Mannan Oligosaccharide Blocks The AFB₁-induced Prmotion Of SIV Replication And Their Mechanisms

Aflatoxin B1(AFB1)is one of the most common mycotoxins in feeds and food.Swine influenza virus(SIV)is a major pathogen of both animals and humans.However,the central contribution of AFB1 in SIV infection remains unclear.Given the differences in morbidity and mortality following SIV infection,we hypothesize that AFB1 promotes SIV replication.Here we investigated the involvement of AFB1 in SIV infection in vivo and in vitro,as well as its underlying mechanism using mouse and multiple cell models.Mannan oligosaccharide(MOS),derived from the cell walls of yeast,is a potent immune-modulator.In this study,we investigated the role of MOS in influenza infection and inflammatory responses in porcine alveolar macrophages(PAMs)and mice exposed to AFB1,and further explored the underlying mechanisms.1.AFB1 promotes H1N1 swine influenza virus replication in vitro and in vivoIn this study,we investigated the involvement of AFB1 in SIV replication in vitro and in vivo using multiple cell lines and mice.At first,to remove the effects of AFB1-induced cytotoxicity on viral replication,the effects of various concentrations of AFB1 on the cell viabilities of MDCK cells,A549 cells and PAMs were determined by MTT and LDH assays.The results suggest that AFB1 at concentrations between 0.01 and 0.25 ?g/ml,0.01 and 0.25 ?g/ml,and 0.01 and 0.05 ?g/ml are not toxic to MDCK cells,A549 cells and PAMs,respectively.Thus,for subsequent experiments,AFB1 was used at concentrations of 0.01,0.05 and 0.25 ?g/ml in both MDCK and A549 cells and at concentrations of 0.01,0.025 and 0.05 ?g/ml in PAMs.Correspondingly,according to the guidelines established by the US Food and Drug Administration and the National Food Safety Standard(GB2761-2017,China),maximum dietary AFB1 concentrations of 20 and 300 ?g/kg are allowed for humans and animals,respectively.However,we did not know whether low-level AFB1 may cause or exacerbate other secondary diseases.Therefore,the concentrations of 10,20 and 40 ?g/kg b.w.were selected and used in the in vivo study.The in vitro studies demonstrated that AFB1 could promote SIV replication as assessed by the increased viral titers,matrix protein(M)mRNA,nucleoprotein(NP)expression levels in SIV-infected cells.The in vivo study showed that AFB1 increased SIV replication and its severity,as assessed by the decreased weight gain,the increased lung virus titers and the increased expression levels of viral M mRNA and NP in lungs from mice,as well as the increased lung index and lung histologic damage.Taken together,our results suggest that AFB1 promotes H1N1 swine influenza virus replication in vitro and in vivo.2.AFB1 promotes H1N1 swine influenza virus replication via activating TLR4-NF?B signalingTo further investigate the involvement of AFB1 in SIV infection in vivo and in vitro,as well as its underlying mechanism,the mouse models and porcine alveolar macrophage(PAM)models were used.Toll-like receptor 4 knockout(TLR4-/-LR4 knockdown and a TLR4 inhibitor,TAK242,were used to remove the effects of TLR4.The in vitro studies demonstrated that AFB1could promote SIV replication as assessed by the increased viral titers,matrix protein(M)mRNA and nucleoprotein(NP)expression levels in SIV-infected cells.The in vivo study showed that AFB1increased SIV replication and its severity,as assessed by the decreased weight gain,the increased lung virus titers and the increased expression levels of viral M mRNA and NP in lungs from mice,as well as the increased lung index and lung histologic damage.Further study showed that AFB1upregulated TLR4,but not other TLRs(TLR1-0),in SIV-infected PAMs.Moreover,AFB1activated TLR4 signaling as demonstrated by the increases of TLR4,phosphorylated NF?B p65(pp65)and TNF-? release in PAMs and mice.In contrast,TLR4 knockdown or the use of BAY 11-7082,a specific inhibitor of NF?B,blocked the AFB1-promoted SIV replication and inflammatory responses in PAMs.Furthermore,a TLR4-specific antagonist,TAK242,and TLR4 knockout both attenuated the AFB1-promoted SIV replication,inflammation and lung damage in mice.We therefore conclude that AFB1 exposure aggravates SIV replication,inflammation and lung damage by activating TLR4-NF?B signaling.3.Low-level AFB1 promotes H1N1 swine influenza virus infection via macrophage polarization to M1/M2To further verify the role AFB1 played in SIV replication,here,TCID50 assays,fluorescence-based quantitative real-time PCR,western blotting,immunofluorescence staining,histopathological examination,flow cytometry and scanning electron microscopy were performed to investigate the involvement and underlying mechanism of AFB1 in SIV infection in vivo and in vitro using mouse models and porcine alveolar macrophage(PAM)models,respectively.The in vivo study showed that low doses of AFBi promoted SIV infection and increased its severity,as demonstrated by the increased mRNA expression levels of viral matrix protein(M);by the increased protein expression levels of nucleoprotein(NP),matrix protein 1 and ion channel protein;and by the increased body weight loss,lung index and lung histologic damage in mice.In addition,the increased occurrence of SIV infection accompanied by increases in the level of IL-10 in sera and lungs,in the spleen index and in the number of CD206-positive mouse alveolar macrophages but decreases in the level of TNF-?.in sera and lungs,in the thymus index and in the number of CD80-positive mouse alveolar macrophages was observed in SIV-infected mice after low-level AFB1 exposure.The in vitro study showed that low concentrations of AFB1 promoted SIV infection,as demonstrated by the increases in viral titers and viral M mRNA and NP expression levels in SIV-infected PAMs as well as by the percentage of cells positive for NP protein expression.Furthermore,AFB1 promoted the polarization switch of SIV-infected PAMs from the M1 phenotype at 8 hpi and to the M2 phenotype at 24 hpi,as measured by the increases in IL-10 expression and in the number of CD206-positive PAMs as well as by the morphological changes observed by scanning electron microscopy and flow cytometry.The administration of the immune stimulant lipopolysaccharide(LPS)reversed the switch in PAM polarization from M2 to M1 and thereby counteracted the promotion of influenza virus infection induced by AFB1.Our results are the first to confirm that low-level exposure to AFB1 promotes SIV infection and modulates a switch in macrophage polarization from M1 to M2.The work reported here provides important data that point to a role for AFB1 in SIV infection,and it opens a new field of study.It is likely that toxic contamination will increase the risk to a range of viral infections beyond influenza.4.?-MOS blocks the AFB1-induced promotion of H1N1 swine influenza virus replicationIn this study,we investigated the role of ?-MOS in influenza infection and inflammatory responses in porcine alveolar macrophages(PAMs)and mice exposed to AFB1.The in vitro and in vivo results indicated that exposures of influenza-infected PAMs and mice to AFB1 alone resulted in increases in the viral infection demonstrated by the increased viral titers,M protein mRNA,nucleoprotein(NP)and the percentage of NP-positive cells,as well as in weight loss,lung index,lung and spleen tisusse damages as compared to the influenza control.Increased influenza infection coupled with an increase in inflammatory responses assessed by the increased mRNA and protein levels of TLR4,phosphorylated NF-?B(pp65)and TNF-?.However,?-MOS given in conjunction with exposure to AFB1 reversed significantly these above changes,blocking the AFB1-promoted influenza infection and inflammatory responses.Taken together,our results indicated that ?-MOS reduced AFB1-promoted SIV infection and its severity.5.Study on the mechanism of ?-MOS blocking the AFB1-induced promotion of H1N1 swine influenza virus replicationIn this experiment,mice and porcine alveolar macrophages(PAMs)were used to further explore the mechanism of ?-MOS blocking the AFB1-induced promotion of H1N1 swine influenza virus replication.The in vitro and in vivo results together showed that ?-MOS reduced the mRNA and protein expression levels of TLR4,NF?B and TNF-? in the spleen and lung of mice and PAMs.Further study indicated that MOS activity was absent in TLR4 knockout mice and was abolished by the overexpression of TLR4 by DNA transfection,suggesting that TLR4 was required and sufficient to alleviate the AFBi-promoted influenza infection and tissue damages.Surprisingly,TNF-?,the downstream pro-inflammatory cytokine of TLR4-NF?B signaling,played no role in the MOS-mediated inhibitory effects demonstrating that no significant changes in viral titer,vital M mRNA and NP expression levels between groups with and without TNF-?.Taken together,our data suggest that MOS alleviates the AFB1-promoted influenza infection and tissue damages in a TLR4-dependent,but not TNF-?-dependent manner.