Development and validation of a method to deliver vitamin A to macrophages

Macrophages are critical players in the development of atherosclerotic lesions, where they promote local and systemic inflammation. Macrophages engulf lipoproteins and cell debris upon entry into the arterial wall, becoming lipid-laden foam cells. While most lipids found in foam cells are triglyceride and cholesterol, these cells accumulate several other lipids with bioactive properties, such as vitamin A and carotenoids. Vitamin A has strong immunomodulatory actions in macrophages and other immune cells. For example, macrophages release vitamin A as retinoic acid to modulate T cell differentiation, but the implication of intracellular vitamin A stores in this process remains elusive due to the lack of an adequate experimental model to load vitamin A into macrophages. The purpose of this study was to develop a reliable method to deliver vitamin A to cultured murine macrophages. Our results show that thioglycolate-elicited peritoneal macrophages fail to take up significant levels of vitamin A when provided as free retinol. Cultured macrophages and macrophages in the peritoneal cavity can take up retinyl esters, either as retinyl ester-loaded serum or retinyl esters infused directly into the peritoneal cavity. HPLC analyses in macrophage lysates revealed that the intraperitoneal injection method results in a fourfold greater vitamin A loading efficiency than retinyl ester-loaded serum added to cultured cells. These two alternative methods provide an efficient and reliable methodology to load macrophages with vitamin A for downstream applications such as studies of gene regulation trafficking of intracellular vitamin A, and vitamin A release from macrophages.