Acute CCL5 Exposure Promotes Megakaryopoiesis through Non-Canonical Pathways
Authors: N. Barrachina Maria , Guo Karen , Freire Daniela , Camacho Virginia , Becker Isabelle C , Nikols Emma , Tilburg Julia , Italiano Joseph E. , Machlus Kellie R 
Published: December 2024

Background: Despite the importance of platelets in thrombosis and hemostasis, the mechanism by which megakaryocytes (MKs) differentiate and initiate platelet release is incompletely understood. Although thrombopoietin (TPO) has been identified as the most important regulator of MK maturation, recent reports indicate that MK maturation and platelet biogenesis can occur independently of TPO. For instance, in inflammatory conditions, platelet counts can rapidly elevate within hours. We previous identified CCL5 [chemokine ligand 5 (CCL5, also known as RANTES)] as an additional regulator of MK differentiation and maturation during inflammation. In an in vitro model, we found that CCL5 enhanced proplatelet production in a CCR5-dependent manner. In addition, other work has shown that exposing isolated hematopoietic stem cells (HSCs) to CCL5 before transplantation can directly affect fate choices of them and their progenitor cells. Building upon these observations, we aimed to investigate whether CCL5 affects megakaryo- and thrombopoiesis using in vivo mouse models, and in a novel human bone marrow organoid.

Results: To determine the effect of CCL5 under steady state, we administered acute CCL5 to mice and verified that cytokine levels were elevated in both plasma and bone marrow fluid 24 hours post administration. Quantification of MKs by flow cytometry revealed an increase in both MK progenitors [CD41+] and mature MKs [CD41+CD42d+] 24 hours after treatment with CCL5. Additionally, immunofluorescence imaging of femoral cryosections confirmed an increase in MK numbers, suggesting that acute exposure to CCL5 enhanced megakaryopoiesis. Expanding our analysis to HSPCs, we observed that CCL5 also induced the expansion of MK-biased CD41+ long-term (LT)-HSCs [Lin-cKithighSca-1+Flt3-CD48-CD150+CD41+] and MkPs [Lin-cKithighCD150+CD41+], 2.8- and 2.1-fold respectively, suggesting a potential non-canonical pathway triggered by CCL5. To elucidate the mechanism responsible for increased MK numbers, we aimed to determine the in vivo expression patterns of the CCL5 receptors CCR1, CCR3, and CCR5 with and without CCL5 treatment. While no differences were found in LT- and short-term (ST)-HSCs, CCR1 and CCR5 expression was increased in MkPs 24 hours after CCL5 administration, suggesting that CCL5 signaling through CCR1 and CCR5 represents a pathway by which CCL5 signals to drive megakaryopoiesis.

To investigate the effect of CCL5 administration upon stress, i.e. during platelet recovery following anti-GPIba antibody-induced platelet depletion, mice were treated with either vehicle or CCL5 48 hours post platelet depletion. After 6 days, platelets counts were higher in CCL5-treated mice compared to the vehicle-treated group. Due to the faster recovery observed in CCL5-treated mice, we aimed to determine if the increase in platelet counts occurred via non-canonical pathways. Quantification of MKs by flow cytometry revealed an increase in MK-biased CD41+ LT-HSCs while we did not observe differences in multipotent progenitors (MPPs) compared to the vehicle-treated group, indicating that CCL5 facilitated the expansion of MKs in the bone marrow post depletion.

To substantiate our findings in a human system, we utilized an iPCs-derived human bone marrow organoid, which recapitulates essential features of the adult human bone marrow and offers a platform to study cellular behavior within a bone marrow microenvironment. We treated bone marrow organoids with CCL5 for 24 hours and quantified populations including endothelial cells, HSPCs, erythroid cells and MKs using flow cytometry. Consistent with our observations in the murine model, the organoids exhibited 2-fold increase in MKs [CD41+].

Conclusions: Using murine and human models, we demonstrate a CCL5-induced expansion of MkPs, revealing a role for CCL5 in promoting megakaryopoiesis in the bone marrow. These data suggest that CCL5 may be one component of the inflammatory milieu that triggers elevated platelet counts in inflammatory thrombocytosis.