Candidemia-Induced Emergency Granulopoiesis Consists of Successive Dual Waves Triggered by the Shift From C/EBPalpha to C/EBPbeta Dependency.

Granulocyte is a major cellular component in the front line of host defense. The number of granulocytes must be tightly tuned to meet the demand, because both the shortage and the excess of granulocytes can be harmful to the host. During emergency situations such as infections, granulocytes are replenished from peripheral pools and bone marrow production. As the half-life of granulocytes is quite short, granulopoiesis, de novo production of granulocytes in bone marrow, plays an important role during emergency. We have previously shown that granulopoiesis at steady state is largely dependent on a transcription factor, C/EBPalpha, whereas emergency granulopoiesis is dependent on C/EBPbeta (Hirai H, et al. Nature Immunol., 2006). However, the precise developmental stage where the shift from C/EBPalpha dependency to C/EBPbeta dependency takes place is almost unknown. The aim of this study is to dissect the process of granulopoiesis by a novel flow cytometric method and to elucidate the molecular mechanisms involved in the regulation of emergency granulopoiesis.

4 ≂ 10⁶ cfu Candida albicans were intravenously injected to induce emergency granulopoiesis. Mouse bone marrow cells were harvested and stained with a combination of fluorescent-conjugated antibodies including anti-c-kit, anti-CD34, anti-Ly6G antibodies and markers for other lineages. Then the stained cells were analyzed or sorted by flow cytometry. After eliminating the cells which lost potential to give rise to granulocytes, the remaining cells were dissected into five subpopulations (#1≂ #5) according to the expression levels of c-kit and Ly6G. #1 is c-kit^high Ly6G^low cells, @ #2: c-kit^int Ly6G^low, #5: c-kit^low Ly6G^high, and the cells residing between #2 and #5 are divided into #3 and #4. Cell number, gene expressions and cell cycle status of each population were analyzed before and after inducing emergency granulopoiesis. @

Wright-Giemsa staining and qRT-PCR for granule proteins (cathepsin G, myeloperoxidase, neutrophil elastase2, lactoferrin and MMP9) in each population indicated that lower c-kit expression and higher Ly6G expression correlated well with granulocytic differentiation and that the granulopoiesis progresses from # 1 to #5 in this order both at steady state and during emergencies (Figure 1). Then we applied this method to candidemia-induced emergency granulopoiesis. In vivo BrdU incorporation analysis showed immediate acceleration of the cell cycle in the most immature population (#1) and in one of the intermediate populations (#2). Chronological monitoring of each population after inducing candidemia revealed that rapid increase in mature granulocytes (#5) preceded the replenishment from the most immature population (#1). These results suggested that there are two distinct gwavesh in granulopoiesis at the early phase of infection, a rapid supply (first gwaveh) of granulocytes from relatively mature population (#2≂ #4), and a further and sustained supply (second gwaveh) originated from more immature populations (#1) including hematopoietic stem/progenitor cells (Figure 1). Transcripts of C/EBPalpha were significantly downregulated in #1≂ #4 at the early phase of infection, while those of C/EBPbeta were maintained in all the subpopulation (Figure 2), suggesting that shift from C/EBPalpha dependency to C/EBPbeta dependency took place at multiple developmental steps in granulopoiesis. C/EBPbeta has less inhibitory effects on cell cycle than C/EBPalpha while their abilities to induce granulocytic differentiation are similar (Hirai H, et al. Nature Immunol., 2006). The shift toward C/EBPbeta dependency may trigger the dual waves in emergency granulopoiesis, which demands both differentiation and proliferation of granulocytic precursors.

View largeDownload slide

View largeDownload slide

Close modal

No relevant conflicts of interest to declare.