**1. Introduction**

Mycotoxins are toxic, secondary metabolites produced by certain filamentous fungi, mainly belonging to the genera of *Fusarium, Aspergillus, Penicillium* and *Alternaria.* Contaminating food and feed pre- and postharvest, mycotoxins pose a potential risk to food safety and thus to human and animal health. After ingestion, the intestinal tract represents the first barrier of the host against food contaminants such as mycotoxins and is therefore the first line for many defense mechanisms. In order to protect the entire organism from the entrance of unwanted solutes, microorganisms and luminal antigens, a proper function of the intestinal barrier and its innate immune response is crucial. As a defense mechanism against external stressors, intestinal epithelial cells are able to secrete cytokines and chemokines, including transforming growth factorα (TGFα), interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8) or interleukin-10 (IL-10) in order to activate the immune response and to

recruit respective immune cells [1]. However, if not controlled properly, excessive immune response and cytokine release can lead to chronic intestinal inflammation and contribute to the progression of inflammatory disorders, such as inflammatory bowel diseases (IBDs) [2].

One of the most prevalent mycotoxins in temperate climate regions is the trichothecene mycotoxin deoxynivalenol (DON, vomitoxin, Figure 1A), which frequently contaminates grain- and cereal-based products [3–5]. Human biomonitoring studies revealed that, due to its ubiquitous occurrence, consumers are chronically exposed to low levels of DON [6–8]. The epoxide moiety at position C12-C13 is considered a key factor in the toxin's main mechanism of action, the inhibition of eukaryotic protein synthesis. By binding to the 60S ribosomal subunit, DON causes a ribotoxic stress response [9,10], resulting in the activation of mitogen-activated protein kinases (MAPKs) and furthermore in the induction of apoptosis and inflammation [11]. Depending on dose, exposure frequency and duration, DON can induce both immunostimulatory and immunosuppressive e ffects [11–13]. Low dose exposure caused transcriptionally and post-transcriptionally upregulation of immunostimulating cytokines and chemokines, whereas high dose exposure was shown to promote apoptosis with concomitant immunosuppressive e ffects [11,13]. Modulatory e ffects of DON on cytokine production in intestinal tissue and intestinal epithelial cancer cell lines have been studied and reviewed extensively during the last decades [11,14,15]. DON was reported to induce the secretion of IL-8 via a nuclear factor "kappa-light-chain-enhancer of activated B cells"-mediated (NF-κB) mechanism in various human intestinal epithelial cells such as Caco-2 or HT-29 [16–18]. When mimicking an inflamed intestinal epithelium, co-exposure experiments with various pro-inflammatory stimuli including interleukin-1β (IL-1β), the tumor necrosis factorα (TNFα) or lipopolysaccharide (LPS) resulted in additive and synergistic e ffects regarding cytokine secretion and NF-κB activation [16]. However, the e ffects of DON on the inflammatory response have so far only been reported in cancer cell models, but not in noncancer intestinal epithelial cells.

**Figure 1.** Chemical structures of the investigated *Fusarium* secondary metabolites: ( **A**) deoxynivalenol (DON), (**B**) type A trichothecene (NX-3) and ( **C**) aurofusarin (AURO).

Moreover, no toxicological characterization of the recently discovered type A trichothecene NX-3 (Figure 1B) regarding immunomodulatory e ffects has been performed to date. Structurally identified by Varga et al. [19] as an analogue of DON lacking the carbonyl moiety at the C8-position, this toxin was found to be produced by *F. graminearum* [20,21]. In vitro translation assays showed a similar inhibitory potency on protein biosynthesis as DON [19]. Likewise, cytotoxicity studies revealed comparable effects of both trichothecenes in human liver cancer (HepG2), nontransformed colon cells (HCEC-1CT) and colon cancer cells (HT-29) [22,23]. However, potential inflammatory properties of NX-3 have not been investigated yet.

Among many other aspects, oxidative stress plays an essential role in the pathogenesis and progression of IBDs [24]. Another *Fusarium* secondary metabolite reported to induce oxidative stress in human colon adenocarcinoma cells HT-29, is the coloring pigment aurofusarin (AURO, Figure 1C) [25]. Even though AURO was already isolated in 1937 from the mycelium of *Fusarium culmorum* [26], toxicological studies are still limited. In HT-29 and HCEC-1CT cells, Jarolim, Wolters, Woelflingseder, Pahlke, Beisl, Puntscher, Braun, Sulyok, Warth and Marko [25] reported AURO to be cytotoxic at concentrations ≥ 1 μM. Although AURO is frequently found at high concentrations in various food commodities [27–29], immunomodulatory effects have not been assessed yet. As *Fusarium* fungi can produce several mycotoxins simultaneously, infested food and feed may be contaminated by a high number of toxins. In recent years, human biomonitoring studies confirmed that humans are exposed to a variety of toxins [30–32]. Thus, the assessment of potential interactions of mycotoxins is crucial for proper risk assessment.

In the present study, we addressed the question whether the *Fusarium* metabolites NX-3 and AURO affect the NF-κB signaling pathway as individual compounds and as binary mixtures. Furthermore, we characterized alterations in the gene expression profiles of the pro-inflammatory cytokines IL-8, IL-6, TNF-α and IL-1β in a cancer and a noncancer colon cell line, mimicking an inflamed intestinal epithelium by IL-1β stimulation.
