IEX‐1 is Required for Vitamin D‐induced Inhibition of Proinflammatory State of Macrophages

Abstract

Vitamin D (Vit‐D) deficiency is linked to several metabolic diseases. However, the underlying mechanism remains elusive. Immediate early response gene x‐1 (IEX‐1)—which is negatively regulated by Vit‐D—plays a pathogenic role in obesity and atherosclerosis. IEX‐1 paucity inhibits macrophage‐mediated inflammation and protects against these diseases. We hypothesize that Vit‐D promotes anti‐inflammatory state of macrophages via inhibiting IEX‐1 expression.We harvested bone marrow cells from wild‐type (WT) and IEX‐1 knockout (KO) mice and cultured them with macrophage‐colony stimulating factor (M‐CSF) to mature into macrophages. The cells were differentiated into pro‐ or anti‐inflammatory macrophages by treatment with LPS (10 ng/ml) or IL‐4 (10 nM), for 24 hours in the absence/presence of Vit‐D (10 nM). Total RNA was extracted and cDNA was synthesized. The mRNA levels of IEX‐1, pro‐, and anti‐inflammatory marker genes were quantified by qRT‐PCR. The TNF‐α protein level was measured in supernatant using ELISA. Further, impact of IEX‐1 deficiency on LPS‐induced pro‐inflammatory macrophage formation and apoptosis was determined using flow cytometry. Vit‐D inhibited LPS‐induced increases in mRNA levels of TNF‐α (42±5 vs. 58±5‐fold, p<0.05) and iNOS (327±29 vs. 523±102‐fold, p<0.05) in WT cells. Vit‐D also suppressed LPS‐induced TNF‐α protein production by macrophages (18±1 vs. 27±1 ng/ml, p<0.05). Conversely, Vit‐D potentiated IL‐4‐induced increases in expression of anti‐inflammatory genes Arg‐1 (15204±2780 vs. 4362±1624‐fold, p<0.01), MRC‐1 (13±3 vs. 6±1‐fold, p<0.05), and Clec10a (8±1 vs. 4±1‐fold, p<0.05). However, these effects of Vit‐D were markedly attenuated in IEX‐1 KO cells. In WT cells, Vit‐D treatment dose‐dependently decreased IEX‐1 expression, showing negative regulation of IEX‐1 by Vit‐D. Flow cytometry analysis demonstrated that IEX‐1 deficiency alone was sufficient to inhibit LPS‐induced pro‐inflammatory macrophage formation (11±1 vs. 26±3%, p<0.05). This effect was concomitant with an increase in apoptosis (10±1 vs. 18±2‐fold, p<0.05) during LPS treatment.These results suggest that Vit‐D favors the anti‐inflammatory state of macrophages in part via repressing IEX‐1 expression.Support or Funding InformationCTRE Faculty Development Seed Grant, Chicago State University