**3. Results**

### *3.1. Impact of Fusarium Secondary Metabolites on LPS-Induced NF-*κ*B Activation*

Modulatory effects of DON and NX-3 and potential combinatory effects with the pro-oxidant co-contaminant AURO on LPS-induced NF-κB pathway activation were assessed in THP-1 NF-κB Luc Reporter Monocytes (Figure 2A). DON (1 μM), as well as NX-3, significantly increased the luciferase signal up to 241% ± 35% (DON) and 207% ± 29% of the LPS-induced signal (solid line, 100%). AURO caused an increase of luciferase signal limited to 139% ± 6% of the LPS-induced signal at 0.01 μM, whereas the other concentrations did not significantly modulate NF-κB activity. Combined incubations of AURO and DON or NX-3 resulted in a slightly, but not significantly reduced luminescence intensity compared to effects caused by DON and NX-3 alone.

Cell viability was monitored using the alamarBlue® assay and all data were normalized to the evaluated metabolic activity (Figure 2B). A pronounced decrease of the fluorescence signal was determined in cells incubated with 5 and 10 μM DON and NX-3 and the respective combinations with 0.5 and 1 μM AURO, in line with a substantially decreased NF-κB activity. In most tested conditions, similar effects of NX-3 and DON could be observed. Only in the case of 5 μM NX-3 and the combination with 0.5 μM AURO, which showed similar effects on cell viability as the respective DON-treated samples, significant differences in NF-κB activity were determined.

**Figure 2.** Activity of nuclear factor kappa B (NF-κB) in (**A**) lipopolysaccharide (LPS)-stimulated human monocytic THP1-Lucia™ NF κB cells. THP1-Lucia NF-κB cells were preincubated with the compounds (DON, NX-3, AURO and their combinations) for 2 h followed by an 18 h LPS challenge (10 ng/mL). Heat-killed *Listeria monocytogenes* (HKLM; 20 × 10<sup>6</sup> cells/well) served as positive control for Toll-like receptor-mediated activation of the NF-κB pathway. Luminescence intensity data are expressed as mean values ± SE normalized to LPS-treated solvent control and to the respective cell viability data, assessed in (**B**) the alamarBlue® cell viability assay of at least five independent experiments. One percent DMSO and 1% water (LC–MS grade) served as solvent control (dotted line). Significant differences to LPS, calculated with one-sample *t*-test, are indicated with \* (*p* < 0.05), \*\* (*p* < 0.01) and \*\*\* (*p* < 0.001), whereas differences between DON and NX-3, calculated with a two-sample *t*-test, are indicated with ◦ (*p* < 0.05).

### *3.2. Modulation of Cytokine Gene Transcription by Fusarium Secondary Metabolites*

In order to assess the effects of DON and NX-3 (1 μM) and also their combination with AURO (0.1 μM) on NF-κB-dependent cytokine transcription, two colon cell lines, the cancer cells HT-29 and the noncancer cells HCEC-1CT, were exposed to the *Fusarium* secondary metabolites in the presence of the pro-inflammatory stimulus IL-1β (25 ng/mL). In HT-29 cells (Figure 3A), both trichothecene mycotoxins DON and NX-3 significantly increased TNF-<sup>α</sup>, IL-1β and IL-8 mRNA levels, whereas AURO did not lead to alterations of the analyzed cytokine transcription levels. Combinatory treatments of DON/AURO or NX-3/AURO resulted again in a slightly decreased signal when compared to the effects of the respective trichothecene single treatments.

**Figure 3.** Relative gene transcription levels of TNF-<sup>α</sup>, IL-1β, IL-8 and IL-6 in (**A**) HT-29 and (**B**) HCEC-1CT cells (calibrator was IL-1β-treated solvent control, which was set to 1). Cells were preincubated with the compounds (DON, NX-3, AURO and their combinations) for 2 h followed by a 3 h IL-1β challenge (25 ng/mL). Relative transcript levels were measured with qRT-PCR. Data are expressed as mean values ± SD normalized to IL-1β-treated solvent control samples of at least five independent experiments. One percent DMSO and 1% water (LC–MS grade) served as solvent control. Significant differences to IL-1β-stimulation, calculated with two-sample *t*-test, are indicated with \* (*p* < 0.05), \*\* (*p* < 0.01) and \*\*\* (*p* < 0.001).

In the noncancer cell line HCEC-1CT, a different cytokine pattern could be identified (Figure 3B). While in HT-29 the strongest induction was found for IL-1β transcription, followed by TNF-α and IL-8, in HCEC-1CT more TNF-α mRNA was present compared to the transcript levels of the other tested cytokines. In addition to IL-8 and IL-1β, IL-6 mRNA also could be identified in HCEC-1CT samples. While DON and NX-3 enhanced significantly the cytokine mRNA levels of all four target genes tested, respective cytokine transcripts were only marginally modulated after AURO treatment. Upon co-incubation of DON or NX-3 with AURO, increased mRNA levels similar to those following DON and NX-3 single substance treatment were identified.

When comparing the TNF-α transcription levels of the two colon cell lines, significant differences were observed (Figure 4A). While in the samples exposed to DON or NX-3 as single compounds no significant differences in TNF-α gene transcription were determined, combination with AURO decreased the TNF-α mRNA levels in HT-29 (DON/AURO: 1.6 ± 0.2 and NX-3/AURO: 1.3 ± 0.3 rel. transcription). These differences reached statistical significance in comparison to levels detected in HCEC-1CT cells. Combinations with AURO in the noncancer cell line resulted namely even in an increase in TNF-α gene transcription compared to the single compound treatments (DON/AURO: 2.7 ± 0.7 and NX-3/AURO: 2.7 ± 0.5 rel. transcription). Regarding IL-1β gene transcription (Figure 4B), significant differences between the two cell lines were already present in the samples exposed to DON and NX-3 as single compounds (DON: 3.1 ± 0.8 in HT-29 and 1.5 ± 0.2 in HCEC-1CT rel. transcription; NX-3: 2.8 ± 1.1 in HT-29 and 1.4 ± 0.3 in HCEC-1CT rel. transcription). Comparable to the effects in HT-29 cells, a slight decrease in IL-1β mRNA levels was observed, reaching statistical significance in the case of co-incubation with DON and AURO. IL-8 transcription levels did not differ between the two cell lines (Figure 4C).

**Figure 4.** Relative gene transcription levels of (**A**) TNF-<sup>α</sup>, (**B**) IL-1β and (**C**) IL-8 in HT-29 and HCEC-1CT cells (calibrator was IL-1β-treated solvent control, which was set to 1). Cells were preincubated with the fungal metabolites (DON, NX-3, AURO and their combinations) for 2 h followed by a 3 h IL-1β challenge (25 ng/mL). Relative transcript levels were measured with qRT-PCR. Data are expressed as mean values ± SD normalized to IL-1β-treated solvent control samples of at least five independent experiments. One percent DMSO and 1% water (LC–MS grade) served as solvent control. Significant differences between the two cell lines, calculated with two-sample *t*-test, are indicated with \* (*p* < 0.05), \*\* (*p* < 0.01) and \*\*\* (*p* < 0.001).

### *3.3. E*ff*ects of Fusarium Secondary Metabolites on Cell Viability*

In order to rule out cytotoxicity potentially compromising the analysis of immunomodulatory effects, the impact of the tested concentrations on cell viability was determined by the SRB (Figure 5A,B) and the alamarBlue® assay (Figure 5C,D). In both cell lines, pronounced effects on the cellular protein content and on the metabolic activity after DON and NX-3 treatment for 5 h (last 3 h co-exposed to 25 ng/mL IL-1β) could be identified at concentrations ≥ 5 μM. AURO did not trigger any significant effects on cell viability except for the highest tested concentration (10 μM), which caused a pronounced decrease of the fluorescence signal in the alamarBlue® assay in both cell lines. Partly significant differences between the samples treated in combination with AURO and the DON- or NX-3-single incubations were determined (Figure 5A–D, highlighted with ◦ symbols). However, due to the fact that the observed effects were of rather limited nature, no appropriate mathematical model for a correct evaluation of the combinatory interactions (e.g., the model of independent joint action [37] or the multiple drug effect equation [38]) could be applied.

**Figure 5.** Effects of DON, NX-3, AURO and their combinations on the cellular protein content (**A,B**) and viability (**C,D**) of the two human colon cell lines HT-29 and HCEC-1CT determined in the sulforhodamine B (HT-29: **A**; HCEC-1CT: **B**) and alamarBlue® assay (HT-29: **C**; HCEC-1CT: **D**). Cells were preincubated with the compounds (DON, NX-3, AURO and their combinations) for 2 h followed by a 3 h IL-1β challenge (25 ng/mL). Data are expressed as mean values ± SD normalized to IL-1β-treated solvent control samples of at least five independent experiments. One percent DMSO and 1% water (LC–MS grade) served as solvent control (dotted line). Significant differences to IL-1β-treated solvent control, calculated with one-sample *t*-test, are indicated with \* (*p* < 0.05), \*\* (*p* < 0.01) and \*\*\* (*p* < 0.001), whereas differences between DON, NX-3 and their combinations with AURO, calculated with a two-sample *t*-test, are indicated with ◦ (*p* < 0.05), ◦◦ (*p* < 0.01) and ◦◦◦ (*p* < 0.001).
