CCL5 Enhances Megakaryocyte Differentiation and Development
Authors: Noetzli Leila , Parvin Somayje , Battinelli Elisabeth , Machlus Kellie 
Published: October 2019

Abstract
The importance of platelets in clot formation is well understood, but the role of platelets in the inflammatory response is becoming increasingly apparent. Platelet count increases during acute inflammatory stress; platelets also facilitate inflammation by releasing chemokines and cytokines from their granules. Our lab has previously shown that platelet derived chemokine ligand 5 (CCL5, RANTES), an inflammatory chemokine that is stored in platelets and released upon activation, acts to increase megakaryocyte maturation and proplatelet production in vitro. Furthermore, our lab found that CCL5 treated megakaryocytes have enhanced phosphorylation of proteins in the AKT pathway, such as BAD, which when phosphorylated induces pro-survival signaling. This indicates that CCL5-induced increase of platelet production is mediated by suppression of apoptosis, but the exact mechanism of CCL5 on megakaryocytes is not understood.
To more completely asses the global impact of CCL5 on megakaryocytes, we performed proteomic analysis of CCL5-treated megakaryocytes and found that, as expected, CCL5 increases the expression of several pro-survival proteins in the AKT pathway. We have now confirmed increased phosphorylation of BAD by western blot in megakaryocytes treated with CCL5. In addition, we found that CCL5 treatment protects megakaryocytes from etoposide-induced apoptosis. Together, our data support the model of CCL5-induced inhibition of mitochondrial apoptosis via BAD phosphorylation. Interestingly, proteomic analysis also revealed an increase in proteins involved in quantity and differentiation of hematopoietic progenitor cells, as well as differentiation of megakaryocytes. To test whether CCL5 can also affect the differentiation of hematopoietic progenitor cells, we cultured mouse bone marrow with 100 ng/mL CCL5 and TPO and found a small but sustained increase in the number of megakaryocytes in vitro. To better understand the effect of CCL5 on skewing of hematopoietic cells in a physiological setting, we serially injected mice with 0.025 mg/kg or 0.05 mg/kg CCL5 for one week. Flow cytometry of bone marrow from these mice showed an increase in the number of Pre-MK E (Lin-/cKit+/Sca-1-/CD41-/FcgR-/CD105-/CD150+) and Pre-CFU E (Lin-/cKit+/Sca-1-/CD41-/FcgR-/CD105+/CD150+) populations and a decrease in the number of Pre GM (Lin-/cKit+/Sca-1-/CD41-/FcgR-/CD105-/CD150-) cells in CCL5 treated mice. Interestingly, the CCL5 treated mice did not have a significant inflammatory response, as was measured by complete blood count, indicating that these hematopoietic changes are independent from inflammation. These data suggest that CCL5 may act to skew hematopoiesis toward a megakaryocyte-erythroid lineage and away from a granulocyte lineage in non-inflamed mice.
Our proteomics data and in vitro live-dead assay suggest that CCL5 treatment protects against apoptosis through increased BAD phosphorylation, potentially leading to increased megakaryocyte maturation and proplatelet formation. Furthermore, CCL5 may have a dual action in both enhancing platelet production via pro-survival signaling and skewing hematopoiesis toward the megakaryocyte-erythroid lineage. Hematopoietic skewing was seen in CCL5 treated mice despite not having increased inflammation, suggesting that CCL5 itself does not cause a significant inflammatory response. Nonetheless, CCL5 has previously been shown to increase in an inflammatory setting, indicating that the effects seen here could be triggered by systemic inflammation in a different model. Further investigation of the mechanisms leading to these phenotypes may reveal novel therapeutic options to enhance megakaryocyte development and increase platelet counts in patients with thrombocytopenia.

Disclosures
Battinelli: Sanofi: Consultancy.