotype and revealed reduced PF4 expression (35% reduction; P=0.0248). Notably, using young (2-month-old) PF4-deficient mice, we observed phenotypes reminiscent of accelerated physiological HSC aging, including decreased lymphoid output (P=0.0023), increased myeloid output (P=0.0014), and DNA damage (2.8-fold increase, P<0.0001) as seen by γH2AX staining, similar to old phenotypes. Young PF4-deficient HSCs also showed reduced polar distribution of Cdc42 and alpha-tubulin, identical to the frequencies seen in old mice. These results suggest that PF4 levels might be critical in preventing the aging of the hematopoietic system.

Next, we tested the effect of short-term PF4 treatment (1 μg/mL) on old HSCs. PF4 blocks the proliferation (37% reduction; P= 0.0105) and decreased γH2AX foci (20% reduction; P=0.0273) of old HSCs in vitro. Importantly, long-term (42 days) continuous recombinant PF4 administration (1 μg/day) by osmotic pumps in 18-month-old mice restored the serum levels of PF4 in old mice to the levels of 2-month-old mice. Notably, this restored phenotypic HSC cell number (P=0.001), HSC polarity (P=0.0464), and decreased DNA damage (P<0.0001). Furthermore, 4 months after competitive transplantation, HSCs from old PF4-treated mice showed a significantly higher reconstitution (17% ± 6.08) compared to old saline-treated mice (2.72% ± 0.54) and a balanced lineage output akin to young HSCs. In agreement, HSCs from old PF4-treated mice exhibited decreased expression of myeloid genes and increased expression of lymphoid genes, mirroring the profile observed in youthful, balanced HSCs.

Mechanistically, we identified CXCR3 (C-X-C Motif Chemokine Receptor 3) and LDLR (Low-Density Lipoprotein (LDL) Receptor) as the HSC receptors transmitting the PF4 signal. In vitro antibody blockade of LDLR (P=0.039) and CXCR3 (P=0.0013) abrogated the anti-proliferative effects of PF4 on myeloid-biased HSCs, the HSC subset that expands with age. LDLR is involved in the endocytosis of LDL cholesterol, with accumulating evidence suggesting a role for cholesterol in myeloid-biased HSC proliferation. Accordingly, our functional studies with fluorescently labeled LDL indicated that PF4 limits LDL uptake by HSCs in a dose-dependent manner, suggesting an important role for PF4-LDLR in old HSC metabolism. In vitro HSC culture experiments also revealed that HSCs from LDLR-deficient and CXCR3-deficient mice are insensitive to PF4 treatment. Notably, only LDLR and CXCR3 double knockout mice showed significantly increased HSC frequency (P=0.009) and number (P=0.0426) in vivo, similar to our previously published results with PF4-deficient mice. Finally, our in vitro culture experiments further indicated that phenotypic human HSCs (Lineage^- CD34⁺ CD38^- CD45RA^- CD90⁺ CD49f⁺) at young, middle-aged, and old ages are also responsive to the PF4 youthful signal in a dose-dependent manner. Altogether, our findings contribute to the development of targeted therapies for HSC rejuvenation and potential strategies to prevent or improve the course of age-related immune dysfunction and hematopoietic diseases.

No relevant conflicts of interest to declare.