Date of Graduation

8-2011

Document Type

Dissertation (PhD)

Program Affiliation

Immunology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Stephanie S. Watowich

Committee Member

Chen Dong

Committee Member

Gary Gallick

Committee Member

Kimberly Schluns

Committee Member

Shao-Cong Sun

Abstract

Among the first white blood cells to respond to bacterial and fungal infections, neutrophils are produced in the bone marrow, released into circulating blood, and recruited to inflamed tissue. The cytokine granulocyte colony-stimulating factor (G􀀎CSF) is used clinically to induce neutrophil mobilization from the marrow. This process was previously demonstrated to require the STAT3 transcription factor (signal transducer and activator of transcription 3), the principal signaling molecule activated upon G-CSF-binding of its receptor, but the mechanism was unknown. The chemokines KC (Cxcl1) and MIP-2 (Cxcl2), and their shared receptor CXCR2 (l8rb), also stimulate neutrophil mobilization, in contrast to SDF-1 (Cxcl12), which contributes to neutrophil retention in the bone marrow, requiring downregulation to promote neutrophil release. Using a murine model with conditional STAT3 deletion in bone marrow, we demonstrate that STAT3 regulates G-CSF-dependent changes in bone marrow chemokine and chemokine receptor expression levels. We found that G-CSF/STAT3 signals increase transcription of Cxcl1, Cxcl2, and Il8rb, and concomitantly suppress Cxcl12. Administration of a MIP-2-neutralizing antibody in vivo suppressed GCSF- stimulated upregulation of circulating neutrophil levels, indicating its critical role in neutrophil mobilization. Consistent with this observation, STAT3-deficient mice were more susceptible than wild type to infection with Listeria monocytogenes, a bacterium that initiates a G-CSF-mediated immune response. STAT3-deficient mice failed to upregulate circulating neutrophils after infection, and demonstrated higher levels of bacterial infiltration in the liver, indicating that impaired neutrophil mobilization contributes to an insufficient immune response. Further analysis of molecular events that transpire upon STAT3-binding to the Cxcl2 and Il8rb promoters indicates that G-CSF induces higher levels of activating trimethylated lysine 4 (H3K4me3) modifications relative to repressive trimethylated lysine 27 (H3K27me3) marks, suggesting G-CSF/STAT3 signals stimulate an open chromatin configuration at these promoters. This is further supported by the observation that pharmacologic inhibition of STAT3 phosphorylation blocked G-CSF-stimulated accumulation of H3K4me3 at target promoters, indicating that STAT3 may contribute to accumulation of H3K4me3 marks. Taken together, our study demonstrates that G-CSF-mediated STAT3 regulation of bone marrow chemokine and chemokine receptor expression may regulate neutrophil mobilization and indicates a potential role for STAT3 in opening of the chromatin structure at target genes in neutrophils.

Keywords

neutrophils, STAT3, mobilization, chromatin, transcription, CXCL2, CXCR2

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