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. 2020 Apr 21;8(4):603.
doi: 10.3390/microorganisms8040603.

Pro-Inflammatory Effects of NX-3 Toxin Are Comparable to Deoxynivalenol and not Modulated by the Co-Occurring Pro-Oxidant Aurofusarin

Lydia Woelflingseder  1 , Nadia Gruber  1 , Gerhard Adam  2 , Doris Marko  1
Affiliations

Pro-Inflammatory Effects of NX-3 Toxin Are Comparable to Deoxynivalenol and not Modulated by the Co-Occurring Pro-Oxidant Aurofusarin

Lydia Woelflingseder et al. Microorganisms. .

Abstract

The type A trichothecene NX-3, produced by certain Fusarium graminearum strains, is similar to the mycotoxin deoxynivalenol (DON), with the exception that it lacks the carbonyl moiety at the C-8 position. NX-3 inhibits protein biosynthesis and induces cytotoxicity to a similar extent as DON, but so far, immunomodulatory effects have not been assessed. In the present study, we investigated the impact of NX-3 on the activity of the nuclear factor kappa B (NF-κB) signaling pathway in direct comparison to DON. Under pro-inflammatory conditions (IL-1β treatment), the impact on cytokine mRNA levels of NF-κB downstream genes was studied in human colon cell lines, comparing noncancer (HCEC-1CT) and cancer cells (HT-29). In addition, potential combinatory effects with the co-occurring Fusarium secondary metabolite aurofusarin (AURO), a dimeric naphthoquinone known to induce oxidative stress, were investigated. NX-3 and DON (1 μM, 20 h) significantly activated a NF-κB regulated reporter gene to a similar extent. Both trichothecenes also enhanced transcript levels of the known NF-κB-dependent pro-inflammatory cytokines IL-8, IL-6, TNF-α and IL-1β. Comparing the colon cancer HT-29 and noncancer HCEC-1CT cells, significant differences in cytokine signaling were identified. In contrast, AURO did not affect NF-κB pathway activity and respective cytokine expression levels at the tested concentration. Despite its pro-oxidant potency, the combination with AURO did not significantly affect the immunomodulatory effects of the tested trichothecenes. Taken together, the present study reveals comparable potency of DON and NX-3 with respect to immunomodulatory and pro-inflammatory potential. Consequently, not only DON but also NX-3 should be considered as factors contributing to intestinal inflammatory processes.

Keywords: NF-κB; combinatory effects; food safety; intestinal inflammation; mycotoxin; trichothecene.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Chemical structures of the investigated Fusarium secondary metabolites: (A) deoxynivalenol (DON), (B) type A trichothecene (NX-3) and (C) aurofusarin (AURO).
Figure 2
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 × 106 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).
Figure 3
Figure 3
Relative gene transcription levels of TNF-α, 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).
Figure 4
Figure 4
Relative gene transcription levels of (A) TNF-α, (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).
Figure 5
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).
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