Chronic calcitriol supplementation improves the inflammatory profiles of circulating monocytes and the associated intestinal/adipose tissue alteration in a diet-induced steatohepatitis rat model

RESEARCH ARTICLE Chronic calcitriol supplementation improves the inflammatory profiles of circulating monocytes and the associated intestinal/ adipose tissue alteration in a diet-induced steatohepatitis rat model a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Su Y-B, Li T-H, Huang C-C, Tsai H-C, Huang S-F, Hsieh Y-C, et al. (2018) Chronic calcitriol supplementation improves the inflammatory profiles of circulating monocytes and the associated intestinal/adipose tissue alteration in a diet-induced steatohepatitis rat model. PLoS ONE 13(4): e0194867. https://doi.org/10.1371/journal. pone.0194867 Editor: Jordi Gracia-Sancho, IDIBAPS Biomedical Research Institute, SPAIN Received: October 28, 2017 Accepted: March 12, 2018 Published: April 23, 2018 Copyright: © 2018 Su et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files (S3 File). Funding: We especially thank Yun-Ru Wang, FanYi Jhan, Che-Jui Chang and Yen-Ling Lin for their excellent technical supports. This work was entirely supported by research grants NSC-102-2314-B010-036-MY3 and from the National Science Yen-Bo Su1,2, Tzu-Hao Li2,3,4,5, Chia-Chang Huang1,2,4, Hung-Cheng Tsai1,2, ShiangFen Huang2,6, Yun-Cheng Hsieh2,7, Ying-Ying Yang1,2,4,7,8☯*, Yi-Hsiang Huang2,7, MingChih Hou2,7, Han-Chieh Lin2,7☯* 1 Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan, 2 Department of Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan, 3 Division of Allergy and Immunology, Taipei Veterans General Hospital, Taipei, Taiwan, 4 Institute of Clinical Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan, 5 Chia-Yi Branch of Taichung Veterans General Hospital, Chiayi, Taiwan, 6 Division of Infection, Taipei Veterans General Hospital, Taipei, Taiwan, 7 Division of Gastroenterology and Hepatology, Taipei Veterans General Hospital, Taipei, Taiwan, 8 Division of General Medicine, Taipei Veterans General Hospital, Taipei, Taiwan ☯ These authors contributed equally to this work. * (YYY); (HCL) Abstract Vitamin D deficiency and up-regulated TNFα-related signals are reported to be involved in abnormalities including intestinal hyper-permeability, bacterial translocation, systemic/portal endotoxemia, intestinal/adipose tissue/hepatic inflammation, and hepatic steatosis in nonalcoholic steatohepatitis (NASH). This study aims to explore the molecular mechanisms and effects of chronic calcitriol [1,25-(OH)2D3, hormonal form of vitamin D] on gut-adipose tissue-liver axis abnormalities using a high-fat diet (HFD)-fed rat model of NASH. In HFD-fed obese rats on a 10-week calcitriol (0.3 μg/kg/TIW) or vehicle treatment (NASH-vit. D and NASH-V rats) reigme, various in vivo and in vitro experiments were undertaken. Through anti-TNFα-TNFR1-NFκB signaling effects, chronic calcitriol treatment significantly restored plasma calcitriol levels and significantly improved vitamin D receptor (VDR) expression in monocytes and the small intestine of NASH-vit. D rats. Significantly, plasma and portal endotoxin/TNFα levels, bacterial translocation to mesenteric lymph nodes, plasma DX4000-FITC, fecal albumin-assessed intestinal hyper-permeability, over-expression of TNFα-related immune profiles in monocytes, inflammation of intestinal/mesenteric adipose tissue (MAT)/liver and hepatic steatosis were improved by chronic calcitriol treatment of NASH rats. Additionally, in vitro experiments with acute calcitriol co-incubation reversed NASH-V rat monocyte supernatant/TNFα-induced monolayer barrier dysfunction in caco-2 cells, cytokine release from MAT-derived adipocytes, and triglyceride synthesis by lean-V rat hepatocytes. Using in vivo and in vitro experiments, our study reported calcitriol signaling in the gut as well as in adipose tissue. Meanwhile, our study suggests that restoration of PLOS ONE | https://doi.org/10.1371/journal.pone.0194867 April 23, 2018 1 / 17 Chronic calcitriol supplementation improves intestinal/adipose tissue alternation in steatohepatitis rats Council, and V106EA-007, VGHUST105-GI-2-2 and by the Taipei Veterans General Hospital. Competing interests: The authors have declared that no competing interests exist. systemic and intestinal vitamin D deficiency using by chronic vitamin D treatment effectively reduces TNFα-mediated immunological abnormalities associated with the gut-adipose tissue-liver axis and hepatic steatosis in NASH rats. Introduction Higher levels of plasma and intestinal lipopolysaccharide (LPS, also called endotoxin) are noted in nonalcoholic steatohepatitis (NASH) patients than in healthy subjects [1,2]. LPS is the main stimulator to induce tumor necrosis factor-α (TNFα) release from immune cells [1]. Compared to healthy controls, significantly high LPS-stimulated TNFα production is observed in cultured whole blood cells from NASH patients [2]. In NASH, increased TNFα can exacerbate intestinal inflammation and mucosal barrier disruption [3–5]. In the inflamed intestinal epithelium, TNFα produced from infiltrated immune cells further results in systemic/portal inflammation and endotoxemia [3–6]. So, NASH is characterized by remarkable intestinal hyper-permeability, epithelial tight junctions disruption and endotoxemia [5,7]. In obese animals, intestinal dysbiosis is associated with increased macrophage infiltration and high cytokine release by mesenteric adipose tissue (MAT), which is positioned near the intestine and is drained by the portal vein [8]. Additionally, intestinal hyper-permeability and MAT inflammation are involved in the pathogenesis of portal endotoxemia and hepatic steatosis [9,10]. Chronic intestinal/MAT inflammation and a disrupted intestinal barrier result in bacterial translocation and the progression of NAFLD to NASH [9,11,12]. Notably, in NASH, intestinal hyper-permeability, systemic/portal endotoxemia, and systemic/intestinal/MAT inflammation are initiated by TNFα-released from activated immune cells [2,4,5, 7,9,10]. So, anti-TNFα agents have the potential to simultaneously ameliorate the aforementioned abnormalities in NASH [9,10, 13,14]. In cultured human peripheral blood monocytes, 1,25-(OH)2D3, the hormonal form of vitamin D, is able to dose-dependently inhibit LPS-stimulated TNFα production [15]. Serum TNFα levels are negatively correlated with serum vitamin D concentrations in healthy women [16]. Among NASH patients, low serum vitamin D concentrations are closely associated with severe hepatic steatosis and inflammation [17]. Vitamin D receptor (VDR) is a nuclear receptor that mediates most of the known functions of vitamin D, including its anti-TNFα effects. Chronic mucosal inflammation and TNFα-indu (...truncated)