Zearalenone disturbs the reproductive-immune axis in pigs: the role of gut microbial metabolites

Abstract

Background: Exposure to zearalenone (ZEN, a widespread Fusarium mycotoxin) causes reproductive toxicity and immunotoxicity in farm animals, and it then poses potential threats to human health through the food chain. A systematic understanding of underlying mechanisms on mycotoxin-induced toxicity is necessary for overcoming potential threats to farm animals and humans. The gastrointestinal tract is a first-line defense against harmful mycotoxins; however, it remains unknown whether mycotoxin (e.g., ZEN)-induced toxicity on the reproductive-immune axis is linked to altered gut microbial metabolites. In this study, using pigs (during the three phases) as an important large animal model, we investigated whether ZEN-induced toxicity on immune defense in the reproductive-immune axis was involved in altered gut microbial-derived metabolites. Moreover, we observed whether the regulation of gut microbial-derived metabolites through engineering ZEN-degrading enzymes counteracted ZEN-induced toxicity on the gut-reproductive-immune axis.

Results: Here, we showed ZEN exposure impaired immune defense in the reproductive-immune axis of pigs during phase 1/2. This impairment was accompanied by altered gut microbial-derived metabolites [e.g., decreased butyrate production, and increased lipopolysaccharides (LPS) production]. Reduction of butyrate production impaired the intestinal barrier via a GPR109A-dependent manner, and together with increased LPS in plasma then aggravated the systemic inflammation, thus directly and/or indirectly disturbing immune defense in the reproductive-immune axis. To validate these findings, we further generated recombinant Bacillus subtilis 168-expressing ZEN-degrading enzyme ZLHY-6 (the Bs-Z6 strain) as a tool to test the feasibility of enzymatic removal of ZEN from mycotoxin-contaminated food. Notably, modified gut microbial metabolites (e.g., butyrate, LPS) through the recombinant Bs-Z6 strain counteracted ZEN-induced toxicity on the intestinal barrier, thus enhancing immune defense in the reproductive-immune axis of pigs during phase-3. Also, butyrate supplementation restored ZEN-induced abnormalities in the porcine small intestinal epithelial cell.

Conclusions: Altogether, these results highlight the role of gut microbial-derived metabolites in ZEN-induced toxicity on the gut-reproductive-immune axis. Importantly, targeting these gut microbial-derived metabolites opens a new window for novel preventative strategies or therapeutic interventions for mycotoxicosis associated to ZEN.

Keywords: Butyrate; Gastrointestinal tract; Immune system; Immunity; Lipopolysaccharides; Mycoestrogen; Mycotoxin; Reproductive system; Sus scrofa; Zearalenone.

Fig. 1

ZEN-contaminated food disturbs reproductive and immune systems. a, b The organ’s index of pigs, during phase-1 (a) and phase-2 (b), between the Ctrl group and zearalenone (ZEN) group (n=4). c, d The uterine size (uterine length, uterine width, and uterine horn’s width) of pigs (n=4). e, f Representative H&E staining of immune organs (liver, spleen, thymus, and inguinal lymph nodes) and reproductive organs (uterus and reproductive tract) from each group under ×40 magnification. The scale bar represents 100 μm. g, h Representative transmission electron microscopy (TEM) images of immune organs and reproductive organs from each group (The yellow arrows show swelling nucleus). The scale bar represents 2 μm. i–l The mRNA expression of oxidative stress markers (i, j) and pro-inflammatory cytokines (k, l) in immune organs (e.g., liver and thymus) and reproductive organs (e.g., uterus) (n=8). Bar values are means ± SEM. *P < 0.05

Fig. 2

RNA-seq analysis unravels ZEN-induced toxicity on the reproductive system. a–i ZEN disturbs the reproductive system (e.g., uterus) in pigs during phase 1/2. a Distribution of differentially expressed genes (DEGs) in the uterus of phase-1/2’s pig between the Ctrl group and ZEN group (n=4). b Gene Ontology (GO) classification of overlapping DEGs. BP represents biological process; CC represents cellular component; MF represents molecular function. c Top10 terms of GO enrichment of overlapping DEGs in the uterus of phase-1 pig, the heatmap of DEGs enriched in marked terms and MGI & Human diseases of representative DEGs. d Statistical analysis of top30 KEGG pathways enriched by DEGs in the uterus. e Pathways related to immune diseases and system in top30 KEGG pathways in the uterus of phase-1 (upper) or phase-2 (down) pig. The arrow referred to different terms between phase-1 pigs and phase-2 pigs. f, g Interactions of genes enriched in immune diseases and system terms in the uterus of phase-1 pigs (f) and phase-2 (g) pigs. h The heatmap of inflammatory cytokines in the uterus of phase-1 and phase-2 pigs based on interactions of genes enriched in immune diseases and system terms. i RPKM value and RT-qPCR results of representative genes detected in the uterus. Bar values are means ± SEM. *P < 0.05

Fig. 3

RNA-seq analysis unravels ZEN-induced toxicity on the immune system. a–f ZEN disturbs the immune system (e.g., liver, spleen, thymus, and inguinal lymph nodes) in pigs, during phase-1/2. a Number of DEGs detected in immune organs. b Statistical analysis of top30 KEGG pathways enriched by DEGs in immune organs. c Venn map of KEGG pathways related to immune disease and system in immune organs. d Number of overlapping KEGG pathways between phase-1 and phase-2 pigs and its corresponding number of common genes in overlapping pathways. e Other top30 KEGG pathways enriched by DEGs in immune organs. f Representative Western blots and its quantifications of key proteins involved in NF-κB (e.g., p-p65), CAMs (e.g., CD54/ICAM-1 and E-cadherin), and phagosome (e.g., CD63 and TLR4) pathways (n=4). Bar values are means ± SEM. *P < 0.05

Fig. 4

ZEN-induced toxicity alters gut microbial metabolites. a, b The community richness (Chao and ACE index) and community diversity (Shannon and Simpson index) in all five gut sections, including the duodenum, jejunum, ileum, caecum, and colon of pigs, during phase-1 (a) and phase-2 (b), between the Ctrl group and ZEN group (n=8). c, d The PLS-DA score plots of phase-1 pigs (c) and phase-2 pigs (d) between the Ctrl group and ZEN group (n=40, pooled samples in five gut sections). e, f Bacterial compositions at the phylum level and its dominant bacterial phyla (relative abundance > 1.00%) of all five gut sections of phase-1 pigs (e) and phase-2 (f) pigs that were exposed to ZEN (n=8). g, h Differential abundance analysis of gut bacteria (at the family level) in pigs (n=40, pooled samples in five gut sections). The bar plot on the left shows the top 20 most abundant bacteria. The bubble plot on right displays its Wilcox test results. i, j The concentrations of short-chain fatty acids (SCFAs) in all five gut sections (n=8). k The levels of plasma lipopolysaccharides (LPS) in pigs (n=8). l, m Spearman’s correlation coefficients between core bacteria (at the family level) and major microbial metabolites, including SCFAs and LPS. Bar values are means ± SEM. *P < 0.05

Fig. 5

ZEN-induced toxicity impairs the intestinal barrier by altered microbial metabolites. a, b Representative Western blots and its quantification of SCFAs receptors (e.g., GPR109A and GPR41) in the jejunum, ileum, and colon of pigs, during phase-1 pigs (a) and phase-2 pigs (b), between the Ctrl group and ZEN group. The GAPDH is used as the loading Ctrl (n=4). c Representative Western blots and its quantification of the ERK1/2 and p38 signalling pathway (n=4). d–f The mRNA expression (d, e, n=8), representative Western blots and its quantification (fn=4) of tight junction markers (e.g., occludin, claudin-1, and ZO-1) in the jejunum, ileum, and colon. g, h The intestinal morphology of pigs during phase-1 (g) and phase-2 (h). The left of panels g and h: representative light micrographs of a cross-section from the jejunum, ileum, and colon under ×40 magnification. The scale bar represents 200 μm. On the right of panels g and h: the quantification of villus height and crypt depth (n=8). i, j Periodic acid–Schiff staining (PAS) staining for goblet cells in the jejunum, ileum, and colon. On the left of panels i and j: representative staining for goblet cells in the intestine (The arrows indicate positive goblet cells). Scale bars: 100 μm. The right of panels i and j: the quantification of immunoreactive goblet cells using digital image analysis (n=8). k, l The mRNA expression of pro-inflammatory cytokines in the jejunum, ileum, and colon (n=8). m, n The levels of plasma pro-inflammatory cytokines in pigs (n=8). Bar values are means ± SEM. *P < 0.05

Fig. 6

Modified gut microbial metabolites by engineering ZEN-degrading enzyme ZLHY-6 counteracts ZEN-induced toxicity on intestinal barrier function. a Generation of recombinant B. subtilis-expressing ZLHY-6 (the Bs-Z6 strain) and its degradation effect on ZEN-contaminated food. a_1–3 Construction and its identification of the recombinant expression vector-pWBZ7 containing ZLHY-6 gene (a₁). a_2–3 The PCR identification and its double enzyme digestion of positive transformants of the recombinant Bs-Z6 strain. Lanes 1-4: positive transformants; M: DNA marker. a₄ SDS-PAGE electrophoresis analysis of purified ZLHY-6 in recombinant Bs-Z6 strain. M: protein ladders; Lanes 1, 3, 5: 0.8, 5, 25-fold target protein purification samples, respectively. Lanes 2, 4: 80μg/mL, 16μg/mL BSA, respectively. a₅ SDS-PAGE electrophoresis of recombinant ZLHY-6 protein expressed in Bs-Z6 at different fermentation duration (6, 9, 12 h). a₆ Stability evaluation of pWBZ7 in the recombinant Bs-Z6 strain. Lanes 1, 2: Enzyme digestion of pWBZ7 plasmid from the first and 60th generation of this recombinant strain, respectively. M: DNA ladder. a₇ The degradation activity of ZEN detected by HPLC for the genetic engineering strains. b The community richness (Chao and ACE index) and community diversity (Shannon and Simpson index) in the colon of phase-3 pigs among the control (Ctrl), zearalenone (ZEN), ZEN supplemented with recombinant Bs-Z6 strain (Bs-Z6) groups (n=8). c Analysis of PLS-DA score plots (n=8). d Bacterial compositions at the phylum level and its dominant bacterial phyla (relative abundance > 1%). e, f Differential abundance analysis of potential butyrate-producing bacteria and LPS-producing bacteria (at the family level) (n=8). g The concentrations of SCFAs in the colon. h The levels of plasma LPS. i–k Representative Western blots and its quantification of SCFAs receptors (GPR109A and GPR41), the ERK1/2 and p38 signalling pathway, and tight junction markers (occludin, claudin-1, and ZO-1) in the colon (n=4). The B-actin is used as the loading control (n=4). l Representative light micrographs of a cross-section of the colon under ×40 magnification and its quantification of villus height, crypt depth (n=8). Scale bar represents 200 μm. m, n The mRNA expression (m) and plasma concentrations (n) of pro-inflammatory cytokines (n=8). o Butyrate supplementation reduces ZEN-induced toxicity on intestinal barrier in porcine intestinal epithelial cell (IPEC-J2). o₁ Representative TEM images of IPEC-J2 cells from each group (The arrows show swelling nucleus and/or mitochondria). The scale bar represents 500 nm. o₂ Representative confocal microscope images of oxidative stress marker ROS content) of IPEC-J2 cells. o_3–4 Representative Western blots and confocal microscope images of tight junction markers (occludin and claudin-1) (n=3). o₅ The mRNA expression of pro-inflammatory cytokines (e.g., IL-12) (n=4). Bar values are means ± SEM. *P < 0.05

Fig. 7

Modified microbial metabolites reduce ZEN-induced toxicity on reproductive and immune systems. a Uterine size (uterine length, uterine width, and uterine horn’s width) of phase-3 pigs among the control (Ctrl), zearalenone (ZEN), ZEN supplemented with recombinant Bs-Z6 strain (Bs-Z6) groups (n=4). b Representative H&E staining of immune organs (e.g., thymus) and reproductive organs (e.g., uterus) from each group under ×40 magnification. The scale bar represents 100 μm. c Representative TEM images of thymus and uterus (The arrows show swelling nucleus). The scale bar represents 2 μm. d The mRNA expression of oxidative stress markers in the thymus and uterus (n=8). e, g Transcriptomic analysis reveals altered gene expression patterns in the thymus (e) and uterus (g). f, h The top 15 enriched Gene Ontology (GO) terms from the biological process in the thymus (f) and uterus (h). i Pathways related to immune diseases and system in top30 KEGG pathways of the thymus and uterus. j Statistical analysis of top30 KEGG pathways enriched by differentially expressed genes (DEGs). k RT-qPCR results of pro-inflammatory cytokines detected in the thymus and uterus. Bar values are means ± SEM. *P < 0.05

Fig. 8

Schematic representation of ZEN disturbs the reproductive-immune axis accompanied by altered gut microbial metabolites. (1) Excessive mycotoxin (e.g., ZEN)-induced toxicity firstly shifted gut bacterial community profiles, and then elevated microbial-derived LPS but lowered microbial-derived butyrate. (2) Reduction of butyrate production resulted in intestinal barrier dysfunction via a GPR109A-dependent manner, and together with increased LPS in plasma aggravated the systemic inflammation. (3) Subsequently, ZEN-induced systemic inflammation impaired immune-related pathways in immune systems, and these changes were also accompanied by pathomorphism and mitochondrial oxidative stress. (4) Meanwhile, ZEN-induced toxicity disturbed the reproductive system through an over-triggered systemic inflammatory response, which was closely related to alterations in specific immune-related genes