ZEN disturbs reproductive and immune systems
To test the toxicity effects of ZEN on both pre-starter (phase-1) and starter (phase-2) pigs, ZEN/its metabolites residues, growth performance, organ’s index, and vulvar areas were measured. Using the HPLC-MS/MS assay, we observed both phase-1 and phase-2 pigs that were exposed to ZEN displayed a higher residue of ZEN/its metabolites in all five gut sections (e.g., duodenum, jejunum, ileum, caecum, and colon), blood, immune system (e.g., liver, spleen, thymus, and inguinal lymph nodes), and reproductive system (e.g., uterus and ovary) (P < 0.05) (Supplemental Table S1). However, no obvious changes in growth performance were observed in pigs between the Ctrl group and ZEN group (Supplemental Table S2). Although no significant differences were found in the relative weight of immune organs in pigs between the Ctrl group and ZEN group, the reproductive tract of pigs that were exposed to ZEN showed a higher relative weight (P < 0.05) (Fig. 1a, b). Next, a larger vulvar area in pigs exposed to ZEN was started on day 7 (P < 0.05) and then on day 14 (P < 0.01) (Supplemental Fig. S1a-b). Also, an obvious increase in uterine size (e.g., uterine length, uterine width, and uterine horn’s width) was observed in pigs exposed to ZEN (Fig. 1c, d). Accordingly, compared with other organs, the reproductive system in pigs is a comparatively high sensitivity to ZEN.
Next, we explored whether ZEN-induced toxicity on the reproductive system was accompanied by morphologic changes, mitochondrial dysfunction, oxidative stress, and inflammation in immune organs. As indicated in Fig. 1e, f, obvious pathomorphism in reproductive and immune systems from pigs that were exposed to ZEN resulted in inflammatory lesions. Also, by using the TEM method, we observed pigs in the ZEN group showed a remarkable swelling nucleus (Fig. 1g, h). Furthermore, ZEN-induced toxicity rapidly contributed to adverse oxidative stress (e.g., increased mRNA expression of Cu/Zn-SOD, Mn-SOD, GSH-Px, and PGC-1α) (Fig. 1i, j; P < 0.05), thus resulting in increased mRNA expression of local pro-inflammatory cytokines (e.g., TNF-α, IFN-γ, IL-1β, and IL-12) (Fig. 1k, l; P < 0.05). Taken together, these data demonstrated that the effect of ZEN-induced toxicity on the reproductive system was linked to an evoked inflammation in the immune system.
RNA-seq reveals ZEN-induced toxicity on the reproductive system
Using RNA-seq analysis, we further revealed the underlying mechanism of ZEN-induced toxicity on the reproductive system, including the uterus (Fig. 2, Supplemental Table S3) and ovary (Supplemental Fig. S2, Supplemental Table S3). Compared with the Ctrl group, ZEN exposure significantly shifted uterus’s gene expression patterns, with 1179 and 3154 differential genes in phase-1 pigs and phase-2 pigs, respectively (Fig. 2a). In the uterus from phase-1 pigs that were exposed to ZEN, upregulated genes were 621 while downregulated ones were 558. Additionally, phase-2 pigs that were exposed to ZEN showed 1315 of upregulated genes but 1839 of downregulated ones. Among these, only a few differential genes altered dramatically [log2(FC)>4 or log2(FC)<−4, 6.36~13.98%] (Fig. 2a). Also, there were 345 upregulated and 216 downregulated overlapping genes between phase-1 and phase-2 pigs, and Gene Ontology (GO) analysis of these genes, including BP (biological process), MF (molecular function), and CC (cellular component), showed a similar term enrichment (Fig. 2b). Furthermore, according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, upregulated overlapping genes were enriched in the cytoskeleton structure, whereas downregulated overlapping genes were involved in protease activity (Fig. 2c). Also, statistical analysis of the top 30 pathways enriched in KEGG demonstrated that the most significant enrichment pathways were related to immune system and infection diseases (Fig. 2d). Among these, both phase-1 pigs (8/13) and phase-2 pigs (8/11) showed many overlaps among most pathways related to immune system, such as autoimmune thyroid disease, primary immunodeficiency, systemic lupus erythematosus, and allograft rejection (Fig. 2d-e).
Next, by using the Cytoscape STRING, we identified that the network of maximal clique centrality (MCC) on coding genes were enriched in immune system and showed three genes (e.g., serpina1, PLG, and FGG) in the first nine genes were consistent between phase-1 pigs and phase-2 pigs (Fig. 2f, g). Although a similar toxicity effect of ZEN exposure on the uterus of pigs during phase 1/2 was shown, its coding genes and underlying mechanism might be different due to age-related changes. As earlier described in Fig. 1, ZEN-induced toxicity caused a swollen vulva and an evoked immune response in the reproductive system, gene expressions of inflammation markers were therefore analyzed in Fig. 2h. Specifically, ZEN exposure significantly lowered the expressions of C-C motif chemokine, macrophage colony-stimulating factor, C-X-C motif chemokine, and interferon, but again dramatically increased those of interleukin, tumor necrosis factor, and transforming growth factor. These results obtained suggested that ZEN exposure aggravated local inflammation in pig’s uterus (Fig. 2h). Accordingly, specific immune-related genes (e.g., IL-1A, IL-20RA, CCL26, FGG, SPLI, WFDC2, PLAUR, and WFIKKN1) were used for verifying the precision and reproducibility of RNA-seq analysis. As expected, the results from RT-qPCR were basically consistent with RNA-seq data, it, therefore, indicated those with high reliability in its results (Fig. 2i).
In addition to the uterus, a similar effect of ZEN-induced toxicity on immune dysfunction was also observed in the ovary of pigs (Supplemental Fig. S2, Supplemental Table S3). Altogether, the RNA-seq analysis from the reproductive system of pigs during phase 1/2 showed that ZEN-induced toxicity contributed to an evoked inflammation and immune dysfunction.
RNA-seq analysis unravels ZEN-induced toxicity on the immune system
Next, by using RNA-seq analysis, we tested whether ZEN exposure significantly altered gene function and gene expression pattern of the immune system, including liver, spleen, thymus, and inguinal lymph nodes (Fig. 3, Supplemental Table S3). Firstly, a higher number of DEG in the immune system was observed in phase-1 pigs than those in phase-2 pigs (Fig. 3a). According to the top 30 of KEGG pathway enrichment, most pathways were significantly enriched and were mainly involved in immune diseases and system (Fig. 3b). Also, as shown in Fig. 3c, the immune system of pigs during phase 1/2 displayed many overlaps among most pathways, which were related to immune diseases and system. Although both phase-1 and phase-2 pigs that were exposed to ZEN displayed many similar pathways (e.g., immune diseases and systems), there were still great differences in corresponding genes (Fig. 3d). This was consistent with the earlier findings from the reproductive system (Fig. 2).
For both phase-1 and phase-2 pigs, six common pathways were enriched in the immune system, such as asthma, rheumatoid arthritis, the intestinal immune network for IgA production, allograft rejection, autoimmune thyroid disease, and systemic lupus erythematosus (Fig. 3c, Supplemental Table S3). Additionally, two other pathways were also enriched in the immune system of phase-2 pigs, such as antigen processing and presentation, and graft-versus-host disease (Fig. 3c, Supplemental Table S3). Thus, the obtained results indicated that ZEN-induced immune abnormalities had a high similarity and a high tissue specificity for immune organs.
To further analyze the top 30 pathways enriched in KEGG, most pathways were related to immune response and inflammation (Fig. 3e). Among these, immune-related pathways, including phagosome, NF-kappa B, and CAMs, were used to verify the precision and reproducibility of RNA-seq analysis (Fig. 3e, f). Briefly, ZEN-induced toxicity on immune system was as follows: (i) an evoked NF-kappa B pathway (e.g., p-p65); (ii) a stimulated phagosome pathway (e.g., CD63 and TLR4); (iii) a loss of E-cadherin-dependent cell-cell adhesion (e.g., CD54/ICAM-1 and E-cadherin) (Fig. 3f).
Taken together, ZEN exposure, on the one hand, aggregated immune response and inflammation in the immune system by activating the NF-kappa B/phagosome pathways, and on the other hand, resulted in inflammatory lesions by inhibiting cell-cell adhesion.
ZEN-induced toxicity alters gut microbial metabolites
Given that the gut is a first-line defense against mycotoxin and its gut microbiota represents a crucial bridge between environmental substances and host health, we tested whether ZEN-induced toxicity on the reproductive-immune axis was accompanied by altered gut microbial-derived metabolites (Fig. 4, Supplemental Figs. S3 and S4). Firstly, both phase-1 and phase-2 pigs that exposed to ZEN significantly reduced the richness (e.g., Chao and ACE index) of caecal and colonic microbiota, whereas alterations of α-diversity (e.g., Shannon and Simpson index) were observed in the jejunum and ileum from phase-1 pigs but not phase-2 pigs (Fig. 4a, b). Next, β-diversity analysis was performed to reveal the differences among multiple samples by analyzing the OTUs (97% similarity), and OTU-based PLS-DA analysis showed a clear separation of gut microbiota composition in pigs between the Ctrl group and ZEN group (Fig. 4c, d, Supplemental Fig. S3a-b).
The structural changes of gut microbiota were then evaluated by cluster analysis (Fig. 4e–h, Supplemental Fig. S4). At the phylum level, Firmicutes and Bacteroidetes were the most abundant phyla in each group (Fig. 4e, f). More specifically, phase-1 pigs that were exposed to ZEN displayed a higher abundance of Bacteroidetes in the jejunum, caecum, and intestine, Actinobacteria in the duodenum and intestine, and Firmicutes in the caecum, respectively (Fig. 4e). Also, significant increases in the relative abundances of Bacteroidetes in the duodenum and caecum, Actinobacteria in the duodenum and intestine, and Cyanobacteria in the caecum and colon were observed in phase-2 pigs that were exposed to ZEN (Fig. 4f). Furthermore, at the family level, compared with the Ctrl group, significant altered gut microbial community (pooled samples in five gut sections) from pigs in the ZEN group were as follows: (1) phase-1 pigs exposed to ZEN showed an increase in Streptococcaceae (P < 0.05) and Clostridiaceae (P < 0.05), but a decrease in Erysipelotrichaceae (P < 0.01) (Fig. 4g); (2) phase-2 pigs exposed to ZEN also displayed an increase in Clostridiaceae (P < 0.01), whereas a decrease in Ruminococcaceae (P < 0.05) and Erysipelotrichaceae (P < 0.01) (Fig. 4h). Collectively, significant changes in α-diversity and β-diversity contributed to an adverse toxicity effect of ZEN on gut bacterial community profiles in both phase-1 and phase-2 pigs (Fig. 4a–h, Supplemental Fig. S4).
In line, we further investigated whether altered gut microbiota affects the production of microbiota-derived metabolites, including short-chain fatty acids (SCFAs) and LPS (an endotoxin derived from the outer membrane of Gram-negative bacteria) (Fig. 4i–k). Intriguingly, although no significant changes were observed in other two SCFAs (acetate and propionate) between the Ctrl group and the ZEN group, reduction of butyrate production was observed in five gut sections from both phase-1 and phase-2 pigs in the ZEN group when compared with those in pigs in the Ctrl group (P < 0.05) (Fig. 4i, j). Additionally, ZEN exposure also significantly increased the level of plasma LPS in both phase-1 and phase-2 pigs (Fig. 4k).
Next, Spearman’s correlation test was used for assessing the relationships between the core bacteria (at the family level) and these microbiota-derived metabolites, including SCFAs and LPS (Fig. 4l, m). In phase-1 pigs, the level of butyrate was negatively associated with the relative abundance of Clostridiaceae (ρ = −0.39, P < 0.05) but positively correlated with the relative abundance of Erysipelotrichaceae (ρ = 0.68, P < 0.01) (Fig. 4l). In phase-2 pigs, the concentration of butyrate was negatively linked to the relative abundances of Lachnospiraceae (ρ = −0.54, P < 0.05) and Clostridiaceae (ρ = −0.56, P < 0.05) while positively correlated with the relative abundances of Ruminococcaceae (ρ = 0.53, P < 0.05) and Erysipelotrichaceae (ρ = 0.63, P < 0.01) (Fig. 4m). Also, the concentration of LPS was negatively related to the relative abundances of Erysipelotrichaceae (ρ = −0.52, P < 0.01) in phase-1 pigs, Ruminococcaceae (ρ = −0.41, P < 0.05) and Prevotellaceae (ρ = −0.45, P < 0.05) in phase-2 pigs, but positively linked with the relative abundances of Clostridiaceae (ρ= 0.66, P < 0.05) in both phase-1 and phase-2 pigs (Fig. 4l, m). Accordingly, these data suggested that both Ruminococcaceae and Erysipelotrichaceae belonged to potential butyrate-producing bacteria, and Clostridiaceae might be potential LPS-producing bacteria.
Altogether, these obtained results indicate that ZEN exposure lowered butyrate production by inhibiting butyrate-producing bacteria (e.g., Ruminococcaceae, and Erysipelotrichaceae), whereas it stimulated LPS production by increasing LPS-producing bacteria (e.g., Clostridiaceae).
ZEN-induced toxicity impairs the intestinal barrier through altered microbial metabolites
To identify whether gut microbial metabolites (e.g., butyrate) affect intestinal barrier function, SCFA receptors, signalling pathways in intestinal integrity (e.g., ERK1/2, p38 MAPK, and tight junction markers), intestinal morphology, goblet cells, and pro-inflammatory cytokines were tested (Fig. 5). Firstly, a lower protein expression of GPR109A (P < 0.05) but not GPR41 was found in the jejunum, ileum, and colon from both phase-1 and phase-2 pigs that were exposed to ZEN (Fig. 5a, b). Subsequently, decreased butyrate production caused by ZEN exposure significantly evoked the phospho-ERK1/2 and phospho-p38 signalling pathway (Fig. 5c; P < 0.05), and then impaired intestinal integrity (e.g., decreased expression of occludin and claudin-1; P < 0.05) (Fig. 5d–f) in a GPR109A-dependent manner. Furthermore, impaired intestinal integrity was accompanied by reduction of villus height (Fig. 5g, h; P < 0.05) and goblet cells (Fig. 5i, j; P < 0.05)]. As a result, ZEN-induced toxicity resulted in intestinal barrier dysfunction, and it then lowered immune defense against ZEN. Indeed, our results also found that ZEN-induced toxicity rapidly increased mRNA expressions of pro-inflammatory cytokines (e.g., TNF-α, IFN-γ, IL-1β, and IL-12) (P < 0.05) (Fig. 5k, l), thus dramatically aggravating the circulating levels of these pro-inflammatory cytokines (P < 0.05) (Fig. 5m, n) and LPS (P < 0.05) (Fig. 4k). Accordingly, these data showed that ZEN-induced toxicity impaired the intestinal barrier through altered gut microbial metabolites (e.g., reduction of butyrate production, and increasement of LPS production).
Taken together, we conclude that ZEN-induced toxicity results in impaired intestinal barrier through altered gut microbial metabolites (e.g., decreased butyrate production and/or increased LPS production), and then triggered the systemic inflammatory response, thus disturbing immune defense in the reproductive-immune axis.
Modified gut microbial metabolites by engineering mycotoxin-degrading enzymes counteract ZEN-induced toxicity on the intestinal barrier
To validate these findings above, we generated recombinant Bacillus subtilis (B. subtilis) 168-expressing ZEN-degrading enzyme ZLHY-6 [28] as an example to investigate the feasibility of enzymatic removal of ZEN from mycotoxin-contaminated food. Briefly, we constructed recombinant expression vector pWBZ7 (Fig. 6a1–a3), which regulated ZEN degradation enzyme gene ZLHY-6 by the promoter PlapS (Supplemental Fig. S5a-c). And, the pWBZ7 plasmid was then electrically transformed into B. subtilis 168, which was designated as Bs-Z6 in this study (Supplemental Fig. S5b). Using BSA as a protein standard, we estimated the concentration of recombinant ZLHY-6 protein expressed in Bs-Z6 and found it could reach the peak concentration of 500 μg/mL at a fermentation time of 12 h (Fig. 6a4–a5). To further evaluate the stability of pWBZ7 in recombinant Bs-Z6 strain, the plasmids on the first passage and the 60th passage were digested with two enzymes: EcoR I and BamH I. The results showed that the plasmids of the two passages were cut into fragments with sizes of 3310 and 1190 bp, respectively, indicating that recombinant pWBZ7 plasmid was highly stable in recombinant Bs-Z6 strain (Fig. 6a6). Of note, after 12 h of incubation at 37° C, the degradation effects of recombinant Bs-Z6 strain on ZEN-contaminated food (e.g., corn) reached above 92% (Fig. 6a7, Supplemental Fig. S5d-e). Also, the relative enzyme activity recorded was 219.02 U mL−1 at the end of 12 h (Supplemental Fig. S5f). Consistent with these data, after optimizing fermentation conditions (e.g., dissolved O2, pH, and temperature) (Supplemental Fig. S5g), the highest protein expression of ZLHY6 in recombinant Bs-Z6 strain was also recorded at 12 h (Supplemental Fig. S5h).
Subsequently, using the pig (phase 3) as an animal model, we identified whether a ZEN-contaminated diet supplemented with recombinant Bs-Z6 strain could counteract ZEN-induced toxicity on the intestinal barrier by regulating colonic microbial metabolites (Fig. 6b–n, Supplemental Fig. S6). When a ZEN-contaminated diet was combined with recombinant Bs-Z6 strain, by using the HPLC-MS/MS assay, we found that ZEN/its metabolites residues were significantly decreased in intestinal contents, blood, liver, and uterus (P < 0.05) (Supplemental Table S4). Thus, these findings further supported the degradation effects of recombinant Bs-Z6 strain on ZEN-contaminated food.
As earlier observed (Fig. 4a, b), ZEN exposure resulted in significant alterations in the richness (e.g., Chao and ACE index) and α-diversity (e.g., Shannon index) (P < 0.05) (Fig. 6b). However, no statistical differences were found in these parameters between the ZEN group and the ZEN/Bs-Z6 group (Fig. 6b). Using OTU-based PLS-DA analysis, we showed ZEN exposure resulted in an obvious change of β-diversity, which was prevented by recombinant Bs-Z6 strain (Fig. 6c). Furthermore, by using cluster analysis, we investigated the toxicity effects of recombinant Bs-Z6 strain on structural changes of gut microbiota (Fig. 6d, Supplemental Fig. S6). As shown in Fig. 6d, ZEN exposure significantly changed the three most dominant phyla (Actinobacteria, Tenericutes, and Bacteroidetes), which was also counteracted by this recombinant strain (P < 0.05). In agreement with earlier descriptions (Fig. 4g, h, l, m), ZEN exposure significantly lowered potential butyrate-producing bacteria (e.g., Ruminococcaceae and Erysipelotrichaceae) (P < 0.05), while increased potential LPS-producing bacteria (e.g., Clostridiaceae) (P < 0.05). Notably, these altered dominant bacteria were preserved by recombinant Bs-Z6 strain (Fig. 6e, f).
Considering that ZEN exposure significant altered gut microbial metabolites, we also observed that recombinant Bs-Z6 strain counteracted ZEN-induced a loss of butyrate (Fig. 6g; P < 0.05), whereas inhibited ZEN-induced an increase of LPS (Fig. 6h; P < 0.05). As previously described (Fig. 5), reduction of butyrate production resulted in a lower protein expression of SCFA receptors (e.g., GPR109A), and an increase in phosphorylation of ERK1/2 and p38, thus leading to impaired intestinal integrity (e.g., decreased expression of occludin and claudin-1) as well as decreased villus height. These abnormalities in the intestinal barrier were all restored upon increased butyrate production, which was achieved by recombinant Bs-Z6 strain (Fig. 6i–l; P < 0.05). Furthermore, this restored intestinal barrier function significantly lowered the mRNA expression and the circulating level of pro-inflammatory cytokines (e.g., TNF-α, IFN-γ, IL-1α, and IL-1β), thus leading to increased immune defense against ZEN (Fig. 6m, n; P < 0.05).
To further confirm these findings above, the porcine small intestinal epithelial cell line, IPEC-J2, is also employed in this study (Fig. 6o). Using the TEM method, we observed that ZEN exposure resulted in mitochondrial dysfunction and increased apoptosis in cells, which was rescued by butyrate supplementation (Fig. 6o1). Additionally, in IPEC-J2 cells, ZEN exposure increased ROS content (Fig. 6o2) and pro-inflammatory cytokines (e.g., IL-12; P < 0.05) (Fig. 6o5), but decreased the expression of tight junction markers (e.g., occludin; P < 0.05) (Fig. 6o3-4). As expected, when ZEN exposure was combined with butyrate, these ZEN-induced abnormalities were also partially preserved (Fig. 6o1-5).
Taken together, both in vitro and in vivo data demonstrate that modified gut microbial metabolites (e.g., increased butyrate production and/ or decreased LPS level) offer the potential strategy to counteract ZEN-induced toxicity on the intestinal barrier and its immune dysfunction.
Modified gut microbial metabolites reduces ZEN-induced toxicity on reproductive and immune systems
Next, we investigated whether gut modified microbial metabolites, which was achieved by the supplementation of recombinant Bs-Z6 strain, counteracted ZEN-induced toxicity on the reproductive-immune axis in pigs during phase-3.
Firstly, no significant changes were observed in pig's growth performance among all groups (Supplemental Table S5). As indicated in Fig. 7a and Supplemental Fig. S7, ZEN exposure resulted in a larger vulvar area and uterine size (uterine length, uterine width, and uterine horn’s width), which was completely restored by recombinant Bs-Z6 strain (P < 0.05). Using the HE staining and TEM methods, we also observed that ZEN exposure contributed to obvious inflammatory lesions, swelling nucleus, mitochondrial oxidative stress in immune organ (e.g., thymus) and reproductive organ (e.g., uterus), which were preserved by recombinant Bs-Z6 strain (Fig. 7b,c). Also, in agreement with earlier findings in Fig. 1k, l, mRNA expression of oxidative stress markers (e.g., Cu/Zn-SOD, Mn-SOD, GSH-Px, and PGC-1α) was significantly increased upon ZEN exposure, while was completely rescued when ZEN exposure was combined with recombinant Bs-Z6 strain (Fig. 7d).
Also, by using RNA-seq analysis, we examined whether ZEN-contaminated food supplemented with recombinant Bs-Z6 strain counteracted ZEN-induced toxicity on the reproductive-immune axis in pigs during phase-3 (Fig. 7e–j, Supplemental Fig. 8). For altered gene expression pattern, we identified that 74 downregulated genes and 100 upregulated genes in immune organ (e.g., thymus) could be preserved by recombinant Bs-Z6 strain (Fig. 7e). Also, 656 downregulated genes and 844 upregulated genes in reproductive organ (e.g., uterus) were rescued by this recombinant strain (Fig. 7g). According to the top 15 enriched GO terms from biological process, most ZEN-induced downregulated genes that were rescued by recombinant Bs-Z6 strain, were closely related to an immune system process, immune response, and defense response (Fig. 7f, h). To further analyze the top 30 pathways enriched in KEGG, most pathways are also related to immune systems and infection disease (9/26 in the thymus; 23/30 in the uterus) (Fig. 7i, j). Among these, immune-related pathways were commonly enriched in both the thymus and uterus, such as, systemic lupus erythematosus, RIG-I-like receptor signalling pathway, autoimmune thyroid disease, antigen processing and presentation, and NOD-like receptor signalling pathway (Fig. 7i, j). In agreement with earlier findings (Fig. 1k, l), mRNA expression of pro-inflammatory cytokines (e.g., TNF-α, IFN-γ, IL-1α, and IL-1β) was significantly increased upon ZEN exposure, but was completely inhibited when ZEN exposure was combined with recombinant Bs-Z6 strain (Fig. 7k).
Taken together, these data obtained further supported that ZEN-induced intestinal barrier dysfunction resulted in the systemic inflammation through altered microbial metabolites, thus disturbing immune defense in the reproductive-immune axis. Notably, the regulation of gut microbial metabolites (e.g., butyrate and LPS), which was achieved by recombinant Bs-Z6 strain, could counteract ZEN-induced toxicity on the reproductive-immune axis.