Effects of mycotoxins on the immune system of animals

Understanding the mechanisms of action is crucial for any curbing strategy and knowledge of their synergism or additivity contributes to possible new limit standards in feed.

As Aflatoxin B1 (AFB1), ochratoxin A (OTA), deoxynivalenol (DON), T-2 toxin (T-2), Fumonisin B1 (FB1), and zearalenone (ZEA) are major mycotoxins that are legislated by the European Food Safety Authority (EFSA), these are often studied and controlled in the food and feed industries. To evaluate the immunosuppression and immunostimulatory mechanisms of the 6 mycotoxins, the researchers reviewed a total of 71 journal articles published in the period 1985 to 2021. In addition, the mycotoxin synergism or additivity was explained through the interaction of deoxynivalenol and Fumonisin B1.

Immunosuppression and immunostimulation

The immunotoxicity of mycotoxins exhibits immune suppression or stimulation, which depends on factors including toxin exposure dose, time, administration route, and immunological stimulants. In general, low mycotoxin doses were found to induce an inflammatory response, whereas elevated levels induced immunosuppression; on the other hand, long-term mycotoxin exposure was immunosuppressive.

Aflatoxin B1

The immunosuppression mechanism of AFB1 is mainly involved in oxidative stress and apoptosis, as well as in immunity-related signal pathways. In vitro, AFB1 increases total reactive oxygen species (ROS) and oxidation of biomolecules to induce oxidative stress, promoting immunosuppression. The studies showed that apoptosis is closely linked to oxidative stress. AFB1 also inhibits anti-CD3-induced lymphocyte proliferation and IL-2 production through oxidative stress mediated by the extracellular signal-regulated kinase1/2 (ERK1/2) signalling pathway. In vivo, studies mainly focus on the immunosuppressive effects of feed additives on AFB1.

In vivo studies show that AFB1 induces inflammatory responses and hepatic injury by upregulating the NF-κB pathway; AFB1 induces inflammation by activating the NF-κB and inhibiting the Nrf2 signalling pathways in broiler chickens. By activating the NF-κB pathway, AFB1 significantly increases pro-inflammatory cytokines including tumour necrosis factor α (TNF-α) and IL-6. In both in vitro and in vivo studies, AFB1 induces liver inflammatory injury by the dephosphorylation of COX-2. In fish, AFB1 induces inflammation injury in the spleen via purinergic signals. In addition, transcriptional analysis indicates that AFB1 also induces oxidative stress which causes inflammatory responses, thus explaining the immunostimulation effects of AFB1.

Ochratoxin A

In vitro studies reviewed that OTA inhibits autophagy via upregulating the p-Akt1 signalling pathway (involved in T cell development and survival), thus inducing immunosuppression. While both in vitro and in vivo studies indicate that OTA promotes porcine circovirus type 2 replication by inducing autophagy. Similarly, OTA promotes porcine circovirus type 2 replication via the oxidative stress-mediated p38 and ERK1/2 MAPK signalling pathways in both in vitro and in vivo observations.

In terms of immunostimulation, in vitro studies show that OTA markedly enhances pro-inflammatory cytokine levels by activating the NF-κB pathway, thus activating specific inflammation-related pathways. It was suggested that OTA may be involved in immunostimulation via activating the unfolded protein response of T cells.

Deoxynivalenol

In in vitro observations, DON can induce immunosuppression via a ROS-mediated mitochondrial pathway; it also activates T lymphocytes via MAPK overactivation mediated by apoptosis, thus decreasing their immune function. Moreover, autophagy may also promote DON immunosuppression; DON exposure shows immunosuppressive effects at high doses by blocking mitophagy in vitro and in vivo. In addition to oxidative stress, apoptosis, and autophagy mechanisms, DON exhibits immunosuppression by directly inhibiting inflammatory mediators and suppressing nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. MAPK subfamily members, including ERK, JNK, and p38 are also key factors that modulate the immunosuppression of DON.

Immune cells are more sensitive to DON than other cells, and DON can activate T-cell responses by increasing intracellular calcium levels. In in vitro studies, DON upregulates pro-inflammatory genes (IL-6, IL-1β, and TNF-α) by activating the JAK/STAT pathway. In both in vitro and in vivo studies, low doses of DON exposure can cause immunostimulation by activating the TLR4/NF-κB pathway; DON also upregulates IL-6 and exhibits immunotoxicity by enhancing COX-2 expression and transcriptional activity that is modulated by ERK and p38 signals.

T-2 toxin

T-2 induces early apoptosis in MOLT-4 (a T lymphoblast cell line) cells, resulting in immunosuppression. In in vivo studies, T-2 is shown to promote the apoptosis of splenic cells and it decreases CD4+/CD8+ T cells in the spleen through the caspase-mediated mitochondrial apoptosis pathway; it also decreases the level of inflammatory cytokines (IL-1β, IL-6, and IL-10) via promoting endoplasmic reticulum stress, while autophagy is reported to promote T-2 immunosuppression.

Like DON, T-2 is also proven to up-regulate IL-6, IL-1β, and TNF-α by activating JAK1/2 and STAT1-3, which is related to apoptosis. T-2 can significantly increase the levels of COX-2 and activate 2 inflammatory signalling pathways, NF-κB and JNK (c-Jun-N-terminal kinase).

Fumonisin B1

The FB1-induced immunotoxicity mechanism is mainly through oxidative stress; in vitro studies indicate that FB1 increases ROS and oxidation of biomolecules, while ROS-dependent activation of ERK1/2 and p38 pathways mediate FB1-triggered neutrophil extracellular traps release. However, the studies that were reviewed in this survey do not distinguish between immunosuppression and immunostimulation.

Zearalenone

In this survey, few studies elaborated on the immunosuppression mechanism of ZEA. Some studies showed that ZEA over-activates T lymphocytes via MAPK over-activation mediated by apoptosis, thus decreasing their immune function. As observed in the studies of Fumonisin B1, there was no distinction between immunosuppression and immunostimulation.

Synergistic effect

In a similar study published in the journal Toxicology Letters, researchers explored the combined toxicity of DON and Fumonisin B1 on the intestine and its underlying mechanisms in vivo and in vitro. The results showed that the intestinal toxicity of DON and FB1 has a synergistic effect, suggesting that mycotoxin limits in future feed standards may need to reconsider the combined effects of mycotoxins.

Challenges for future studies

• It is difficult to distinguish between immunosuppression and immunostimulation as many factors are involved.

• There are few quantified dose-response relationships between in vivo and in vitro studies, making it difficult to assess the effects of the administration route.

• It is also difficult to assess the effects of exposure duration and frequency due to the absence of a uniform standard for distinguishing acute and chronic mycotoxin exposure between in vitro and in vivo trials. Therefore, it was concluded that more comprehensive and systematic tests are needed to investigate the effects of mycotoxins on the immune system of animals, including innate and acquired immunity, as well as the proliferation, maturation and differentiation of various immune cells. Tests are also needed to rigorously compare differences in animal species, age, sex, duration, dose, and the frequency and synergism of mycotoxin exposure.