Lycopene modulates THP1 and Caco2 cells inflammatory state through transcriptional and nontranscriptional processes

Abstract

We revisited the action of a carotenoid, the lycopene, on the expression of proinflammatory genes, reactive oxygen species (ROS) production, and metalloprotease (MMP9) activity. THP1 and Caco2 cell lines were used as in vitro models for the two main cell types found in intestine tissue, that is, monocytes and epithelial cells. Proinflammatory condition was induced using either phorbol ester acetate (PMA), lipopolysaccharide (LPS) or tumor necrosis factor (TNF). In THP1 cells, short term pretreatment (2 h) with a low concentration (2 μM) of lycopene reinforce proinflammatory gene expression. The extent of the effect of lycopene is dependent on the proinflammtory stimulus (PMA, LPS or TNF) used. Lycopene enhanced MMP9 secretion via a c-AMP-dependent process, and reduced ROS production at higher concentrations than 2 μM. Cell culture media, conditioned by PMA-treated monocytes and then transferred on CaCo-2 epithelial cells, induced a proinflammatory state in these cells. The extent of this inflammatory effect was reduced when cells has been pretreated (12 h) with lycopene. At low concentration (2 μM or less), lycopene appeared to promote an inflammatory state not correlated with ROS modulation. At higher concentration (5 μM-20 μM), an anti-inflammatory effect takes place as a decrease of ROS production was detected. So, both concentration and time have to be considered in order to define the exact issue of the effect of carotenoids present in meals.

Figure 1

Lycopene modulates basal and LPS-induced inflammatory gene expressions in THP1 cells. ICAM-1 (a), IL-1β (b), IL-8 (c), and MMP9 (d) gene expression was studied in THP1 cells treated with LPS. Modulation of this expression was studied in THP1 cell cultures pretreated for 2 h with or without lycopene (2 μM). Then, according to assays, LPS (100 ng/mL) was added for 6 h and mRNA expression was analyzed by RT-qPCR. Values are means ± SD (n = 8). Assays treated with lycopene were compared to corresponding assays without lycopene. ***P < 0.01; **P < 0.05; ∗, nonsignificantly different.

Figure 2

Lycopene modulates gene expression induced by PMA and TNF in THP1 cells. ICAM-1 ((a), (e)), IL-1β ((b), (f)), IL-8 ((c), (g)), and MMP9 (d) gene expression was studied in THP1 cells treated with either PMA (a,b,c, and d) or TNFα ((e), (f), and (g)). Modulation of this expression was studied in THP1 cell cultures pretreated for 2 h with or without Lycopene (2 μM). Then, according to assays, PMA (100 nM) or TNFα (10 ng/mL) was added for 6 h and mRNA expression was analyzed by RT-qPCR. Values are means ± SD (n = 8). Assays treated with lycopene were compared to corresponding assays without lycopene. ***P < 0.01; **P < 0.05; ∗, nonsignificantly different.

Figure 3

Modulation by lycopene of reporter gene activity in THP1 cells. THP1 cells were transfected using one of the luciferase reporter genes controlled by the promoter/enhancer sequence derived from either the IgK gene (pGL2-IgK-luc) (a) or the IL-8 gene (pGL2-IL-8-luc) (b). The plasmid pGL2-AP1-luc (c) contains the AP1 specific sequence. After 12 h transfection, cells were washed and fresh medium with or without lycopene at the indicated concentration was added, and culture continued for 2 h. Then, according to assays, TNFα ((a), (b)) or PMA (c) was added and culture was resumed for 6 h. Cells were harvested, lysed, and placed at −80°C. Luciferase activity was recorded. Values are means ± SD (n = 8). Assays treated with lycopene were compared to corresponding assays without lycopene. ***P < 0.01; **P < 0.05; ∗, nonsignificantly different.

Figure 4

Lycopene modulates PMA-induced ROS production in THP1 cells. THP1 cell suspension was distributed into multiwell plates and, according to the assays, different concentrations of lycopene were added for 2 hours. Then, PMA (100 nM) together with lucigenin was added, and ROS-induced luminescence was immediately recorded for 60 min (a). Data were collected on computer and kinetics integration was performed (b). A representative experiment is shown. Values are means ± SD (n = 8).

Figure 5

Lycopene-induced modulation of IL-8 and IL-1 gene expression in Caco2 cells. (a) Lycopene induced upregulation of basal and LPS-induced IL-8 gene expression in Caco2 cells. IL-8 gene expression was studied in Caco2 cells treated with LPS. Modulation of this expression was studied in Caco2 cell cultures pretreated for 2 h with or without vectorized lycopene. Then, according to assays, LPS (100 ng/mL) was added for 6 h and mRNA expression was analyzed by RT-qPCR. (b) Lycopene induced no modification of gene expression induced by THP1-derived conditioned medium (CM) in Caco2 cells. THP1 cells were or not exposed to PMA for 2 hours. Then, the cell culture medium was discarded and the cells were extensively washed and a fresh medium without PMA was added and culture was resumed for 6 h. At the end of the culture period, the conditioned media (CM) were collected and then added to Caco2 cell cultures pretreated or not with lycopene for 12 h. The final treatment of Caco2 cells with the conditioned medium (CM) was for 6 h and, finally, expression of inflammatory genes was assessed by qRT-PCR. (c) Lycopene downregulates IL-1 gene expression induced by THP1-derived conditioned medium (CM) in Caco2 cells. THP1 cells were or not exposed to PMA for 2 hours. Then, the cell culture medium was discarded and the cells were extensively washed and a fresh medium without PMA was added and culture was resumed for 6 h. At the end of the culture period, the conditioned media (CM) were collected and then added to Caco2 cell cultures pretreated or not with lycopene for 12 h. The final treatment of Caco2 cells with the conditioned medium (CM) was for 6 h and, finally, expression of inflammatory genes was assessed by qRT-PCR. Values are means ± SD (n = 8). Assays treated with lycopene were compared to corresponding assays without lycopene. ***P < 0.01; **P < 0.05; ∗, nonsignificantly different.

Figure 6

MMP9 activity is induced by lycopene in THP1 cells. THP1 cells were cultured for 12 h in the presence or not of lycopene (2 μM) together with or without different effectors acting specifically on the c-AMP metabolism (FSK, MDL, and IBMX). Culture medium was collected and zymography ((a), (b)) was performed as described under Materials and Methods. Densitometry analysis of zymograms (A) and (B) was performed and results are reported on graphs (c) and (d), respectively. One representative experiment out of three is shown.

Figure 7

Lycopene-induced enhancement of c-AMP level in THP1 cells. THP1 cells were cultured for indicated times in the presence or not of lycopene (2 μM). The cells were then harvested and analyzed for c-AMP content as indicated under Materials and Methods. Values are means ± SD (n = 6).