Stat3 Is Required for In Vivo G-CSF-Responsiveness and Neutrophil Function.
Authors: Panopoulos Athanasia D. , Yoon Donghoon , Zhang Ling , Watowich Stephanie S. 
Published: October 2019

Abstract
Granulocyte-colony stimulating factor (G-CSF) and its receptor (G-CSFR) are essential for neutrophil development. The G-CSF/G-CSFR pathway supplies normal circulating neutrophil levels and supports neutrophil terminal maturation. These activities have been exploited clinically, where G-CSF is used frequently to treat congenital or therapeutically induced neutropenia, or to mobilize hematopoietic stem cells for bone marrow transplant. Previous work from our laboratory and others has shown a role for the major G-CSF-responsive signaling molecule STAT3 in granulopoiesis. It is well established that STAT3 has an important negative function in controlling neutrophil production. Importantly, our studies have elucidated a critical positive regulatory role for STAT3 in G-CSF-responsiveness and development of functional neutrophils in vivo. Mice lacking STAT3 in the bone marrow (TIE2cre/Stat3f/Δ) fail to upregulate circulating neutrophil levels in response to a single dose of G-CSF administered subcutaneously, while their wild type littermates demonstrate a two-fold increase in circulating neutrophil levels (e.g., Gr-1+/CD11b+ cells) under these conditions. Inspection of bone marrow responses revealed an increase in the Gr-1lo/CD11bhi population and a corresponding decrease in the Gr-1hi/CD11bhi population of littermate control animals following G-CSF administration. Strikingly, mice that lack functional STAT3 in the bone marrow fail to respond to G-CSF similarly; these animals do not demonstrate an induction in the Gr-1lo/CD11bhi population, or a decrease in the Gr-1hi/CD11bhi population within the bone marrow following G-CSF treatment. These results indicate that in vivo G-CSF administration recruits early progenitors (e.g., Gr-1lo/CD11bhi cells) into the granulocytic lineage while concomitantly decreasing levels of more mature granulocytes (e.g., Gr-1hi/CD11bhi) within the marrow, which are likely mobilized to the peripheral circulation, and suggest an essential role for STAT3 in these responses. Furthermore, neutrophils isolated from TIE2cre/Stat3f/Δ mice demonstrate defective chemotaxis in response to MIP-2. This deficiency appears to be cell autonomous since granulocytes derived from an ex vivo differentiation system show a similar phenotype. Defective chemotaxis may be due in part to improper actin rearrangement dynamics, since STAT3 deficient neutrophils show enhanced actin polymerization in response to MIP-2 compared to wild type controls. To examine potential pathways by which STAT3 may function, we investigated the expression of the PU.1 transcription factor in TIE2cre/Stat3f/Δ mice. PU.1 is known to control expression of genes that are essential for mature neutrophil functions. Our results demonstrate that PU.1 is expressed at high levels in the Gr-1lo/CD11bhi population relative to the Gr-1hi/CD11bhi population isolated from bone marrow of wild type mice. Ex vivo G-CSF treatment of wild type bone marrow stimulates an increase in the proportion of Gr-1lo/CD11bhi cells within the culture, as well as the total PU.1 protein level of the population. By contrast, induction of Gr-1lo/CD11bhi cells and total PU.1 expression by G-CSF treatment ex vivo is abrogated in bone marrow from TIE2cre/Stat3f/Δ mice. Importantly, these effects are not due to significant changes in cell survival or selection in the ex vivo culture system. Collectively, our results indicate that STAT3 is essential for G-CSF-mediated neutrophil responses and function in vivo, and suggest that STAT3-dependent regulation of PU.1 may be an important intermediate in this pathway.