Olive oil bioactives protect pigs against experimentally-induced chronic inflammation independently of alterations in gut microbiota

March Olive oil bioactives protect pigs against experimentally-induced chronic inflammation independently of alterations in gut microbiota Martin Liehr 0 1 Alessandro Mereu 1 Jose Javier Pastor 1 Jose Carlos Quintela 1 Stefanie Staats 0 1 Gerald Rimbach 0 1 Ignacio Rodolfo Ipharraguerre 0 1 0 Institute of Human Nutrition and Food Science, University of Kiel , Kiel, Germany, 2 Lucta S.A., Montorn eÂs del Vall eÂs, Barcelona, Spain, 3 ProNutra Solutions SL, Madrid , Spain 1 Editor: Hauke Smidt, Wageningen University , NETHERLANDS Subclinical chronic inflammation (SCI) is associated with impaired animal growth. Previous work has demonstrated that olive-derived plant bioactives exhibit anti-inflammatory properties that could possibly counteract the growth-depressing effects of SCI. To test this hypothesis and define the underlying mechanism, we conducted a 30-day study in which piglets fed an olive-oil bioactive extract (OBE) and their control counterparts (C+) were injected repeatedly during the last 10 days of the study with increasing doses of Escherichia coli lipopolysaccharides (LPS) to induce SCI. A third group of piglets remained untreated throughout the study and served as a negative control (C-). In C+ pigs, SCI increased the circulating concentration of interleukin 1 beta (p < 0.001) and decreased feed ingestion (p < 0.05) and weight gain (p < 0.05). These responses were not observed in OBE animals. Although intestinal inflammation and colonic microbial ecology was not altered by treatments, OBE enhanced ileal mRNA abundance of tight and adherens junctional proteins (p < 0.05) and plasma recovery of mannitol (p < 0.05) compared with C+ and C-. In line with these findings, OBE improved transepithelial electrical resistance (p < 0.01) in TNF-α-challenged Caco-2/ TC-7 cells, and repressed the production of inflammatory cytokines (p < 0.05) in LPS-stimulated macrophages. In summary, this work demonstrates that OBE attenuates the suppressing effect of SCI on animal growth through a mechanism that appears to involve improvements in intestinal integrity unrelated to alterations in gut microbial ecology and function. - Funding: This work was funded by Kiel University (Kiel, Germany), Lucta S.A. (Barcelona, Spain) and ProNutra Solutions S.L. (Madrid, Spain). Funders provided support in the form of salaries for authors [ML, AM, JJP, JCQ, SS, GR and IRI]. In addition, Lucta S.A. provided partial funding support and research material (olive-oil bioactive extract). Introduction Inflammation is a protective mechanism of higher organisms that aids in coping with stressors and harmful environmental stimuli [ 1 ]. Despite being tremendously complex and involving a variety of immune cells, blood vessels, and molecular mediators, inflammatory processes can be distinguished into two somewhat different types [2; 3] termed herein clinical and subclinical Funding organizations did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the indicated forms. The specific roles of the authors are articulated in the 'author contributions' section. Competing interests: The authors have declared that no competing interests exist. Abbreviations: APP, acute-phase protein; BW, body weight; C-, negative control; C+, positive control; CDH1, E-cadherin; ELISA, Enzyme-linked Immunosorbent Assay; GAPDH, glyceraldehyde-3phosphate dehydrogenase; HPLC, highperformance liquid chromatography; i.m., intramuscular; i.p., intraperitoneal; IL1B, interleukin 1 beta; IL8, interleukin-8; iNOS, nitric oxide synthase 2; LPS, lipopolysaccharide; Mip1a, macrophage inflammatory protein 1 alpha; OBE, olive-oil bioactive extract; OCLN, occludin; qRTPCR, quantitative RT-PCR; TEER, transepithelial electrical resistance; TJ, tight junction; TMB, 3, 3', 5, 5'±tetramethylbenzidin; TNF-α, tumor necrosis factor alpha; ZO-1, zonula occludens 1. chronic inflammation (SCI). In contrast to clinical inflammation resulting for instance from injury or bacterial infection, SCI appears as a much milder but persistent response of the host's immune system [ 4 ]. Initial steps following immune system activation involve the release of pro-inflammatory cytokines to counteract migration and spreading of potential antigens. Tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL1B) belong to the group of pro-inflammatory first order cytokines, released in the early stages of inflammation [ 5 ]. Immediate effects of these cytokines are numerous and include fever as well as recruitment, activation and differentiation of immune cells at the site of ongoing inflammation [6; 7; 8]. Usually the period of cytokine action is tightly regulated through various control mechanisms and thereby strictly limited to the initial phases of the inflammation process [ 9 ]. However, under certain circumstances elevated plasma levels of pro-inflammatory mediators are main (...truncated)