Antiviral Effect of Melatonin on Caco-2 Cell Organoid Culture: Trick or Treat?

Abstract

Melatonin is a hormone naturally produced by the body that has recently been found to have antiviral properties. However, its antiviral mechanisms are not entirely understood. Using Caco-2 cells, we developed a gastrointestinal organoid model to investigate the impact of melatonin on cellular organoid culture response to Poly I:C-induced viral inflammation in the gastrointestinal tract. Melatonin was found to have different effect when applied as a pretreatment before the induction of viral inflammation or as a treatment after it. Melatonin pretreatment after Poly I:C stimulation did not protect organoids from size reduction but enhanced cell proliferation, especially when lower (1 and 10 µM) melatonin concentrations were used. On the other hand, treatment with melatonin after the induction of viral inflammation helped to maintain the size of the organoids while reducing cell proliferation. In pretreated cells, reduced IFNLR1 expression was found, while melatonin treatment increased IFNLR1 expression and reduced the production of viral cytokines, such as IFNλ1 and STAT1-3, but did not prevent from apoptosis. The findings of this study emphasize the importance of type III IFNs in antiviral defense in epithelial gastrointestinal cells and shed more light on the antiviral properties of melatonin as a potential therapeutic substance.

Keywords: Caco-2 cell; IFNLR1; IFNλ1; antiviral effect; gastrointestinal organoids; melatonin.

Figure 1

Brightfield microscopy of Caco-2 cell culture (A) Day 0, (B) Day 7, (C) Day 14, (D) Day 21 showing the formation of gastrointestinal organoids (indicated by arrows). Brightfield and immunohistochemistry images of Caco-2 showing different markers: (E) vimentin, (F) Zo-1 (zonula occludens-1), and (G) EpCAM (epithelial cell adhesion molecule).

Figure 2

(A) Caco-2 cell viability after stimulation with different concentrations of Poly I:C for 24 h (n = 5). (B) Caco-2 cell viability after stimulation with different concentrations of melatonin for 24 h (n = 3). (C) Organoid size after stimulation with different concentrations of Poly I:C or melatonin for 24 h (means with 95% confidence interval (CI)). (D) Number of organoids after stimulation with different concentrations of Poly I:C or melatonin for 24 h compared to control (means with min and max values). (E) Brightfield images of Caco-2 cells monolayer culture after stimulation with different concentrations of Poly I:C or melatonin for 24 h alone in comparison to stimulation with 1 and 100 μM of melatonin before (pretreatment) and after (treatment) application of 1, 10 and 50 µg/mL Poly I:C; reducing size and number of organoids when stimulated with higher Poly I:C concentrations are seen, while higher number of organoids when pretreated with melatonin and stimulated with high Poly I:C concentrations in comparison to control cells are observed. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and ns indicates p > 0.05. Abbreviations: µg/mL—micrograms per milliliter, µm—micrometer, µM—micromolar, Poly I:C — polyinosinic polycytidylic acid.

Figure 2

(A) Caco-2 cell viability after stimulation with different concentrations of Poly I:C for 24 h (n = 5). (B) Caco-2 cell viability after stimulation with different concentrations of melatonin for 24 h (n = 3). (C) Organoid size after stimulation with different concentrations of Poly I:C or melatonin for 24 h (means with 95% confidence interval (CI)). (D) Number of organoids after stimulation with different concentrations of Poly I:C or melatonin for 24 h compared to control (means with min and max values). (E) Brightfield images of Caco-2 cells monolayer culture after stimulation with different concentrations of Poly I:C or melatonin for 24 h alone in comparison to stimulation with 1 and 100 μM of melatonin before (pretreatment) and after (treatment) application of 1, 10 and 50 µg/mL Poly I:C; reducing size and number of organoids when stimulated with higher Poly I:C concentrations are seen, while higher number of organoids when pretreated with melatonin and stimulated with high Poly I:C concentrations in comparison to control cells are observed. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and ns indicates p > 0.05. Abbreviations: µg/mL—micrograms per milliliter, µm—micrometer, µM—micromolar, Poly I:C — polyinosinic polycytidylic acid.

Figure 3

The levels of interferons produced by Poly I:C-stimulated Caco-2 cells detected by ELISA. (A) The levels of IFNλ1 after stimulation of Caco-2 cells with different concentrations of Poly I:C after 24 h (n = 3). (B) The levels of IFNλ1 at different timepoints after stimulation with Poly I:C (n = 3). (C) The levels of IFNβ after stimulation of Caco-2 cells with different concentrations of Poly I:C after 24 h (n = 3). (D) The levels of IFNα after stimulation of Caco-2 cells with different concentrations of Poly I:C after 24 h (n = 3). *** indicates p < 0.001, ns indicates p > 0.05. Abbreviations: µg/mL—micrograms per milliliter, ng/mL—nanograms per milliliter, h—hour, IFN—interferon.

Figure 4

Comparison of the effect of 1, 10, 50 and 100 μM of melatonin before (pretreatment) and after (treatment) stimulation with different concentrations of Poly I:C compared to control cells: (A) cell viability (n = 3), (B) the number of organoids, (C) organoid size, (D) apoptosis (n = 3). (E) Scatter plots showing the comparison of the effect of 1 and 100 µM of melatonin on apoptosis in Poly I:C-stimulated cells in contrast to the controls. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Abbreviations: µg/mL—micrograms per milliliter, µm—micrometer, µM—micromolar.

Figure 5

The effect of melatonin on the expression of IFNλ1 and IFNLR1. (A) IFNλ1 expression by ELISA in Caco-2 cells treated with different concentrations of melatonin before and after Poly I:C stimulation in comparison to control non-infected cells treated with melatonin (only statistically significant differences in melatonin-pretreated or -treated cells in comparison to melatonin-naïve Poly I:C-stimulated cells are shown) (n = 3). (B) IFNLR1 expression by flow cytometry in Caco-2 cells treated with different concentrations of melatonin before and after Poly I:C stimulation in comparison to control non-infected cells treated with melatonin as well as those stimulated by Poly I:C only (n = 3). (C) Histograms comparing the effect of 1 and 100 µM of melatonin on the expression of IFNLR1 in Poly I:C-stimulated cells in contrast to the controls (red histogram shows unstained control, blue—IFNLR1 expression). * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001, ns indicates p > 0.05. Abbreviations: µg/mL—micrograms per milliliter, ng/mL—nanograms per milliliter, µM—micromolar, IFNLR1—IFNλ receptor 1.

Figure 6

Expression of STAT1-3 proteins by flow cytometry in Caco-2 cells. (A) STAT1 expression in control and melatonin-pretreated Caco-2 cells before Poly I:C stimulation in comparison to treated cells (n = 3). (B) STAT2 expression in control and melatonin-pretreated Caco-2 cells before Poly I:C stimulation in comparison to treated cells (n = 3). (C) STAT3 expression in control and melatonin-pretreated Caco-2 cells before Poly I:C stimulation in comparison to treated cells (n = 3). * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001, ns indicates p > 0.05. Abbreviations: µg/mL—micrograms per milliliter, µM—micromolar, STAT — signal transducer and activator of transcription.

Figure 7

A summary of the research findings. Melatonin, when applied after Poly I:C stimulation, reduces the expression of IFNλ1 and slightly increases the production of IFNLR1 receptor. Furthermore, it reduces the expression of STAT1-3 proteins. In contrast, when used as a pretreatment, it does not have a significant effect on IFNλ1 but reduces IFNLR1 expression, though it does not influence the levels of STAT1-3.

Figure 8

Outline of the research study.