Figure 1.
Expression of the Sigma-1 receptor in human monocytes, and differentiated monocyte-derived macrophages (moMACs) and dendritic cells (moDCs).
A–B: Expression of sigmar-1 protein in monocytes, and differentiated moMACs and moDCs measured by Western blot. In Fig1A a typical experiment out of four is demonstrated. Densitometry data of Fig1B show the Mean ± SEM values of four independent donors. C: Time kinetics of sigmar-1 gene expression in non-activated control (ctrl), 20 µg/ml polyI:C (polyI:C) or 500 ng/ml LPS (LPS) treated cells. Data of triplicates of four independent measurements are show as Mean ± SEM. D–E: Sigmar-1 protein expression in non-activated control, 20 µg/ml polyI:C or 500 ng/ml LPS treated cells following 24 hours of activation. In Fig1D, results of a typical experiment out of four is shown. Densitometry data of Fig1E show the Mean ± SEM values of four independent donors. (*) represents p values<0.05. (n.s.: “non-significant”).
Figure 2.
Effects of NN-DMT and 5-MeO-DMT treatment on the gene expression and secretion of cytokines and chemokines in LPS-stimulated moDCs.
A: MoDCs were incubated with 100 µM NN-DMT (NN-DMT) or 5-MeO-DMT (5-MeO-DMT) for 8 h or left untreated (ctrl) were used as controls. Additionally, cells were either activated with 500 ng/ml LPS alone for 8 h, or pre-treated with tryptamines for 1 hour and subsequently were activated with LPS for 8 hours (LPS+NN-DMT; LPS+5-MeO-DMT). The expression of IL-1β, TNFα, IL-6, IL8, and IL-10 was assessed by real-time Q-PCR and shown as relative mRNA expression. Results represent the Mean ± SEM of 4 independent experiments. B: Cytokine profile of cells treated as above. Supernatants of DC cultures were collected after 24 h and were subjected to ELISA measurements. Concentration of the secreted cytokines and chemokines are shown as Mean ± SEM values of 4 independent donors. (*) means statistical significance as p<0.05.
Figure 3.
Effects of NN-DMT and 5-MeO-DMT treatment on the gene expression and secretion of cytokines and chemokines in polyI:C-activated moDCs.
A: MoDCs were treated as in Figure 2. In this case, cell activation was induced with 20 µg/ml polyI:C for 8 h (alone, or subsequently to an 1 h 100 µM NN-DMT or 5-MeO-DMT pre-treatment). The expression of IL-1β, TNFα, IL-6, IL8, and IL-10 was assessed by real-time Q-PCR and shown as relative mRNA expression. Results represent the Mean ± SEM of 4 independent experiments. B: Cytokine profile of cells treated as in Fig3A. Supernatants of DC cultures were collected after 24 h and were measured by ELISA. Concentration of the secreted cytokines and chemokines are shown as Mean ± SEM values of 4 independent donors. (*) means statistical significance as p<0.05.
Figure 4.
NN-DMT and 5-MeO-DMT pre-treatment of pathogen-activated human dendritic cells effectively inhibit their capacity to prime autologous naive T helper 1 and T helper 17 cells.
Dendritic cells were activated either by heat-killed E. coli (E. coli) or inactivated influenza virus (IV) for 24 h, washed, and then co-cultured with naive autologous CD4+ T lymphocytes for 4 days. The number of primed, IFNγ or IL-17 secreting T cells was assessed by ELISPOT assay. T cells alone (T-cell ctrl), and non-activated DCs treated with DMT and co-cultured with autologous naive CD4+ T lymphocytes (NN-DMT, 5-MeO-DMT) were used as controls. A–B: Induction of IFNγ (A) or IL-17 (B) production of autologous naive CD4+ T cells induced by moDC loaded by E. coli. Bacteria alone (E. coli; red bars) or bacteria in combination with 1 h DMT pre-treatment (E. coli + NN-DMT, E. coli+5-MeO-DMT; empty bars) were used to activate moDCs as written above. C–D: Cells were activated as in Fig4A–B; in this case inactivated influenza virus (IV) was added to the moDCs alone for 24 h (blue bars), or in combination with an 1 h NN-DMT or 5-MeO-DMT pre-treatment (IV + NN-DMT, IV + 5-MeO-DMT; empty bars). Data represent Mean + SEM values of triplicate measurements of three independent donors. Asterisk indicates statistical significance (p<0.05).
Figure 5.
Effect of sigmar-1 gene silencing on the DMT-modified cytokine profile of LPS or polyI:C-activated moDCs.
A: Validation of siRNA knockdown by Western blot. MoDCs were transfected with negative control siRNA (ctrl siRNA) or with gene-targeting siRNA (sigmar-1 siRNA), or left untreated (non-transfected, NT). B–C: Non-treated, 24 h ctrl siRNA-only, and 24 h targeting siRNA-only treated cells were used as negative controls (black bars). Red bars represent 24 h 500 ng/ml LPS-treated cells, while white bars show ctrl siRNA and 1 h DMT pre-treated cells activated with LPS for one day. Grey (NN-DMT) and checkered bars (5-MeO-DMT) demonstrate 1 h DMT pre-treated and then 24 h LPS activated sigmar-1 knockdown cells. D–E: MoDCs were treated as in Fig5B–C. Here, cell activation was performed with a 24 h 20 µg/ml polyI:C treatment. Blue bars represent polyI:C-only stimulation as positive control. Results are shown as Mean ± SEM of three independent donors. (*) represents p values <0.05. Differences are significant (p<0.05) in all cases of specific activation (LPS or polyI:C) versus control cells (no treatment, ctrl siRNA, sigmar-1 siRNA).
Figure 6.
Sigmar-1 gene knockdown abrogates the inhibitory effect of tryptamines on the Th1/Th17 cell-activating capacity of pathogen-activated moDCs.
Cells were co-cultured and activated, and the number of IFNγ or IL-17 secreting T cells was assessed by ELISPOT as in Figure 4. A–B: Red bars represent E. coli-loaded DCs co-cultured with autologous naive CD4+ T cells. White bars show ctrl siRNA and 1 h tryptamine pre-treated cells prior to a 24 h E. coli activation and subsequent T cell co-culturing. Grey (NN-DMT) and checkered bars (5-MeO-DMT) demonstrate 1 h tryptamine pre-treated and then 24 h E. coli activated sigmar-1 knockdown cells subsequently co-cultured with T cells as in Materials and Methods. C–D: The same setup was applied as in Fig6A–B, however, in this case inactivated influenza virus (IV) was added to moDCs as antigen stimulation. Blue bars show moDCs incubated with influenza-only for 24 h and then co-cultured with T cells (positive control). Data represent Mean + SEM values of triplicate measurements of three independent donors. Asterisk shows statistical significance (p<0.05). Differences are significant (p<0.05) in cases of specific activation (E. coli or IV) versus T-cell controls.