Defining the Contribution of Cholesterol Biosynthesis to the Molecular and Biochemical Phenotype of Macrophages — 6p — Amelia G. Lawver^1,2, Jazmine D. W. Yaeger², Reagan E. Fisher^2,4, Sonali Sengupta², Kevin R. Francis^2,3 

1 Sanford Program for Undergraduate Research (SPUR) Scholar from Bethel University, Arden Hills, MN, USA 

2 Cellular Therapies and Stem Cell Biology Group, Sanford Research, Sioux Falls, SD, USA

3 Department of Pediatrics, Sanford School of Medicine, University of South Dakota, Sioux Falls, SD, USA 

4 South Dakota Biomedical Research Infrastructure Network (BRIN) Scholar from Augustana University, Sioux Falls, SD, USA 

As cholesterol is a vital component of cell membranes, allowing for membrane fluidity and changes in membrane structure, proper cholesterol levels are paramount for the function of immune cells. Immune cells such as macrophages rely on membrane structural change to remove dead material or harmful pathogens through endocytic processes. How cholesterol levels affect immune cell phenotypes and function remains unclear. To better understand the importance of cholesterol homeostasis in immune cell biology, we isolated mouse bone marrow-derived macrophages (BMDMs) and assessed their biochemical profiles using gas chromatography in tandem with mass spectrometry (GC-MS) after exposure to inhibitors of cholesterol biosynthesis. Cholesterol removal alone from the cell media attenuated cellular cholesterol levels. However, cholesterol synthesis inhibitors had little biochemical effect, suggesting BMDMs utilize cholesterol release from esterified cholesterol stores following endogenous uptake. Next, we assessed if blockade of cholesterol biosynthesis impacts cell proliferation or survival. Using Ki67, which is expressed in dividing cells, we found inhibition of Dhcr7, which catalyzes the final step in cholesterol synthesis, with AY9944 significantly inhibiting proliferation. Cholesterol inhibition did not affect cell survival. Next, we analyzed how impaired cholesterol biosynthesis affected surface marker expression, which denotes the macrophage immunotypic profile. Using flow cytometry, we analyzed surface marker antibody expression, including CD11b, CD115, CD64, CD16/32, and CD80. Inhibition of Dhcr7 significantly reduced expression of these markers. Finally, we analyzed BMDMs from a Dhcr7 mouse model of SLOS. Though GC-MS determined that cellular cholesterol is only mildly impacted, Dhcr7 BMDMs exhibit enhanced reactivity (CD68 expression) and elevated lipid droplets relative to controls. Our results suggest loss of cholesterol homeostasis induces an immunological phenotype in BMDMs which may be exacerbated in environmentally isolated immune cells such as microglia. These findings are likely relevant for immunological phenotypes within disorders of cholesterol metabolism and cholesterol biosynthesis affected surface marker expression, which denotes the macrophage immunotypic profile. Using flow cytometry, we analyzed surface marker antibody expression, including CD11b, CD115, CD64, CD16/32, and CD80. Inhibition of Dhcr7 significantly reduced expression of these markers. Finally, we analyzed BMDMs from a Dhcr7 mouse model of SLOS. Though GC-MS determined that cellular cholesterol is only mildly impacted, Dhcr7 BMDMs exhibit enhanced reactivity (CD68 expression) and elevated lipid droplets relative to controls. Our results suggest loss of cholesterol homeostasis induces an immunological phenotype in BMDMs which may be exacerbated in environmentally isolated immune cells such as microglia. These findings are likely relevant for immunological phenotypes within disorders of cholesterol metabolism and cholesterol biosynthesis.

Sanford Research
Kevin Francis