The probiotic mixture VSL#3 attenuates colitis in patients with Inflammatory Bowel Disease (IBD) and in animal models of this condition, but the mechanisms involved are incompletely understood. VSL#3 alters macrophage morphology and secretory profile in vitro in a polarization-dependent manner. We examined the effect of VSL#3 on macrophages in acute trinitrobenzene sulfonic acid-induced colitis. Rats were randomized to normal, colitis, or colitis+VSL#3 groups. After sacrifice, the colons were evaluated for macroscopic and microscopic damage. Serum cytokine levels were measured, and microbiome analysis undertaken. Total and M1 colonic macrophages, and total and proliferating hepatic macrophages were assessed by double immunofluorescence staining. Colitis+VSL#3 rats had lower macroscopic damage, with less microscopic damage in the proximal colon, compared with colitis alone. Colitis significantly increased colonic macrophage infiltration, which was significantly reduced by VSL#3 treatment. VSL#3 did not decrease the colitis-induced surge of colonic M1 macrophages or hepatic macrophages. VSL#3 reduced colitis-induced serum cytokine levels, and induced restoration of colonic transcript levels for pro-inflammatory, anti-inflammatory, and barrier proteins to, or past, normal levels. Fecal bacteria distribution changed between groups. In summary, the probiotic VSL#3 reduces colitis severity, colonic macrophage infiltration, and serum cytokine levels, but does not dampen the pro-inflammatory phenotype of M1 macrophages.

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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.AIMInvestigate the impact of stress on vitamin D levels, cytokine levels, and VDR expression in vesicles and vesicle macrophages.METHODSEndometriosis 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.RESULTSThe 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.CONCLUSIONStress 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 InformationR15AT006373, R25GM096955 and R25GM082406