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

Abstract

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.

Keywords: Atherosclerosis; Cardiovascular disease; Chylomicrons; Retinoic acid; Retinoids.

Fig. 1

The enzyme β-carotene oxygenase 1 catalyzes the conversion of β-carotene to retinal, the first vitamin A intermediate. This scheme highlights the metabolic pathway of vitamin A formation and the main vitamin A metabolites.

Fig. 2

(A) Experimental design—Pilot Study. Eight-week-old C57/B6 mice were fed a purified, carotenoid-free diet for 4 days before receiving a single gavage containing 500mg of retinyl palmitate/kg of body weight dissolved in olive oil. Mice were sacrificed at the indicated time points to isolate serum from the blood. (B) HPLC quantifications. (C) Representative chromatograms of mouse serum collected at the second hour after gavaging vitamin A (continuous black line) and a mixture of retinyl palmitate and retinol standards (dotted blue line). The insert show the spectrum for retinyl palmitate.

Fig. 3

(A) Experimental design—Pilot Study. Two twenty-week-old pigs fasted for 8h and then fed a single dose of retinyl acetate, at a ratio of 500mg/kg body weight, mixed with feed. After this period, blood was drawn at the time point zero (baseline), 5, 6, 7, 8, 9, 10, 12, and 30h after vitamin A supplementation (see Section 2.3.3) for serum isolation. (B) Retinyl ester levels and free retinol in sera of two pigs determined by HPLC described in Section 2.3.2 (Pilot Study I and Pilot Study II). (C) Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in pooled serum.

Fig. 4

Results of the Final Study in pigs. (A) Vitamin A measurements in serum of control pigs fed a control standard diet and serum from retinyl ester-fed pigs at 7h after the feeding. (B) Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in sera of Control and vitamin A-fed pigs showed no significant differences (t-test analysis) in transaminase levels. (C) Retinyl ester (left) and free retinol (right) in isolated chylomicrons of mice fed vitamin A. Data are represented relative to the vitamin A content in total serum. (D) Immunoblot of total serum (TS) and isolated chylomicrons (IC) from the two pig samples fed retinyl ester. A mouse sample was used to identify Apo B100 and Apo B48 (top panel). Albumin (medium panel) and RBP4 (lower panel) blots were used to highlight the purity of the IC fractions. ApoB and RBP4 proteins were detected in samples by immunoblotting the membranes with with the corresponding antibodies (reagent 37 and 38, respectively).

Fig. 5

(A) Endotoxin levels measured in various sera samples. Fetal bovine serum (FBS) treated with activated charcoal to eliminate endotoxins was utilized as a control (red). (B) Ccl2 mRNA expression in mouse and pig sera levels relative to FBS treated with activated charcoal. Cells exposed to pure endotoxin were utilized as positive control. Hprt was utilized as housekeeping control. Experiments were carried out in triplicates. **** P < 0.0001 using One-way ANOVA with Dunnett’s multiple comparison testing.

Fig. 6

Comparison of different methodologies to deliver vitamin A to macrophages. The final concentration of retinoids in the medium was 4μM during the 24h before harvesting. (A) Intracellular retinyl ester and (B) and intracellular retinol levels (ROL) were measured by HPLC(as described in Section 2.3.2). Retinoid levels were standardized to total protein content in pellets. (C) Accumulation of the retinoic-acid sensitive Cyp26a1 and Rarß mRNAs relative to Hprt transcripts in peritoneal macrophages (as described in Section 2.6). For all experiments, retinoic acid in DMSO was utilized as a positive control and DMSO only as a negative control. Data represent n = 2 experiments in triplicate. Statistical differences were evaluated using one-way ANOVA with Dunnett’s multiple comparison testing (****, P < 0.001).

Fig. 7

Schematic representation of the two experimental approaches to load macrophages with vitamin A.