Mycotoxins Modulate the Inflammatory Response?
Mycotoxins are secondary metabolites of fungi that produce a wide spectrum of toxicological effects. Mycotoxins have the capacity to alter immune functions and intestinal barrier. Immune cells are vulnerable to mycotoxins effects due to the capacity of continuously proliferating and differentiating, whereas enterocytes are the first target to the toxic effects. Mycotoxins induce immunosuppression but also a proinflammatory reaction. Some of these changes are modulated by mitogen-activated protein kinases. Activation of this pathway induces a decreased expression of cell junction proteins resulting in changes in intestinal barrier function and permeability. In addition, some mycotoxins contribute to invasion of pathogens, potentiating intestinal inflammation. The ability of mycotoxins to interfere with intestinal epithelial and immune cells contributes to the acute and chronic toxic effects in vivo.
Mycotoxins are secondary metabolites produced by different fungi genus as Aspergillus, Fusarium and Penicillium. These metabolites are commonly found in food and feed at all stages of the food/feed chain. In a survey with 17 316 feed and feed raw samples, 72% were contaminated with one mycotoxin and 38% were co-contaminated (Streit et al., 2013). Actually, besides improvement of agricultural practices, it is considered that mycotoxin contamination cannot be avoided and continuous ingestion of some level of a variety of mycotoxins is expected in animals and humans. Furthermore, climate change is an increasing concern for feed and food safety impacting in the levels of mycotoxins contamination worldwide (Battilani et al., 2016).
Based on their known and suspected effects on human and animal health, aflatoxins, fumonisins, deoxynivalenol, ochratoxin A and zearalenone are recognized as the five most important agricultural mycotoxins (Chu, 1998). In a long term survey (2004 to 2011) the most prevalent mycotoxins were deoxynivalenol (DON) and fumonisins (FB) contaminating 55% and 54% of the samples, respectively (Streit et al., 2013). DON is produced by Fusarium graminearum and F. culmorum mainly in wheat, barley and maize. Acute high-dose exposure to DON is characterized by emesis, diarrhea, vomiting and gastrointestinal haemorrhage. Chronic exposure to DON induces digestive problems, reduced food intake and food refusal. The main clinical sign is reduction in growth performance, most often related to decreased feed intake and decreased weight gain. DON acts to inhibit protein synthesis by binding to the 28S ribosomal RNA peptidyltransferase site, inducing the phosphorylation of mitogen-activated protein kinases (MAPKs), promoting apoptosis, and inducing changes in cytokine gene expression (Pinton and Oswald, 2014).
Fumonisins (FB) are toxic and carcinogenic mycotoxins produced by Fusarium verticillioides and F. proliferatum, common pathogens of corn. At high concentrations, FB1 causes a variety of species-specific acute toxicological effects in domestic animals including leukoencephalomalacia in horses and pulmonary edema in pigs (Casteel et al., 1993). Studies analyzing the effects of chronic ingestion of low levels of FB indicate that it does not induce major clinical signs in pigs (Grenier et al., 2011). Nevertheless, ingestion of low levels of FB revealed pulmonary lesions and an increase in intestinal colonization by pathogenic bacteria in piglets (Halloy et al., 2005, Burel et al., 2013). At the cellular level, FB1 inhibits ceramide synthase, blocking the synthesis of sphingolipids, a class of membrane lipids that plays an important role in cell signaling transduction pathways and cell growth, differentiation and death (Soriano et al., 2005).
Among farm animals, pigs were one of the most sensitive to mycotoxins, including DON and FB. In addition, pigs are considered a good model for extrapolation to humans due to the similarity with the digestive, cardiovascular and immune system (Basso and Bracarense, 2013). In the actual system of animal production, the main route of exposure to mycotoxins is the gastrointestinal tract. This system has two major roles: the absorption of nutrients and the physical barrier function, limiting the entry of pathogens. However, intestinal epithelial cells have also a role in the innate immune response, producing antimicrobial peptides, mucus, and cytokines (Oswald, 2006). Considering these aspects, this paper will focus on the main effects on intestinal mucosa and on inflammatory response induced by deoxynivalenol and fumonisins in pigs.
Effects of mycotoxins on the intestinal mucosa
The gut, as the first tissue to have contact with food contaminants, is considered a target organ for the action of mycotoxins. The gastrointestinal tract is the first barrier to ingested mycotoxins, but is also the first line of defense against intestinal infection. This system is lined by a continuous monolayer of epithelial cells. Intestinal epithelial cells (IECs) form a crucial physical and functional barrier, which regulates the movement of water, electrolytes, nutrients and xenobiotics (Peterson and Artis, 2014). IECs regulate the selective permeability from the intestinal lumen into the circulation through two major routes: transcellular and paracellular permeability. Transcellular permeability is associated with solute transport through the epithelial cell, whereas paracellular permeability is related to transport via the space between IECs and is regulated by membrane junctional complexes (Pinton and Oswald, 2014).
The adherens junctions (AJ), together with tight junctions (TJ) and desmosomes, form an apical junction complex that controls epithelial cell-to-cell adherence and barrier function as well as regulation of the actin cytoskeleton, intracellular signaling pathways and transcriptional regulation (Mehta et al., 2015). These structures play a fundamental role in preserving cell membrane selectivity. The TJs are intercellular, multiprotein complexes located at the apical ends of the lateral membranes of IECs, and comprise both transmembrane (occludin, claudins, juctional adhesion molecules and tricellulin) and peripheral membrane proteins, as zonula occludens (ZO) and cingulin (Qasim et al., 2014). In a state of homeostasis, the apical TJs modulate the paracellular uptake of water, nutrients and electrolytes; however TJ dysfunction can lead to the disruption of the intestinal barrier integrity (Landy et al., 2016). Furthermore, disintegrated intestinal TJs allow the paracellular infiltration of luminal antigens and are considered as a pivotal pathogenic factor in the onset and promotion of intestinal inflammation.
Following ingestion of mycotoxin-contaminated feed, enterocytes may be exposed to a high concentration of toxins (Bouhet and Oswald, 2007) affecting the structure and function of IECs. The effects of mycotoxins on intestinal permeability were evaluated using in vitro, ex vivo and in vivo models by several research groups. Firstly, we are interested in evaluating the effects of DON on pigs’ intestinal cells. We have used the cell line IPEC-1, derived from the small intestine of a newborn unsuckled piglet. The paracellular permeability was evaluated by measuring the trans-epithelial electrical resistance (TEER). TEER is representative of the degree of organization of tight junctions over the cell monolayer. A common early marker of an impairment of epithelial barrier integrity is a decrease in the TEER (Oswald, 2006). IPEC-1 cells exposed to DON (0, 10, 20 and 50 µM) showed a significant decrease in TEER values and an increase in 4 KDa dextran permeability. Cells exposure to these different concentrations of DON resulted in a significant decrease in claudin-3 and claudin-4 expression. Also, an increase in paracellular passage of dextran was observed in pigs’ jejunal explants mounted in an Ussing chamber (Pinton et al., 2009). These results clearly indicate that DON affects the paracellular permeability.
As well as claudins, E-cadherin plays a fundamental role in maintaining cell–cell adhesion and contributing to the integrity of the gut epithelium. E-cadherin compounds the adherens junctions, but is also found extrajunctionally, forming intercellular cement on the lateral cell membrane of IECs (Sanders, 2005). In a previous experiment using pigs’ jejunal explants exposed to DON (10 µM) or FB1 (100 µM) a significant decrease in E-cadherin expression was observed. Furthermore, this decrease was accompanied by ultrastructural changes such as increased intercellular spaces, decrease in the size and number of microvilli and loss of junction complexes (Basso et al., 2013).
These data indicate that mycotoxins induce changes in junctional complexes resulting in impaired enterocyte permeability. A consequence of this increased permeability is translocation to intestinal lamina propria of antigens and bacteria that are normally restricted to the gut lumen. In fact, we verified that DON induced a dose-dependent translocation of a pathogenic strain of Escherichia coli across the porcine IPEC-1 epithelial cell monolayers (Pinton et al., 2009). In addition, IPEC-J2 cells exposed to low doses of DON showed an increased translocation of Salmonella Typhimurium, with undifferentiated cells being more sensitive than the differentiated ones (Vandenbroucke et al., 2011).
The results of in vivo experiments also showed that the intestine is a target for mycotoxins such as DON and FB. Pigs fed chronically a DON (2.8 mg/kg) or FB (5.9 mg/kg) contaminated diet showed an increase in intestinal histological changes compared to animals fed a non-contaminated diet. The changes include enterocyte flattening, villi atrophy, apical enterocytes necrosis and bacterial adhesion in areas of enterocyte necrosis. These changes were more severe in pigs exposed to DON. A significant decrease in E-cadherin and occludin expression was also observed (Bracarense et al., 2012). A dose-related response was observed in pigs receiving increasing concentrations of FBs (3.7 mg FB/kg feed, 8.1 mg FB/kg feed and 12.2 mg FB/kg feed). Flattening of enterocytes and apical necrosis were the main histological changes in jejunum and were significantly more severe in pigs fed the highest dose (manuscript in preparation).
We are also interested in evaluating a possible association between the histological changes and cell apoptosis. Using the jejunal explant model we observed that exposure to DON (10 µM) and FB1 (70 µM) induced a significant increase in caspase-3 expression indicating enterocyte apoptosis (83% for DON and 47% for FB1). In addition, a significant decrease in enterocyte proliferation and a tendency to increasing apoptosis was reported in pigs’ ileal loops exposed to DON (10 µM) (Cheat et al., 2015). The increase in enterocyte apoptosis may explain the decrease in villi height reported both in in vivo and ex vivo models exposure to DON and FB. Also, an increased expression (71%) of cicloxigenase-2 (COX-2) was verified in explants exposed to DON (Silva et al., 2014). Increased levels of COX-2, an enzyme that plays an important role in inflammatory reactions, were associated with increased cytoplasmic levels of reactive oxygen species (ROS). It is established that DON induces cell apoptosis through a ribotoxic stress response and generation of ROS (Pinton and Oswald, 2014). The mechanisms for apoptosis induction by FB1 involve the disruption of many regulatory cell pathways, including the inhibition of protein kinase C and the activation of MAPKs and caspase-3 due to the intracellular accumulation of sphingolipids and consequent lipid peroxidation (Soriano et al., 2005). The present data indicate that in this apoptotic pathway induced by DON and FB, caspase-3 is activated (Silva et al., 2014).
Besides the production of ROS, the up-regulation of COX-2 is also associated with mitogen activated protein kinase (MAPKs) activation. The regulation of the expression of tight junction proteins may be associated to the mitogen activated protein kinase (MAPKs) signaling pathway. Indeed, the ribotoxic stress induced by trichothecenes leads to the activation of members of the family of Src tyrosine kinases implicated as upstream regulators of a large number of intracellular signaling pathways. Jejunal explants exposed to DON (10 µM) and jejunal samples from piglets fed a DON-contaminated (2.8 mg/kg) diet showed a MAPK ERK1/2 and p38 activation (Lucioli et al., 2013). In in vitro studies using IPEC-1 cells exposed to DON, it was observed an increase in MAPK phosphorylation that was associated with a decreased expression of claudin and a reduction in the barrier function of the intestine (Pinton et al., 2010). Furthermore, fumonisin B1 is able to stimulate MAPKs resulting in cytoplasmic phospholipase A2 activity and increased arachidonic acid release (Pinelli et al., 1999). MAPKs are components of the signaling cascade that regulates cell survival in response to stress. These kinases modulate numerous physiological processes including cell growth, differentiation and apoptosis and are crucial for signal transduction in the immune response (Petska, 2010).
Effects of mycotoxins on inflammatory response
In the previous section we have focused on the effects of DON and FB on intestinal mucosa. Available data confirm that both DON and FB impair intestinal permeability. Besides their role in the barrier function, IECs may also be considered as a part of the immune system, modulating non-specific acute inflammatory response. IECs can produce cytokines and chemokines, crucial for the recruitment and activation of immune system cells. Tumor necrosis factor-α (TNF-α), interleukin (IL)-1, IL-6 and IL-8 and CC-chemokine ligand (CCL)-20 can be produced by IECs as defense mechanism, modulating the recruitment and activation of immune cells from the lamina propria (Oswald, 2006).
Deoxynivalenol has the capacity to upregulate proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and IL-1 in in vitro, ex vivo and in vivo models. An increase in the expression levels of IL-8, Il-1 α and TNF- α was observed in IPEC-1 cells and jejunal explants exposed to DON (10 µM) (Cano et al., 2013). Pigs fed a contaminated diet with DON (2.8 mg/kg) or FB (5.9 mg/kg) showed an increased expression of TNF-α, IL-1 and IL-6 levels in the intestine (Bracarense et al., 2012). In addition, FB induced a significant decrease in the expression of IL-8 mRNA in both in vitro and in vivo models that may impair neutrophil recruitment in an inflammatory response to pathogens (Bouhet and Oswald, 2007). TNF-α, an important inflammatory cytokine, increases enterocyte shedding in mice, producing microerosions that cannot be sealed by tight junction protein redistribution (Watson and Hughes, 2012). Our hypothesis is that, besides the known apoptotic mechanisms (Burel et al., 2013, Pinton and Oswald, 2014), FB and DON could induce TNF-mediated apoptosis, affecting both immune and epithelial cells. Interestingly, an association between increased levels of TNF-α, IL-1β and anorexia induction following oral exposure to DON was reported in mice (Wu and Zhang, 2014).
In the ileal loop model, co-exposure of DON (1 µg/L) and Salmonella Typhimurium induced a significant higher expression of IL-12 and TNF- α, and a clear trend to increased expression of IL-1β and IL-8. These results indicate that DON enhances the inflammatory response to Salmonella Typhimurium in the intestine of pigs (Vandenbroucke et al., 2011). An increased colonization of pathogenic Escherichia coli was reported in jejunum, ileum and colon of piglets fed a FB-contaminated diet (0.5 mg/kg/body weight) (Bouhet and Oswald, 2007). In addition, pigs that received a crude extract of FB (0.5 mg FB1/kg body weight/day for 7 days) and were exposed to Pasteurella multocida intratracheally presented a significant increase in lung lesions and in TNF-α, IFN-γ and IL-18 mRNA expression compared to animals exposed only to P. multocida (Halloy et al., 2005).
Recently it was demonstrated in in vitro (IPEC-J2) and in vivo (2.2 to 2.9 mg DON/kg) models that ingestion of DON induces an increase in the number of pores in the lamina propria of jejunum (but not in the ileum) as a result of a marked reduction in the production of laminin, the most abundant non-collagenous protein in basement membrane (Nossol et al., 2013). A probable consequence of this change in the basement membrane is the reported increase in the number of CD16+ cells migrating from the lamina propria into the epithelium of the jejunum, leading to a change in the antigen sampling in the gut (Nossol et al., 2013).
In conclusion, mucosal integrity is fundamental to maintain the intestinal barrier function. The available data reinforce the concept that both enterocytes and immune cells of the intestinal mucosa are important targets for the toxic effects of deoxynivanelol and fumonisins. In addition, these common food contaminants are a predisposing factor to tissue inflammation. Taken together, these results support that consumption of mycotoxins contaminated food or feed must be regarded as an increasing risk for human and animal health.