Mycotoxins effects and mechanisms
of immune modulation in swine

Many are the harmful effects of mycotoxins on swine health, including immunosuppression.

Assist. Prof. Panagiotis Tassis

Assistant Professor of Swine Medicine and Reproduction, Clinic of Farm Animals, School of Veterinary Medicine, Aristotle University of Thessaloníki, Greece

Mycotoxin menace in grains worldwide

Mycotoxins are secondary metabolites produced by fungi (genera Aspergillus, Penicillium, Fusarium, Alternaria, and Claviceps) that can be found in grains (e.g., maize, wheat, barley) worldwide.

Mycotoxins are secondary metabolites produced by fungi (genera Aspergillus, Penicillium, Fusarium, Alternaria, and Claviceps) that can be found in grains (e.g., maize, wheat, barley) worldwide. (FBs, FB1-FB3), (ZEN), aflatoxins (AFs, mainly AFB1),ochratoxin A (OTA) and T-2 toxins are the most significant
for swine health and production2.

Recent studies have suggested that up to 80% of feed and food crops are contaminated with mycotoxins globally (occurrence above the detectable levels up to 60–80%), whereas co-contamination of grains with multiple mycotoxins is a common finding3.

A 10-year survey with samples from 100 countries reported that DON, FBs, and ZEN were most prevalent mycotoxins and were detected in 64%, 60%, and 45% of all samples, respectively. The median concentrations were 723 μg/ kg, 388 μg/kg and 55 μg/kg for FBs, DON and ZEN, respectively1.

Pigs and poultry are very susceptible and sensitive to the effects of mycotoxins4.

The effects of mycotoxins on pigs are multiple and they depend on5:

  • The type of mycotoxin
  • The level and duration of exposure
  • The age of the animal

Ingestion of great dosage levels can induce acute cases of mycotoxicosis with well-described clinical symptoms, such aso2,4,6:

  • Reproductive disorders and hyperestrogenism syndrome in the case of ZEN
  • Vomiting and growth retardation in the case of DON
  • Pulmonary edema after FBs ingestion
  • Reduced feed intake and weight gain in acute AF cases
  • Polydipsia, polyuria, and reduced growth in OTA cases

However, chronic consumption of low mycotoxins levels and the induction of vague clinical symptoms seems more probable under field conditions.

The chronic toxic effects of mycotoxins in swine include hepatotoxicity, genotoxicity, nephrotoxicity, neurotoxicity, reprotoxicity, immunotoxicity as well as other effects such as neuroendocrine disorders6,7.

The pig’s immune system

The pig’s immune system is the main defense mechanism against infectious and other agents. Its response is complicated, but the basic aspects of the immune system response are8:

  • Inflammation
  • Cellular response
  • Humoral response

Briefly, after engagement of the immune system (e.g. after contact with an infectious agent) a first defense multiple-mechanism takes action.

INNATE IMMUNITY

This mechanism includes the innate immune response with phagocytic cells and the production of various cytokines, chemokines, and proteins that provide antimicrobial protection, recruit T cells through the inflammatory process, and further activate the adaptive or acquired immune response.

The innate system also includes natural killer (NK) cells that present a dual function including an innate response to attack infectious agent-infected cells and production of cytokines for assisting in the activation of acquired immunity9-11.

Additionally, pattern recognition receptors, including Toll-like receptors (TLRs), participate in monitoring pathogen-associated molecular patterns and induce signaling pathways, that will enable activation of the immune system against infection11.

ACQUIRED IMMUNITY

Further on, the adaptative system uses B cells, T cells, cytokines, and antibodies in order to provide pathogen-specific memory for protection from subsequent infections with the same pathogen.

Taken together, the innate defense mechanisms that
neither require previous exposure to antigen nor have
an immunological “memory” provide the first and
almost immediate response to the infectious agent
and control infection, while at the same time assist
in the activation of the adaptive immune system,
which has immunological “memory”, and will produce
antibody and cell-mediated immune responses11.

The innate immune system – First line of defense

There are major parts of the innate immune system that act as the first line of defense or “barriers” to different types of infections (physical, chemical, microbial), such as epithelial cells, bactericidal fatty acids, normal flora, and the mucus layer, as well as cells with phagocytic abilities such as granular leukocytes (neutrophils, basophils, mast cells, and eosinophils), and mononuclear phagocytes (circulating blood monocytes and tissue macrophages).

Natural killer cells (NK) and other parts of the innate immune system such as defensins (host defense peptides), complement system, toll-like receptors (TLR), type I interferons (IFNs), tumor necrosis factor-α (TNF-α), IL-6, and IL-8 (proinflammatory cytokines)11:

  • Defend against pathogens
  • Control infections
  • Activate the cascade of events of inflammation and adaptive immunity response

Parts that play a significant role in this immune defense system and the response to pathogens are the mucosal epithelium (e.g. intestinal and respiratory tract), the microbiome (intestinal microbial ecosystem) and the swine lymphoid system consisting of the lymph nodes, lymphoid follicles, tonsils, thymus, and spleen12,13.

Additionally, an optimal microbiota14:

  • Prevents colonization of the intestinal epithelium by pathogens and penetration of the gut barrier
  • Modulates the gut-associated lymphoid tissue (GALT) and systemic immunity
  • Influences gastrointestinal development

Effects of mycotoxins on the swine immune system

The effects of the abovementioned mycotoxins in swine are multiple and vary significantly.

Teniendo en cuenta que los cerdos ingieren alimentos contaminados con micotoxinas, la capa de células epiteliales gastrointestinales es el primer lugar de contacto e interacción14,15.

From that point and after, immunomodulation and a sequence of immunological reactions take place, and they can be altered due to the mycotoxin’s effects at a molecular and cellular level.

Fusarium mycotoxins can either result in immunostimulatory or immunosuppressive effects, depending on the age of the host, exposure dose and duration7,16, whereas AFs and OTA induce immunosuppression17,18.

The health and economic implication of the effects of mycotoxins on the immune defense system of pigs is significant. Three major outcomes have been described19-21:

  • Increased susceptibility to infectious diseases
  • Reactivation of chronic infections
  • Decreased vaccination efficacy

A sensitivity of the immune system to mycotoxin-induced immunosuppression has been suggested, due to the vulnerability of the continually proliferating and differentiating cells that take part in immune-mediated activities and regulate the communication between cellular and humoral components8.

Moreover, it should be stated that effects of mycotoxin mixtures in pig feed on the immune system can also increase variability of the outcome and cannot be easily predicted, since they could have antagonistic, additive or synergistic interaction and increase the impact of each mycotoxina20.

The present review will focus on evidence regarding underlying mechanisms of mycotoxin-induced immunomodulation.

Special reference is given to trials on pigs or porcine cell lines, however evidence from other farm animals, laboratory animals and cell lines are also selectively presented.

Major part of the facts regarding the effects of mycotoxins on vaccinal efficacy and disease susceptibility will be presented in a separate review.

TRICHOTHECENES

DON effects on the immune system of the pig

DON and other Fusarium mycotoxins directly affect globulin synthesis in the liver and compromise the immune response of pigss22.

Type B trichothecenes, including DON, have the capacity to up- and  down-regulate immune functions by disrupting intracellular signaling among leukocytes23.

DON immunostimulatory or immunosuppressing effects depend on the dose, frequency and duration of exposure24, whilst few research efforts have demonstrated the effects of 3-Ac-DON, 15-Ac-DON and DON-3-glucoside on immune response.

As regards the acetylated forms of DON, there is evidence that at least 15-Ac-DON elicits similar general chronic toxicity as DON, whereas the immunotoxicity of 3-Ac-DON and 15-Ac-DON might be less expressed25.

As observed in vivo, trichothecenes can be stimulatory in some leukocyte models but inhibitory in others; paradoxically, these activities sometimes co-occur26.

Immune cells (macrophages, B and T lymphocytes and natural killer (NK) cells) are sensitive to DON, 3-Ac-DON and 15-Ac-DON, and dose-dependent immunostimulatory/ inflammatory or immunosuppressive effects can be observed7,23,24,27,28.

Differential inflammatory gene expression and DON-induced apoptosis are mechanisms that play a significant role in those immune effects.

The most prominent molecular target of trichothecenes is the 60S ribosomal subunit suggesting that one underlying mechanism is translational inhibition29.

However, it is known that trichothecenes and other translational inhibitors which bind to ribosomes can also rapidly activate mitogen-activated protein kinases (MAPKs), eliciting expression of inflammation-related genes as pro-inflammatory cytokines23,24, and induce apoptosis in a process known as the “ribotoxic stress response“30,31.

MAPKs modulate physiological processes including cell growth, differentiation, and apoptosis32 and are critical for signal transduction in the immune response33.

Toxic effects of DON on farm animals have been extensively reviewed25, with anorectic and immune-modulatory effects being the most pronounced in pigs.

Feed refusal and reduced feed intake after ingestion of DON contaminated feed have been associated with the hormonal and immunotoxic effects of DON as changes in satiety hormones (e.g. cholecystokinin and peptide tyrosine tyrosine) and changes of proinflammatory cytokines (e.g. IL-1β, IL-6, TNF-α) have been observed to be related to DON-induced anorexia25.

Furthermore, it has been suggested that DON predominantly affects vigorously proliferating cells such as intestinal epithelial cells (IEC), liver and immune cells, and the order of system sensitivity to DON is immune >neuroendocrine> intestinal7,14.

IMMUNE EFFECTS OF DON IN PIGLETS

As previously reportede34, low DON concentrations (up to 840 μg/kg feed for 4 weeks) do not affect piglet immune responses for immunoglobulin concentration, lymphocyte proliferation, and cytokine production.

  • However, in other studies DON has been shown to increase IgA concentration in blood, whereas nonspecific lymphocyte proliferation can be either increased or decreasedr19,35-38.
  • On the other hand, Ferrari et al.39 did not demonstrate significant immune effects after 6 weeks of oral DON exposure in pigs, confirming the variability of DON immune effects.
  • According to Frankic et al.37, DON (4 mg/kg feed for 14 days in weaned pigs) significantly increased the amount of DNA damage in lymphocytes by 28%.

As stated by Döll y Dänicke40, according to previous investigations on pigs, other farm animals and humans there are usually only minor (up to 1.5-fold), insignificant, or no effects of DON on IgA.

HEPATOTOXIC EFFECTS OF DON

In the in vitro study (hepatocytes exposed to 500 or 2000nM DON with or without 1μg lipopolysaccharides (LPS)/ml; incubation for 48 hours) from Doll et al.41, it was suggested that DON has the potential to provoke and modulate the immunological reactions of porcine liver cells. The study provided evidence that:

  • DON and LPS were synergistic for increased mRNA expression of TNF-α in hepatocytes.
  • DON stimulated a dose-dependent induction of IL-6 mRNA.
  • Supernatant concentrations of LPS-induced IL-6 were significantly decreased.
  • mRNA expression of the anti-inflammatory IL-10 was increased.

VACCINAL IMMUNE RESPONSE

In a study of DON effects on vaccinal immune responses (2.2–2.5 mg DON/kg feed, weaned pigs for 9 weeks)19, increased ovalbumin-specific (OVA) IgA and IgG were reported, whereas lymph nodes from treated pigs had reduced expression of TGF-β and IFN-γ mRNA, thus supporting the possibility of DON-induced reduction of vaccinal response.

The toxin had a biphasic effect on the OVA-specific lymphocyte proliferation, suggesting an up-regulation in the days after OVA immunization but a down-regulation in the weeks following.

Another research effort on pigs immunized with OVA, suggested an increase of anti-OVA IgG titers, after 42 days of exposure to a DON contaminated diet.

Simultaneously, the expression of chemokines involved in inflammatory reactions [(IL-8, chemokine (C-X-C motif) ligand 20 (CXCL20), interferon-g (IFN-g)] were up-regulated.

Deoxynivalenol also up-regulated the gene expression of antioxidant glutathione peroxidase 2 (GPX-2) and down-regulated expression of genes encoding enzymatic antioxidants including GPX-3, GPX-4 and superoxide dismutase 3 (SOD-3), involved in oxidative stress42.

Reduced or delayed antibody response to thymus-dependent antigens was observed also in growing pigs fed DON-contaminated grains22,43.

EFFECTS ON MACROPHAGES

The macrophage and innate immune system appear sensitive to trichothecenes26, 44. Macrophages are considered to be cells with greater (10 to 100-fold) sensitivity to DON when compared to fibroblasts, lymphocytes, IEC or astrocytes.

Based on some hypothesis, increased sensitivity can be attributed to increased DON ability to enter macrophage cells or increased apoptosis of macrophages after DON-induced activation of JAK/STAT pathway4,45.

↓DON

Low dose DON exposure results in:

Macrophage stimulation and activation (human, mouse, murine and porcine macrophages).

Secretion of inflammatory cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6 and TNFα).

Expression of intracellular COX-2 and iNOS proteins (selective activation of ERK, NFκB and activator protein-1)24,36,46-48, nitric oxide synthase49 and
numerous chemokines50,51.

↑DON

High dose DON exposure DON induces suppressive effects on macrophage-involved processes, such as cytokine secretion and phagocytosis, and induces their apoptosis (through p38 kinase activation), thus increase host susceptibility to pathogens and reduce activation of B and T lymphocytes (macrophages failing to act as antigen-presenting cells)52-54.

However, studies in primary porcine macrophages provide evidence for a lack of COX-2 and IL-6 activation by DON in porcine macrophages, suggesting a distinct mode of action in this species36.

EFFECTS ON DENDRITIC CELLS

Modulation of dendritic cells (DC) function probably contributes to DON-induced immunosuppressive effects. In vitro and in vivo investigation revealed DON and DC interaction.

Findings after feeding pigs with 5.3 ppm DON in feed for 5–11 weeks (in vivo) or/ and after 100–800ng/mL DON treatment of monocyte-derived DC (in vitro), included55:

  • Decreased endocytic activity
  • Inhibition of IL-10 secretion
  • Impairment of DC capacity for antigen-uptake

DON effects on the intestine of pigs are well reviewed by Pinton y Oswald56, suggesting multiple negative effects on integrity of the intestinal epithelium and barrier, as well as modulation of intestinal epithelium immune responsiveness, since type B trichothecenes can affect cytokine production by intestinal or immune cells and interfere with communication between epithelial cells and other intestinal immune cells.

Cano et al.57, investigated in vitro effects of purified DON [porcine IPEC-1 and porcine jejunal explants (ex vivo model)], and suggested that DON can:

  • Potentiate the expression of immune genes
  • Increase protein concentration in differentiated IPEC-1 cells in a time-dependent manner
  • Cause an early intestinal inflammatory response
  • Disrupt the intestinal homeostasis
  • Promote the intestinal immune system towards a Th17 response

T-2 effects on the immune system of the pig

According to EFSA58, the domestic pig is amongst the most sensitive species to the immunotoxic and haematotoxic effects of T-2 and HT-2 toxins.

T-2 toxin is reported to be immunotoxic, either by its cytotoxic, apoptotic or immunosuppressive attributes.

Like other trichothecenes, T-2 toxin can be both immunosuppressive and immunostimulatory depending on the dose and timing of exposure.

Effects of T-2 toxin on both humoral and cellular immune response have been demonstrated in various studies59. Moreover, T-2 toxin60,61:

  • Induces lipid peroxidation, affecting cell membrane integrity
  • Causes cell depletion in lymphoid tissue
  • Inhibits inflammatory cell function
  • Decreases humoral and cell-mediated immune responses, leading to an increased susceptibility to infection

PROTEIN SYNTHESIS INHIBITION

The most prominent molecular target of trichothecenes includes the 60S ribosomal unit, where it prevents polypeptide chain initiation66.

In vitro studies suggest that T-2 toxin interacts with the peptidyl transferase, which is an integral of the 60S ribosomal subunit, thus inhibiting the transpeptidation of peptide-bond formation, resulting in an inhibition of prolongation and termination of protein synthesis62,63.

The toxic effects exerted by T-2 toxin and HT-2 toxin include the inhibition of protein synthesis (through binding and inactivation of peptidyl-transferase activity at the transcription site), affecting also the synthesis of immunoglobulins and, in turn, the humoral immunity67-69.

T-2 toxin exposure results in leukopenia and cell depletion in lymphoid organs, significantly impairing antibody production, reducing the proliferative response of lymphocytes and hindering the development of dendritic cells70.

Moreover, it can disrupt DNA polymerases, terminal deoxynucleotidyl transferase, monoamine oxidase and several other proteins involved in the coagulation pathway71.

A time- and dose-dependent DNA damaging effect of T-2 toxin could be demonstrated using peripheral blood mononuclear cells from pigs (incubation with 0.1-1 μM for 24 or 42 hours)72.

Dendritic cells, the most potent antigen-presenting cells (APCs) of the immune system, have demonstrated sensitivity to trichothecene mycotoxins, and T-2 toxin disturbed their maturation process64.

Moreover, in a previous in vitro study with primary porcine alveolar macrophages, pre-exposure of macrophages to 3 nM of T-2 toxin decreased the production of inflammatory mediators (IL-1β, TNF-α, nitric oxide) in response to LPS and the decrease of the pro-inflammatory response was associated with a decrease of TLR mRNA expression.

Thus, ingestion of low concentrations of T-2 toxin can affect the TLR activation by decreasing pattern recognition of pathogens and interfere with the start of inflammatory immune response against pathogens65.

Acute T-2 toxicity (1.2 mg/kg body weight intravenously) has been characterized by emesis, posterior paresis, listlessness and lethargy, as well as severe damage to actively dividing cells in bone marrow, lymph nodes, spleen, thymus and intestinal mucosa. However, within 24 hours, surviving pigs recovered and appeared normal60,73.

In a feeding study with pigs, (0.5-3.0 mg T-2/kg feed) immunosuppression was observed.

Pigs were immunized with horse globulin and synthesis of antibodies towards this globulin was reduced, whereas a dose dependent depletion of lymphoid elements in the thymus and spleen, was also reported.

Leukocyte counts and the portion of T lymphocytes were decreased in all exposure groups74.

In pigs immunized with OVA, subclinical doses of T-2 toxin-induced an early and transient increase of total IgA plasma concentration but a decrease in the anti-OVA IgG titer.

Pigs fed 1.324 or 2.102 mg T-2 toxin/kg exhibited reduced anti-ovalbumin antibody production on day 21 without significant alteration to specific lymphocyte proliferation75.

Frankic et al.37 reported that, T-2 toxin (3 mg/kg feed for 14 days in weaned pigs) increased the amount of DNA damage in lymphocytes by 27% and decreased total serum IgG.

Similarly to the effect on lymphocyte proliferation, low amounts of T-2 toxin were found to increase antibody levels, whereas high amounts were found to be immunosuppressive60, therefore increased susceptibility to infectious diseases can be observed (e.g. Mycobacterium, Staphylococcus, Listeria, Toxoplasma and Herpes simplex virus (HSV-1) – effects seen in rats, mice and chicken)58.

Li et al.76 discussed that suppression of IFN-γ by T-2 toxin is probably one of the factors responsible for the decreased anti-viral immunity in the presence of T-2 toxin. The suppression of IFN-γ may be due to increased IL-6 (interleukin 6) expression.

Interesting facts about the trichothecenes – immune system interaction

In conclusion, macrophages, IgA, and pro-inflammatory cytokines have a significant role in the immunomodulatory effects of trichothecenes77.

Underlying mechanisms of trichothecenes effects on the immune system, as suggested by reviews from Wu et al.77-79 y Liao et al.62, son:

1. Activation of mitogenactivated protein kinases (MAPKs)

The most recognized theory is that DON and other ribosome-binding translational inhibitors can activate mitogen-activated protein kinases (MAPKs) through a mechanism known as “ribotoxic stress response” process30.

MAPKs, which are crucial for signal transduction in the immune response, mediate transcriptional and post-transcriptional gene upregulation caused by DON.

DON binds to the ribosome 28s peptidyl transferase locus, which activates ribotoxic stress response and induces the phosphorylation of protein kinase (PKR) and hematopoeitic cell kinase (Hck), then rapidly triggers MAPKs signaling pathway (as well as NF-kB, and JAK/STAT pathways), eventually leading to cell apoptosis and the expression of pro-inflammatory cytokines80.

Additionally, trichotechenes regulate apoptosis-related signal molecules such as IL-6, IL-1β, and TNF-α.

MAPK phosphorylation can be activated after these toxins bind to the peptidyl of ribosomes to regulate immune responses and apoptosis23,88.

2. Trigger endoplasmic reticulum stress and calcium-mediated signalling

DON can also induce endoplasmic reticulum (ER) stress81, as well as increasing ATF3 and DDIT3 (two major ER stress markers) protein expression within 3 hours81.

As it has been reported, the over-expression of ATF3 and DDIT3 could result in cell cycle arrest and/or apoptosis82,83.

DON can induce the phosphorylation of protein kinases JNK in Jurkat cells to trigger T-cell activation response and cleavage of caspase-3, an event known to mediate apoptosis, within 3 hours after its exposure81.

Activated caspase-12 can induce the activation of caspase-9 through the direct cleavage of caspase-9, which in turn induces the activation of caspase-3 and finally apoptosis takes place84.

3. Induction of mitochondrial signaling pathways and apoptosis

DON also induces apoptosis by involving the mitochondrial intrinsic pathway through the following mechanisms85,86:

  • Opening of the mitochondrial permeability transition pore (mPTP)
  • Loss of the mitochondrial transmembrane potential
  • ⇰  Increase of O2 (superoxide anion)
  • Release of cytochrome C

Thus, mitochondrial dysfunction, pursuant release of cytochrome C into the cytoplasm and serial activation of caspases contribute to DON-induced apoptosis, which is possibly modulated by Bcl-2 family62.

4. Influence the pathway for protein synthesis in cells, like RNA synthesis, ribosome functioning and translation

DON can also upregulate microRNAs (miRNA) which are responsible for downregulation of selective genes and ribosome synthesis87.

Additionally, trichothecenes significantly downregulate IFN-γ expression in pigs and mice, thereby reducing the host resistance to viruses and repairing ability19,93, whereas DON reduces IFN-b expression and promotes cell apoptosis48.

OXIDATIVE STRESS

Oxidative stress is an important mechanism of trichothecene toxicity, since they disrupt the normal function of mitochondria and generate free radicals, including ROS.

These compounds induce lipid peroxidation, change the antioxidant status of the cells, and reduce the activity of antioxidant enzymes such as glutathione-S-transferase (GST), superoxide dismutase (SOD), and catalase (CAT)77,88.

DNA damage is also associated with the generation of ROS and lipid peroxidation. Some signaling pathways, including MAPK, JAK/ STAT, and NF-κB, are subsequently induced by oxidative stress, and the caspase-mediated apoptosis pathways are also activated89.

Oxidative stress (generation of ROS as early as 30 min after exposure) is the mechanism by which T-2 toxin causes DNA damage and apoptosis90-92.

IMMUNOMODULATION

The immunomodulatory effect of trichothecenes may be determined by a balance between cell-survival and death-signaling pathways26,88,94.

T-2 toxin-induced cell apoptosis is simultaneously inhibited by the JNK1– STAT3 pathway (which maintains the normal function of mitochondria) and promoted by the STAT1 pathway in the same cell94.

DON initiates both a survival pathway (ERK/AKT/p90Rsk/ Bad) and a competing apoptotic pathway (p38/p53/ Bax/Mitochondria/Caspase-3) in RAW 264.7 macrophages26.

IMMUNOSTIMULATION

The immunostimulatory effect of trichothecenes may be partly mediated by autophagy95,96.

According to Tang et al.96, DON-induced autophagy inhibits the DON-induced apoptosis of intestinal epithelial cells by ameliorating the damage caused by oxidative stress, thereby causing the cell stress response to fail.

According to Bin-Umer et al.95, autophagy of damaged mitochondria (mitophagy) plays a key role in the resistance of cells to trichothecenes.

IMMUNE EVASION

Trichothecenes have an “immune evasion” mechanism that suppresses host and vaccine-induced immune defenses.

This mechanism interferes with anti-apoptotic genes, promoting oxidative stress-induced apoptosis93,97,98, allowing the toxins to escape host resistance and immune repair78.

ZEARALENONE

Due to its estrogenic properties, ZEN binds to estrogen receptors (ERs) and is typically associated with reproductive disorders in swine99.

However, ZEN has also been known to exhibit hepatotoxicity, hematotoxicity, immunotoxicity and genotoxicity45,100.

Since, immune cells also express ERs such as ERα in NK cells, macrophages and T cells, as well as ERβ in monocytes and B cells101, ZEN can also bind to such ERs and regulate a variety of metabolic pathways of the immune response100.

ZEN not only activates immune response-related genes, but also interferes with the immune system of the spleen, changes the phenotypes of spleen lymphocytes, and even causes lymphocyte atrophy in mice or rats102,103.

In addition, ZEN can induce immunosuppression by reducing immunoglobulins in serum and cytokines in lymphoid organs104.

On the other hand, RNA sequencing on liver samples from piglets fed with ZEN and DON-contaminated feed, indicated an effect on the expression and network of immune-related transcripts105.

HUMORAL IMMUNE RESPONSE

As regards ZEN effects on humoral immune response, a study performed with rats (5.0 mg/kg of ZEN for 36 days) revealed that ZEN alone (without immune challenge) can decrease the production of immunoglobulins106.

In addition, an in vitro study with peripheral blood mononuclear cells (PBMC) of piglets also showed a decrease in immunoglobulin levels107.

On the other hand, in an in vivo study by Swamy et al.108, increased serum immunoglobulin concentrations (IgM and IgA were increased, but not IgG) were observed in pigs fed grains contaminated with DON, fusaric acid (FA), ZEN, and 15-acetyldeoxynivalenol (15-acetylDON).

According to another in vivo study by the same group109 with different concentrations of the same mycotoxins in pigs, absence of effect of diet on the IgM and IgG antibody levels was reported.

The inconsistent effects of ZEN on humoral immune response could be related to receptor-specific effects, since ZEN is an agonist toward ERα and a mixed agonist-antagonist of ERβ110, with possible full antagonism of the ERβ expressed by B cells103.

CYTOTOXICITY AND OXIDATIVE DAMAGE

In vitro experiments using Vero and Caco-2 cells suggested that ZEN induces cytotoxicity and oxidative damage in addition to its estrogenic potential111.

Additionally, an in vitro study by Taranu et al.112, investigating the effects of ZEN (10 mM) on gene expression of porcine intestinal cells (IPEC-1), supported that even though such ZEN concentrations do not affect cell viability, 70% out of 190 differentially expressed genes were up-regulated.

 

Genes coding for glutathione peroxidase enzymes (GPx6, GPx2, GPx1) were among those up-regulated, providing evidence for mycotoxins inducing oxidative damage, whereas increased expression of cytokines involved in inflammation (e.g. TNF-α, IL-6, IL-8) and immune cell recruitment (e.g. IL-10) was also revealed, thus demonstrated that ZEN modulates intestinal cell immune and/or cellular repair pathways.

ZEN METABOLITES

Marin et al.113 investigated the effects of ZEN and its metabolites, α- zearalenol (α-ZEL), β-zearalenol (β-ZEL), and zearalanone (ZAN), on several neutrophil functions such as proliferation, cytokine synthesis and oxidative stress in a porcine polymorphonuclear (PMN) cells model.

It was observed that the parental toxin was less toxic, whilst ZEN derivatives induced a significant decrease of the IL-8 synthesis in swine PMNs.

It was concluded that ZEN and its derivatives may have divergent effects on important parameters of swine innate immunity, such as cell viability, IL-8 and superoxide anion synthesis. In another study by the same group107 with PBMC, 5 and 10 μM of ZEN and ZAN significantly decreased the TNF-α synthesis in the supernatant from the PBMC cell culture, and 10 μM of ZAN decreased also the IL-8 synthesis, while ZEN and its metabolites at concentrations higher than 5 μM also induced a significant decrease in IgG, IgA or IgM concentration.

Further in vitro evaluation of the toxicity of α-ZEL and β-ZEL on RAW264.7 macrophages114 showed that β-ZEL had a stronger inhibitory effect on the viability of macrophages than α-ZEL.

β-ZEL also induced cell death, mainly by apoptosis rather than necrosis, whereas the other ZEN metabolites induced:

  • Loss of mitochondrial membrane potential (MMP)
  • Mitochondrial changes in Bcl-2 and Bax proteins
  • Cytoplasmic release of cytochrome c and apoptosis-inducing factor (AIF)

Explaining the observed alterations in macrophages, it was concluded that the activation of p53, JNK or p38 kinase by ZEN metabolites is the main upstream signal required for the mitochondrial alteration of Bcl-2 (anti-apoptotic)/Bax (pro-apoptotic) signaling pathways and intracellular reactive oxygen species (ROS) generation, while mitochondrial membrane potential loss and nuclear translocation of apoptosis-inducing factor are the critical downstream events for ZEN metabolite-mediated apoptosis in macrophages.

FUMONISIN B

FB toxicosis, depending on contamination level and time of exposure, could result in porcine pulmonary oedema syndrome due to cardiovascular toxic effects, as well as increased sphinganine/ sphingosine (Sa/So) ratio in serum and tissues, liver and kidney toxicity, delay in sexual maturity and reproductive functionality alterations, impairment of innate and acquired immune response, histological lesions in internal organs, as well as alterations of brain physiology.

Due to a structural resemblance with ceramide, fumonisins competitively inhibit ceramide synthases (CerS), a group of key enzymes in the biosynthesis of ceramide and more complex sphingolipids, resulting in the disruption of the de novo synthesis of ceramide as well as sphingolipid metabolism and, as a consequence, alterations in lipid pathways115.

INFLAMMATION

FBs, especially B1 (FB1), influence the inflammatory response21,116.

A reduced expression of cytokines (IL-6, IL-1β, IL-12p40 and IL-8) in spleen and a significant upregulation of IL-1β, IL-6, IFN-γ, and TNF-α in the small intestine of piglets fed with contaminated diets [either DON (3 mg/kg) or FB (6 mg/kg), or both for 35 days] was reported117.

Following ingestion of 2.8 μM FB1/kg body weight (37–44 mg FB1/kg feed), a decreased expression of most of the cytokines was found in the different parts of the intestine segments after 14 days of exposure118.

Moreover, 8 mg FB1/kg feed decreased the gene expression of Th2 cytokines IL-4, IL-6 and IL-10 in blood of pigs116,119. Some of the changes in the mRNA expression of IL1α, IL1β, IL6, IL8, TNFα and MCP-1 induced by FB or other Fusarium toxins could be also cytotoxicity-related120.

INTESTINAL FUNCTION

As regards the intestinal morphology and function, FBs have been associated with21:

  • Intestinal villous fusion and atrophy
  • Decrease of transepithelial electrical resistance (TEER), globet cell density, occludin and E-cadherin expression
  • Greater bacterial translocation to other organs and proliferation of intestinal opportunistic bacteria

Significant negative effects have been demonstrated on intestinal immune system (1ppm FB oral exposure for 10 days, followed by Escherichia coli challenge), showing reduced intestinal expression of IL-12p40, impaired function of intestinal antigen presenting cells (APC), decreased upregulation of Major Histocompatibility Complex Class II molecule (MHC-II) and reduced T cell stimulatory capacity upon stimulation121.

HUMORAL IMMUNE RESPONSE

In pigs exposed to FB1 and vaccinated against Aujeszky’s disease virus (Suid Herpesvirus 1 [SuHV1]), the humoral immune response was greatly disturbed, with a strong decrease in observed antibodies122.

Similarly, in vivo exposure (28 days) of weanling piglets to feed contaminated with 8 m FB1/kg significantly decreased the expression of IL-4 mRNA by porcine whole blood cells and diminished the specific antibody titer after vaccination against Mycoplasma agalactiae116.

In a similar study with FB1 and vaccination against Mycoplasma agalactiae, significantly decreased specific antibody levels after vaccination as well as the mRNA expression level of IL-10 was demonstrated119.

Another study reported decreased expression of IL-8 in the gut of pigs following the oral administration of 0,5 mg/kg de FB1, although other cytokines were unaffected123.

According to in vitro and in vivo experiments, FB1modifies the Th1/Th2 (T-helper 1/T-helper 2) cytokine balance in pigs similar to an impaired humoral response116,119, as well as influencing the inflammatory response.

Incubation of swine alveolar macrophages with FB1 led to a significant reduction of the number of viable cells and cell death by apoptosis124.

An in vivo experiment on pigs [either DON (3 mg/kg) or FB (6 mg/kg), or both for 35 days] demonstrated that IL-8, IL-1β, IL-6 and macrophage inflammatory protein-1β were significantly decreased in the spleen of piglets exposed to multi-contaminated diet (DON and FB), whereas animals that received only FB-contaminated feed demonstrated a significant decrease in mRNA encoding for IL-1b and IL-6125.

AFLATOXINS

Aflatoxins have hepatotoxic, carcinogenic, and immunotoxic properties, impairing both the innate and the acquired immune responses126.

Ingestion of aflatoxins (140 and 280 ppb for 4 weeks) resulted in a biphasic effect on total white blood cell number, thus a low dose of AF (140 ppb) decreased the total number of white blood cells, whereas the high dose (280 ppb) had the opposite effect, while decreased proinflammatory (IL-1β, TNF-alpha) and increased anti-inflammatory (IL-10) cytokine mRNA expression was also observed127.

In that study, a reduced immune response induced by Mycoplasma agalactiae in the 280-ppb-treated group was also observed. Additionally, regarding the effects of AFB1 on the inflammation process, in vitro exposure of swine alveolar macrophage to this toxin has been shown to result in a time- and dose-dependent decreased viability and phagocytic activity of primary cultures cells124.

Evaluation of 25-days old piglets blood samples, born from sows that received AFs through feed during gestation and lactation, demonstrated reduction of lymphoproliferative response to mitogens and failure of monocyte-derived macrophages to efficiently produce superoxide anions after oxidative burst stimulation in vitro, whereas their ability to phagocytose red blood cells was not compromised.

Granulocytic cells showed a reduction of chemotactic response to chemoattractant bacteria factor and casein128.

AFB1 interferes with the development of acquired immunity in swine following vaccination against erysipelas with bacterin preparation (a suspension of killed bacteria) of E. rhusiopathiae and increases the severity of infection with E. rhusiopathiae129.

On the other hand, in a pig model vaccinated with ovalbumin (OVA), AFB1 non humoral immunity (concentrations of total IgA, IgG and IgM and specific anti-OVA IgG), but impaired lymphocyte activation was reported130.

Findings in another investigation131 on the involvement of AFB1 Swine Influenza Virus (SIV) replication in vitro and in vivo, supported that AFB1 exposure aggravates SIV replication, inflammation and lung damage by activating TLR4-NFkB signaling

A study on porcine splenocytes132 provided evidence of underlying mechanisms implicated in inmunosupresión inducida por AFB1-induced immunosuppression

In that study, la AFB1 inhibited the production of IL-2 when exposed to porcine splenocytes, leading to immunotoxicity in a dose-dependent manner.

Moreover, AFB1 decreased the level of reduced glutathione (GSH) and increased lipid peroxidation in porcine splenocytes, which is accompanied by increased phosphorylation of ERK1/2.

Therefore, it was concluded that AFB1 inhibits anti-CD3- induced lymphocyte proliferation and IL-2 production by the oxidative stress-mediated ERK1/2 MAPK signaling pathway.

Furthermore, AFB1 impairs cell-mediated immunity, probably through dysregulation of the antigen-presenting capacity of dendritic cells133.

On the other hand, exposure to AF increases the T-cell proliferation-inducing capacity of porcine monocyte-derived dendritic cells, thus enhances presenting capacity of cells134.

OCHRATOXIN A

OTA is a major nephrotoxic agent, whereas it has also liver toxicity properties, as well as immunotoxic, neurotoxic and teratogenic properties21.

HUMORAL IMMUNE RESPONSE

Disturbance in humoral immune response was reported in an in vivo study with pigs (500 μg OTA/kg feed for 3 months)122, since a strong decrease in antibody titer was observed after immunization against Morbus Aujeszky (Pseudorabies).

Gilts fed OTA-contaminated feed had135:

  • Reduced cutaneous basophil hypersensitivity response to phytohemagglutinin
  • Reduced delayed hypersensitivity to tuberculin
  • Decreased stimulation index for lymphoblastogenesis
  • Decreased IL-2 production when lymphocytes were stimulated with concanavalin A
  • Decreased number and phagocytic activity of macrophages

Spontaneous occurrence of dose-related clinical Salmonella choleraesuis infection occurred in piglets fed 1 and 3 mg OTA/kg feed dietary136.

In a further experiment by the same group, piglets were vaccinated against S. choleraesuis, OTA ingestion (1mg OTA / kg feed) lead to spontaneous Brachyspira hyodysenteriae and Campylobacter coli infections which were associated with OTA immunosuppression, showing delayed response to antigen and reduced humoral response136.

On the contrary, in a previous study135, OTA (2.5 mg of OTA/kg feed for 35 days) had no effect on total and specific immunoglobulin concentrations.

CYTOKINE EXPRESSION

OTA also affects cytokine expression.

An experiment on weaned pigs that ingested an OTA contaminated diet (181 ng/g of feed) has shown an increased level of TNF-α and IL-10 in plasma, with a decreased capacity to respond with cytokine expression in an ex vivo challenge with lipopolysaccharides (LPS)137.

There is evidence mainly from in vitro studies, for effects of OTA on neutrophils and macrophages including oxidative stress, apoptosis, phosphorylation of the ERK1/2 and release of TNFα via NF-kB pathways138.

In a study that investigated the toxicity of Penicillium mycotoxins on mitogen-induced lymphocyte proliferation, it was reported that OTA was the most potent (50% inhibition at 1.3 mM) cell proliferation inhibitor139.

Moreover, it has been reported that OTA induces the phosphorylation of P38 and ERK1/2 in porcine alveolar macrophages, causing immunotoxicity through an increase in Toll-like receptor 4 (TLR4)-mediated inflammatory signaling pathway proteins and elevated intracellular ROS production140.

OTA can induce altered expression of genes involved in cellular growth/proliferation, cell death/survival, and immune function in the kidney, as well as altered expression of molecules involved in immune response and antioxidant self-defense in pig gut141,142.

OTA can also modulate the expression of microRNAs, in kidney cells in vivo and in vitro, whilst many of the altered miRNAs are involved in the MAPK signaling pathways138.

OTA also induces the phosphorylation of P38 and ERK1/2 in porcine splenocytes, leading to nephrotoxicity and immunotoxicity, respectively143.

According to proteomic approaches, enhanced expression of mitochondrial proteins involved in electron transport, protein synthesis, stress response and cell death and modulation of proteins involved in inflammation are factors related to OTA toxicity144,145.

Moreover, a decrease in TCR-induced T lymphocyte viabilities in peripheral blood lymphocytes and splenocytes (400, 800 μg/kg diet) was reported in pigs146. Thus, it was suggested that nephrotoxicity and immunotoxicity of OTA may involve ER stress, activation of MAPK signaling and autophagy143,146.

Concluding remarks and practical notes

Fungi are proposed to be the greatest threat to animal and plant health among all the taxonomic classes of pathogens147.

Research up to today has provided clear evidence that mycotoxins affect the immune system of pigs.

The intestine is undoubtedly the key link between ingested mycotoxins and detrimental effects on the animal.

Negative effects of mycotoxins on the intestine (e.g. reduced barrier integrity) and immune system mean that they can play a critical role in the initiation, progression and duration of intestinal (and systemic) infections.

Therefore, compromising the integrity of the intestine will also increase the likelihood of microbes or microbial products, or mycotoxins, entering circulation and inducing systemic disease148-150.

The broad immunosuppressive effects of mycotoxins may decrease host resistance to infectious diseases20, whereas, vaccine immune response is also altered at mycotoxin doses that do not alter the global immune response75,116,130.

Such disruption in vaccine immunity may lead to the occurrence of disease even in properly vaccinated groups. Such cases are of utmost importance when investigating effectiveness of on-farm vaccination programs.

Furthermore, cases of feed contamination with mycotoxin mixtures should be accounted for as more probable than contamination with only one mycotoxin under field conditions.

Effects of mycotoxin mixtures on the immune system of pigs have not been fully clarified yet.

As regards the reduction of lymphocyte proliferation in in vivo studies, additivity has been suggested after co-exposure to AF and FB or OTA and T-2 toxin, and synergism after co-exposure to FB and DON151.

To further understand the complexity of interactions, in vivo co-exposure to FB and DON resulted in synergistic interaction on lymphocytes proliferation upon mitogenic stimulation, additive interaction on cytokines expression (IL-8; IL-1b, IL-6 and macrophage inflammatory protein 1b) and antagonistic interaction on levels of specific IgA and cytokine expression125.

When such observations are combined with various immunomodulating properties of mycotoxins, it becomes obvious that vaccine responses, susceptibility to infections and use of antimicrobials, as well as productivity and financial outcome can be altered due to mycotoxins ingestion, under field conditions. Such issues should be considered when assessing the necessity of a regular preventive program for mycotoxins on farms.

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