Stress Influences the Vitamin D‐Vitamin D Receptor System and Macrophages in an Animal Model of Endometriosis

Although the role of vitamin D in endometriosis is still unclear, vitamin D signaling through the vitamin D receptor (VDR) is associated with anti‐proliferative and anti‐inflammatory effects. We previously reported differential VDR expression in our endometriosis model when the animals were exposed to stress. Vitamin D can induce an anti‐inflammatory phenotype in macrophages, phagocytic cells that can increase inflammation and stimulate the immune system. Therefore, we hypothesized that stress might affect the vitamin D‐VDR system, thus influencing macrophage behavior and the local inflammatory milieu. AIM Investigate the impact of stress on vitamin D levels, cytokine levels, and VDR expression in vesicles and vesicle macrophages. METHODS Endometriosis was surgically induced in female Sprague‐Dawley rats. Sham controls received sutures only (sham). Endometriosis rats were exposed to either uncontrollable, controllable or no stress for 10 days. At sacrifice (day 60) all rats were examined for vesicles, blood taken, and tissues collected for mRNA extraction and histology. Vitamin D levels were measured by ELISA and cytokine levels were measured by multiplex assay and PCR. VDR expression and macrophage numbers were assessed by immunohistochemistry and immunofluorescence. RESULTS The endometriosis animals exposed to stress had more vesicles which were larger, with a significant increase in vesicle area in those rats exposed to uncontrollable stress (p<0.05; n=12–14). This was attenuated by controllable stress. Serum vitamin D levels were higher in endometriosis animals compared to sham (p<0.01) with no significant effect of stress. Peritoneal fluid vitamin D levels were higher in uncontrollable stress animals compared to all other groups (p=0.06 vs endo no stress). Uncontrollable stress increased macrophage infiltration (p<0.01) and VDR expression in vesicles, which was attenuated by controllable stress. Macrophage infiltration correlated with vesicle area (r=0.67, p<0.05), and peritoneal vitamin D levels correlated with VDR expression in vesicles (r=0.81, p<0.01). VDR expression in the uterine glands of endometriosis animals was significantly increased compared to sham (p<0.05), but down‐regulated by stress at the protein level. No differences were found in uterus macrophage infiltration between groups. VDR expression per macrophage in the vesicles was not significantly different between groups. Serum levels of MCP‐1, IL‐10, IL‐12, IL‐1β, IL‐6 and IFNγ were higher in endometriosis animals, and further increased in those animals receiving controllable stress. In contrast, in uterine tissue we found decreased levels of chemokine ligand 2 (p<0.05), TGFβ and IL‐4 in endometriosis animals with uncontrollable stress compared to sham at the transcriptional level. CONCLUSION Stress exacerbates development of cysts in an animal model of endometriosis through mechanisms that include macrophage recruitment, regulation of the vitamin D‐VDR system, and cytokine changes, suggesting an impact on the local inflammatory environment. Support or Funding Information R15AT006373, R25GM096955 and R25GM082406