The Novel Role of the NLRP3 Inflammasome in Mycotoxin-Induced Toxicological Mechanisms

Simple Summary Mycotoxins pose a serious threat to human and animal health by causing acute poisoning and chronic effects. However, the toxicological mechanism of mycotoxins is complicated and unclear. Recent reports have revealed that activation of the nucleotide-binding, oligomerization domain (NOD)-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome is linked with the tissue damage and inflammation induced by mycotoxin exposure. Through a comprehensive literature review, this study illuminates the dysregulated expression of NLRP3 inflammasome responses to mycotoxin exposure. This study not only advances our knowledge of the role of the NLRP3 inflammasome in mycotoxin exposure but also offers valuable insights for future studies of novel anti-inflammatory agents used in cases of mycotoxin exposure. Abstract Mycotoxins are secondary metabolites produced by several fungi and moulds that exert toxicological effects on animals including immunotoxicity, genotoxicity, hepatotoxicity, teratogenicity, and neurotoxicity. However, the toxicological mechanisms of mycotoxins are complex and unclear. The nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome is a multimeric cytosolic protein complex composed of the NLRP3 sensor, ASC adapter protein, and caspase-1 effector. Activation of the NLRP3 inflammasome plays a crucial role in innate immune defence and homeostatic maintenance. Recent studies have revealed that NLRP3 inflammasome activation is linked to tissue damage and inflammation induced by mycotoxin exposure. Thus, this review summarises the latest advancements in research on the roles of NLRP3 inflammasome activation in the pathogenesis of mycotoxin exposure. The effects of exposure to multiple mycotoxins, including deoxynivalenol, aflatoxin B1, zearalenone, T-2 toxin, ochratoxin A, and fumonisim B1, on pyroptosis-related factors and inflammation-related factors in vitro and in vivo and the pharmacological inhibition of specific and nonspecific NLRP3 inhibitors are summarized and examined. This comprehensive review contributes to a better understanding of the role of the NLRP3 inflammasome in toxicity induced by mycotoxin exposure and provides novel insights for pharmacologically targeting NLRP3 as a novel anti-inflammatory agent against mycotoxin exposure.


Introduction
Mycotoxins are toxic secondary metabolites produced by fungi and are widely occurring in crops.In past years, mycotoxin contamination has been considered the most hazardous public health problem in food and feed, attracting significant attention worldwide [1].Approximately 500 mycotoxins from toxic fungal metabolites have been estimated to have the potential to contaminate food and feed.Common mycotoxins include aflatoxins (AFTs), fumonisins B, deoxynivalenol (DON), ochratoxin A (OTA), T-2 toxin, zearalenone (ZEA), Alternaria toxins, HT-2 toxin, citrinin, enniatins, ergot alkaloids, nivalenol, cyclopiazonic acid, and patulin [1].These mycotoxins can induce significant harm to the kidneys, livers, and reproductive organs of animals by triggering immune responses, inflammation, and oxidative stress (OS), consequently leading to substantial economic losses within the livestock industry [2,3].Mycotoxins have long been recognized as a source of human food-borne illness and may be associated with several chronic diseases.The specific mechanism of mycotoxin-induced toxicity is unclear; however, previous research has shown that it is closely related to DNA damage, cell-cycle arrest, OS, the inflammatory response, and apoptosis.
In recent years, a number of investigators have reported that the toxic effects of mycotoxins are related to pyroptosis [4][5][6].The term "pyroptosis" was first used in 2001 to describe a type of inflammatory cell death characterized by the appearance of pores in the plasma membrane, cell swelling and rupture, and leakage of cell contents [7].Pyroptosis is a key component of the immune response and is widely associated with the occurrence and progression of infectious diseases, tumors, nervous system-related diseases, metabolic diseases, and chronic inflammation [8].The mechanisms of pyroptosis can be divided into classic and nonclassic pathways [9,10].In the classic pathway, the NOD-, LRR-family and pyrin domain-containing protein 1 (NLRP1), NLRP3, NLR-family and caspase activation recruitment domain-containing protein 4 (NLRC4), absent in melanoma 2 (AIM2), pyrin, or other inflammasomes are activated through several specific ligands.For instance, the NLRC4 inflammasome is activated by flagellar and rod proteins that make up the components of the type III secretory apparatus.The AIM2 and IFN-gamma-inducible protein-16 (IFI16) inflammasomes recognize double-stranded DNA via the hemopoietic IFNinducible nuclear proteins (HIN)-200 domain.Subsequently, the inflammasome complex is assembled to activate caspase-1, and activated caspase-1 cleaves gasdermin D (GSDMD) to generate active N-and C-termini.GSDMD-N causes cell membrane perforation, leading to cell death.Moreover, activated caspase-1 induces the conversion of pro-IL-1β to IL-1β, which is then released into the extracellular space where it amplifies inflammation.The nonclassic pyroptosis pathway mainly depends on the function of caspase-4/5 in humans (murine caspase-11) [11].The ligand lipopolysaccharide (LPS) of Gram-negative bacteria directly activates caspase-4/5/11 without cleaving the precursors of interleukin-1β (IL-1β) and IL-18 to cleave GSDMD, and GSDMD-N is subsequently transferred to the plasmalemma.Activation of caspase-4/5/11 actuates the pannexin-1 channel, thereby releasing ATP and opening the membrane channel P2X7 to induce cell membrane lysis and pyroptosis [12].
As a key factor of pyroptosis, the NLRP3 inflammasome has been associated with numerous diseases, such as inflammatory bowel disease, infectious disease, and cancer [13].Moreover, the application potential of targeting the NLRP3 inflammasome is supported by observations regarding therapies for autoimmune diseases [14].NLRP3 inflammasomemediated pyroptosis is instrumental in the process of mycotoxin-induced pathological damage [4].Elucidating the mechanisms of inflammasome induction caused by mycotoxin exposure is crucial for circumventing mycotoxin-mediated pathology.In this review, we summarize the evidence for mycotoxin-induced damage and stimulation of the NLRP3 inflammasome.Furthermore, we highlight the role of the NLRP3 inflammasome in mycotoxin-induced toxicity.Finally, the possibility of targeting the NLRP3 inflammasome in cases of mycotoxin exposure is discussed.

Mycotoxicosis
Most mycotoxins are chemically stable under harsh conditions.Warm and humid environments are usually conducive to the growth of molds, further contributing to pollution caused by mycotoxins.There are hundreds of known mycotoxins, among which the most common harmful mycotoxins are mostly produced by Aspergillus spp., Fusarium spp., and Penicillium spp., among others.Mycotoxin contamination may occur at any stage of crop growth, harvest, storage, or feed processing.As noted by Eskola et al., up to 60-80% of food crop samples worldwide are threatened by mycotoxin contamination [15].Ingestion of these mycotoxins can rapidly cause disease symptoms and may be life-threatening in severe cases.Mycotoxins can also cause long-term harm to humans, leading to cancer and immunodeficiency diseases [16].Therefore, mycotoxin contamination poses a serious challenge to food safety and animal husbandry development and is a danger to human and animal health [17].
Food safety has received increasing attention, and research into the prevention and control of mycotoxin contamination has gradually become a greater focus of academic attention.Scientists have explored in depth the toxicity mechanisms of mycotoxins, which exert toxicological effects on animals through immunotoxicity, genotoxicity, OS, hepatotoxicity, cytotoxicity, teratogenicity, and neurotoxicity [18].In vitro, mycotoxins induce programmed cell death such as apoptosis, as well as irreversible cell damage [19].In addition, mycotoxins prevent the formation of mitochondrial complex I in cells, hindering the normal function of mitochondria and leading to increased generation of reactive oxygen species [20].Moreover, mycotoxins have potassium-specific ionophore activity, which allows potassium ions to flow into the mitochondrial matrix and cause mitochondrial swelling [21].Additionally, mycotoxins can bind to the 60S subunit of eukaryotic ribosomes and inhibit the production of peptide transferases [22].Researchers have also found that mycotoxins competitively inhibit the activities of metabolic enzymes, thereby inhibiting carbohydrate and lipid metabolism [23].Mycotoxins are structurally similar to sex hormones, affecting hormone receptors and altering hormone levels [24].In addition, mycotoxins have a great impact on deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).Aflatoxin B1 (AFB1) can indirectly inhibit the activity of RNA polymerase, thereby interfering with RNA synthesis [25].Moreover, AFB1 undergoes irreversible covalent interactions with DNA, leading to the formation of N7-guanine adducts [26].More importantly, a few mycotoxins can change DNA methylation levels [27].In vivo studies have shown that mycotoxins induce different degrees of inflammatory responses and toxicological effects, resulting in organ lesions and even death in pigs, rats, chickens, mice, and other animals [28].The diversity of mycotoxins and the complexity of toxicity mechanisms indicate that the control of mycotoxins activity is very difficult.It is crucial to investigate the toxicity of mycotoxins and elucidate their mechanisms of action to improve food safety and promote the development of animal husbandry.Numerous studies have demonstrated that NLRP3 inflammasome-mediated pyroptosis plays a critical role in mycotoxin-induced toxicity [4][5][6].

Mycotoxins Activate the NLRP3 Inflammasome
In 2002, Martinon and colleagues first described a caspase-activating complex using the "inflammasome" [29].Inflammasomes are large multi-molecular arrays with important roles in regulating innate immunity and inflammation.Pyroptosis is an inflammatory type of programmed cell death caused by the activation of inflammasomes.Unlike cells undergoing apoptosis and necrosis, pyroptotic cells continue to swell until the cell membranes burst, releasing cellular debris and producing a strong inflammatory response [30].Recently, as a novel method of programmed cell death, pyroptosis has been shown to be associated with many diseases [31].The occurrence of pyroptosis depends on a variety of inflammasomes, among which the NLRP3 inflammasomes are one of the best-studied types.The NLRP3 inflammasomes are composed of an NLRP3 sensor, an apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC) adaptor and a caspase-1 effector.Data increasingly indicate that NLRP3 inflammasomes are widely found in epithelial and immune cells and are important in host defence [32].The three main signaling pathways involved in the activation of NLRP3 inflammasomes are known as canonical, non-canonical, and alternate activation [32].Activation of the canonical and non-canonical NLRP3 inflammasomes requires two steps: priming and activation.However, the alternative activation pathway requires only one step.
In the priming step for canonical activation, PRRs (TLRs or NLRs) or cytosolic receptors (PAMPs or DAMPs) activate nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and post-translational modifications (ubiquitination or phosphorylation) of NLRP3 promote its expression and that of IL-1β [33].In the activation step, numerous molecular or cellular events occur, including ionic flux (efflux of potassium and chloride ions, influx of sodium, and calcium ion mobilization), mitochondrial dysfunction, processes involving mitochondrial DNA (mtDNA), reactive oxygen species (ROS) [34], and mitochondrial ROS (mtROS), trans-Golgi disassembly, lysosomal disruption, and metabolic changes [32].Subsequently, the assembly and activation of NLRP3 inflammasomes stimulates caspase-1 activity and triggers the release of the cytokines IL-1β and IL-18, together with the subsequent activation of GSDMD, inducing an inflammatory response and cell death by lysis [35].However, non-canonical activation is closely related to caspase-4 and -5 in humans and caspase-11 in mice [36].In this signaling pathway, extracellular LPS activates toll-like receptor 4 (TLR4) and inducible type-I interferon, along with the complementary C3-C3aR system, upregulating caspase-11 expression [32].GSDMD cleavage by activated caspases-4, -5, and -11 results in caspase oligomerization, autoproteolysis, and pyroptosis [37][38][39].Along with the 2-step activation model, a 1-step NLRP3 inflammasome activation has been observed.Alternative inflammasome activation occurs in monocytes from humans and pigs and macrophages from bone marrow of mice.This pathway does not involve ASC speck formation, pyroptosis induction, or K + efflux [32].In this pathway, the TLR ligand alone is sufficient to activate maturity and release of caspase-1 or IL-1β from human or porcine monocytes via the TRIF/RIPK1/FADD caspase-8 pathway [33].This one-step rapid response might be helpful in sustaining blood sterility in the early stages of infection and reducing bacterial septicaemia.
The intestines are the critical target organ of DON, which can cause gut impairment, inflammatory reactions, and dysfunction; therefore, intestinal porcine epithelial cells (IPEC-J2) were employed as models for validating DON-induced intestinal damage.DON (2 µg/mL) activated NLRP3 inflammasomes by regulating OS and induced the production of proinflammatory cytokines (IL-18 and IL-1β), resulting in pyroptosis in IPEC-J2 cells [46,47].Stress from accumulated ROS was instrumental in DON-inducible NLRP3-mediated pyroptosis; thus, caveolin-1 is a therapeutic target for overcoming DON-induced enterotoxicity [48].Interestingly, DON exposure can aggravate LPS-induced cellular inflammatory responses and increase NLRP3 and procaspase-1 in IPEC-J2 cells through NF-κB signaling and the autophagy-related protein LC3B [49].Thus, DON exposure could induce apoptosis and inflammation through ROS accumulation, NF-κB activation, and apoptosis in IPEC-J2 cells.However, incubation with non-toxic doses of DON (2 µM for IPEC-J2 cells) did not change the levels of NLRP3 or procaspase-1 [50].
DON-mediated activation of NLRP3 inflammasomes has been studied in different cells.Song et al. reported that incubation with DON increased the expression of caspase-1 and GSDMD in donkey (Equus asinus) endometrial epithelial cells [51].However, determination of NLRP3 expression was not performed in their study.The synthesis of pro-IL-1β and NLRP3 by the action of NF-κB are the primary signals of NLRP3 inflammasome activation.DON can upregulate IL-1β in murine BV2 microglia and promote the formation of ASC/NLRP3 inflammasomes by activating the NF-κB signaling pathway [52].Another study indicated that lymphocytes from the spleens of carp (Cyprinus carpio L.) incubated with DON had higher levels of mtROS, disrupting the balance of mitochondrial homeostasis and activating the mtROS/NF-κB/NLRP3 axis, and inducing pyroptosis [53].
In human keratinocytes (HaCaT cells), DON exposure also significantly induced excess mtROS production, disrupted mitochondria, and increased OS, apoptosis, and pyroptosis by activating MAPK/NF-κB and NLRP3 signaling [54].Moreover, DON induced OS and activated the Keap1/Nrf2 and TLR4/NF-κB signaling pathways that contribute to the production of inflammatory cytokines.The above data show that OS is a primary signal in the DON-induced activation of NLRP3.Deafness autosomal dominant 5 (DFNA5), also called gasdermin D (GSDME), belongs to the gasdermin family.A study by Roger et al. [55] revealed a novel mechanism by which GSDME activated caspase-3-mediated regulated pyroptosis.After exposure to 32 µM or 64 µM DON for 24 h, HepaRG cells exhibited typical pyroptotic characteristics, including the release of IL-1β and IL-6 and the activation of caspase-3 and GSDME [56].In addition, at concentrations approximating those in bovine follicular fluids, DON activated NLRP3 inflammasomes in ovarian thecal cells.Because of the association between inflammation of the ovaries, ageing and infertility, the effect of DON on dairy cow fertility should be studied further [57].
In vivo, DON-induced NLRP3-dependent pyroptosis was detected in mice and piglets.Several studies have reported that chronic and subacute oral administration of DON induced liver inflammatory injury and intestinal damage in mice by activating caspase-3/GSDME-dependent pyroptosis [56,58].DON exposure activated NLRP3 inflammasomes, increased OS, and induced the expression of pyroptosis-related factors (GSDMD, ASC, caspase-1 P20, and IL-1β) in the mouse jejunum [48].DON treatment enhanced OS in mice and piglets infected with enterotoxic Escherichia coli (ETEC) and increased the activation of NLRP3 inflammasomes in the jejunum and the expression of NLRP3, caspase-1, and ASC [50].In addition, DON exposure aggravated porcine epidemic diarrhea virus (PEDV)-induced immunosuppression by inhibiting TLR4/NF-κB/NLRP3 signaling in weaned piglets, thereby causing intestinal mechanical/immune barrier dysfunction [59].Although DON exposure has been confirmed to activate the NLRP3 inflammasome and cause host inflammatory damage in vitro and in vivo, the comprehensive mechanism of NLRP3 activation needs to be further investigated.
The regulatory effect of DON on the activity of NLRP3 inflammasomes is directly related to host immune status.Humans and animals are generally exposed to low doses of DON in the natural environment, and an exposure dose of less than 1 µg/kg BW does not cause harmful effects.When the host is in a state of pathogen infection, sensitivity to the DON dose increases.The production of NLRP3 inflammasomes, key regulators of inflammation, is also increased.Low-dose DON was able to increase pro-IL-1β, NLRP3, and caspase-1 in IPEC-J2 cells infected with ETEC K88 and enhance activation of NLRP3 inflammasomes [50].Exposure to a low dose of DON (2 µM) enhanced NLRP3 inflammasome activation and induced pyroptosis-mediated toxicity.Surprisingly, a high dose of DON (16 µM) inhibited the chemotaxis and phagocytosis of macrophages [60].Macrophages are a key link in inflammation leading to tissue damage.DON exposure aggravated PEDVinduced immunosuppression in a porcine alveolar macrophage model by inhibiting the TLR4/NF-κB/NLRP3 signaling pathway [59].Sertoli cells are immune-privileged cells in the testes.Song et al. [61] reported that incubation with 10 µM DON significantly increased the expression of pyroptosis-associated genes for caspase-1 and GSDMD-N in Sertoli cells of E. asinus.Therefore, DON has a bidirectional effect on activation of NLRP3 inflammasomes.Whether high-level exposure to DON leads to immunosuppression via the NLRP3 inflammasome remains unclear.
The effect of AFB1 on NLRP3 inflammasome activity has been studied in vivo and in vitro.The liver is the primary detox organ and is also the main site of AFB hepatotoxicity.AFB1 induced NLRP3 inflammasome-mediated pyroptosis in hepatocytes through dephosphorylation of cyclooxygenase-2 (COX-2) [64].AFB1 enhanced the expression of NLRP3 and pro-IL-1β by regulating NF-κB and downstream caspase-1 activity, p10, IL-1β, and GSDMD, in HepaRG cells and primary Kupffer cells (KCs).The enhanced proinflammatory signaling of KCs then activated the NLRP3 inflammasome in hepatocytes and upregulated the expression of COX-2 and proteins involved in assembly (NLRP3, ASC, and p10) and activation (IL-1β and GSDMD) of NLRP3 inflammasomes in primary hepatocytes [64].Consistent with this report, Lv et al. [65] also studied the role of AFB1-induced severe pyroptosis-dependent hepatotoxicity through NLRP3/caspase-1/GSDMD signaling in AML12 cells.
In vivo, AFB1 induced pyroptosis by enhancing NLRP3 inflammasome assembly and activation (NLRP3, caspase-1 and GSDMD) and the release of IL-1β and IL-18 in the livers of BALB/c and C57BL/6 mice [66].NLRP3-dependent pyroptosis in hepatocytes and liver injury in C57BL/6 mice were triggered by AFB1 via upregulation of COX-2 expression and an increase in mitochondrial damage [65,67].Excessive ROS production activates the thioredoxin-interacting protein (TXNIP) upon dissociation from thioredoxin, which then binds to NLRP3 to promote inflammasome activation [68].mtDNA synthesis induced by binding to TLRs is essential for activation of NLRP3 inflammasomes.TLR signaling triggers IRF1-dependent CMPK2 transcription and induces mtDNA synthesis through MyD88 and the TRIF adaptor, which activates the NLRP3 inflammasomes [69].In addition, Ca 2+mediated mitochondrial damage can trigger the activation of NLRP3 inflammasomes [70].PINK1/Parkin-mediated mitophagy is important in decreasing AFB1-induced liver injury in mice [62].Therefore, the mechanism of how mitochondrial damage affects NLRP3 inflammasome activation via AFB1 needs further investigation.Moreover, AFB1-induced liver and splenic pyroptosis in mice can be mediated by disturbing the gut microbiota-immune axis [71,72].
Furthermore, AFB1 induced GSDMD-mediated pyroptosis and inflammatory cytokine expression in primary microglia from C57BL/6 mice by activating the NLRP3 inflammasome [80].In addition, 100 µg/kg AFB1 in C57BL/6J mice and 50 µM AFB1 in primary microglia inhibited the proliferation and neuronal differentiation of neural stem/precursor cells through upregulation of NLRP3, caspase-1, GSDMD-N, and IL-1β in the hippocampus [6].In rats, AFB1 (0.15 and 0.3 mg/kg) caused increased OS and myocardial structural damage by activating the NLRP3 signaling pathway [81].Therefore, AFB1 exposure causes various pathological changes in the host through the activation of NLRP3-mediated inflammation.Interestingly, chronic mild stress aggravated these changes in mice.
Studies have shown that the inflammatory response is the primary player in ZEAinduced intestinal toxicity, via interfering with the TLR4/NF-κB pathway [84,85].In IPEC-J2 cells, ZEA exposure activated the ROS-mediated NLRP3 inflammasomes, which increased the secretion of caspase-1-dependent inflammatory factors and initiated the intestinal inflammatory cascade response [86,87].Dextran sulfate sodium (DSS) is a chemical colitogen with anticoagulant properties that has been widely used to induce colitis.This model is especially helpful for delving into the role of innate immune mechanisms in intestinal inflammation because of its similarity to human ulcerative colitis.DSS increased the expression levels of NLRP3, ASC, caspase-1, pro-IL-1β, and pro-IL-18 in IPEC-J2 cells and in mouse colon tissues, consistent with the findings that ZEA induced caspase-1 activation via the NLRP3 inflammatory complex [84].However, ZEA has a surprising degree of protection against inflammatory responses.The reason for this might be that the structure of ZEA is similar to that of oestradiol-17β [85].After coadministration of DSS and ZEA, inflammatory cell infiltration and tissue damage were significantly restored.Oestradiol-17β is an ovarian oestrogen with anti-inflammatory properties, and its anti-inflammatory activity can be mediated through the oestrogen receptors ERα and ERβ.Intestinal ERβ expression has a protective effect on colitis-associated colorectal cancer.The expression of ERβ was extremely high in the colons of mice treated with DSS and ZEA [84].Additionally, administration of up to 10 µM ZEA did not significantly upregulate ASC, IL-1β, or IL-18 expression in bovine primary theca cells; the expression levels of NLRP3 and IL-1β were weakly altered [57].However, whether high-level exposure to ZEA activates the NLRP3 in-flammasome in reproductive organs remains unclear [57].Interestingly, ZEA suppressed the LPS-induced macrophage immune response by decreasing proinflammatory mediators and cytokines [88].
The autophagy-mediated regulation of the activity of NLRP3 inflammasomes is essential for immune homeostasis.In a rat insulinoma cell line, ZEA promoted activation of NLRP3 inflammasomes by modulating NF-κB/p65 signaling to induce NLRP3-dependent pyroptosis and inflammation.However, the increase in autophagy prevented ZEA-induced NLRP3 inflammasome activation and inflammatory responses [5].
Research has shown that IL-1β secretion induced by the fungal trichothecene mycotoxin roridin A is dependent on the NLRP3 inflammasome through P2X7R and Src tyrosine kinase signaling in human primary macrophages [91].To date, there have only been two studies on the mechanisms by which T-2 toxin affects NLRP3 activation and pyroptosis.Yang et al. [92] reported that T-2 toxin induced ROS that activated NLRP3 inflammasomes and caused testicular inflammatory damage, thereby leading to disruption in TM4 cells and severe reproductive disorders in male mice [92].Kidneys are the primary targets of T-2 toxin, which activated NLRP3 inflammasomes causing fibrosis via the mtROS/NLRP3/Wnt/β-catenin axis in a T-2 animal model [93].

OTA and the NLRP3 Inflammasome
OTA mycotoxins are widely found in several species of Aspergillus spp.including A. ochraceus, A. carbonarius, A. niger, A. steynii, A. subramanianii, and A. westerdijkiae and Penicillium spp.including P. brevicompactum, P. chrysogenum, P. commune, P. cyclopium, P. nordicum, P. polonicum, P. verrucosum, and P. viridicatum.OTA can contaminate a variety of foodstuffs, such as wheat, maize, and beans.The detection rate of OTA over a large range of values is 5-31% in animal feeds [42,43].The molecular formula of OTA ((2S)-2-[[(3R)-5-chloro-8-hydroxy-3-methyl-1-oxo-3,4-dihydroisochromene-7-carbonyl]amino]-3-phenylpropanoic acid) is C 20 H 18 ClNO 6 .Due to its stable chemical properties and strong tolerance of OTA, it easily survives in feed and retains its strong toxicity.OTA has a half-life of more than a month in humans and animals.It has now been shown that OTA conveys strong nephrotoxic, hepatotoxic, immunotoxic, carcinogenic, and teratogenic effects.OTA is listed as a group 2B carcinogen by the IARC.As kidneys are the target organ of OTA, renal injury, renal fibrosis, interstitial nephritis, and other nephropathies are common in the kidneys of rodents, pigs, and humans [94].OTA induces nephrotoxicity both in vivo and in vitro by promoting apoptosis, inducing OS, regulating autophagy, and inhibiting mitosis [95].
In addition, studies have reported that mitochondrial damage is one of the mechanisms of OTA-induced cytotoxicity in various cell lines [98,99].It has been shown that OTA can lead to disruption of mitochondria and cell death via apoptosis and autophagy in human gastric epithelial cells [100].Additionally, OTA upregulated the expression of TFAM (an mtDNA transcription factor) in ducklings and enhanced mitochondrial OS in the jejunal mucosa [101].Mitochondrial damage is an important pathway activating NLRP3 inflammasomes.Whether OTA induces the activation mechanism of NLRP3 inflammasomes through mitochondrial damage remains to be further studied.
Ma et al. found that FB1 caused pyroptosis in IPEC-J2 cells by upregulating preprotein translocation factor (Sec62) to activate the PKR-like ER kinase protein pathway [102].FB1 also promoted inflammatory cytokine secretion and upregulated the expression of NLRP3 and caspase-1, causing damage in IPEC-J2 cells and in intestinal epithelial cells in mice [103].However, FB1-induced NLRP3-dependent pyroptosis is shielded by autophagy.FB1-induced pyroptosis increases the level of autophagy to a certain extent, which is related to the inhibition of mechanistic target of rapamycin (mTOR) phosphorylation.The mTOR inhibitor, rapamycin, inhibited the expression of NLRP3 and downregulated FB1-induced pyroptosis in IPEC-J2 cells, indicating the capacity to alleviate intestinal inflammatory injury.This indicates that increasing the level of autophagy may be used as a method to against tissue damage induced by FB1 exposure in practice.
In addition to the abovementioned mycotoxins, patulin (PAT) can activate NLRP3 inflammasomes [104].PAT (4-hydroxy-4H-furan [3, 2c] pyran-2 [6H]-ketone) is a common mycotoxin in fruits and vegetables, and its molecular formula is C 7 H 6 O 4 .Although PAT is considered a noncarcinogen, it is genotoxic, cytotoxic, and neurotoxic, mainly causing liver and kidney damage.PAT also induced pyroptosis and inflammatory damage through activation of NLRP3 inflammasomes in mouse livers and HepG2 cells [104].Mycophenolic acid (MPA) is an in vivo metabolic product of mycophenolate mofetil.Although MPA alone does not affect the levels of NF-κB p-p65 or pro-IL-18 in THP-1 cells, it synergizes with LPS to greatly promote the secretion of IL-18 through activation of NLRP3 inflamma-somes [105].However, it is unknown whether other mycotoxins, such as Alternaria toxins, citrinin, enniatins, ergot alkaloids, nivalenol, and HT-2 toxin, are capable of triggering NLRP3 inflammasome activation.

Targeting the NLRP3 Inflammasome for Mycotoxin Exposure
Much research on pharmacological interference with the activation of NLRP3 inflammasomes has been recently published [33].Theoretically, common inflammasome components or signaling molecules activating NLRP3 inflammasomes could be therapeutic targets for inhibiting NLRP3-dependent inflammation.To our delight, the development of NLRP3 inhibitors has been vigorously promoted, and a range of NLRP3 inhibitors have been discovered and have shown promising therapeutic potential in clinical trials with a wide array of autoinflammatory and chronic inflammatory diseases [33].Mycotoxininduced nephrotoxicity, hepatotoxicity, and immunotoxicity are closely related to the NLRP3 inflammasome, making it a likely target for pharmacotherapy.Inhibiting the activation of NLRP3 inflammasomes can effectively reduce the toxic effects of mycotoxins.Therefore, targeting NLRP3-mediated inflammation represents a novel therapeutic option for against mycotoxin-induced toxicity [33,106,107].MCC950 (originally described as CRID3 and CP-456,773), a diaryl sulfonylureacontaining compound, was first identified by Gabel et al. [34].MCC950 is the beststudied small-molecule inhibitor of NLRP3 and does not affect the NLRP1, NLRC4, or AIM2 inflammasome-or TLR-mediated priming signals.MCC950 directly interacts with the Walker B motif in the NACHT domain of NLRP3, thereby preventing ATP hydrolysis, NLRP3 activation, and inflammasome generation [108].MCC950 has since been widely studied for its therapeutic effects on autoimmune, cardiovascular, metabolic, and other diseases and has entered phase-II clinical trials [109].MCC950 has also been shown to attenuate DON-induced pyroptosis and inflammatory responses by preventing activation of NLRP3 inflammasomes.In TM4 cells, inflammatory damage and upregulation of NLRP3, ASC, caspase-1, IL-1β, and IL-18 caused by 4 nM T-2 toxin were markedly lower in the presence of 20 nM MCC950 [92].However, MCC950 (20 nM) did not lower ROS levels in TM4 cells [92].The fibrosis caused by T-2 toxin and the structural and functional damage through activation of NLRP3 inflammasomes and the Wnt/β-catenin pathway were mitigated by MCC950 in an HK-2 cell model (10 µM) and a C57BL/6N mouse model (10 mg/kg) [93].MCC950 (10 µM) blocked FB1-induced pyroptosis by abolishing the expression of GSDMD and the release of proinflammatory factors, IL-1β and IL-18, induced by the combination of FB1 and DON in IPEC-J2 cells [58,103].The above phenomena have also been observed in DON-treated IPEC-J2 cells and HaCaT cells [48,54] and in patulin-treated HepG2 cells.Therefore, MCC950 can serve as a potential treatment for alleviating toxicity-induced mycotoxins [104].
Luteolins (3,4,5,7-tetrahydroxy flavones) are flavonoids mainly found in fruits, vegetables, and herbs.Luteolin has multiple useful effects, including antitumour, antiinflammatory, antiviral, antioxidant, and immune regulatory effects.Luteolin inhibits NLRP3 inflammasome activation by preventing the interaction of NLRP3 with ASC.To date, the protective effect of luteolin on inflammatory injury induced by mycotoxin exposure following NLRP3 inflammasome activation has not been directly reported.However, luteolin mitigated DON-induced toxicity in broilers by reducing oxidative damage [114] and alleviated AFB1-induced apoptosis and OS in mouse [115] and rat liver [116].Luteolin (50 and 100 µg/mL) also reduced OTA-induced damage on Vero cells and rat lymphocytes [117].Luteolin reduced OTA-induced OS by reducing ROS levels, reversing the reduction in mitochondrial membrane potential, activating antioxidant enzymes, and regulating the Nrf2 and HIF-1α pathways in NRK-52E rat kidney cells (100 µM) and IPEC-J2 cells (8.7 µM) [118,119].Luteolin (8,16, and 32 µg/mL) also reduced FB1-induced damage to the intestine from inflammation, through inhibiting NF-κB and ERK signaling and reducing IL-6 and IL-1β expression in IPEC-J2 cells [120].It has been reported that luteolin could serve as a target for limiting mycotoxin-induced neuroinflammation and improving neuropsychiatric diseases [121].Thus, luteolin could alleviate mycotoxin-induced toxicity through a reducing OS and ameliorating the reduction in the mitochondrial membrane potential.
Nonsteroidal anti-inflammatory drugs (NSAIDs) can reduce inflammation, acute fever, and pain.Piroxicam is an NSAID that suppresses NLRP3 activation via reversible inhibition of volume-regulated anion (chloride) channels and the NF-κB pathway.To date, the mitigating effect of NSAIDs on inflammatory injury induced by mycotoxin exposure following NLRP3 inflammasome activation has not been described.Interestingly, piroxicam (5 mg/kg/48 h for 14 d) proved useful in preventing the chronic toxic effects of OTA (289 µg/mL), particularly nephrotoxicity, in rats [122].However, indomethacin, phenylbutazone, and aspirin were not effective in preventing mortality in mice administered with T-2 toxin [123].Therefore, whether NSAIDs can alleviate mycotoxin toxicity via inhibiting the NLRP3 inflammasome requires further research.
Tanshinone I (Tan I) and Tan IIA are the active ingredients in Salvia miltiorrhiza plants.Tanshinone exerts anticancer, antioxidant, and anti-inflammatory effects via different mechanisms.Tan I prevents the formation and activation of NLRP3 inflammasomes by blocking the NLRP3-ASC interaction.To date, there has been no report that Tan I can reduce the inflammatory injury from mycotoxin exposure following NLRP3 inflammasome activation.However, Tan IIA (45 µg/mL) did show a protective effect against DON-mediated toxicity in IPEC-J2 cells by inhibiting the expression of NLRP3, caspase-1, GSDMD, IL-1β, and IL-18 [47] and restoring mitochondrial function via quality control [124].
Autophagy negatively regulates NLRP3 inflammasome activation, inhibits the inflammatory response, and reduces inflammatory damage [125].The mechanism by which autophagy inhibits NLRP3 inflammasomes may be related to a reduction in the expression of ASC protein, NLRP3 phosphorylation, and the clearance of mtROS.Hemin and its derivative, cobalt protoporphyrin, inhibited assembly of NLRP3 inflammasomes by enhancing autophagy, resulting in increased degradation of ASC [126].Phosphorylation of NLRP3 promoted entry of the NLRP3 protein into autophagosomes and inhibiting the activation of NLRP3 inflammasomes [127].Rapamycin, an inhibitor of mTOR, reduced NLRP3 inflammasome activation by attenuating the mTOR/NF-κB signaling pathway in macrophages [128].Notably, rapamycin (50 nM) decreased damage to the intestine from FB1-induced inflammation and inhibited the upregulation of pyroptosis-related genes in IPEC-J2 cells [103].Injection (i.p.) of 5 mg/kg rapamycin protected animals from FB1-created enteritis by inhibiting the expression of pyroptosis-related genes.PINK1/Parkin-induced mitophagy alleviated mitochondrial damage from exposure to T-2 toxin by decreasing the activation of NLRP3 inflammasomes [129].Another study showed that PINK1/Parkin activation alleviated AFB1-induced liver injury, and that Parkin expression deficiency aggravated NLRP3 inflammasome activation and mitochondrial damage in AFB1-exposed mice [67].However, 3-methyladenine (1 mM), an autophagy inhibitor, was shown to attenuate the LDH release, activation of NLRP3 inflammasomes, pyroptosis, and inflammatory responses induced by patulin [104].Therefore, the role of autophagy during pyroptosis needs to be verified through further experiments [125].
The above studies reveal that inhibiting the activation of NLRP3 inflammasomes can significantly reduce the toxic effects of mycotoxins (Table 1).In the future, we will focus on identifying NLRP3 inhibitors that alleviate the injurious effects of fungal toxins.↓ represents downregulated levels.

Conclusions and Future Perspectives
Mycotoxins are harmful compounds synthesized by specific filamentous fungi that pose a danger to human and animal health.Contamination with mycotoxins causes wastage of food and animal feed and harms the world's commerce in farm crops.The control of mycotoxins is based on two strategies: prevention of toxin production and detoxification.Biodegradation of mycotoxins as a detoxification method has become a popular subject for research.The use of specific microbes to reduce mycotoxin contamination of crops is highly effective and broadly applicable.In recent years, our group has screened and identified bacteria with the capability of digesting mycotoxins including ZEA-degrading Bacillus velezensis [131], AFB1-degrading Bacillus subtilis [132], and DON-degrading Bacillus cereus [133].Probiotics are safe and effective alternatives to fungicides for controlling mycotoxin contamination, but the toxicological mechanisms of mycotoxins are complex and unclear and need to be further studied to clarify their pathogenic activity.
The innate inflammatory immune response protects the body from pathogenic attack, but chronic inflammation can lead to diseases such as cancers.In recent years, the toxicological mechanisms of mycotoxins, including DON, AFB1, ZEA, T-2 toxin, OTA, and FB1, have been associated with the systemic NLRP3 inflammasome, which contributes to immunotoxicity, hepatotoxicity, intestinal toxicity, and neurotoxicity.Here we have summarized the recent research on the dysregulated expression of the NLRP3 inflammasome in response to mycotoxin exposure and the roles of NLRP3 inflammasome inhibitors in alleviating tissue damage and the inflammatory response induced by mycotoxins.and ZEA, whereas studies of OTA, T-2 toxin, FB1, mycophenolic acid, and patulin are scarce; alternaria, HT-2 toxins, citrinin, enniatins, ergot alkaloids, and nivalenol have not been studied at all.Based on the existing research, it is currently not possible to ascribe a set of common factors to the activation of NLRP3 inflammasomes triggered by all 500 mycotoxins.Therefore, a wide range of mycotoxins as a subject of future study is necessary to improve understanding of the general rules for mycotoxininduced NLRP3 inflammasome activation.(3) In recent years, more than 20 pharmacological inhibitors of NLRP3 inflammasomes have been reported, some of which exhibit promising therapeutic potential for treating NLRP3-related diseases in the clinic.Undeniably, all studies have clearly indicated that MCC950, BAY 11-7082, curcumin, and luteolin ameliorate tissue damage and inflammatory responses in models of mycotoxin exposure.Theoretically, any molecule or signal involved in the activation of NLRP3 inflammasomes, such as active caspase-1, GSDMD cleavage, proteins involved in inflammasome assembly, and inflammatory cytokines, can potentially inhibit the NLRP3 inflammasome.These are the two major mechanisms of small molecule inhibitors: they directly interact with the NLRP3 protein and bind to the ATP-binding motifs of the NACHT domain of NLRP3, subsequently inhibiting ATPase activity.In addition to the inhibitors mentioned above, other kinds of NLRP3 inflammasome inhibitors should be designed to investigate their effects on mycotoxin exposure.It will be worth examining whether the occurrence of chemical reactions between these inhibitors and mycotoxins reduces their toxic effects.For example, the carbon-carbon double bonds in BAY 11-7082 can react with the amino group in FB1 via Michael addition, and the sulfonyl group in MCC950 can react with the carboxyl group in FB1 via amide bond-forming reactions.
In summary, the activation of NLRP3 inflammasomes by mycotoxins via oxidative stress and the NF-κB pathway exacerbates the pathological conditions of the host.Our data improve the understanding of mycotoxin toxicity by demonstrating that the activation of NLRP3 inflammasomes has a critical role in the toxic effects of mycotoxins.Therefore, targeting the NLRP3 inflammasome could be an effective strategy for alleviating my-cotoxin-induced toxicity.However, there is still a long way to go before exploiting NLRP3 inflammasome inhibitors can be confidently applied to the treatment of disease.Future research should focus on the development of safe, specific, efficient, stable, nontoxic, inexpensive, and simple preparation processes for NLRP3 inflammasome inhibitors.We believe that the continuing advancement in biomolecular medicine and related technologies will result in a substantial number of new NLRP3 inflammasome inhibitors that will steadily be applied clinically to mitigate mycotoxin-induced toxicity and treat NLRP3related inflammatory diseases.

Figure 1 .
Figure 1.Depiction of activation of an NLRP3 inflammasome by mycotoxin (DON, AFB1, ZEA, OTA, and FB1) exposure (created in Figdraw).In the case of DON exposure, the first signal comes from TLR4 and NF-κB and increases the intracellular transcripts of pro-IL-1β, pro-IL-18, and NLRP3.

( 1 )
As mentioned above, mycotoxins trigger NLRP3 inflammasome activation via the canonical activation pathway.Although evidence suggests that exogenous stimuli such as DON, AFB1, ZEA, OTA, T-2 toxin, FB1, or patulin can trigger NLRP3 inflammasome activation, whether these mycotoxins act directly on NLRP3 remains unclear.Previous studies have investigated alterations in the expression of factors involved in generation of NLRP3 inflammasomes in response to mycotoxin exposure, but the exact mechanisms and signaling pathways by which mycotoxins induce NLRP3 inflammasome activation have remained elusive.(2)Most of the research on NLRP3 inflammasome activation has focused on DON, AFB1,

Table 1 .
Literature summary of the effects of NLRP3 inflammasome inhibitors on mycotoxin exposure.