Listeria monocytogenes Inhibits Serotonin Transporter in Human Intestinal Caco-2 Cells

Abstract

Listeria monocytogenes is a Gram-positive bacterium that can cause a serious infection. Intestinal microorganisms have been demonstrated to contribute to intestinal physiology not only through immunological responses but also by modulating the intestinal serotonergic system. Serotonin (5-HT) is a neuromodulator that is synthesized in the intestinal epithelium and regulates the whole intestinal physiology. The serotonin transporter (SERT), located in enterocytes, controls intestinal 5-HT availability and therefore serotonin's effects. Infections caused by L. monocytogenes are well described as being due to the invasion of intestinal epithelial cells; however, the effect of L. monocytogenes on the intestinal epithelium remains unknown. The main aim of this work, therefore, was to study the effect of L. monocytogenes on SERT. Caco2/TC7 cell line was used as an enterocyte-like in vitro model, and SERT functional and molecular expression assays were performed. Our results demonstrate that living L. monocytogenes inhibits serotonin uptake by reducing SERT expression at the brush border membrane. However, neither inactivated L. monocytogenes nor soluble metabolites were able to affect SERT. The results also demonstrate that L. monocytogenes yields TLR2 and TLR10 transcriptional changes in intestinal epithelial cells and suggest that TLR10 is potentially involved in the inhibitory effect observed on SERT. Therefore, L. monocytogenes, through TLR10-mediated SERT inhibition, may induce increased intestinal serotonin availability and potentially contributing to intestinal physiological changes and the initiation of the inflammatory response.

Keywords: 5-HT; Intestinal epithelium; Listeriosis; SERT; TLR.

Fig. 1

L. monocytogenes effects on 5-HT uptake. Cells were infected with alive or inactivated L. monocytogenes at MOI 50, 100, and 200 for 1 or 2 h. 5-HT uptake was measured after 6 min incubation with 0.2 μM 5-HT. Control condition corresponds to untreated cells. a Effect of live L. monocytogenes on 5-HT uptake. The results are expressed as the percentage of the uptake control and are the mean ± SE of six biological replicates in seven independent experiments (n = 42). ***P < 0.001 compared with the control. b Effect of heat-killed L. monocytogenes on 5-HT uptake. The results are expressed as the percentage of the uptake control and are the mean ± SE of six biological replicates in seven independent experiments (n = 42). c Effect of L. monocytogenes inactivated by PEF on 5-HT uptake. The results are expressed as the percentage of the uptake control and are the mean ± SE of three biological replicates in four independent experiments (n = 12). d Effect of L. monocytogenes supernatant on 5-HT uptake. The results are expressed as the percentage of the uptake control and are the mean ± SE of three biological replicates in four independent experiments (n = 12)

Fig. 2

L. monocytogenes inhibit SERT mRNA and protein expression. a Quantitative RT-PCR analysis of SERT mRNA expression in cells infected with L. monocytogenes for 2 h at MOI 200. Relative quantification was performed using comparative Ct (2^−ΔΔCt) of three biological replicates in five independent experiments (n = 15). Results are expressed as arbitrary units of control = 1. ***P < 0.001 compared with the control value. b Immunodetection of SERT by western blot in cell lysate and brush border from Caco-2/TC7 cells infected with L. monocytogenes for 2 h at MOI 200. c Quantification of SERT protein in both cell lysate and brush border using β-actin as an internal control of the protein load (SERT/β-actin ratio). The results are expressed as a percentage of the control value and are the mean ± SEM of two biological replicates in five independent experiments (n = 10). **P < 0.01 compared with the control value

Fig. 3

MyD88 involvement on L. monocytogenes effect on 5-HT uptake. Cells were infected with L. monocytogenes at MOI 100 and 200 for 2 h and/or MyD88 inhibitor (100 μM 1 h previous and during the infection with Listeria) and compared with untreated cells (control). Uptake of 5-HT was measured after 6 min of incubation, and 5-HT concentration was 0.2 μM. The results are expressed as the percentage of the uptake control and are the mean ± SE of three biological replicates in four independent experiments (n = 12). ***P < 0.001 compared with the control. ^### P < 0.001 and ^## P < 0.01 compared with the corresponding MOI effect without MyD88 inhibitor

Fig. 4

TLR2 involvement on L. monocytogenes effects. a Cells were infected with L. monocytogenes at MOI 200 for 2 h and/or TLR2 antibody (1 μg 30 min previous the infection) and compared with untreated cells (control). Uptake of 5-HT was measured after 6 min of incubation, and 5-HT concentration was 0.2 μM. The results are expressed as the percentage of the uptake control and are the mean ± SE of three biological replicates in four independent experiments (n = 12). ***P < 0.001 compared with the control. b Quantitative RT-PCR analysis of TLR2 mRNA expression in cells infected with L. monocytogenes for 2 h at MOI 200. Relative quantification was performed using comparative Ct (2^−ΔΔCt) of three biological replicates in five independent experiments (n = 15). Results are expressed as arbitrary units of control = 1. ***P < 0.001 compared with the control value. c Expression and quantification of TLR2 protein levels in cell lysate using β-actin as an internal control of the protein load (TLR2/β-actin ratio). The results are expressed as a percentage of the control value and are the mean ± SEM of two biological replicates in four independent experiments (n = 8). **P < 0.01 compared with the control value

Fig. 5

TLR10 involvement on L. monocytogenes effects. a Cells were infected with L. monocytogenes at MOI 200 for 2 h and/or TLR10 antibody (1 μg 30 min previous the infection) and compared with untreated cells (control). Uptake of 5-HT was measured after 6 min of incubation, and 5-HT concentration was 0.2 μM. The results are expressed as the percentage of the uptake control and are the mean ± SE of three biological replicates in four independent experiments (n = 12). ***P < 0.001 compared with the control. ^### P < 0.001 compared with MOI 200 effect without antibody. b Quantitative RT-PCR analysis of TLR10 mRNA expression in cells infected with L. monocytogenes for 2 h at MOI 200. Relative quantification was performed using comparative Ct (2^−ΔΔCt) of three biological replicates in five independent experiments (n = 15). Results are expressed as arbitrary units of control = 1. ***P < 0.001 compared with the control value. c Expression and quantification of TLR10 protein in cell lysate using β-actin as an internal control of the protein load (TLR10/β-actin ratio). The results are expressed as a percentage of the control value and are the mean ± SEM of two biological replicates in four independent experiments (n = 8)

Fig. 6

5-HT effect on growth curve of L. monocytogenes. L. monocytogenes growth was evaluated adding increasing concentrations of 5-HT 10⁻⁸ M and 10⁻⁴ M to the bacterial medium from 0 to 24 h. Results are expressed as the mean of three independent experiments in duplicate