Vitamin D Promotes Anti-inflammatory Phenotype as Opposed to Proinflammatory State of Macrophages

Vitamin D (Vit‐D) deficiency is linked to several metabolic diseases such as atherosclerosis and obesity. However, the underlying mechanism remains elusive. Immediate early response gene x‐1 (IEX‐1)—which is negatively regulated by Vit‐D—is recently shown to play a crucial role in the pathogenesis of obesity and atherosclerosis. Its paucity prevents anti‐to‐pro‐inflammatory shift in macrophage phenotype and thereby protecting against these diseases. We hypothesize that Vit‐D protects against the metabolic disease partly by inhibiting pro‐inflammatory macrophages via repressing IEX‐1. We cultured RAW 264.7 cells, a murine macrophage cell line in DMEM, and pretreated them with different doses of Vit‐D (0.1, 1, and 10 nM) for 8 hours. The cells were then differentiated into pro‐ or anti‐inflammatory macrophages by treatment with LPS (10 ng/ml) and IL‐4 (10 nM), respectively, for 24 hours in the absence/presence of Vit‐D. Total cellular RNA was extracted using Trizole and cDNA was synthesized. The mRNA levels of IEX‐1, pro‐, and anti‐inflammatory genes were determined using quantitative RT‐PCR. The protein levels of TNF‐α and IL‐10 were measured in culture supernatant using ELISA. LPS treatment increased mRNA levels of proinflammatory genes IL‐6 (60±6‐fold, p<0.001), TNF‐α (8±0.7‐fold, p<0.01), and iNOS (10±0.7‐fold, p<0.01) in RAW cells. Pretreatment with 10 nM Vit‐D but not 0.1 or 1 nM significantly inhibited LPS‐induced increases in expression levels of IL‐6 (24±2‐fold, p<0.01), TNF‐α (4±0.3‐fold, p<0.05), and iNOS (4.7±0.3‐fold, p<0.05 versus LPS alone). Vit‐D also suppressed LPS‐induced TNF‐α protein production by macrophages (18±0.8 versus 27±1.4 ng/ml, p<0.05). On the other hand, IL‐4 treatment induced expression levels of anti‐inflammatory genes Arg‐1 (93±18‐fold, p<0.01), MRC‐1 (2±0.1‐fold, p<0.05), and Clec10a (2.2±0.2‐fold, p<0.001), suggesting the formation of alternatively activated macrophages. Interestingly, pretreatment with Vit‐D dosedependently potentiated IL‐4‐induced increases in Arg‐1, MRC‐1 , and Clec10a mRNA levels. Specifically, Vit‐D at 10 nM markedly increased expression levels of Arg‐1 (287±51‐fold, p<0.01), MRC‐1 (6±0.8‐fold, p<0.05), and Clec10a (6±0.8‐fold, p<0.001 versus IL‐4 alone) induced by IL‐4. Vit‐D also increased IL‐4‐induced IL‐10 protein production (510±41 versus 748±36 pg/ml, p<0.05). Concurrently, Vit‐D treatment dose‐dependently repressed IEX‐1 expression in RAW cells, confirming negative regulation of IEX‐1 by Vit‐D. These results suggest that Vit‐D promotes the anti‐inflammatory state of macrophages likely in part, by repressing IEX‐1 expression. Support or Funding Information None This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .