| Hematopoiesis is a highly regulated process that produces billions of new blood cells every day for an entire lifetime. In order to fully characterize the mRNA and microRNA expression profiles of the hematopoietic stem and progenitor cells (HSPCs), that are responsible for the daily production of blood cells, we optimized a highly effective protocol for isolating rare HSPC subsets by fluorescent activated cell sorting (FACS). These methods have provided preliminary data for a number of projects in the lab. We also applied these methods to fully characterize the role of Tristetraprolin (TTP), a destabilizing RNA binding protein, in maintaining hematopoietic homeostasis. We found that TTP KO mice display a spontaneous phenotype that closely resembles that described in reactive granulopoieisis; increased frequency and numbers of mature granulocytes, granulocyte-monocyte progenitors (GMPs), multipotent progenitors (MPPs) and short-term hematopoietic stem cells (LT-HSCs). Interestingly, we found no effect on LT-HSC frequency, number or cell cycle status. By creating reverse chimeras, WT bone marrow was transplanted into WT and KO recipients, we found that WT marrow in a KO environment phenocopied the entire phenotype. These results proved conclusively that the TTP KO phenotype was caused by a non-cell autonomous mechanism. Recently, the inflammatory cytokine, and TTP target, IL-1beta was shown to be necessary and sufficient for the process of reactive granulopoiesis. We found elevated levels of IL-1beta, as well as the TTP targets IL-6 and TNFalpha, in the serum of KO animals compared to WT littermates; indicating an association between a lack of regulation of their transcripts and the spontaneous granulocyte hyperplasia seen in TTP KO mice. Together, these results highlight the importance of mRNA half-life regulation in maintaining appropriate levels of inflammatory cytokines. In addition, LT-HSCs, whose relative quiescence is necessary for life-long blood production, were not affected by the type of inflammatory signals we observed in TTP KO mice. |
