Development of an Advanced Multicellular Intestinal Model for Assessing Immunomodulatory Properties of Anti-Inflammatory Compounds

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.

Keywords: anatabine; immune competent cells; in vitro co-culture; inflamed intestine; inflammatory bowel disease; intestinal inflammation; nicotine; tobacco alkaloids.

FIGURE 1

Single and repeated Caco-2/HT29-MTX pro-inflammatory induction after days 14 and 21. TEER (A,B), permeability (C), and IL-8 release (D,F) in a differentiated Caco-2/HT29-MTX coculture basolaterally treated with different concentrations of TNFα (0–100 ng/ml) on day 21 (single) or days 14 and 21 (repeated). Each inflammatory stimulus (red dashed line) was left for up to 24 h; this was followed by a recovery period of up to 120 h without an inflammatory inducer. TEER values are represented in percentage; zero-hour treatment was used as reference (100%). Permeability is expressed as fold change over the untreated control (CTL). Data are presented as the average of three technical replicates with the corresponding standard deviations (n = 1; m > 3). On the day at the end of the simulation and the last day recorded, statistical differences between the control without TNFα and each of the three concentrations of TNFα were determined by a two-tailed t-test, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns, p > 0.05. Equivalence comparisons and linear regression analysis are provided in Supplementary Tables S1 and S2. Abbreviations: TEER, transepithelial electrical resistance; TNF, tumor necrosis factor; CTL, control.

FIGURE 2

Effects of different PMA concentrations on THP-1 post-differentiation cytokine release. (A) IL-8 and (B) TNFα release by THP-1 cells differentiated for 65 h with increasing concentrations of PMA (10, 20, and 40 ng/ml) and rested for 24 or 48 h after PMA removal. (C) IL-8 and (D) TNFα release by THP-1 cells differentiated with increasing concentrations of PMA (10, 20, and 40 ng/ml) for 65 h, rested for 24 or 48 h after PMA removal, and treated with 10 ng/ml LPS for 24 h. Data are presented as the average of three technical replicates with corresponding standard deviation. Statistical differences were calculated between the 65-h induced sample and the rested samples (A,B) or between LPS-treated and untreated cultures (C,D) by means of two-tailed t-tests. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns, p > 0.05 (A,B). Equivalence comparisons and linear regression analysis are provided in Supplementary Tables S3 and S4. R24h = cells rested for 24 h; R48h = cells rested for 48 h. Abbreviations: PMA, phorbol 12-myristate.

FIGURE 3

Effect of pro-inflammatory stimulation on optimized triculture. Pro-inflammatory effect of 10 ng/ml LPS added basolaterally to a triculture containing (A–D) 12 × 10⁴ and (E–H) 24 × 10⁴ THP-1 cells in 400 µl of cell culture medium in the basolateral compartment. (A,E) TEER (B,F) membrane permeability, and (C,D,G,H) cytokine release (IL-8 and TNFα, respectively) of the triculture. For each THP-1 seeding condition, different PMA concentrations (10 and 20 ng/ml) were tested for optimizing the triculture response. TEER values are represented in percentage; zero-hour treatment was used as the reference (100%). Permeability is expressed as fold change over the untreated control without LPS. Data are presented as the average of three technical replicates with the corresponding standard deviations. Statistical differences between the LPS- and PMA-treated cultures and the control culture without the compounds (with 12 × 10⁴ THP-1 cells) were determined by a two-tailed t-test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns, p > 0.05. Abbreviations: PMA, phorbol 12-myristate; TNF, tumor necrosis factor; IL, interleukin; CTL, control.

FIGURE 4

Schematic representation of the triculture assembly protocol.

FIGURE 5

Reference item assessment and verification of the anti-inflammatory protocol. Tricultures were treated basolaterally with increasing concentrations of (A,C) TPCA-1 and (B,D) budesonide for 6 h and subsequently stimulated with 10 ng/ml LPS for 18 h. TEER, epithelial layer permeability, cell viability of differentiated Caco-2/HT29-MTX cells (A,B) and cytokine release and cell viability of differentiated THP-1 cells (C,D) were measured. TEER values are normalized to control (CTL; 100%) and LPS treatment (0%). Permeability is normalized by using LPS treatment as reference (100%). Basolateral IL-8 and TNFα release are expressed as percentage of control (cells treated with LPS only; CTL; 100%). Cell viability values of Caco-2-HT29-MTX cocultures and THP-1 cells are expressed as percentage of control (cells treated with LPS only, CTL; 100%). n = 3 independent experiments. Statistical differences between reference compound-treated vs. LPS-treated-only cultures were determined by a two-tailed t-test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns, p > 0.05. Abbreviations: LPS, lipopolysaccharide; TEER, transepithelial electrical resistance; TNF, tumor necrosis factor; IL, interleukin.

FIGURE 6

Test item assessment. Tricultures were treated basolaterally with increasing concentrations of (A,D) nicotine (B,E) (R/S)-anatabine, or (C,F) (S)-anatabine for 6 h and subsequently stimulated with 10 ng/ml LPS for 18 h. TEER, epithelial layer permeability, cell viability of differentiated Caco-2/HT29-MTX cells (A–C) and cytokine release and cell viability of differentiated THP-1 cells (D–F) were measured. TEER values are normalized to control (CTL; 100%) and LPS treatment (0%). Permeability is normalized by using LPS treatment as the reference (100%). Basolateral IL-8 and TNFα release are expressed as percentage of control (cells treated with LPS only; CTL; 100%). Cell viability values of Caco-2–HT29-MTX cocultures and THP-1 cells are expressed as percentage of control (cells treated with LPS only; CTL; 100%). n = 3 independent experiments. Statistical differences between the reference compound-treated vs. LPS-treated-only cultures were determined by a two-tailed t-test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns, p > 0.05. Abbreviations: LPS, lipopolysaccharide; TEER, transepithelial electrical resistance; TNF, tumor necrosis factor; IL, interleukin; CTL, control.