1SCIENTIFIC REPORTS | (2019) 9:13661 | https://doi.org/10.1038/s41598-019-50038-x www.nature.com/scientificreports The Bacterial Product Violacein Exerts an Immunostimulatory Effect Via TLR8 Francisco A. Venegas1,2,3,4, Gabriele Köllisch5, Kerstin Mark6, Wibke E. Diederich6,7, Andreas Kaufmann8, Stefan Bauer8, Max Chavarría1,2,9, Juan J. Araya1,2 & Alfonso J. García-Piñeres1,3 Violacein, an indole-derived, purple-colored natural pigment isolated from Chromobacterium violaceum has shown multiple biological activities. In this work, we studied the effect of violacein in different immune cell lines, namely THP-1, MonoMac 6, ANA-1, Raw 264.7 cells, as well as in human peripheral blood mononuclear cells (PBMCs). A stimulation of TNF-α production was observed in murine macrophages (ANA-1 and Raw 264.7), and in PBMCs, IL-6 and IL-1β secretion was detected. We obtained evidence of the molecular mechanism of activation by determining the mRNA expression pattern upon treatment with violacein in Raw 264.7 cells. Incubation with violacein caused activation of pathways related with an immune and inflammatory response. Our data utilizing TLR-transfected HEK- 293 cells indicate that violacein activates the human TLR8 (hTLR8) receptor signaling pathway and not human TLR7 (hTLR7). Furthermore, we found that the immunostimulatory effect of violacein in PBMCs could be suppressed by the specific hTLR8 antagonist, CU-CPT9a. Finally, we studied the interaction of hTLR8 with violacein in silico and obtained evidence that violacein could bind to hTLR8 in a similar fashion to imidazoquinoline compounds. Therefore, our results indicate that violacein may have some potential in contributing to future immune therapy strategies. Violacein (Fig.  1) is a natural purple pigment produced by several Gram-negative bacteria1, such as Chromobacterium violaceum2 and Janthinobacterium lividum3. This secondary metabolite is an alkaloid formed by three structural units: a 5-hydroxyindole, an oxindole and a 2-pyrrolidone4. A variety of biological activities have been reported for this compound, including antibacterial1,5, anti-viral6, anti-inflammatory7, antitumor8, antileukemic9, as well as antifungal, antiparasitic, antiprotozoal, antioxidant and antiulcerogenic1. This wide range of biological activities has attracted interest to understand its mechanism of action with the purpose of finding a potential application as a therapeutic agent. The effect of violacein on immune cells and inflammation has been previously studied. For instance, it was reported that violacein exhibits immunosuppressive, analgesic and antipyretic effects in mice and rats7. In another study, violacein showed a gastroprotective effect in rats mediating the maintenance of the balance between pro- and anti-inflammatory cytokines and inhibiting TNF-α production10. Violacein also showed anti-inflammatory and anti-tumor activity through inhibition of metalloproteinase in the MCF-7 breast cancer cell line11. In a recent work12, it was shown that violacein treatment in a mouse model of acute inflammation was able to modulate production of several cytokines: IL-6 and TNF-α levels were reduced, and IL-10 levels were increased compared to untreated mice. A cytotoxic effect towards macrophages was also observed. Moreover, violacein treatment in a mouse model of experimental autoimmune encephalomyelitis (EAE) led to an amelioration of symptoms 1Escuela de Química, Universidad de Costa Rica, 11501-2060, San José, Costa Rica. 2Centro de Investigaciones en Productos Naturales (CIPRONA), Universidad de Costa Rica, 11501-2060, San José, Costa Rica. 3Centro de Investigación en Biología Celular y Molecular (CIBCM), Universidad de Costa Rica, 11501-2060, San José, Costa Rica. 4Present address: Institute for Immunology, Philipps-University Marburg, BMFZ, 35043, Marburg, Germany. 5Department of Parasitology, Philipps University Marburg, 35043, Marburg, Germany. 6Department of Pharmaceutical Chemistry and Center for Tumor Biology and Immunology (ZTI), Philipps University Marburg, 35043, Marburg, Germany. 7Core Facility Medicinal Chemistry, Philipps University Marburg, 35043, Marburg, Germany. 8Institute for Immunology, Philipps-University Marburg, BMFZ, 35043, Marburg, Germany. 9Centro Nacional de Innovaciones Biotecnológicas (CENIBiot), CeNAT-CONARE, 1174-1200, San José, Costa Rica. Correspondence and requests for materials should be addressed to A.J.G.-P. (email: alfonso.garciapineres@ucr.ac.cr) Received: 13 November 2017 Accepted: 19 July 2019 Published: xx xx xxxx OPEN https://doi.org/10.1038/s41598-019-50038-x mailto:alfonso.garciapineres@ucr.ac.cr 2SCIENTIFIC REPORTS | (2019) 9:13661 | https://doi.org/10.1038/s41598-019-50038-x www.nature.com/scientificreportswww.nature.com/scientificreports/ compared to placebo-treated mice. This study found that violacein exerts this effect by increasing regulatory T-cell counts12. In contrast to the above-mentioned studies suggesting that violacein has an inhibitory effect on TNF-α expression, other studies related to the mechanism of the antileukemic activity of violacein in HL60 cells have found an increase in the pro-inflammatory cytokine TNF-α and the activation of TNF receptor 1 signaling upon incubation with violacein. These results suggest that violacein induces apoptosis of immune cells by TNFR1 acti- vation9. Further work by Antonisamy et al.13 reports that violacein induces apoptosis in human breast cancer cells through upregulation of TNF-α expression and the p53-dependent mitochondrial pathway. In summary, the evidence shows that violacein exerts an anti-inflammatory effect in vivo that involves a reduc- tion in TNF-α production, but these results appear to be in contradiction with some in vitro results, where an induction of the pro-inflammatory cytokine TNF-α is observed. TNF-α is a pro-inflammatory cytokine that is produced by a wide range of cells and regulates several processes like cell proliferation, differentiation and apoptosis14. TNF-α signaling is mediated by binding to one of two receptors, TNFR1 or TNFR215. TNFR1 is expressed in most tissues while TNFR2 is exclusively found in immune cells15. TNF-α signaling through TNFR1 involves induction of apoptosis, and this implies recruitment of the adaptor protein FADD and caspase-816. TNFR1 also induces nuclear translocation of NF- κB, a transcription factor that promotes cell survival through the induction of anti-apoptotic and inflammatory gene expression15,16. TNF-α is relevant to physiological processes such as apoptosis and inflammation, and it has been shown that vio- lacein affects TNF-α expression. However, the evidence of induction or suppression of TNF-α by violacein is not clear. Therefore, further work is needed to clarify the role of this cytokine in the activity of violacein on cells9,11–13. Macrophages are cells of the innate immune system and are able to detect invading microorganisms by rec- ognizing associated molecular patterns (PAMPs) through pattern recognition receptors (PRR) such as Toll-like and NOD-like Receptors (TLR and NLR)17,18. Binding of PAMPs to their cognate receptor leads to the production of several mediators of inflammation, such as inflammatory cytokines and chemokines. As a consequence, acti- vation of macrophages and phagocytes leads to chemotaxis of immune cells, inflammation and activation of an adaptive response, with a global effect of protection of the organism against microbes18. In this study, we investigated the effect of violacein obtained from C. violaceum on different immune-related cell lines. We found that treatment with violacein was able to induce TNF-α expression in Raw 264.7 and ANA-1 cells. In addition, we determined the gene expression profile of Raw 264.7 cells incubated with this substance. Our results indicate the induction of inflammatory cytokines and suggest the activation of the TLR signaling pathway and in consequence an induction of inflammatory cytokines and negative regulators of TLR signaling. Using TLR-transfected HEK-293 cells, we determined that violacein significantly activates hTLR8 at 15 µmol/L or higher. Moreover, we found that the immunostimulatory activity of violacein in PBMCs was suppressed by the specific and potent hTLR8 antagonist, CU-CPT9a. Finally, we studied the interaction of violacein with hTLR8 in silico through molecular docking and obtained evidence that this binding could occur in a similar fashion to syn- thetic agonists of hTLR8. Our results show that violacein presents an immune stimulating effect in some murine cell lines and in PBMCs, and that this effect could be associated with signaling through TLR8. Results Purity of violacein. After purification, violacein was identified by comparison of 1H-NMR signals with pre- viously reported data19. Additional signals were observed in the spectrum at very low intensity compared to violacein and they were consistent with common fatty acid impurities (δ 0.90, t, CH3 and δ 1.29, br s, (CH2)n)20. Characteristic signals related with Lipopolysaccharide contamination21,22 were not detected. Both HPLC-UV and 1H-NMR confirmed that violacein was obtained at highly purity (90–93%, measured by HPLC-UV). Effect of violacein on different murine cell types. The effect of violacein on cell viability was evaluated in the Raw 264.7 and ANA-1 cell lines, as well as in murine bone marrow-derived macrophages (BMM), plas- macytoid dendritic cells (pDC) and myeloid dendritic cells (mDC) from wild-type (wt), and from TLR7−/− and TLR2/4−/− knock-out mice. Figure 1. Chemical structure of violacein (3-(1,2-dihydro- 5-(5-hydroxy-1H-indol-3-yl)-2-oxo-3H-pyrrol-3- ilydene)-1,3-dihydro-2H-indol-2-one). https://doi.org/10.1038/s41598-019-50038-x 3SCIENTIFIC REPORTS | (2019) 9:13661 | https://doi.org/10.1038/s41598-019-50038-x www.nature.com/scientificreportswww.nature.com/scientificreports/ To determine the effect of violacein on the Raw 264.7 cell line, cells were treated with different concentra- tions of the compound and cell viability was evaluated using the MTT assay. A cytotoxic effect of violacein was observed after 24 hours, with an IC50 value of 12 ± 2 µmol/L (mean ± standard deviation, n = 4, Fig. 2). The effect of violacein on macrophage activation was initially determined by measuring nitric oxide production with the Griess reagent23. In macrophages, nitric oxide is produced by the inducible nitric oxide synthase, after cell stimulation. Raw 264.7 cells were incubated with increasing sub-toxic concentrations of violacein (1–12 µmol/L). As a positive control, lipopolysaccharide (LPS) from E. coli was added to a final concentration of 1.1 µg/mL. After 24 hours, 100 µl of supernatant were harvested and the production of nitric oxide was measured (data not shown). In contrast to LPS, treatment of cells with violacein did not produce a significant change in the nitric oxide levels when compared to unstimulated cells. Raw 264.7 cell activation by violacein was then investigated by measuring TNF-α expression using real-time qRT-PCR. Cells were treated with different violacein concentrations, and total RNA extracts were obtained after 4 hours. As shown in Fig. 3A, violacein at a 2 µmol/L or higher induces the expression of TNF-α mRNA in Raw 264.7 cells (stimulation index = 1.5). TNF-α production induced by violacein in Raw 264.7 and ANA-1 cells was also measured by ELISA, and consistent results were obtained (Fig. 3B,C): TNF-α production was increased at concentrations higher than 4 µmol/L in Raw 264.7 cells (SI = 3.1) or 2 µmol/L in ANA-1 cells (SI = 3.6), and a cytotoxic effect was observed for ANA-1 cells at 12 µmol/L. To study the effect of violacein on primary murine cells, BMM, mDC and pDC from wt, TLR7−/− and TLR2/4−/− mice were incubated with violacein and TNF-α, IL-6 and IFN-α (only for pDC) production was measured by ELISA (data not shown). Production of the investigated cytokines was not induced, and we observed cytotoxicity at a concentration of 12 µmol/L (Table 1). Gene expression pattern induced by violacein in Raw 264.7 cells. Microarray analysis was per- formed for total RNA extracts obtained from Raw 264.7 cells that were cultured for 4 hours in the presence or absence of violacein (4 µmol/L). As shown in Table 2, of 41.346 genes analyzed, 129 showed a differential expression (≥1.5-fold change and LPE p-value < 0.05) when violacein-treated cells were compared to untreated cells. Of these, 46.5% of genes (60/129) showed an increase in expression, and 53.5% of genes (69/129) showed a decrease in expression. 26.4% of genes (34/129) were orphan genes and 9.3% of genes (12/129) corresponded to non-coding RNA. To confirm the gene expression profile obtained with the microarray analysis, four genes were chosen (TNF-α, IRG1, CCL2 and CXCL2) for confirmation with real-time qRT-PCR. Selected genes showed >2.0-fold change in the microarray experiment. As shown in Table 3, all four genes showed an increase in expression in both microarray and real time PCR, and in both cases, up-regulation was highly significant as compared to untreated control cells. The list of genes whose expression significantly changed after incubating Raw 264.7 cells with violacein was analyzed using the DAVID database, to understand the effect of violacein on biological pathways. Table 4 shows a list of the identified differentially expressed genes grouped by functional annotation. According to these results, violacein induced changes in expression of genes in biological pathways related with an immune and inflamma- tory response, apoptotic pathway and regulation of cell proliferation. Figure 2. Cytotoxic effect of violacein on Raw 264.7 cells. Cells were incubated with the indicated concentrations of violacein for 24 h, and cell viability was evaluated using the MTT assay. The experiment was performed in a 96-well plate. Cell viability was defined as the percent ratio of absorbance in treated cells and that of control (amount of reduced MTT observed in the absence of compounds). Each data point represents one independent experiment run in triplicate and the center line indicates the mean. https://doi.org/10.1038/s41598-019-50038-x 4SCIENTIFIC REPORTS | (2019) 9:13661 | https://doi.org/10.1038/s41598-019-50038-x www.nature.com/scientificreportswww.nature.com/scientificreports/ These results suggest that in murine cells, violacein activates Toll like receptor signaling (8 genes involved) and NOD-like receptor (5 genes involved) pathways. Effect of violacein on different human cell lines and PBMCs. To study the effect of violacein on human cells, TNF-α and IL-6 production by MonoMac 6, undifferentiated THP-1 cells and PBMCs was measured using ELISA. Neither cell type showed an induction of cytokine production at violacein concentrations up to 12 µmol/L . For this reason, TNF-α and IL-6 production was evaluated at higher concentrations with PBMCs from two donors. In this case, an induction of IL-6 production but not of TNF-α was observed (Table 1). Figure 3. Effect of violacein on TNF-α production in Raw 264.7 and ANA-1 cells. (A) Raw 264.7 cells were treated with the indicated concentrations of violacein (V) and TNF-α was determined in total RNA extracts by real-time qRT-PCR. Relative TNF-α expression using GAPDH as a reference gene is shown. (B) Raw 264.7 cells or (C) ANA-1 cells were treated with the indicated concentration of violacein or incubated with various stimuli and TNF-α production was determined by ELISA. Each data point represents one experiment run in duplicate and the center line indicates the mean. **p < 0.01 compared to the untreated control, ***p < 0.001 compared to the untreated control. https://doi.org/10.1038/s41598-019-50038-x 5SCIENTIFIC REPORTS | (2019) 9:13661 | https://doi.org/10.1038/s41598-019-50038-x www.nature.com/scientificreportswww.nature.com/scientificreports/ Effect of violacein on HEK-293 cells transfected with hTLR8 or hTLR7. To analyze the possibility that violacein could act as a hTLR7 or hTLR8 agonist, we measured NF-κB activation in HEK-293 cells expressing either hTLR7 or hTLR8 and a NF-κB-luciferase reporter plasmid. We observed that at 15 and 30 µmol/L, violacein caused a significant NF-κB induction (1.7 and 8.7-fold respectively) in hTLR8 transfected HEK-293 cells. Cell death was also observed at the highest concentration (Fig. 4A). In contrast, highest concentration of violacein caused a cytotoxic effect with no NF-κB induction in hTLR7 transfected HEK-293 cells (Fig. 4B). R848 (both TLR7 and TLR8 agonist) was used as a positive control. These results suggest that violacein can act as an agonist for hTLR8 and not for hTLR7. Blockade of violacein effect on PBMCs by a specific hTLR8 antagonist. To explore the possibil- ity that violacein acts via hTLR8, we investigated the effect of CU-CPT9a (4-(7-methoxyquinolin-4-yl)-2-met hylphenol), a specific hTLR8 antagonist24,25, on its immunostimulatory effect on PBMCs. In the absence of the antagonist, we observed that at 30 µmol/L of violacein, PBMC viability was very low (13 ± 4%; mean ± standard deviation, n = 3) and no IL-6 production was detected. However, at 15 µmol/L viability was higher (48 ± 16%; mean ± standard deviation, n = 3) and there was a significant increase in IL-6 production in comparison to the untreated control (Fig. 5A). We also observed a significant production of IL-6 in presence of other stimuli, namely R848 or RNA-40 (TLR7/8 agonist) and LPS (TLR4 agonist) (Fig. 5A). In order to assess the effect of CU-CPT9a on cell activation, PBMCs from five donors were incubated with differ- ent concentrations of CU-CPT9a (0, 0.02, 0.2, 2, 20 µmol/L), before addition of R848 (1 µmol/L), LPS (50 ng/mL), violacein (15 µmol/L) or RNA-40 (5 µg/mL). PBMC treatment with R848 or RNA-40 induced IL-6 production, and this activation was suppressed by CU-CPT9a in a dose-dependent manner (Fig. 5C,F, respectively). In the case of R848, this suppression was sig- nificant at 2 and 20 µmol/L, whereas for RNA 40, a reduction was observed at all used antagonist concentrations. When PBMCs were stimulated with LPS, IL-6 induction was not suppressed at any concentration of the antag- onist (Fig. 5D). These results are evidence for antagonist specificity. A dose-dependent suppression of IL-6 production by CU-CPT9a was also observed when cells were stimu- lated with violacein. A significant reduction was observed at 2 and 20 µmol/L in comparison to the no antagonist control (Fig. 5E). Interestingly the cytotoxic effect of violacein was not abolished by the antagonist. In addition, when using IL-1β, as a marker, cytokine secretion was stimulated by violacein (30 and 15 µmol/L) and R848 (1 µmol/L) (Fig. 5B). However, previous addition of CU-CPT9a only blocked IL-1β stimulated by R848 and had no effect on violacein-induced IL-1β stimulation (data not shown). Molecular docking of violacein on TLR8. The molecular docking of hTLR8 with violacein or CL097, a synthetic hTLR8 agonist26,27, was performed using AutoDock Vina. Different binding models were obtained, and the one with the biggest change in binding free energy was selected (Fig. 6B,C). This model was compared to the crystal structure of hTLR8 bound to CL09728 (Fig. 6A). The simulated docking models of CL097 and violacein with TLR8 (Fig. 6) show that violacein and CL097 share similar binding modes by means of interactions with amino acids within the dimer interface as observed in the crystal structure of hTLR8 bound to CL097 (Fig. 6D,E). If violacein can interact with amino acids that are at a distance of 5 Å or lower within the hTLR8-hTLR8* dimer interface, the following amino acids could be in contact with the ligand: F261, N262, Y348, G351, S352, Y353, V378, F405, D545*, N546*, A547*, G572*, V573*, T574* and H576*. Discussion Violacein is a natural product derived from bacteria, for which an array of biological effects have been described in the literature, such as immunosuppressive, analgesic, antipyretic and anti-inflammatory7. Moreover, treatment with violacein has been reported to reduce acute and chronic inflammation through the stimulation of regulatory T cells12. Contradictory evidence has been reported on whether violacein induces or inhibits TNF-α expression: while an induction has been observed in cell culture (MCF-7 breast cancer cells13 and HL-60 cells9), a reduction was observed on gastric mucosa in violacein-treated rats in which gastric lesions were induced with indometha- cin10. A reduction of TNF-α expression was also observed upon violacein treatment in lumbar spinal cords of the EAE mouse model12. In this study, we researched the effect of violacein in different cell lines and primary cultured Cells Activation Raw 264.7 ≥2 µmol/La and ≥4 µmol/Lb ANA-1 4 and 6 µmol/Lb PBMC 15 µmol/Lc Murine BMM (wt; TLR7−/−; TLR2/4−/−) N.D.d,e Murine mDC (wt; TLR7−/−; TLR2/4−/−) N.D.d,e Murine pDC (wt; TLR7−/−; TLR2/4−/−) N.D.d,e MonoMac 6 N.D.d,f THP-1 N.D.d,f Table 1. Activation of different cell lines by violacein. aData of real time qRT-PCR experiment for TNF-α. bData of ELISA experiment for TNF-α. cData of ELISA experiment for IL-6. dN.D. = Not detected. eCell death was observed at 6 and 12 µmol/L. fNo cell death was observed at 12 µmol/L. https://doi.org/10.1038/s41598-019-50038-x 6SCIENTIFIC REPORTS | (2019) 9:13661 | https://doi.org/10.1038/s41598-019-50038-x www.nature.com/scientificreportswww.nature.com/scientificreports/ Gene Symbol Gene name mRNA Accession Fold change p-valuea UP-REGULATED GENES Egr1 Early growth response 1 NM_007913 3.92 <1.52E-26 Plaur Plasminogen activator, urokinase receptor ENSMUST00000002284 2.79 <1.52E-26 Irg1 Immunoresponsive gene 1 ENSMUST00000022722 2.64 <1.52E-26 Gprc5a G protein-coupled receptor, family C, group 5, member A NM_181444 2.58 2.95E-11 Plk2 Polo-likekinase 2 NM_152804 2.55 <1.52E-26 Osm Oncostatin M ENSMUST00000075221 2.54 1.87E-11 Ccdc85b Coiled-coil domain containing 85B NM_198616//NM_198616 2.48 <1.52E-26 Tnf Tumor necrosis factor NM_013693 2.46 <1.52E-26 Il7r Interleukin 7 receptor NM_008372 2.46 3.18E-12 Pmepa1 Prostate transmembrane protein, Androgen induced 1 NM_022995 2.35 <1.52E-26 Rcan1 Regulator of calcineurin 1 NM_019466 2.28 <1.52E-26 Ccl2 Chemokine (C-C motif) ligand 2 NM_011333 2.27 8.48E-11 Cxcl2 Chemokine (C-X-C motif) ligand 2 ENSMUST00000075433 2.27 6.65E-07 Rgs16 Regulator of G-protein signaling 16 NM_011267 2.19 <1.52E-26 Plk3 Polo-like kinase 3 NM_013807 2.1 2.58E-08 Serpine1 Serine (or cysteine) peptidase inhibitor, clade E, member 1 NM_008871 1.99 2.22E-07 Rhob Ras homolog gene family, member B NM_007483 1.97 1.18E-05 Pdgfb Platelet derived growth factor, B polypeptide NM_011057 1.96 <1.52E-26 Gpr84 G protein-coupled receptor 84 ENSMUST00000079824 1.94 <1.52E-26 Traf1 TNF receptor-associated factor 1 NM_009421 1.93 9.26E-08 Dusp5 Dual specificityphosphatase 5 ENSMUST00000038287 1.92 1.79E-05 Itga5 Integrinalpha 5 (fibronectin receptor alpha) NM_010577 1.9 <1.52E-26 Egr2 Earlygrowth response 2 NM_010118 1.9 2.74E-09 Dusp1 Dual specificity phosphatase 1 ENSMUST00000025025 1.88 <1.52E-26 Rgs1 Regulator of G-protein signaling 1 ENSMUST00000172388 1.88 3.90E-08 Slc6a8 Solute carrier family 6 (neurotransmitter transporter, creatine), member 8 NM_133987 1.82 <1.52E-26 Slc20a1 Solutecarrierfamily 20, member 1 NM_015747 1.79 <1.52E-26 Cxcl10 Chemokine (C-X-C motif) ligand 10 NM_021274 1.77 4.22E-05 UP-REGULATED GENES Egr3 Earlygrowth response 3 NM_018781 1.76 0.001266 Tm4sf19 Transmembrane 4 L six family member 19 NM_001160402 1.76 9.76E-09 Trib1 Tribbleshomolog 1 (Drosophila) ENSMUST00000067543 1.74 0.000143 Tmem26 Transmembraneprotein 26 NM_177794 1.74 5.44E-09 Ier3 Immediateearly response 3 NM_133662 1.73 2.72E-10 Ptgs2 Prostaglandin-endoperoxidesynthase 2 NM_011198 1.71 3.14E-08 Nfkbia Nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha NM_010907 1.71 4.76E-12 Kdm6b KDM1 lysine (K)-specific demethylase 6B NM_001017426 1.7 4.11E-06 Myc Myelocytomatosisoncogene ENSMUST00000160009 1.69 0.000582 Skil SKI-like NM_011386 1.68 1.21E-06 Clec4e C-type lectin domain family 4, member e NM_019948 1.67 2.07E-11 Dusp4 Dual specificityphosphatase 4 ENSMUST00000033930 1.66 4.84E-08 Ccl4 Chemokine (C-C motif) ligand 4 NM_013652 1.66 4.94E-08 Zfp36 Zinc finger protein 36 ENSMUST00000051241 1.66 1.10E-09 Vegfc Vascular endothelial growth factor C NM_009506 1.66 0.003692 Nfkbie Nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, epsilon NM_008690 1.62 0.00102 Tfrc Transferrin receptor NM_011638 1.61 1.37E-09 Ehd1 EH-domaincontaining 1 ENSMUST00000025684 1.61 8.10E-08 Tnfaip3 Tumor necrosis factor, alpha-inducedprotein 3 NM_009397 1.59 0.02444 Tgm2 Transglutaminase 2, C polypeptide NM_009373 1.58 0.000104 Nfkb2 Nuclear factor of kappa light polypeptide gene enhancer in B cells 2, p49/p100 NM_001177369 1.58 4.68E-07 Nr4a1 Nuclear receptor subfamily 4, group A, member 1 NM_010444 1.55 0.001768 Junb Jun-B oncogene NM_008416 1.55 4.67E-08 Continued https://doi.org/10.1038/s41598-019-50038-x 7SCIENTIFIC REPORTS | (2019) 9:13661 | https://doi.org/10.1038/s41598-019-50038-x www.nature.com/scientificreportswww.nature.com/scientificreports/ immune cells (human and murine) and obtained evidence for a cell-type specific activation and for the molecular mechanism of this activation. We first studied whether violacein induced Raw 264.7 cell activation by measuring nitric oxide production. Our results show that violacein did not induce iNOS expression in Raw 264.7 cells after 24 h incubation. These observations are in agreement with previous reports that found that violacein decreases iNOS activity in gastric ulcers induced by indomethacin in rats10 and also that violacein does not cause significant changes in gene expres- sion of iNOS in mouse lumbar spinal cord12. However, these results do not clarify whether violacein exerts an Gene Symbol Gene name mRNA Accession Fold change p-valuea Csrnp1 Cysteine-serine-rich nuclear protein 1 NM_153287 1.54 0.046319 Lims2 LIM and senescent cell antigen like domains 2 NM_144862 1.53 0.022744 Fos FBJ osteosarcomaoncogene NM_010234 1.53 5.79E-05 Rai14 Retinoicacidinduced 14 NM_030690 1.52 2.91E-05 Map2k3 mitogen-activated protein kinase kinase 3 NM_008928 1.52 1.36E-05 Fam129b Family with sequence similarity 129, member B NM_146119 1.51 5.29E-06 Plau Plasminogenactivator, urokinase NM_008873 1.51 3.01E-06 DOWN-REGULATED GENES Ccl3 Chemokine (C-C motif) ligand 3 NM_011337 1.51 8.85E-12 Rn5s20 5 S RNA 20 NR_046144//NR_046144 −1.87 1.52E-26 Adm Adrenomedullin NM_009627 −1.87 0.003717 Bex6 Brain expressed gene 6 NM_001033539 −1.82 0.046036 Gm5431 Predicted gene 5431 ENSMUST00000109212 −1.79 0.010583 S1pr1 Sphingosine-1-phosphate receptor 1 NM_007901 −1.79 0.000124 Tlr8 Toll-like receptor 8 ENSMUST00000112170 −1.77 0.004444 Rps20 Ribosomal protein S20 ENSMUST00000130128 −1.75 0.003227 Gm5771 Predicted gene 5771 NM_001038997 −1.74 0.027193 Trp53inp1 Transformation related protein 53 inducible nuclear protein 1 NM_001199105 −1.74 0.000304 Mxd4 Max dimerization protein 4 ENSMUST00000042701 −1.69 0.003729 Ccng2 Cyclin G2 ENSMUST00000121127 −1.68 0.005001 Snord58b Small nucleolar RNA, C/D box 58B NR_028552 −1.68 1.15E-11 Scel Sciellin NM_022886 −1.67 0.000661 Klhl24 Kelch-like 24 (Drosophila) NM_029436 −1.66 7.08E-09 Gm7429//Gm6109//Rpl30 Predicted pseudogene 7429//predicted gene 6109// ribosomal protein L30 ENSMUST00000135417 −1.64 0.000191 Lrp2bp Lrp2 bindingprotein ENSMUST00000066451 −1.62 0.002288 Olfr820 Olfactory receptor 820 ENSMUST00000059244 −1.58 0.00979 Fbxl20 F-box and leucine-rich repeat protein 20 NM_028149 −1.58 0.016514 Ighm Immunoglobulin heavy constant mu AB067787//AB067787 −1.57 3.14E-08 9930111J21Rik2 RIKEN cDNA 9930111J21 gene 2//RIKEN cDNA 9930111J21 gene 2 BC066104//BC066104 −1.56 0.000145 Rny3 RNA, Y3 small cytoplasmic (associated with Ro protein) NR_024202//NR_024202 −1.56 0.007455 Cysltr1 Cysteinylleukotriene receptor 1 ENSMUST00000113480 −1.54 0.00261 Bnip3 BCL2/adenovirus E1B interactingprotein 3 NM_009760 −1.54 5.28E-05 Clec7a C-type lectin domain family 7, member a NM_020008 −1.53 4.12E-06 Snord1b Small nucleolar RNA, C/D box 1B NR_028567 −1.52 9.76E-09 Dpep2 Dipeptidase 2 ENSMUST00000150001 −1.52 0.000407 Table 2. Differentially expressed genes in Raw 264.7 cells treated for 4 hours with 4 µmol/L of violacein, compared to untreated control cells. aLPE p-value < 0.05 is considered significant. Gene name Gene symbol mRNA Accession Microarray Real time PCR FCa p-valueb FC p-valueb Tumor necrosis factor alpha TNF-α NM_013693 2.46 <1.52E-26 5.19 0.003c Immune responsive gene 1 IRG1 NM_008392 2.64 <1.52E-26 5.84 0.019d Chemokine (C-C motif) ligand 2 CCL2 NM_011333 2.27 8.48E-11 6.52 0.002e Chemokine (C-X-C motif) ligand 2 CXCL2 NM_009140 2.27 6.65E-07 10.53 1.87E-05c Table 3. Confirmation of microarray results by comparison with real-time qRT-PCR for selected differentially expressed genes. aFC = Fold change. bp < 0.05 is considered significant. cn = 6. dn = 4. en = 5. https://doi.org/10.1038/s41598-019-50038-x 8SCIENTIFIC REPORTS | (2019) 9:13661 | https://doi.org/10.1038/s41598-019-50038-x www.nature.com/scientificreportswww.nature.com/scientificreports/ effect on macrophages. For this reason, we studied if violacein induces the production of TNF-α, which is related with macrophage activation. TNF-α is a cytokine whose up-regulation is related with inflammatory activity, immune response and mac- rophage activation15,16. In this study, we determined that violacein induced TNF-α expression at sub-toxic con- centrations in Raw 264.7 cells, at the mRNA (Fig. 3A) and protein level (Fig. 3B). Induction of TNF-α by violacein has been previously described in HL-60 cells, where the evidence suggests that TNF-α activates TNFR1, medi- ating apoptosis in leukemia cells9. Violacein was also shown to induce TNF-α gene expression in human breast cancer cells13. Violacein also induced a weak production of TNF-α in ANA-1 cells at concentrations higher than 2 µmol/L (Fig. 3C). ANA-1 cells and Raw 264.7 cells are murine macrophages, and both cells lines were established by ret- roviral infection (Abelson murine leukemia virus for Raw 264.7, J2 retrovirus for ANA-1)29,30. Cytokine production (TNF-α, IL-6 and IFN-α only for pDC) in murine BBM, mDC and pDC from wild-type mice, or TLR7−/− and TLR2/4−/− knock-outs were not induced after treatment with violacein. Thus, our results indicate that violacein only presents an activity in virus-transformed murine macrophages such as Raw 264.7 and ANA-1 cells. In both cases, up-regulation of TNF-α but not IL-6 was observed. Due to our observation of a TNF-α up-regulation by violacein in Raw 264.7 cells, we decided to study the gene expression profile in this cell line. According to our results (Table 4), the global changes in gene expression that were caused by incubation with violacein agree with an activation of macrophages. We observed differential expression of genes involved in an inflammatory response and signaling (TNF-α, MAP2K3, KDM6B, TLR8), immune response (BNIP3, NFKB2, IL7R, TLR8, OSM) and chemotaxis (CCL2, CCL3, CXCL2, CCL4, CXCL10). The expression of genes associated with regulation of cell proliferation (TNF-α, PTGS2, PDGFB, VEGFC) and apoptosis (TNF, TGM2, TRP53INP1, BNIP3, MYC) was also significantly affected. We also found an association with signaling pathways through PAMP-receptors such as Toll-like receptor (FOS, CCL3, TNF-α, MAP2K3, NFKBIA, CCL4, TLR8, CXCL10) and NOD-like receptor (TNF-α, CCL2, CXCL2, NFKBIA, TNFAIP3). These observations suggest that violacein could activate a Toll-like receptor in murine cells. Given that we found the expression of mTLR8 to be downregulated, we think that the effect of violacein in murine macrophages is related with this receptor. TLR8 is involved in the recognition of single stranded RNA (ssRNA) and initiates an immune response that can signal via two distinct mechanisms involving different adapter proteins, namely MyD88 or TRIF. The Term Gene count p-valuea Genesb SP_PIR_KEYWORDS Inflammatory response 8 3.28E-08 CCL3, CCL2, CXCL2, CLEC7A, CCL4, KDM6B, TLR8, CXCL10 Cytokine 7 9.24E-05 OSM, CCL3, TNF, CCL2, CXCL2, CCL4, CXCL10 Chemotaxis 5 1.26E-04 CCL3, CCL2, CXCL2, CCL4, CXCL10 GOTERM_BP_FAT GO:0006954~inflammatory response 10 1.30E-06 CCL3, TNF, CCL2, MAP2K3, CXCL2, CLEC7A, CCL4, KDM6B, TLR8, CXCL10 GO:0006955~immune response 13 2.35E-06 CCL3, TNF, CCL2, CXCL2, BNIP3, NFKB2, IL7R, CCL4, TLR8, CXCL10, OSM, CLEC4E, CLEC7A GO:0009611~response to wounding 11 6.13E-06 CCL3, TNF, CCL2, MAP2K3, CXCL2, CLEC7A, CCL4, KDM6B, TLR8, PLAUR, CXCL10 GO:0042127~regulation of cell proliferation 13 9.16E-06 TNF, CCL2, PTGS2, PDGFB, NFKBIA, CXCL10, VEGFC, S1PR1, ADM, SERPINE1, TGM2, MYC, PLAU GO:0006952~defense response 11 5.54E-05 CCL3, TNF, CCL2, MAP2K3, CXCL2, BNIP3, CLEC7A, CCL4, KDM6B, TLR8, CXCL10 GO:0008284~positive regulation of cell proliferation 9 6.63E-05 VEGFC, TNF, S1PR1, CCL2, PDGFB, ADM, TGM2, MYC, CXCL10 GO:0006935~chemotaxis 6 1.83E-04 CCL3, CCL2, CYSLTR1, CXCL2, CCL4, CXCL10 GO:0045944~positive regulation of transcription from RNA polymerase II promoter 9 3.25E-04 OSM, EGR1, FOS, TNF, EGR2, S1PR1, CSRNP1, NR4A1, MYC GO:0006917~induction of apoptosis 6 1.29E-03 TNF, TGM2, TRP53INP1, NR4A1, BNIP3, MYC GOTERM_MF_FAT GO:0008009~chemokine activity 5 2.47E-05 CCL3, CCL2, CXCL2, CCL4, CXCL10 KEGG_PATHWAY mmu04620: Toll-like receptor signaling pathway 8 2.99E-06 FOS, CCL3, TNF, MAP2K3, NFKBIA, CCL4, TLR8, CXCL10 mmu04010: MAPK signaling pathway 11 5.59E-06 DUSP5, FOS, DUSP4, TNF, TM4SF19, PDGFB, DUSP1, MAP2K3, NR4A1, NFKB2, MYC mmu04060: Cytokine-cytokine receptor interaction 10 2.17E-05 OSM, VEGFC, CCL3, TNF, CCL2, PDGFB, CXCL2, IL7R, CCL4, CXCL10 mmu04621: NOD-like receptor signaling pathway 5 6.93E-04 TNF, CCL2, CXCL2, NFKBIA, TNFAIP3 Table 4. Biological terms significantly associated with differential gene expression. aModified Fisher exact P-value, EASE Score; p < 0.05 is considered significant. bDown-regulated genes are written in bold. https://doi.org/10.1038/s41598-019-50038-x 9SCIENTIFIC REPORTS | (2019) 9:13661 | https://doi.org/10.1038/s41598-019-50038-x www.nature.com/scientificreportswww.nature.com/scientificreports/ MyD88-dependent pathway results in the activation of NF-κB and activated protein-1 (AP-1or FOS) transcrip- tion factors, while the TRIF-dependent pathway results in the activation of type I interferons17,28. hTLR8 can recognize single stranded RNA (ssRNA) and imizadoquinolines and induce an immune response. In contrast, mTLR8 is not activated by ssRNA or imizadoquinolines31. However, mTLR8 can be activated by imidazoquino- lines in combination with polyT oligonucleotides, causing NF-κB activation and TNF-α expression in HEK-293 cells31. Moreover, mTLR8 overexpression can also induce NF-κB activation and TNF-α production but does not activate AP-1 and interferon-α32. These two studies show that mTLR8 is indeed functional. Our data indicate that violacein activates hTLR8 and suggest that violacein could act via mTLR8 signaling in Raw 264.7 cells. Moreover, the gene expression results related with the activation of mTLR8 do not show differ- ential expression of interferons, this observation is in agreement with previous report of activation of mTLR831. Expression of mTLR8 in the microarray data was found to be down regulated. This could be the consequence of a negative feedback mechanism to shut down TLR signaling. This possibility is supported by the observation that IκBα and IκBε were up-regulated. Both proteins are related with TLR signaling inhibition, specifically by inhibition of the transcription factor NF-κB. Furthermore, the expression of IRG1 leads to negative regulation of TLR-mediated signaling by stimulating A20 via the induction of reactive oxygen species production33. A20 is also involved in feedback inhibition of NF-κB activation34. Both genes (IRG1 and A20) were up-regulated upon violacein incubation, and the activation of IRG1 was also confirmed by real-time qRT-PCR. When assessing the effect of violacein on cytokine expression in human cells, no induction was observed with THP-1 and MonoMac 6 human macrophage cell lines. PBMCs did not produce TNF-α or IL-6 at violacein concentrations of 12 µmol/L or lower. However, IL-6 expression was observed at higher violacein concentrations (Table 1, Fig. 5A). IL-6 is produced by several cell types such as macrophages, dendritic cells and B cells35. It is also involved in the control of T-helper differentiation, where it promotes Th2 differentiation and simultaneously inhibits Th136. Owing to the structural similarity between mTLR8 and hTLR8 and between hTLR8 and hTLR7, we decided to research the possibility that violacein could act as an agonist of hTLR8 or hTLR7. To do this, we studied the ability of violacein to induce NF-κB in hTLR7 or hTLR8 transfected HEK-293 cells. Our results indicate that violacein is acting through hTLR8, and not via hTLR7 (Fig. 4). Interestingly, NF-κB activation in hTLR8 transfected HEK-293 cells could only be observed at cytotoxic concentrations of violacein. According to this, cell death and activation via TLR8 are associated in HEK-293 cells. To obtain further evidence that violacein is acting via hTLR8, we used CU-CPT9a, a known specific hTLR8 antagonist but not of hTLR724,25. We observed that the immunostimulatory effect of violacein in PBMCs was abolished with the highest concentration of the antagonist and the same behavior was observed when the cells were stimulated with R848 or RNA-40 (TLR7 and TLR8 agonists) but not with LPS (TLR4 agonist). These results support the idea that violacein is activating hTLR8 directly or indirectly. Furthermore, we observed that PBMC Figure 4. Effect of violacein on the induction of NF-κB in TLR-transfected HEK-293 cells with a NF-κB- luciferase reporter plasmid. (A) hTLR7 transfected HEK-293 cells were treated with indicated concentrations of violacein (V) and induction of NF-κB was determined by luciferase activity. (B) hTLR8 transfected HEK- 293 cells were treated with indicated concentrations of violacein and induction of NF-κB was determined by luciferase activity. Each data point represents one replicate and the center line indicates the mean. **p < 0.01 compared to the untreated control, ***p < 0.001 compared to the untreated control. https://doi.org/10.1038/s41598-019-50038-x 1 0SCIENTIFIC REPORTS | (2019) 9:13661 | https://doi.org/10.1038/s41598-019-50038-x www.nature.com/scientificreportswww.nature.com/scientificreports/ Figure 5. Effect of violacein and its antagonist on PBMCs (A). PBMCs from five donors were treated with 15 µmol/L violacein and the indicated controls and IL-6 production was determined by ELISA. (B) PBMCs from four donors were treated with 30 or 15 µmol/L violacein (V) or 1 µmol/L R848 and IL-1β production was determined by ELISA. (C–F). PBMCs were treated with indicated concentrations of CU-CPT9a and then stimulated with 1 µmol/L R848, 50 ng/mL LPS, 15 µmol/L violacein or 5 µg/mL RNA-40. Cell activation was evaluated by the production of IL-6. Residual IL-6 percentage was defined as the percent ratio of IL-6 in cells treated with the antagonist and the stimulator compared to control cells (amount of IL-6 production observed in the absence of the antagonist). Each data point represents an individual donor (n = 5A, C, D, E and F; n = 4 B), the center line indicates the mean. *p < 0.05 compared to the untreated control, **p < 0.01 compared to the untreated control, ***p < 0.001 compared to the untreated control. https://doi.org/10.1038/s41598-019-50038-x 1 1SCIENTIFIC REPORTS | (2019) 9:13661 | https://doi.org/10.1038/s41598-019-50038-x www.nature.com/scientificreportswww.nature.com/scientificreports/ treatment with violacein induced IL-1β secretion, which suggests the activation of inflammasome and pyropto- sis37,38. In contrast to NF-κB activation, IL-1β expression was not blocked by CU-CPT9a. Due to our observation that violacein can induce signaling through hTLR8, we decided to address the possibil- ity that violacein could interact with hTLR8. To do this, we performed molecular docking calculations to simulate the interaction between violacein and hTLR8. Our results (Fig. 6) show that violacein could interact with hTLR8 in a similar manner to the imidazoquinoline CL097 (a derivative of resiquimod), based on the X-ray structure of hTLR8 bound to this synthetic agonist28. In the best binding model of violacein to hTLR8, this ligand presents a higher affinity (∆G = −9.4 kcal/mol) for the receptor than the best model obtained for CL097 (∆G = −8.8 kcal/ mol). According to this model, violacein could interact with the following amino acids in the dimer interface of TLR8-TLR8*: F261, N262, Y348, G351, S352, Y353, V378, F405, D545*, N546*, A547*, G572*, V573*, T574*, H576* and T600*. In comparison, CL097 was found to interact with twelve amino acids (F346, Y348, G376, V378, I403, F405, V520*, D543*, D545*, T574*, G572*, V573*)28. In summary, we found that violacein could interact with six amino acids that are also involved in ligand binding in the X-ray structure of CL097 bound to hTLR8. In detail, we observed the following interactions to be analogous to those found for CL097: a hydrogen bond between the amino group of the 2-pyrrolidone ring of violacein with Thr574*, and a hydrophobic interac- tion of the 5-hydroxyindole ring of violacein with a hydrophobic pocket (Y348, V378, F405, G572* and V573*). Furthermore, specific residues (F405, Y348, T574*, D545* and D543*) have shown to be important in the acti- vation of a transduction pathway that leads to NF-κB activation28. In our molecular docking model, we observed an interaction of violacein with the majority (5 out of 6) of these residues. Regarding the effect of violacein on murine cell lines and PBMCs, we observed that treatment leads to the stim- ulation of a pro-inflammatory response. In this sense, our results are in agreement with previous data obtained with other immune cell lines9,13. Moreover, our results also offer an explanation for the anti-inflammatory effect that is observed in the animal models7,10,12: according to our gene-expression results, incubation with violacein could lead to an induction of negative feedback of TLR signaling, and promotion of pro-apoptotic processes (TNF-α, TGM2, TRP53INP1, NR4A1, BNIP3, MYC). This is also in agreement with previous results that show that violacein induces programmed cell death9,13,39–43. Apoptosis due to violacein has been studied previously in different cancer cell lines and there is no agree- ment on which cell death mechanism is activated by this substance9,39–44. These previous studies suggest that the programmed cell death activated by violacein is specific for the cancer cell lines. However, the study of the Figure 6. (A) Crystal structure of TLR8 bound to CL097 (PDB ID: 3W3J). (B–E) Docking results of ligand binding to hTLR8. (B) Docking of CL097 to hTLR8. (C) Docking of violacein to hTLR8. (D) Overlay of (A) (red), (B) (yellow) and (C) (blue). (E) Close-up view of overlay in (D). https://doi.org/10.1038/s41598-019-50038-x 1 2SCIENTIFIC REPORTS | (2019) 9:13661 | https://doi.org/10.1038/s41598-019-50038-x www.nature.com/scientificreportswww.nature.com/scientificreports/ mechanism of programmed cell death in PBMCs and in immune cell lines used in this study is beyond the scope of this manuscript. In conclusion, the current study found that violacein induces TNF-α activation at non-cytotoxic concen- trations in two murine cell lines (Raw 264.7 and ANA-1) established by retroviral infection, and cell death was observed at the highest concentration tested (12 µmol/L). Activation of PBMCs by violacein was detected at higher concentrations than used for murine cells, and was abolished by CU-CPT9a, a specific hTLR8 antagonist. Based on gene expression analysis, we found that violacein induces activation of biological processes such as an immune response, an inflammatory response, signaling through MAPK pathway, cytokine-cytokine receptor interaction and Toll-like receptor signaling in Raw 264.7 cells. Our results suggest that the observed response in PBMCs implies activation of hTLR8 signaling. Finally, according to in silico analysis, violacein could bind to hTLR8 in a similar fashion to imidazoquinoline compounds. TLR8 agonists show promise in immune therapy. For example, resiquimod, has been useful in the treatment of skin cancer45, viral skin lesions46 or as a vaccine adjuvant47,48. For this reason, we propose that violacein could have potential contribution in future immunotherapy strategies. Methods Culture of C. violaceum. C. violaceum was grown aerobically in Erlenmeyer flasks containing 500 mL of LB medium at 25 °C and shaking. Cultures were inoculated at an initial optical density (OD600) of 0.05 and grown for 17 hours. Extraction, purification and characterization of violacein. The culture of C. violaceum (1 L) was cen- trifuged (4 000 rpm at 18 °C, 15 min) and the bacterial pellet was extracted with ethanol at room temperature for 1 h. Then, the extract was sonicated for 6 min in an ultrasonic bath, centrifuged to remove cellular debris and the supernatant dried to yield a crude extract. The crude extract was washed once with water and three times with hexane, followed by sonication in an ultrasonic bath. Violacein was separated from the crude extract by crystallization with MeOH:H2O (30:70), and a violet solid was obtained. Then, the solid was loaded on a solid phase extraction (SPE) column (C8), washed with a series of MeOH:H2O mixtures of increasing polarity, and the purple fractions were combined and dried. Finally, the purple solid was purified by high performance liquid chromatography (HPLC-UV) using the following conditions: room temperature; mobile phase: 15 min 35:65 H2O:MeOH, 2 min 100% methanol and 2 min 35:65 H2O:MeOH; detector wavelength: 230 nm; stationary phase: Phenomenex Luna column C18 (250 × 4.6 mm, 10 µm). The purple fractions for each injection were combined and the final solution was dried. Violacein yield was 0.6 ± 0.1 mg. Violacein was characterized by 1H-NMR and 13C-NMR and UV-Vis. The purity of violacein was determined by reverse-phase HPLC-UV using the follow- ing conditions: room temperature; mobile phase: 0–30 min 35:65 H2O:MeOH (isocratic); detector wavelength: 230 nm; stationary phase: Phenomenex Luna semi-preparative column C18 (250 × 4.6 mm, 10 µm). The purity of violacein was 91 ± 2%. Ethics statement. The use of anonymous blood samples for this study has been approved by the local ethic committees of the Justus-Liebig-University Giessen and Philipps-University Marburg. The human samples (buffy coats from blood donors) were provided by the Institute for Clinical Immunology and Transfusion Medicine, Justus-Liebig-University Giessen, Germany. We confirm that all methods for drawing blood and preparation of buffy coats were performed in accordance with local guidelines and regulations. We also confirm that blood prod- ucts were obtained only after informed consent from the blood donors. Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by Ficoll density gradient centrifugation with LSM 1077 (PAA). The experiments with murine tissue were performed in accordance with the National German welfare law §4 (3) TierSchG and §2 and Annex 2 (TierSchVerV) of the National Order for the use of animals in research. They were approved by the Philipps-University Marburg and supervised by the corresponding animal welfare officer. Mice and cells. TLR7-deficient, TLR2/4-double deficient and C57BL/6 WT mice were kept under specific pathogen free (SPF) conditions in the animal facility of the Philipps-University of Marburg. Mouse bone marrow cells were differentiated into macrophages, myeloid or plasmacytoid DC. Primary macrophages and mDC were cultivated in RPMI supplemented with 10% FCS, 1% L-glutamine, penicillin and streptomycin, 0.1% mercap- toethanol and cultured with 20 ng/mL M-CSF (primary macrophages) or 20 ng/mL GM-CSF (mDC) in a 5% CO2 humidified atmosphere at 37 °C. pDC were cultured in Optimem supplemented with 1% FCS, 100 U/mL penicillin, 100 µg/mL streptomycin 0.05 mmol/L mercaptoethanol and with Flt-3 ligand in a 5% CO2 humidified atmosphere at 37 °C. Macrophages, mDC and pDC were seeded at 2 × 105 cells/well. Raw 264.7 and ANA-1 cells were cultured in DMEM medium supplemented with 10% heat-inactivated FBS, 1% penicillin and streptomycin in a 5% CO2 humidified atmosphere at 37 °C. Raw 264.7 and ANA-1 cells were seeded at 1 × 105 cell/well. MonoMac 6, non-differentiated THP-1 and PBMCs were cultured in RPMI medium supplemented with 10% inactivated FBS, 1% penicillin and streptomycin in a 5% CO2 humidified atmosphere at 37 °C and were seeded at 2 × 105 cell/well. Cell stimulation. Violacein was dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 2.9 mmol/L or 30 mmol/L. The desired concentration of violacein in the experiments was attained by diluting with culture media. For cell viability and cytokine induction experiments, cells were incubated for 16–20 h with positive controls or different concentrations of violacein. https://doi.org/10.1038/s41598-019-50038-x 13SCIENTIFIC REPORTS | (2019) 9:13661 | https://doi.org/10.1038/s41598-019-50038-x www.nature.com/scientificreportswww.nature.com/scientificreports/ Cell viability. Cell viability was assessed using a MTT assay according to Mosmann, T.49, with modifi- cations. Raw 264.7 cells were seeded in triplicate on a 96-well plate and incubated for 24 h at 37 °C, 5% CO2. Different concentrations of violacein were added and the cells were incubated for 24 h at 37 °C, 5% CO2 (Final volume = 200 µL). Subsequently, MTT was added to each well and the incubation was continued for 4 h at 37 °C. The media was discarded, and the formazan crystals were dissolved in acidified-isopropanol. Absorbance was read in a microplate reader (MRX revelation DYNEX Magellan Biosciences) at 570 nm with a reference wave- length of 630 nm. Cytokine measurement. Concentration of different human and murine cytokines (TNF-α, INF-α, IL-6 and IL-1β) in the culture supernatant were measured by ELISA according to the manufacturer’s instruc- tions (R&D Biosystems for murine IL-6 and human 1L-1β, BD bioscience for murine and human TNF-α and Pharmingen for human IL-6). Murine INF-α was analyzed with PBL interferon source. Cytokine production significantly above untreated control was interpreted as an activation of gene expression. Real-time qRT-PCR. Real-time qRT-PCR was performed according to Sripanidkulchai, et al.50, with modi- fications. Raw 264.7 cells were cultured in DMEM as described above and seeded (500 µL of 2 × 106 live cells/mL per well) on a 24 well plate and were incubated for 24 h at 37 °C, 5% CO2. After this, different concentrations of violacein were added and cells were incubated for another 4 h (Final volume = 1000 µL). Total RNA was extracted using RNeasy Mini kit (QIAGEN) following the manufacturer’s instructions. RNA quality was determined with the ratio of absorbance 260/280 nm in a NanoDrop spectrophotometer. Reverse transcription was performed using RevertAid RT Reverse Transcription kit (Life Technologies) following the manufacturer’s instructions. Quantitative real-time PCR was performed using an Applied Biosystems 7500 Real Time PCR system, using SYBR Green master mix (Applied Biosystems) following the manufacturer’s instructions. The temperature was 95 °C for 10 min, followed by 40 cycles of amplification (95 °C for 15 s, 60 °C for 60 s) followed by the measurement of a melting curve. The analyzed genes were TNF-α, CCL2, CXCL2, IRG1 and GAPDH as a reference gene. All primers were designed, and specific sequences and product sizes are summarized in Table 5. TNF-α (200 units per well) was used as a positive control and primers for TNF-α, CCL2, CXCL2, IRG1 were used for microarray validation. Table 5 presents the primers used in the real-time qRT-PCR. Gene expression profile. Raw 264.7 cells were seeded on a 24-well plate (1 × 106 live cells/mL per well) and were incubated for 24 h. Violacein was then added at a final concentration of 4 µmol/L per well. For the negative control, the same volume of fresh medium was added. Cells were incubated for 4 h, and all conditions were setup in triplicate. Total RNA was extracted using the RNeasy Mini kit for total RNA isolation (QIAGEN) as per the manufactur- er’s instructions. RNA samples were sent to Macrogen Inc. (Seoul, South Korea) for carrying out the microarray experiment and analysis. Genes with greater than or equal to 1.5-fold change and LPE (Local-pooled error) p-value less than 0.05 were considered significantly differentially expressed. A total of 129 genes were found to be differentially expressed. Functional and pathway analysis for genes with differential expression was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID)51,52. Pathway enrichment was determined by a Fisher exact test. A p-value of less than or equal to 0.05 and a minimum of 5 genes in the pathway were required to consider that this pathway is involved in the response to violacein. Gene expression data were deposited in NCBI’s Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo) with accession number GSE82136. TLR-transfected HEK-293 cell assay. HEK-293 cells stably expressing TLR7 or TLR8 (Invivogen, Toulouse, France) were additionally stably transfected with an NF-κB-luciferase reporter plasmid (pGL3-Gluc, Thomas Zillinger, University of Bonn, Germany) by cotransfection with the expression plasmid pMSCVpuro. After clonal expansion, clones were selected and tested. For violacein stimulation experiments, cells were seeded at 3 × 104 live cell/well in 96 well plates, and incubated with different concentrations of violacein (0.9 to 30 µmol/L) or R848 as a positive control. After 24 h of stimulation, the supernatant was collected and discarded. 50 µL of Lysis Juice (PJK, Kleinblittersdorf, Germany) was added to each well and cells were lysed by freezing mRNA Accession Gene symbol Primer 5′ to 3′ Product size (bp) NM_001289726.1 GAPDH F: TGACGTGCCGCCTGGAGAAA 98 R: AGTGTAGCCCAAGATGCCCTTCAG NM_013693 TNF-α F: CGGGCAGGTCTACTTTGGAG 166 R: ACCCTGAGCCATAATCCCCT NM_011333 Ccl2 F: CACTCACCTGCTGCTACTCA 117 R: GCTTGGTGACAAAAACTACAGC NM_009140 Cxcl2 F: TGAACAAAGGCAAGGCTAACTG 118 R: CAGGTACGATCCAGGCTTCC NM_008392 Irg1 F: CAACATGATGCTCAAGTCTGTC 101 R: TCCTCTTGCTCCTCCGAATG Table 5. Gene-specific primers used for real-time PCR. https://doi.org/10.1038/s41598-019-50038-x http://www.ncbi.nlm.nih.gov/geo 1 4SCIENTIFIC REPORTS | (2019) 9:13661 | https://doi.org/10.1038/s41598-019-50038-x www.nature.com/scientificreportswww.nature.com/scientificreports/ at −80 °C for at least 20 min. 10 µL of the lysate was mixed with 30 µL of Gaussia luciferase buffer (1.43 µmol/L Coelenterazin, 2.2 mmol/L Na2EDTA, 0.22 mol/L KxPO4, 0.44 mg/mL BSA, 1.1 mol/L NaCl, 1.3 mmol/L NaN3) and measured with a Berthold luminometer (Pforzheim, Germany). The n-fold induction was obtained by divid- ing the value of the stimulus by the media control. Synthesis of CU-CPT9a. General. All reagents and solvents were commercially available and used with- out further purification. All reactions were carried out under an argon atmosphere using Schlenck-technique. 1H-NMR spectra were recorded at 250 MHz on a Bruker Avance 250 spectrometer at 20 °C. Chemical shifts (δ) are given in ppm with the residual solvent signal used as reference. Coupling constants are reported in Hertz (Hz) using the following abbreviations for signal multiplicity: br (broad), s (singlet), d (doublet) and m (multiplet). Thin layer chromatography (TLC) was performed on precoated plates (silica gel 60 F254, Merck). Flash column chromatography was performed on prepacked columns (PF-30SIHP-JP/40 G; Interchim) using a Büchi separa- tion system. Quantitative NMR (qNMR) measurements for compound CU-CPT9a (Fig. 7 compound 5) were recorded on a Jeol ECA-500 spectrometer using maleic acid, purchased from Sigma-Aldrich (99.94% purity), as internal reference standard. 2-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-phenol (Fig. 7 compound 3) was prepared via a slightly modified literature procedure24 as follows: In a three-necked flask 5.88 g potassium acetate (60.0 mmol, 3.00 eq) were dried in situ (200 °C, 4 × 10−3 mbar) and suspended in anhydrous 1,4-dioxane (300 mL). 3.74 g 4-bromo-2-methylphenol (20.0 mmol, 1.00 eq), 6.10 g bis-(pinacolato)-diboron (24.0 mmol, 1.20 eq) and 820 mg [PdCl2(dppf)]*CH2Cl2 (1.00 mmol, 0.05 eq) were added and the orange-red suspension was stirred under argon atmosphere at 90 °C for 17 h. After cooling down to room temperature, 300 mL water were added and the suspen- sion was extracted with ethyl acetate (3 × 150 mL). The combined organic layers were washed with brine, dried over magnesium sulfate, filtered and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography (silica gel, eluent: dichloromethane) to give 4.29 g of the title compound as a beige solid (18.3 mmol, 92% yield). 1H-NMR (250 MHz, CDCl3) δ 7.60 (s, 1H), 7.56 (d, J = 7.9 Hz, 1H), 6.76 (d, J = 7.9 Hz, 1H), 4.95 (s, br, 1H), 2.25 (s, 3H), 1.33 (s, 12H) ppm. All recorded spectra are in accordance to literature24. 4-(7-Methoxyquinolin-4-yl)-2-methylphenol (CU-CPT9a, Fig.  7 compound 5) was prepared via a slightly modified literature procedure24 as follows: In a nitrogen-flask 2.66 g 2-methyl-4-(4,4,5,5-tetramethyl- 1,3,2-dioxaborolan-2-yl)-phenol (11.4 mmol, 1.10 eq), 2.00 g 4-chloro-7-methoxyquinoline (10.3 mmol, 1.00 eq) and 4.27 g potassium carbonate (30.9 mmol, 3.00 eq) were flushed with argon. The mixture was suspended in 1,4-dioxane (120 mL) and water (20 mL) and degassed. Afterwards, 425 mg [PdCl2(dppf)]*CH2Cl2 (0.52 mmol, 0.05 eq) were added and the orange-red suspension was heated at 100 °C until TLC indicated completion of the reaction (18 h). Subsequently, the mixture was concentrated to 50 mL under reduced pressure and filtered over Celite®. The filtrate was diluted with water (100 mL) and extracted with ethyl acetate (3 × 50 mL). The com- bined organic layers were dried over magnesium sulfate, filtered and the solvent was removed under reduced Figure 7. Synthesis of CU-CPT9a. Structures of 4-bromo-2-methylphenol (1), bis-(pinacolato)-diboron (2), 2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-phenol (3), 4-chloro-7-methoxyquinoline (4) and 4-(7-methoxyquinolin-4-yl)-2-methylphenol (CU-CPT9a, 5). https://doi.org/10.1038/s41598-019-50038-x 1 5SCIENTIFIC REPORTS | (2019) 9:13661 | https://doi.org/10.1038/s41598-019-50038-x www.nature.com/scientificreportswww.nature.com/scientificreports/ pressure. The residue was purified by flash column chromatography (silica gel, eluent: dichloromethane → dichloromethane/methanol 99:1) to give 2.17 g of the title compound as a white solid (8.16 mmol, 79% yield). Purity: 97.4 ± 0.3% (1H-qNMR). 1H-NMR (250 MHz, DMSO-d6) δ 9.69 (s, 1H), 8.79 (d, J = 4.6 Hz, 1H), 7.85 (d, J = 9.3 Hz, 1H), 7.44 (d, J = 2.3 Hz, 1 H), 7.25–7.20 (m, 3 H), 6.95 (d, J = 8.2 Hz, 1H), 3.92 (s, 3H), 2.21 (s, 3H) ppm. All recorded spectra are in accordance to literature24. Inhibition of hTLR8 activity by CU-CPT9a. Human PBMCs were cultured (50 µL of 6 × 106 live cells/ mL) in a 96-well plate. Cells were treated with different concentrations of CU-CPT9a (0.02, 0.2, 2 and 20 µmol/L) or medium as a negative control and were incubated for 1 h at 37 °C, 5% CO2. After this, cells were treated with violacein (30 and 15 µmol/L), R848 (1 µmol/L), LPS (50 ng/mL) or RNA-40 (5 µg/mL). After incubating for 20 h, the culture supernatants were collected and used to measure the concentration of IL-6 or IL-1β by ELISA accord- ing to the manufacturer’s instructions. Molecular docking. Molecular docking studies were performed using AutoDock Vina53. The three dimen- sional structure of hTLR8 complexed to 2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-4-amine (CL097) was extracted from the Protein Data Bank (PDB ID 3W3J,28). The structure of the ligand was drawn with Marvin Sketch. The structures of ligand and receptor were prepared using AutoDock Tools 1.5.6 (ADT). In brief, only the dimer interface of hTLR8-hTLR8* (leucine-rich repeat 11- leucine-rich repeat 14 and leucine-rich repeat 16*- leucine-rich repeat 18*) that is known to be involved in the interaction with the ligands was selected (center x = 18, y = −22 and z = 22 and size x = 20, y = 20 and z = 20), to facilitate docking calculations. Additionally, solvent and ligand molecules were deleted and polar hydrogen and charges were added to the structure. Docking calculations were performed for the interaction between hTLR8 and violacein or 2-(ethoxyme- thyl)-1H-imidazo[4,5-c]quinolin- 4-amine (CL097). All protein and ligand structures were built and saved as pdbqt format, and docking was performed with the selected region of the protein, as mentioned above. Visualization and generation of images of possible binding models for violacein or CL097 with hTLR8 were per- formed using UCSF Chimera54. Statistical analysis. Statistical significance of the effect of incubation with violacein (qRT-PCR and ELISA) and NF-κB fold induction was determined by analysis of variance (ANOVA). When a significant effect was found, Dunnett’s post-hoc test was used to contrast the effect of treated cells with the negative control. In cases where the data did not satisfy the normality and homoscedasticity assumptions, the data were log transformed and statis- tical significance was determined by ANOVA and Dunnett’s post-hoc test. A global p value lower than 0.05 was considered statistically significant. Results of microarray confirmation were analyzed as fold change compared to untreated cells, and a t-test was performed for each gene. A two-tailed p-value lower than 0.05 was considered statistically significant. References 1. Durán, M. et al. Potential applications of violacein: a microbial pigment. Med. Chem. Res. 21, 1524–1532 (2012). 2. Lopes, S. C. P. et al. Violacein Extracted from Chromobacterium violaceum Inhibits Plasmodium Growth In Vitro and In Vivo. Antimicrob. Agents Chemother. 53, 2149–2152 (2009). 3. Pantanella, F. et al. Violacein and biofilm production in Janthinobacterium lividum. J. Appl. Microbiol. 102, 992–999 (2007). 4. Hoshino, T. Violacein and related tryptophan metabolites produced by Chromobacterium violaceum: biosynthetic mechanism and pathway for construction of violacein core. Appl. Microbiol. Biotechnol. 91, 1463–1475 (2011). 5. Dodou, H. V. et al. Violacein antimicrobial activity on Staphylococcus epidermidis and synergistic effect on commercially available antibiotics. J. Appl. Microbiol. 123, 853–860 (2017). 6. Andrighetti-Frohner, C., Antonio, R., Creczynski-Pasa, T., Barardi, C. & Simoes, C. Cytotoxicity and Potential Antiviral Evaluation of Violacein Produced by Chromobacterium violaceum. Mem. Inst. Oswaldo Cruz 98, 843–848 (2003). 7. Antonisamy, P. & Ignacimuthu, S. Immunomodulatory, analgesic and antipyretic effects of violacein isolated from Chromobacterium violaceum. Phytomedicine 17, 300–304 (2010). 8. Durán, N. et al. Violacein: properties and biological activities. Biotechnol. Appl. Biochem. 48, 127–133 (2007). 9. Ferreira, C. V. et al. Molecular mechanism of violacein-mediated human leukemia cell death. Blood 104, 1459–1464 (2004). 10. Antonisamy, P. et al. Gastroprotective activity of violacein isolated from Chromobacterium violaceum on indomethacin-induced gastric lesions in rats: investigation of potential mechanisms of action. ScientificWorldJournal. 2014, 616432 (2014). 11. Platt, D. et al. Violacein inhibits matrix metalloproteinase mediated CXCR4 expression: Potential anti-tumor effect in cancer invasion and metastasis. Biochem. Biophys. Res. Commun. 455, 107–112 (2014). 12. Verinaud, L. et al. Violacein Treatment Modulates Acute and Chronic Inflammation through the Suppression of Cytokine Production and Induction of Regulatory T Cells. PLoS One 10, 1–16 (2015). 13. Alshatwi, A. A., Subash-Babu, P. & Antonisamy, P. Violacein induces apoptosis in human breast cancer cells through up regulation of BAX, p53 and down regulation of MDM2. Exp. Toxicol. Pathol. Off. J. Gesellschaft für Toxikologische Pathol. 68, 89–97 (2016). 14. Rath, P. C. & Aggarwal, B. B. TNF-Induced Signaling in Apoptosis. J. Clin. Immunoly 19, 350–364 (1999). 15. Wajant, H., Pfizenmaier, K. & Scheurich, P. Tumor necrosis factor signaling. Cell Death Differ. 10, 45–65 (2003). 16. Bradley, J. R. TNF-mediated inflammatory disease. J. Pathol. 214, 149–160 (2008). 17. Fukata, M., Vamadevan, A. S. & Abreu, M. T. Toll-like receptors (TLRs) and Nod-like receptors (NLRs) in inflammatory disorders. Semin. Immunol. 21, 242–253 (2009). 18. Moresco, E. M. Y., LaVine, D. & Beutler, B. Toll-like receptors. Curr. Biol. 21, R488–93 (2011). 19. Rettori, D. & Durán, N. Production, extraction and purication of violacein: an antibiotic pigment produced by Chromobacterium violaceum. World J. Microbiol. Biotechnol. 14, 685–688 (1998). 20. Fulmer, G. R. et al. NMR Chemical Shifts of Trace Impurities: Common Laboratory Solvents, Organics, and Gases in Deuterated Solvents Relevant to the Organometallic Chemist. Organometallics 29, 2176–2179 (2010). 21. Agrawal, P. K., Bush, C. A., Qureshi, N. & Takayama, K. 1H and 13C NMR assignments of a lipopolysaccharide obtained from the deep rough mutant of Escherichia coli D31m4. Magn. Reson. Chem. 36, 1–7 (1998). 22. Gisch, N. et al. NMR-based Structural Analysis of the Complete Rough-type Lipopolysaccharide Isolated from Capnocytophaga canimorsus. J. Biol. Chem. 289, 23963–23976 (2014). https://doi.org/10.1038/s41598-019-50038-x 1 6SCIENTIFIC REPORTS | (2019) 9:13661 | https://doi.org/10.1038/s41598-019-50038-x www.nature.com/scientificreportswww.nature.com/scientificreports/ 23. Tsikas, D. Analysis of nitrite and nitrate in biological fluids by assays based on the Griess reaction: Appraisal of the Griess reaction in the L-arginine/nitric oxide area of research. J. Chromatogr. B 851, 51–70 (2007). 24. Hu, Z. et al. Small-Molecule TLR8 Antagonists via Structure-Based Rational Design. Cell Chem. Biol. 25, 1–6 (2018). 25. Zhang, S. et al. Small-molecule inhibition of tlr8 through stabilization of its resting state. Nat. Chem. Bio 14, 58–66 (2018). 26. Salio, M. et al. Modulation of human natural killer T cell ligands on TLR-mediated antigen-presenting cell activation. Proc. Natl. Acad. Sci. USA 104, 20490–20495 (2007). 27. Butchi, N. B., Pourciau, S., Du, M., Morgan, T. W. & Peterson, K. E. Analysis of the Neuroinflammatory Response to TLR7 Stimulation in the Brain: Comparison of Multiple TLR7 and/or TLR8 Agonists. J. Immunol. 180, 7604–7612 (2008). 28. Tanji, H., Ohto, U., Shibata, T., Miyake, K. & Shimizu, T. Structural Reorganization of the Toll-Like Receptor 8 Dimer Induced by Agonistic Ligands. Science (80-.). 339, 1426–1430 (2013). 29. Chen, W., Yuan, F., Wang, K., Song, D. & Zhang, W. Modulatory effects of the acid polysaccharide fraction from one of anamorph of Cordyceps sinensis on Ana-1 cells. J. Ethnopharmacol. 142, 739–745 (2012). 30. Ralston, N. V. C. & Hunt, C. D. Transmembrane Partitioning of Boron and Other Elements in RAW 264.7 and HL60 Cell Cultures. Biol. Trace Elem. Res. 98, 181–191 (2004). 31. Gorden, K. K. B., Qiu, X. X., Christine, C. A., Vasilakos, J. P. & Alkan, S. S. Cutting Edge: Activation of Murine TLR8 by a Combination of Imidazoquinoline Immune Response Modifiers and PolyT Oligodeoxynucleotides. J. Immunol. 177, 6584–6587 (2006). 32. Li, T., He, X., Jia, H., Chen, G. & Zeng, S. Molecular cloning and functional characterization of murine toll-like receptor 8. Mol. Med. Rep. 13, 1119–1126 (2016). 33. Li, Y. et al. Immune Responsive Gene 1 (IRG1) promotes endotoxin tolerance by increasing A20 expression in macrophages through reactive oxygen species. J. Biol. Chem. 288, 16225–16234 (2013). 34. Shembade, N. & Harhaj, E. W. Regulation of NF-κB signaling by the A20 deubiquitinase. Cell. Mol. Immunol. 9, 123–130 (2012). 35. Scheller, J., Chalaris, A., Schmidt-arras, D. & Rose-john, S. The pro- and anti-in fl ammatory properties of the cytokine interleukin-6. Biochim. Biophys. Acta 1813, 878–888 (2011). 36. Diehl, S. & Rincón, M. The two faces of IL-6 on Th1/Th2 differentiation. Mol. Immunol. 39, 531–536 (2002). 37. He, Y., Hara, H. & Núñez, G. Mechanism and Regulation of NLRP3 Inflammasome Activation. Trends Biochem. Sci. 41, 1012–1021 (2016). 38. Vanaja, S., Rathinam, V. K. & Fitzgerald, K. A. Mechanisms of inflammasome activation: recent advances and novel insights. Trends Cell Biol. 25, 308–315 (2016). 39. Ahmadi Fakhr, F. An investigation of antileukemia activity of violacein-loaded dendrimer in Jurkat cell lines. African J. Microbiol. Res. 6, 6235–6242 (2012). 40. Hoover, R. B., Yusuf, N. & Bej, A. K. The antiproliferative function of violacein-like purple violet pigment (PVP) from an Antarctic Janthinobacterium sp. Ant5-2 in UV-induced 2237 fibrosarcoma. Int J Dermatol 50, 1223–1233 (2013). 41. de Carvalho, D. D., Costa, F. T. M., Duran, N. & Haun, M. Cytotoxic activity of violacein in human colon cancer cells. Toxicol. Vitr. 20, 1514–1521 (2006). 42. Leal, A. M. D. S., de Queiroz, J. D. F., de Medeiros, S. R. B., Lima, T. K. D. S. & Agnez-Lima, L. F. Violacein induces cell death by triggering mitochondrial membrane hyperpolarization in vitro. BMC Microbiol. 15, 115 (2015). 43. Queiroz, K. C. S. et al. Violacein Induces Death of Resistant Leukaemia Cells via Kinome Reprogramming, Endoplasmic Reticulum Stress and Golgi Apparatus Collapse. PLoS One 7, 1–8 (2012). 44. Kodach, L. L. et al. Violacein synergistically increases 5-fluorouracil cytotoxicity, induces apoptosis and inhibits Akt-mediated signal transduction in human colorectal cancer cells. Carcinogenesis 27, 508–516 (2006). 45. Micali, G., Lacarrubba, F., Nasca, M. R. & Schwartz, R. A. Topical pharmacotherapy for skin cancer Part I. Pharmacology. J. Am. Acad. Dermatol. 70, 965.e1–965.e12 (2014). 46. Meyer, T., Surber, C., French, L. E. & Stockfleth, E. Resiquimod, a topical drug for viral skin lesions and skin cancer. Expert Opin. Investig. Drugs 22, 149–59 (2013). 47. Smits, E. L. J. M., Ponsaerts, P., Berneman, Z. N. & Van Tendeloo, V. F. I. The use of TLR7 and TLR8 ligands for the enhancement of cancer immunotherapy. Oncologist 13, 859–875 (2008). 48. Vasilakos, J. P. & Tomai, M. A. The use of Toll-like receptor 7/8 agonists as vaccine adjuvants. Expert Rev. Vaccines 12, 809–819 (2013). 49. Mosmann, T. Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays. J. Immunol. Methods 65, 55–63 (1983). 50. Sripanidkulchai, B., Junlatat, J., Wara-aswapati, N. & Hormdee, D. Anti-inflammatory effect of Streblus asper leaf extract in rats and its modulation on inflammation-associated genes expression in RAW 264.7 macrophage cells. J. Ethnopharmacol. 124, 566–570 (2009). 51. Huang, D. W., Sherman, B. T. & Lempicki, Ra Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009). 52. Huang, D. W., Sherman, B. T. & Lempicki, Ra Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1–13 (2009). 53. Trott, O. & Olson, A. J. AutoDock Vina: Improving the Speed and Accuracy of Docking with a New Scoring Function, Efficient Optimization, and Multithreading. J. Comput. Chem. 31, 455–461 (2009). 54. Pettersen, E. F. et al. UCSF Chimera–A Visualization System for Exploratory Research and Analysis. J. Comput. Chem. 25, 1605–12 (2004). Acknowledgements We thank M.Sc. Luis M. Quirós and PhD. Giselle Tamayo from Universidad de Costa Rica for their advice and help in the purification of violacein. This work was supported by projects UCR 801-B2-519 (F.V., A.G.P.), MICITT FI-497-11 (F.V., A.G.P.), DFG-TR84 (F.V., S.B.) and DFG-KFO325 (W.D., S.B.). Author Contributions F.V. conducted the experiments of culture of C. violaceum, extraction, purification and characterization of violacein, cell culture, cell viability, Real-time qRT-PCR, gene expression profile experiments, cell stimulation and ELISA experiments. G.K. conducted and planned the experiments with TLR-transfected HEK-293 cells. K.M. conducted the synthesis of CU-CPT9a. W.D. helped in the design and supervised the synthesis of CU-CPT9a. A.K. planned and supervised ELISA experiments. S.B. helped in the design and supervised TLR-transfected HEK-293 cells and ELISA experiments. M.C. helped in the design and analysis of culture of C. violaceum and extraction experiments. J.J.A. helped in the design and analysis of purification and identification of violacein experiments. A.G.P. helped in design and analysis of cell culture, cell viability, Real-time qRT-PCR and gene expression profile experiments. F.V. and A.G.P. wrote the main manuscript text and prepared figures. All authors reviewed the manuscript. https://doi.org/10.1038/s41598-019-50038-x 17SCIENTIFIC REPORTS | (2019) 9:13661 | https://doi.org/10.1038/s41598-019-50038-x www.nature.com/scientificreportswww.nature.com/scientificreports/ Additional Information Competing Interests: The authors declare no competing interests. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2019 https://doi.org/10.1038/s41598-019-50038-x http://creativecommons.org/licenses/by/4.0/ The Bacterial Product Violacein Exerts an Immunostimulatory Effect Via TLR8 Results Purity of violacein. Effect of violacein on different murine cell types. Gene expression pattern induced by violacein in Raw 264.7 cells. Effect of violacein on different human cell lines and PBMCs. Effect of violacein on HEK-293 cells transfected with hTLR8 or hTLR7. Blockade of violacein effect on PBMCs by a specific hTLR8 antagonist. Molecular docking of violacein on TLR8. Discussion Methods Culture of C. violaceum. Extraction, purification and characterization of violacein. Ethics statement. Mice and cells. Cell stimulation. Cell viability. Cytokine measurement. Real-time qRT-PCR. Gene expression profile. TLR-transfected HEK-293 cell assay. Synthesis of CU-CPT9a. Inhibition of hTLR8 activity by CU-CPT9a. Molecular docking. Statistical analysis. Acknowledgements Figure 1 Chemical structure of violacein (3-(1,2-dihydro- 5-(5-hydroxy-1H-indol-3-yl)-2-oxo-3H-pyrrol-3-ilydene)-1,3-dihydro-2H-indol-2-one). Figure 2 Cytotoxic effect of violacein on Raw 264. Figure 3 Effect of violacein on TNF-α production in Raw 264. Figure 4 Effect of violacein on the induction of NF-κB in TLR-transfected HEK-293 cells with a NF-κB-luciferase reporter plasmid. Figure 5 Effect of violacein and its antagonist on PBMCs (A). Figure 6 (A) Crystal structure of TLR8 bound to CL097 (PDB ID: 3W3J). Figure 7 Synthesis of CU-CPT9a. Table 1 Activation of different cell lines by violacein. Table 2 Differentially expressed genes in Raw 264. Table 3 Confirmation of microarray results by comparison with real-time qRT-PCR for selected differentially expressed genes. Table 4 Biological terms significantly associated with differential gene expression. Table 5 Gene-specific primers used for real-time PCR.