Abstract
Intestinal inflammation is the collective term for immune system-mediated diseases of unknown, multifactorial etiology, with often complex interactions between genetic and environmental factors. To mechanistically investigate the effect of treatment with compounds possessing immunomodulating properties in the context of intestinal inflammation, we developed an immunocompetent in vitro triculture intestinal model consisting of a differentiated intestinal epithelial layer (Caco-2/HT29-MTX) and immunocompetent cells (differentiated THP-1). The triculture mimicked a healthy intestine with stable barrier integrity. Lipopolysaccharide treatment triggered a controlled and reversible inflammatory state, resulting in significant impairment of barrier integrity and release of pro-inflammatory cytokines and chemokines, which are known hallmarks of intestinal inflammation. Treatment with known anti-inflammatory reference compounds (TPCA-1 and budenoside) prevented the induction of an inflammatory state; the decreasing triculture responses to this treatment measured by cytokine release, transepithelial electric resistance (TEER), and epithelial layer permeability proved the suitability of the intestinal model for anti-inflammatory drug screening. Finally, selected tobacco alkaloids (nicotine and anatabine (R/S and S forms)) were tested in the in vitro triculture for their potential anti-inflammatory properties. Indeed, naturally occurring alkaloids, such as tobacco-derived alkaloids, have shown substantial anti-inflammatory effects in several in vitro and in vivo models of inflammation, gaining increasing interest. Similar to the anti-inflammatory reference compounds, one of the tobacco alkaloids under investigation partially prevented the decrease in the TEER and increase in permeability and reduced the release of pro-inflammatory cytokines and chemokines. Taken together, these data confirm that our in vitro model is suitable for screening potential anti-inflammatory compounds in the context of intestinal inflammation.
Highlights
The gastrointestinal tract is composed of an epithelial layer, which is exposed to an overwhelming load of foreign antigens arising from commensal microorganisms, dietary products, and occasional pathogens (Tilg and Moschen, 2015)
As a proof of concept, after establishing and assessing the model with known antiinflammatory drugs, we evaluated the putative antiinflammatory effects of two well-known tobacco alkaloids, namely nicotine and anatabine
transepithelial electric resistance (TEER) values reached a maximum of ∼900 Ω*cm2 12 days after the cells reached full confluence, after which they remained stable in the range of 700–900 Ω*cm2 up to day 26 (Supplementary Figure S1A)
Summary
The gastrointestinal tract is composed of an epithelial layer, which is exposed to an overwhelming load of foreign antigens arising from commensal microorganisms, dietary products, and occasional pathogens (Tilg and Moschen, 2015). The gastrointestinal mucosa forms a unique immunological environment in which tolerance to commensal microbes and/or food antigens and specific immunity to pathogens coexist in a fine-tuned immune equilibrium (Schenk and Mueller, 2008; Honda and Littman, 2012; Tilg and Moschen, 2015). When this tightly regulated immune homeostasis becomes altered, pathological inflammation and disruption of the epithelial barrier might occur (Peterson et al, 2015). No unique cause has been determined for inflamed intestine, environmental factors, including medication, stress, diet, and smoking, are known to impact disease onset and progression (Abraham et al, 2017)
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