Neutrophils and macrophages are critical mediators of innate immunity and share protein expression patterns important to their functions as phagocytes. They also share changes in their nuclear features during early development as their nuclei become indented or kidney-shaped. Neutrophil nuclei develop further as they form multiple lobules in mature cells. The physiological role of these unique nuclear features is unclear, but deficient nuclear lobulation in circulating neutrophils, termed Pelger-Hut anomaly (PHA), can be associated with myelodysplasias that often progress to myelogenous leukemias, and certain drugs can cause pseudo-PHA in patients undergoing chemotherapy. An understanding of how nuclear structural changes occur in myeloid cells is also unclear, but neutrophil nuclear lobulation requires expression of the inner nuclear envelope (NE) protein called the lamin B receptor (LBR), loss of which causes PHA. Additional nuclear components also have been implicated in supporting myeloid cell nuclear structural features, including a component of the nuclear lamina, lamin A. The long-term objectives of this proposal are to elucidate the roles that NE proteins perform in mediating both nuclear structural features and functional responses of mature neutrophils and macrophages, focusing on the two A-type lamins, lamin A and C, plus an inner nuclear envelope protein called Sun2. To achieve these objectives, the specific aims are: i) to explore the molecular mechanisms that result in aberrant neutrophil cell functions previously shown to be caused by changes in the expression of either A-type lamins, and identify if similar changes occur in cells with aberrant Sun2 expression, ii) to identify critical and previously unknown roles that these proteins perform in supporting nuclear maturation and functional responses of macrophages, and iii) to identify which transcription factors previously shown to regulate Lbr expression also control Lmna or Sun2 gene expression in each myeloid lineage. To achieve these aims, myeloid cell lines with ectopic or deficient expression of either lamin A, lamin C or Sun2 will be analyzed for molecular pathways that are disrupted and lead to identified aberrant functional responses in mature cells. Immunofluorescence laser confocal microscopy will be used to identify changes to the localization of both nuclear and cytoplasmic proteins that interact with either lamin or Sun2 proteins. Molecular signaling pathways critical to functional responses will be assessed, as will changes to the gene expression patterns using Taqman gene array studies. Altered macrophage functions in genetically manipulated cells will be characterized further, and changes to gene or protein expression patterns identified. A combination of promoter reporter, electrophoretic mobility shift and chromatin immunoprecipitation assays will be used to identify transcriptional regulation of Lmna and Sun2 genes. These studies will provide a deeper understanding of the importance of nuclear maturation to both neutrophil and macrophage functions in innate immunity, and provide better insight as to why aberrant nuclear structure is associated with hematologic diseases.
The objective of this proposal is elucidate the roles that three nuclear proteins, two that comprise the A-type lamins, lamin A and C, plus an inner nuclear envelope protein that associates with these lamins, Sun2, perform in mediating nuclear maturation and functional responses of two white blood cell types essential to innate immunity, neutrophils and macrophages. These studies are relevant to public health because they will provide a better understanding of the molecular mechanisms governing the functions of professional phagocytes in innate immune responses, and better explain why aberrant nuclear structure in mature myeloid cells can be associated with life-threatening hematologic disorders, including myelodysplasias and chronic inflammatory disease.
