Mycotoxins effects and mechanisms of immune modulation in swine

MYCOTOXINS EFFECTS AND MECHANISMS of immune modulation IN SWINE

Assist. Prof. Panagiotis Tassis Assistant Professor of Swine Medicine and Reproduction, Clinic of Farm Animals, School of Veterinary Medicine, Aristotle University of ThessalonĂ­ki, Greece

1

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.

Studies have proven their variability in

Recent studies have suggested that up

distribution among regions and climate zones

to 80% of feed and food crops are

globally1. Among numerous mycotoxins, it has

contaminated with mycotoxins globally

been suggested that deoxynivalenol (DON),

(occurrence above the detectable

fumonisins (FBs, FB1-FB3), zearalenone (ZEN),

levels up to 60–80%), whereas

aflatoxins (AFs, mainly AFB1), ochratoxin A

co-contamination of grains with multiple

(OTA) and T-2 toxins are the most significant

mycotoxins is a common finding3.

for swine health and production . 2

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

DON 64% samples 723 μg/kg

FBs 60% samples 388 μg/kg

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

ZEN 45% samples 55 μg/kg

Mycotoxin contamination in crops

are multiple and they depend on5: The type of mycotoxin The level and duration of exposure The age of the animal

2

ZEN

Ingestion of great dosage levels

Reproductive disorders Hyperestrogenism syndrome

can induce acute cases of mycotoxicosis with well-described clinical symptoms, such as2,4,6:

FBs Pulmonary edema

Reproductive disorders and hyperestrogenism syndrome in the case of ZEN

ACUTE MYCOTOXICOSIS

Vomiting and growth retardation in the case of DON

DON

Pulmonary edema after FBs ingestion

AF ↓Feed intake ↓Weight gain

Vomiting Growth retardation

Reduced feed intake and weight gain in acute AF cases Polydipsia, polyuria, and

OTA

reduced growth in OTA cases However, chronic consumption of

The chronic toxic effects of mycotoxins

low mycotoxins levels and the

in swine include hepatotoxicity,

induction of vague clinical symptoms

genotoxicity, nephrotoxicity,

seems more probable under ield

neurotoxicity, reprotoxicity,

conditions.

immunotoxicity as well as other effects

Polydipsia & Polyuria ↓Growth

such as neuroendocrine disorders6,7.

HEPATOTOXICITY

NEPHROTOXICITY

GENOTOXICITY

CHRONIC MYCOTOXICOSIS

NEUROTOXICITY

IMMUNOTOXICITY REPROTOXICITY

NEUROENDOCRINE DISORDERS

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INNATE IMMUNE RESPONSE

The pig’s immune system

Phagocytic cells

Toll-like receptors (TLR)

Cytokines, chemokines, and proteins

Monitor pathogenassociated molecular patterns and induce signalling pathways

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.

Antimicrobial protection

This mechanism includes the innate immune response with phagocytic cells

Recruit T cells

Activate acquired immune response

and the production of various cytokines, chemokines, and proteins that provide antimicrobial protection, recruit T cells through the inf ammatory process, and further activate the adaptive

Attack infectious agent-infected cells Cytokines

INNATE IMMUNITY

or acquired immune response. The innate system also includes natural killer (NK) cells that present

Natural Killer (NK) cells

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.

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ACQUIRED IMMUNITY

ACQUIRED IMMUNE RESPONSE Further on, the adaptative

B cells & Antibodies

system uses B cells, T cells,

T cells

Cytokines

cytokines, and antibodies in order to provide pathogenspecific memory for protection from subsequent infections with the same pathogen.

Pathogen-specific memory

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 complement

system,

microbial ecosystem) and the swine

There are major parts of the

peptides),

innate immune system that act

toll-like receptors (TLR), type I

lymphoid system consisting of the

as the first line of defense or

interferons (IFNs), tumor necrosis

lymph nodes, lymphoid follicles,

“barriers” to different types of

factor-α (TNF-α), IL-6, and IL-8

tonsils, thymus, and spleen12,13.

infections (physical, chemical,

(proinflammatory cytokines) :

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,

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Defend against pathogens

Additionally, an optimal microbiota14:

Control infections

Prevents colonization of the

Activate the cascade of

intestinal epithelium by pathogens

events of inflammation and

and penetration of the gut barrier

adaptive immunity response

Modulates the gut-associated lymphoid tissue (GALT)

and eosinophils), and mononuclear phagocytes (circulating blood

Parts that play a significant role

and systemic immunity

monocytes and tissue macrophages).

in this immune defense system

Influences gastrointestinal

and the response to pathogens

development

Natural killer cells (NK) and other parts of the innate immune system such as defensins (host defense

are the mucosal epithelium (e.g. intestinal and respiratory tract), the microbiome (intestinal

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ELEMEMENTS OF THE IMMUNE DEFENSE SYSTEM

Mucosal epithelium (intestinal & respiratory tract)

Microbiome (intestinal microbial ecosystem) Phagocytic & NK cells, complement system, TLR, IFNs, proinflammatory cytokines

Lymphoid system (lymph nodes, lymphoid follicules, tonsils, thymus and spleen)

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

Effects of mycotoxins on the swine immune system The effects of the above-

Fusarium mycotoxins can either

mentioned mycotoxins in swine are

result in immunostimulatory

multiple and vary significantly.

or immunosuppressive effects, depending on the age of the host,

Considering that pigs ingest

exposure dose and duration7,16,

mycotoxin-contaminated feed,

whereas AFs and OTA induce

the gastrointestinal epithelial

immunosuppression17,18.

cell layer is the first site of contact and interaction14,15.

From that point and after, immunomodulation and a sequence of immunological reactions take place, and

Fusarium mycotoxins Immunostimulatory or immunosuppressive effects AFs & OTA Immunosuppression

they can be altered due to the mycotoxin’s effects at a molecular and cellular level.

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The health and economic implication

Major part of the facts regarding

of the effects of mycotoxins on

the effects of mycotoxins on

the immune defense system of

vaccinal efficacy and disease

pigs is significant. Three major

susceptibility will be presented

outcomes have been described19-21:

in a separate review.

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

TRICHOTHECENES

activities and regulate the communication between cellular and humoral components8. Moreover, it should be stated that

As regards the acetylated

DON effects on the H

immune system of the pig

O

O OH

HO

effects of mycotoxin mixtures

O HO

forms of DON, there is evidence that at least 15-Ac-DON elicits similar general chronic toxicity as

in pig feed on the immune

DON and other Fusarium mycotoxins

DON, whereas the immunotoxicity

system can also increase

directly affect globulin synthesis

of 3-Ac-DON and 15-Ac-DON

variability of the outcome and

in the liver and compromise the

might be less expressed25.

cannot be easily predicted, since

immune response of pigs22.

they could have antagonistic,

As observed in vivo, trichothecenes

additive or synergistic

Type B trichothecenes, including

can be stimulatory in some

interaction and increase the

DON, have the capacity to up-

leukocyte models but inhibitory

impact of each mycotoxin20.

and down-regulate immune

in others; paradoxically, these

functions by disrupting intracellular

activities sometimes co-occur26.

The present review will focus on evidence regarding underlying mechanisms of mycotoxininduced immunomodulation.

signaling among leukocytes23. DON immunostimulatory or immunosuppressing effects depend on the dose, frequency

Special reference is given to

and duration of exposure24,

trials on pigs or porcine cell

whilst few research efforts have

lines, however evidence from

demonstrated the effects of

other farm animals, laboratory

3-Ac-DON, 15-Ac-DON and DON-

animals and cell lines are

3-glucoside on immune response.

also selectively presented.

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.

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IMMUNOTOXIC EFFECTS

Differential inflammatory gene

Toxic effects of DON on farm

expression and DON-induced

animals have been extensively

apoptosis are mechanisms

reviewed25 with anorectic and

that play a signi icant role in

immune-modulatory effects being

those immune effects.

the most pronounced in pigs.

The most prominent molecular target

Feed refusal and reduced feed

of trichothecenes is the

intake after ingestion of DON-

60S ribosomal subunit suggesting

contaminated feed have been

that one underlying mechanism

associated with the hormonal

is translational inhibition29.

and immunotoxic effects of DON as changes in satiety hormones

However, it is known that

(e.g. cholecystokinin and peptide

trichothecenes and other translational

tyrosine tyrosine) and changes

inhibitors which bind to ribosomes can also rapidly activate mitogen-

of proinflammatory cytokines (e.g. IL-1β, IL-6, TNF-α) have

activated protein kinases

been observed to be related to

(MAPKs), eliciting expression of

DON-induced anorexia25.

inflammation-related genes as

Changes in proinflammatory cytokines

DON O

H

O OH

HO

O HO

DON-contaminated feed

Changes in satiety hormones

Furthermore, it has been

pro-inflammatory cytokines23,24, and

suggested that DON

induce apoptosis in a process known as the “ribotoxic stress response”30,31.

predominantly affects vigorously proliferating cells such as intestinal epithelial cells (IEC), liver and immune cells, and

FEED REFUSAL AND REDUCED FEED INTAKE (ANOREXIA)

the order of system sensitivity to DON is immune >neuroendocrine>intestinal7,14. MAPKs modulate physiological processes including cell growth, differentiation, and apoptosis32 and are critical for signal transduction in the immune response33.

INFLAMMATORY GENE EXPRESIÓN AND DON-INDUCED APOPTOSIS

DON O

60S ribosomal subunit H

O OH

HO

O HO

Translational inhhibition Expression of pro-inflammatory cytokines

Ribotoxic stress response (apoptosis)

8

As previously reported34, low

In the in vitro study (hepatocytes

The toxin had a biphasic

DON concentrations (up to

exposed to 500 or 2000nM

effect on the OVA-specific

840 μg/kg feed for 4 weeks)

DON with or without 1μg

lymphocyte proliferation,

do not affect piglet immune

lipopolysaccharides (LPS)/ml;

suggesting an up-regulation

responses for immunoglobulin

incubation for 48 hours) from

in the days after OVA

concentration, lymphocyte

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:

immunization but a

However, in other studies DON has been shown to increase IgA concentration in blood, whereas nonspecific lymphocyte proliferation can be either increased or decreased19,35-38. 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.

HEPATOTOXIC EFFECTS OF DON

IMMUNE EFFECTS OF DON IN PIGLETS

cytokine production.

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.

pigs immunized with OVA, suggested an increase of anti-OVA IgG titers, after 42 days of exposure to a

mRNA expression of

DON contaminated diet.

TNF-α in hepatocytes.

Simultaneously, the

DON stimulated a dose-dependent induction

of LPS-induced IL-6 were significantly decreased. mRNA expression of the anti-inflammatory IL-10 was increased.

VACCINAL IMMUNE RESPONSE

As stated by Döll and Dänicke , 40

Another research effort on

synergistic for increased

Supernatant concentrations

al .37, DON (4 mg/kg feed for 14 days in weaned pigs) significantly increased the amount of DNA damage in lymphocytes by 28%.

weeks following.

DON and LPS were

of IL-6 mRNA.

According to Frankic et

down-regulation in the

expression of chemokines VACCINAL IMMUNE RESPONSE

proliferation, and

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

In a study of DON effects on

antioxidant glutathione

vaccinal immune responses

peroxidase 2 (GPX-2)

(2.2–2.5 mg DON/kg feed,

and down-regulated

weaned pigs for 9 weeks)19,

expression of genes

increased ovalbumin-specific

encoding enzymatic

(OVA) IgA and IgG were

antioxidants including

reported, whereas lymph nodes

GPX-3, GPX-4 and

from treated pigs had reduced

superoxide dismutase

expression of TGF-β and IFN-γ

3 (SOD-3), involved

mRNA, thus supporting the

in oxidative stress42.

possibility of DON-induced reduction of vaccinal response.

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

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immune system appear

induces suppressive effects

sensitive to trichothecenes26, 44.

on macrophage-involved

Macrophages are considered

processes, such as cytokine

to be cells with greater (10 to

secretion and phagocytosis, and

100-fold) sensitivity to DON

induces their apoptosis (through

when compared to fibroblasts,

p38 kinase activation), thus

Based on some hypothesis, increased sensitivity can be attributed to increased DON ability to enter macrophage cells or increased apoptosis of macrophages after

EFFECTS ON MACROPHAGES

High dose DON exposure

lymphocytes, IEC or astrocytes.

increase host susceptibility to pathogens and reduce activation of B and T lymphocytes (macrophages failing to act as antigen presenting cells)52-54.

porcine macrophages provide

JAK/STAT pathway

evidence for a lack of COX-2

4,45.

and IL-6 activation by DON

↓DON

Low dose DON exposure results in:

in porcine macrophages, suggesting a distinct mode of action in this species36.

Macrophage stimulation and activation (human,

Modulation of dendritic

mouse, murine and

DON effects on the intestine of pigs are well reviewed by Pinton and 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.

However, studies in primary

DON-induced activation of

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

cells (DC) function probably

Increase protein concentration

contributes to DON-induced

in differentiated IPEC-1 cells

Secretion of inflammatory

immunosuppressive

in a time-dependent manner

cytokines (IL-1β, IL-2, IL-4,

effects. In vitro and in vivo

IL-5, IL-6 and TNFα).

Cause an early intestinal

investigation revealed DON

inflammatory response

porcine macrophages).

Expression of intracellular COX-2 and iNOS proteins (selective activation of ERK, NFκB and activator , nitric

protein-1)

24,36,46-48

oxide synthase , and 49

numerous chemokines50,51.

EFFECTS ON DENDRITIC CELLS

EFFECTS ON MACROPHAGES

DON

The macrophage and innate

and DC interaction. Findings after feeding pigs

Disrupt the intestinal homeostasis

with 5.3 ppm DON in feed

Promote the intestinal immune

for 5–11 weeks (in vivo) or/

system towards a Th17 response

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

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T-2 effects on the immune

The most prominent molecular

T-2 toxin exposure results in

system of the pig

target of trichothecenes

leukopenia and cell depletion

includes the 60S ribosomal

in lymphoid organs, significantly

According to EFSA58,

unit, where it prevents

impairing antibody production,

the domestic pig is

polypeptide chain initiation66.

reducing the proliferative

amongst the most sensitive species to the 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

PROTEIN SYNTHESIS INHIBITION

immunotoxic and haematotoxic

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.

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

The toxic effects exerted by T-2

toxin could be demonstrated

depending on the dose

toxin and HT-2 toxin include the

using peripheral blood

and timing of exposure.

inhibition of protein synthesis

mononuclear cells from

(through binding and inactivation

pigs (incubation with 0.1-1

Effects of T-2 toxin on

of peptidyl-transferase activity

ÎźM for 24 or 42 hours)72.

both humoral and cellular

at the transcription site),

and immunostimulatory

affecting also the synthesis of

immune response have been demonstrated in various studies . 59

Moreover, T-2 toxin

:

60,61

immunoglobulins and, in turn, the humoral immunity67-69.

Induces lipid peroxidation, affecting cell membrane integrity Causes cell depletion in lymphoid tissue Inhibits inflammatory cell function

Lipid peroxidation

Decreases humoral and cell-mediated immune responses, leading to an

TOXIN T2

increased susceptibility

Cell depletion in lymphoid tissue

to infection

Inhibition of prolongation & termination of protein synthesis

↓humoral & cellmediated immune responses

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Dendritic cells, the most potent antigen-presenting cells (APCs) of the immune system, have

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

demonstrated sensitivity to trichothecene mycotoxins,

Pigs were immunized with horse globulin and synthesis of

and T-2 toxin disturbed their

antibodies towards this globulin was reduced, whereas

maturation process64.

a dose dependent depletion of lymphoid elements in the thymus and spleen, was also reported.

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

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.

to LPS and the decrease of the

Pigs fed 1.324 or 2.102 mg T-2 toxin/kg exhibited reduced

pro-inflammatory response

anti-ovalbumin antibody production on day 21 without

was associated with a decrease

significant alteration to specific lymphocyte proliferation75.

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

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 antiviral immunity in the presence of T-2 toxin. The suppression of IFN-γ may be due to increased IL-6 (interleukin 6) expression.

in bone marrow, lymph nodes, spleen, thymus and intestinal mucosa. However, within 24 hours, surviving pigs recovered and appeared normal60,73.

12

Interesting facts about the trichothecenes – immune system interaction In conclusion, macrophages,

1. Activation of mitogen-activated protein kinases (MAPKs)

O

IgA, and pro-in lammatory cytokines have a signifiicant 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 and Liao et al. 62, are:

DON

3. Induction of mitochondrial signaling pathways and apoptosis

H

O OH

HO

2. Trigger endoplasmic reticulum stress and calcium-mediated signalling

O HO

UNDERLYING MOLECULAR MECHANISMS OF DON

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

1. Activation of mitogenactivated protein kinases (MAPKs) The most recognized theory is that DON and other ribosomebinding 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

rapidly triggers MAPKs signaling pathway (as well as NF-kB,

upregulation caused by DON.

and JAK/STAT pathways), eventually leading to cell apoptosis

DON binds to the

and the expression of pro-inflammatory cytokines80.

ribosome 28s peptidyl transferase locus, which

Additionally, trichotechenes regulate apoptosis-related

activates ribotoxic stress

signal molecules such as IL-6, IL-1β, and TNF-α.

response and induces the phosphorylation of protein kinase (PKR)

MAPK phosphorylation can be activated after

and hematopoeitic cell

these toxins bind to the peptidyl of ribosomes to

kinase (Hck), then

regulate immune responses and apoptosis23,88.

13

Oxidative stress is an important

2. Trigger endoplasmic reticulum stress and calcium-mediated signalling

mechanism of trichothecene toxicity, since they disrupt the normal function

DON can also induce endoplasmic reticulum (ER) stress ,

of mitochondria and generate

as well as increasing ATF3 and DDIT3 (two major ER

free radicals, including ROS.

81

stress markers) protein expression within 3 hours . 81

These compounds induce lipid peroxidation, change

As it has been reported, the over-expression of ATF3 and

the antioxidant status of the

DDIT3 could result in cell cycle arrest and/or apoptosis82,83.

cells, and reduce the activity of antioxidant enzymes such

DON can induce the phosphorylation of protein kinases JNK in Jurkat 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

as glutathione-S-transferase OXIDATIVE STRESS

cells to trigger T-cell activation response and cleavage of caspase-3, an

(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

DON also induces apoptosis by involving the mitochondrial intrinsic pathway through the following mechanisms

pathways are also activated89.

:

85,86

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

Oxidative stress (generation of ROS as early as 30

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.

min after exposure) is the mechanism by which T-2 toxin causes DNA damage and apoptosis90-92.

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.

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The immunomodulatory

The immunostimulatory effect

Trichothecenes have

effect of trichothecenes may

of trichothecenes may be partly

an “immune evasion”

be determined by a balance

mediated by autophagy

mechanism that suppresses

between cell-survival and

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)

inhibits the DON-induced apoptosis of intestinal epithelial cells by ameliorating the damage caused by oxidative stress,

IMMUNE EVASION

inhibited by the JNK1–

DON-induced autophagy IMMUNOSTIMULATION

IMMUNOMODULATION

apoptosis is simultaneously

host and vaccine-induced

According to Tang et al. , 96

death-signaling pathways26,88,94. T-2 toxin-induced cell

.

95,96

immune defenses. This mechanism interferes with anti-apoptotic genes, promoting oxidative stress-induced

thereby causing the cell

apoptosis93,97,98, allowing

stress response to fail.

the toxins to escape

According to Bin-Umer

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

host resistance and immune repair78.

in RAW 264.7 macrophages26.

GENOTOXICITY ZEARALENONE

HEMATOTOXICITY

HEPATOXICITY

Due to its estrogenic properties, ZEN binds to estrogen receptors (ERs) and is typically associated with

ZEA

reproductive disorders in swine . 99

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

ER

BIND TO ESTROGEN RECEPTORS

Since, immune cells also express ERs such as ERα in NK cells, macrophages and T cells, as

NK cells, macrophages & T cells → ERα

well as ERβ in monocytes and B cells101, ZEN can also bind to such ERs and regulate a variety

Monocytes & B cells → ERβ

of metabolic pathways of the immune response100.

REPRODUCTIVE DISORDERS IMMUNOTOXICITY

15

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.

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.

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,

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

HUMORAL IMMUNE RESPONSE

In addition, an in vitro study with peripheral

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

since ZEN is an agonist toward ERα

involved in inflammation (e.g. TNF-α, IL-6,

and a mixed agonist-antagonist of

IL-8) and immune cell recruitment (e.g.

ERβ , with possible full antagonism

IL-10) was also revealed, thus demonstrated

of the ERβ expressed by B cells103.

that ZEN modulates intestinal cell immune

110

and/or cellular repair pathways.

16

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.

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

It was observed that the parental toxin

Cytoplasmic release of cytochrome c

was less toxic, whilst ZEN derivatives

and apoptosis-inducing factor (AIF)

induced a significant decrease of the

ZEN METABOLITES

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 Explaining the observed alterations in

as cell viability, IL-8 and superoxide anion synthesis. In another study by the same group

macrophages, it was concluded that the

with PBMC, 5 and 10 μM of ZEN and ZAN

activation of p53, JNK or p38 kinase by

significantly decreased the TNF-α synthesis in

ZEN metabolites is the main upstream

the supernatant from the PBMC cell culture, and

signal required for the mitochondrial

10 μM of ZAN decreased also the IL-8 synthesis,

alteration of Bcl-2 (anti-apoptotic)/Bax

while ZEN and its metabolites at concentrations

(pro-apoptotic) signaling pathways and

higher than 5 μM also induced a significant

intracellular reactive oxygen species (ROS)

decrease in IgG, IgA or IgM concentration.

generation, while mitochondrial membrane

107

potential loss and nuclear translocation of 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

apoptosis-inducing factor are the critical downstream events for ZEN metabolitemediated apoptosis in macrophages.

on the viability of macrophages than α-ZEL.

17

FUMONISIN B FB toxicosis, depending on contamination level and time of

FUMONISIN B

exposure, could result in porcine

Cardiovascular effects & Pulmonary oedema

pulmonary oedema syndrome due

Liver & Kidney toxicity

to cardiovascular toxic effects, as well as increased sphinganine/ sphingosine (Sa/So) ratio in serum

Disruption of lipid synthesis pathways

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.

Reproductive disorders

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.

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

INFLAMMATION

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.

18

As regards the intestinal

In pigs exposed to FB1 and

morphology and function, FBs

vaccinated against Aujeszky's

have been associated with21:

disease virus (Suid Herpesvirus 1 [SuHV1]), the humoral

Intestinal villous

immune response was greatly

fusion and atrophy

disturbed, with a strong decrease in observed antibodies122.

Decrease of transepithelial electrical resistance (TEER), globet cell density, occludin

Similarly, in vivo exposure (28

and E-cadherin expression

days) of weanling piglets to feed contaminated with 8 mg FB1/

INTESTINAL FUNCTION

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

Greater bacterial

kg significantly decreased the expression of IL-4 mRNA by porcine whole blood cells and

According to in vitro and in

vivo experiments, FB1 modifies 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.

diminished the specific antibody

An in vivo experiment on pigs

titer after vaccination against Mycoplasma agalactiae 116.

[either DON (3 mg/kg) or FB

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.

(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

Another study reported

demonstrated a significant

decreased expression of IL-8

decrease in mRNA encoding

in the gut of pigs following

for IL-1b and IL-6125.

the oral administration of 0.5 mg/kg FB1, although other cytokines were unaffected123.

19

AFLATOXINS

HEPATOTOXICITY Aflatoxins have hepatotoxic, carcinogenic, and immunotoxic

AFLATOXIN B1

IMMUNOTOXICITY

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

CARCINOGENICITY

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

stimulation in vitro, whereas their

Findings of another investigation131

(IL-10) cytokine mRNA expression

ability to phagocytose red blood

on the involvement of AFB1 in Swine

was also observed127.

cells was not compromised.

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.

In that study, a reduced

Granulocytic cells showed

immune response induced by

a reduction of chemotactic

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 timeand dose-dependent decreased viability and phagocytic activity of primary cultures cells124.

response to chemoattractant

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

bacteria factor and casein128. AFB1 interferes with the development of acquired immunity in swine following vaccination against erysipelas with bacterin

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

preparation (a suspension of

In that study, AFB1 inhibited

killed bacteria) of E. rhusiopathiae

the production of IL-2 when

and increases the severity of

exposed to porcine splenocytes,

infection with E. rhusiopathiae129.

leading to immunotoxicity in

On the other hand, in a pig model vaccinated with ovalbumin (OVA), AFB1 exposure had no major effect on humoral immunity (concentrations of total IgA, IgG and IgM and specific anti-OVA IgG), but impaired lymphocyte activation was reported130.

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.

anions after oxidative burst

20

Therefore, it was concluded that AFB1 inhibits anti-CD3induced lymphocyte proliferation

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

and IL-2 production by the

On the other hand, exposure to AF increases the T-cell

oxidative stress mediated ERK1/2

proliferation-inducing capacity of porcine monocyte-derived

MAPK signaling pathway.

dendritic cells, thus enhances presenting capacity of cells134.

OCHRATOXIN A

NEPHROTOXICITY OTA is a major nephrotoxic agent, whereas it has also liver

OCHRATOXIN A

IMMUNOTOXICITY

toxicity properties, as well as immunotoxic, neurotoxic and teratogenic properties21. Disturbance in humoral immune response was reported in an in vivo study with pigs (500 Îźg OTA/kg feed for 3 months)122, since a strong decrease in antibody titer was observed after

LIVER TOXICITY TERATOGENICITY

NEUROTOXICITY

HUMORAL IMMUNE RESPONSE

immunization against Morbus Aujeszky (Pseudorabies). Gilts fed OTA-contaminated

Decreased number and phagocytic activity of macrophages

feed had135: Reduced cutaneous basophil hypersensitivity response to phytohemagglutinin Reduced delayed hypersensitivity to tuberculin

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

Decreased stimulation index

with OTA immunosuppression, showing delayed response

for lymphoblastogenesis

to antigen and reduced humoral response136.

Decreased IL-2 production

On the contrary, in a previous study135, OTA (2.5 mg

when lymphocytes

of OTA/kg feed for 35 days) had no effect on total

were stimulated with

and specific immunoglobulin concentrations.

concanavalin A

21

OTA also affects

CYTOKINE EXPRESSION

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

OTA can induce altered expression of genes involved in cellular

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.

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

In a study that investigated the

miRNAs are involved in the MAPK signaling pathways138.

toxicity of Penicillium mycotoxins on mitogen-induced lymphocyte

OTA also induces the phosphorylation of P38

proliferation, it was reported

and ERK1/2 in porcine splenocytes, leading to

that OTA was the most potent

nephrotoxicity and immunotoxicity, respectively143.

(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

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.

alveolar macrophages, causing

Moreover, a decrease in TCR-induced T lymphocyte

immunotoxicity through an

viabilities in peripheral blood lymphocytes and

increase in Toll-like receptor 4

splenocytes (400, 800 μg/kg diet) was reported in

(TLR4)-mediated inflammatory

pigs146. Thus, it was suggested that nephrotoxicity

signaling pathway proteins

and immunotoxicity of OTA may involve ER stress,

and elevated intracellular

activation of MAPK signaling and autophagy143,146.

ROS production . 140

22

Concluding remarks and practical notes Fungi are proposed to be the

Such disruption in vaccine

greatest threat to animal and

immunity may lead to the

plant health among all the

occurrence of disease even in

taxonomic classes of pathogens147.

properly vaccinated groups. Such cases are of utmost importance

Research up to today has provided

when investigating effectiveness

clear evidence that mycotoxins

of on-farm vaccination programs.

affect the immune system of pigs. Furthermore, cases of feed The intestine is undoubtedly

contamination with mycotoxin

the key link between ingested

mixtures should be accounted for

mycotoxins and detrimental

as more probable than

effects on the animal.

contamination with only one

Negative effects of mycotoxins on the intestine (e.g. reduced

mycotoxin under field conditions. Effects of mycotoxin mixtures

barrier integrity) and immune

on the immune system of pigs

system mean that they can

have not been fully clarified yet.

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

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.

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.

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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