Effect And Mechanism Of ZEA,DON And Their Combination On Anti-Listeria Infection Mediated By CD4 T Cell Activation And Differentiation In Mice

Zearalenone(ZEA)and deoxynivalenol(DON)are frequently occurring mycotoxins in the environment that simultaneously contaminate food and feed.Both ZEA and DON are immunotoxic,however,their immune toxicity and related mechanisms especially the combined effects are not fully understood caused by them,Unlike other toxic effects,the immunotoxicity caused by mycotoxin,especially the chronic toxicity,usually does not show obvious clinical symptoms.Nonetheless,it can affect important defense mechanisms of the body against invading pathogens,which weakens the body’s ability to resist and eliminate infection.The body produces different immune responses to different types of pathogens.As one of the common foodborne pathogens,Listeria monocytogenes infection mainly occurs in cells,and cellular immunity is the key for the body to resist and eliminate its invasion.This involves the activation of T cells and the differentiation of Th1 subtypes.In this study,a mouse model of combined exposure to ZEA,DON along with L.monocytogenes infection was-established,and Naive CD4 T cells exposed to the toxins were induced to activate and differentiate into Th1 subtype in vitro.Flow cytometry,western blot,cytokine antibody array,proteomics and other techniques were used to detect the changes of Naive CD4 T cell activation,anddiffe rentiation-related immune indicators after bacterial infection and stimulation in vitro.This study investigated the effect and mechanism of ZEA,DON and their combination on Th1 cell-mediated immunity after intracellular bacterial infection.This study can provide a new theoretical basis for further revealing the immunotoxicity and molecular mechanism of these two mycotoxins.1.Effects of ZEA,DON and their mixture on spleen injury and inflammation in L.monocytogenes infected miceAfter 7 days of intragastric administration of ZEA(10 mg/kg),DON(1 mg/kg)and their combination(ZEA+DON,10 mg/kg+1 mg/kg),C57BL/6 mice were inoculated with L.monocytogenes(5×104 CFU)by tail vein injection on day 8.The infection lasted for 7 days,during which time the toxin was maintained by continuous gavage.After the model was established,the following tests were conducted:① The proportion of monocytes in spleen on the 3rd,5th and 7th day of bacterial infection was detected by flow cytometry to reflect the effect of L.monocytogenes infection;②The changes of daily food intake and body weight of mice were observed,and the bacterial load in spleen on-the 3rd,5th and 7th day of bacterial infection was detected by flat colony counting method;③Pathological injury of spleen was ob served by HE staining;④Flow cytometry was used to detect Ml and M2 polarization of splenic macrophages;⑤Cytokine microarray was used to detect the secretion levels of 40 inflammatory cytokines such as IFN-γ,TNF-α,IL-1β and IL-12 in serum on the fifth day of infection.The results showed that:① The proportion of monocytes in spleen increased significantly during L.monocytogenes infection(p<0.01),DON significantly decreased the proportion of monocytes in all stages of infection(p<0.01);Th e proportion of monocytes was significantly decreased by ZEA on day 7 and by ZEA+DON on day 5 and 7 of infection(p<0.05 or p<0.01).②The mice in Listeria group began to lose weight on the third day of infection.ZEA,DON and their combination accelerated the weight loss of mice before and after infection.ZEA induced a significant-increase in spleen bacterial load on the 3rd and 5th day of infection(p<0.01);The bacterial load of spleen in DON and combination groups increased significantly on the 3rd,5th and 7th day of infection(p<0.01).Compared with the toxin alone group,the combined toxin group had a synergistic effect on the increase of bacterial load on days 3 and 5 of infection.③ZEA,DON and their combination aggravated various pathological changes of mouse spleen during bacterial infection,such as extramedullary hematopoiesis,an increase in multinucleated giant cells,lymphocyte necrosis and connective tissue hyperplasia,and the toxin combination group was the most serious.④The proportion of macrophages decreased in all stages of infection caused by toxin;The polarization of M1 macrophages in ZEA group was significantly decreased on the fifth day of infection(p<0.05).The polarization of M1 macrophages in DON group was significantly decreased at all stages of infection(p<0.01).The polarization effect of M1 macrophages in toxin combined group was significantly decreased on the 3rd and 5th day of infection(p<0.05 or p<0.01).There was no synergistic decrease in M1 polarization of macrophages in the combined toxin group.⑤Compared with the blank control group,RANTES and MIG were differentially up-regulated in the Listeria group.Compared with Listeria group,TIMP-1 was differentially up-regulated and FasL and IL-1β were differentially down-regulated after ZEA treatment;After DON treatment,up-regulated KC,BLC,Eotaxin-2,TIMP-1 and MIG and down-regulated IL-1β,IL-12P40/P70,LIX and FasL were identified.Upregulated BLC,TIMP-1 and MIG and downregulated IL-12P40/P70 were identified after toxin combination treatment.The differential proteins in all toxin groups were significantly enriched in cellular immune response,inflammation and cell chemotaxis.These results indicated that ZEA,DON and their combination could inhibit the normal inflammatory response after L.monocytogenes infection and aggravate the spleen injury caused by bacteria.2.Effects of ZEA,DON and their combination on CD4 T cell activation and Th1 subtype differentiation in mice infected with L.monocytogenesTo further characterize the effects of ZEA,DON and their mixture on Thl-mediated cellular immunity in mice infected with L.monocytogenes,flow cytometry was used to detect the related immune indexes in each period of infection.①Expression of CD4 T cell activation markers CD69,CD25,CD71;②Expression of CD4 T cell co-stimulatory-molecule CD28 and GD152;③Depletion of CD62L and expression of CD44 in CD4 T cells;④Expression of IL-12 receptor in CD4 T cells;⑤Differentiation of IFN-γ-secreting T cells.The results showed that:①The expression of CD69 in CD4-T cells of DON group was significantly decreased on the 3rd day of infection(p<0.01).The combined toxin resulted in a significant decrease in CD71 expression in CD4 T cells on day 7 of infection(p＜0.05).② DON significantly decreased the ratio of CD28 to CD 152 in CD4 T cells(p<0.05).③ The expression.of CD62L on CD4 T cells decreased significantly on the 5th day of infection(p<0.01),and then increased significantly on the 7th day of infection(p<0.01).Toxin combination resulted in a significant increase in CD62L expression on day 7 of infection(p<0.01).ZEA induced a significant decrease in the expression of CD44 in CD4+CD62L-cells on the 3rd day of infection(p<0.01).DON significantly decreased the expression of CD44 in all stages of infection(p<0.05 or p<0.01).④The expression of IL-12 receptor in CD4 T cells was decreased by toxin alone and in combination(p<0.05 or p<0.01).⑤The CD4+IFN-γ+(Th1)differentiation effect was significantly decreased by toxin combination,ZEA and DON on the 3rd,5th and 7th day of infection respectively(p<0.05);ZEA significantly decreased the differentiation effect of CD8+IFN-γ+ on day 5 and day 7(p<0.05).In this study,almost no synergistic effect was observed in.the toxin combination group.These results indicated that ZEA,DON and their combination could inhibit the normal activation and differentiation of CD4 T cells into Th1 subtype in mice infected with L.monocytogenes.3.Effects of ZEA,DON and their combination on spleen proteome in mice infected with L.monocytogenesIn order to characterize the effects of ZEA,DON and their combination on the changes induced by L.monocytogenes infection on the proteome,the spleen of mice on the 7th day of infection was analyzed by Label-free proteomics.A functional analysis of the differentially expressed proteins between the infection group and the toxin+infection groups,conducted using GO annotation,KEGG annotation and COG annotation.Finally,the differential proteins related to ribosome function were verified by western bolt.The results showed that compared with the Listeria group,125 differentially expressed proteins were identified in the ZEA treatment group,of which 89 were up-regulated and 36 were down-regulated.136 differentially expressed proteins were identified in DON treatment group,of which 48 were up-regulated and 88 were down regulated.A total of 556 differentially expressed proteins wereidentified in toxin combined treatment group,among which 323 were up-regulated and 233 were down-regulated.With the treatment of ZEA,DON and their combination,differential protein GO annotations in BP ontology were highly enriched in cellular processes,metabolic processes,biological regulation,localization-and multicellular biological-processes;In CC ontology,it was highly enriched in cells,cell parts,organelles,extracellular regions and membrane-closed lumen.It was highly enriched in binding activity,catalytic activity and transporter activity in MF ontology.Compared with Listeria group,the differentially expressed proteins caused by ZEA were mainly involved in ribosome function(35.19%)and metabolic pathways(14.81%).The down-regulated differential proteins were mainly involved in metabolic pathways(12%)and infectious diseases such as malaria(32%)and amoeba(8%).The differential proteins caused by DON were mainly involved in ribosome function(29.33%),metabolic pathway(10.67%)and spliceosome function-(10.67).The down-regulated proteins were mainly involved in metabolic pathways(22.86%)and hematopoietic cell lineage(17.14%).The differentially upregulated proteins caused by ZEA+DON were mainly involved in metabolic pathways(17.53%)and ribosome function(14.34%).The down-regulated proteins were mainly involved in metabolic pathways(30.05%)and hematopoietic cell lineage(10.93%).Three differentially expressed ribosomal subunits(RPL13,RPL24 and RPS24)were identified in all three toxin treatments.The results of western blot verification were consistent with the omics results.Compared with the Listeria group,RPS24 was not only significantly increased in-the toxin-combined group,the expressions of three proteins were signifi cantly increased in all toxin treatment groups(p<0.05 or p<0.01).These results indicated that ZEA,DON and their combination could affect the protein function of mice infected with L.monocytogenes to different degrees,and the effect on ribosomal pathway was mainly reflected in the over-activation of ribosomal protein,thus contributing to the multiplication of bacteria in the cell.4.Effects of ZEA,DON and their combination on activation of Naive CD4 T cells and Th1 subtype differentiation in vitroIn order to confirm the conclusions drawn from in vivo experiments and explore the specific mechanisms,high-purity mouse spleen Naive CD4 T cells were extracted by immunomagnetic beads and exposed to different concentrations of ZEA,DON and their combination.Anti-CD3+anti-CD28+anti-IL-4 antibody + recombinant cytokines IL-2 and IL-12 were used to induce cell activation and differentiation in vitro.Then the relevant immune markers were monitored.In the study of cell activation process,the cell activity of toxin alone and combined groups decreased significantly at 48 and 72 hours(p<0.05 or p<0.01).Different concentrations of toxin could inhibit lymphocyte transformation effect to different extent,which showed that cell dispersion and aggregation ability were weakened,accompanied by cell lysis and so on;Compared with Anti CD3 + Anti CD28 positive control,CD25 was significantly decreased in DON(1 μmol/L),ZEA(10 μmol/L)+ DON(1μmol/L)and ZEA(20 μmol/L)+DON(0.5 μmol/L)groups(p<0.01).Compared with the positive control,CD71 in the all toxin treatment groups was significantly decreased except for ZEA(10 μmol/L)group(p<0.01).Compared with toxin alone group,ZEA(20 μmol/L)+DON(0.5 μmol/L)had a synergistic effect on CD25 and CD71(p<0.01).Except for ZEA(10 μmol/L)group,the ratio of CD28/CD152 in the other toxin treatment groups was significantly decreased(p<0.01).Compared with the positive control group,DON(0.5 μmol/L)and ZEA+DON(20+0.5 μmol/L)significantly decreased the expression of LAT and ZAP-70(p<0.01).ZEA+DON(20+0.5 μmol/L)also significantly decreased the phosphorylation level-of Zap-70(p<0.05).The expression of Lck was not significantly changed in all toxin treatment groups,but the phosphorylation level of Lck was decreased in ZEA(20 μmol/L),DON(0.5 μmol/L)and ZEA+ DON(20+0.5 μmol/L).Compared with the toxin alone group,synergistic effect was observed only in the inhibition of p-Lck expression in the toxin combination group.Compared with the positive control group,the expression level of Ki-67 was significantly inhibited in all toxin treatment groups(p<0.01).In the study of cell differentiation process,compared with positive control group,ZEA(10,20 μmo1/L),DON(1 μmol/L),ZEA+DON(10+1 μmol/L)and ZEA+DON(20+0.5 μmol/L)significantly inhibited the expression of IL-12 receptor(p<0.05 or p＜0.01).Compared with toxin group alone,synergistic effect was observed only in ZEA+DON(10+1 μmol/L)group(p<0.01);In addition,all toxin treatment groups significantly inhibited the expression level of T-bet(p<0.01).Western blot was used to detect the expression and phosphorylation levels of TCR signaling pathway proteins.such as PI3K,Akt,PDK1 and mTOR after 4 days of cell induction.Compared with the positive control group,except ZEA(10 μmol/L)had no significant effect on the expression of PI3K,the expression of all proteins was significantly inhibited in all toxin groups(p<0.05 or p<0.01).Compared with the positive control group,all toxin-treated groups significantly inhibited the differentiation of CD4+ IFN-γ+(Th1)subtype cells(p<0.01).Finally,the secretion levels of Th1 subtype specific cytokines IFN-γ and TNF-α were significantly inhibited in all toxin-treated groups(p<0.01).Synergistic inhibition of TNF-α was observed in ZEA+DON(10+1 μmol/L)group compared with toxin alone group(p<0.01).Altogether,these results indicate that ZEA,DON and their combination affect the activation of Naive CD4 T cells and the differentiation to Th1 subtype by inhibiting the activation of TCR signaling pathway,and ultimately hinder the execution of Thl subtype cell-related immune functions.In conclusion,the combined as well as individual exposure to ZEA and DON showed to affect the innate immune response and to inhibit the cellular immune effect mediated by Th1 cells,thus aggravating the infection of L.monocytogenes in mice.The mechanism identified was that ZEA,DON and their combined exposure inhibit the TCR-regulated signal transduction for activation and differentiation of Naive CD4 T cells.These results indicate that the immunotoxic effects induced by ZEA and DON result in the inhibition of activation and differentiation of specific immune cells.