Doxycycline Supresses The Production Of Interferon-? In IPEC-J2 Cells

With the rapid development of the breeding industry,an increasing number of antibiotics are being used as veterinary drugs and feed additives.However,the serious abuse of antibiotics has attracted people's attention.The abuse of antibiotics not only led to the super bacteria,but also pollute the environment affects human health.Doxycycline(DOX)is a second-generation tetracycline derivative used widely.It occupies the A-site of the bacterial 30S ribosomal subunit and blocks recruitment of aminoacy-tRNA into the bacterial ribosome,which inhibits protein synthesis.In the pig industry,mainly through the use of DOX added to the feed and drinking water,direct effect on intestinal epithelial cells.As the main part of intestinal epithelial nutrient absorption,need to consume a large amount of energy,cells containing abundant mitochondria.A variety of pathogens or chemicals can affect the dynamic balance of intestinal epithelial mitochondria,destroy cell homeostasis.So far,the effect of DOX on intestinal epithelial cells has not been reported.Therefore,in order to investigate whether DOX used in pig industry can affect the normal function of intestinal epithelium,we used porcine jejunum epithelial cells IPEC-J2 as a model of intestinal epithelial cells in vitro.Firstly,we investigated whether DOX can inhibit IPEC-J2 cell viability and the effect on mitochondrial dynamic balance.Secondly,DOX can induce autophagy and complete mitochondrial autophagy.Furtherly,the effects of DOX induced mitophagy on the production of IFN?.Finally,the effect of DOX induced intestinal epithelial cell innate immune suppression on TGEV(transmissible gastroenteritis virus)replication was studied.Specific content as follows:1.Effects of DOX on IPEC-J2 cell viability and mitochondriaDOX can inhibit the proliferation of many kinds of tumor cells and induce cell apoptosis.The main reason is that DOX targets the mitochondrial ribosome and inhibits mitochondrial protein synthesis.To investigate the sensitivity of IPEC-J2 cells to DOX,we determined the cytotoxic effect by testing cell viability.No inhibitory effect of DOX at therapeutic levels used in animal feed were detected in IPEC-J2 cells after a 24-hour incubation.In order to study the effect of DOX on apoptosis.We used flow cytometry to detect the apoptosis treated by 1,50,100(?g/ml)of DOX for24h or 50 ?g/ml DOX treated for 24h,48h 72h.The flow cytometric analysis did not reveal any distinct apoptotic changes in DOX-treated IPEC-J2 cell.In order to study whether DOX affects mitochondrial state,the changes of abnormal mitochondria(MitoTracker Red)and total Green(MitoTracker)in IPEC-J2 cells,total(reactive oxygen species)ROS(DCFH-DA)and mitochondrial ROS(MitoSox)and the changes of mitochondrial membrane potential(Rh123)were detected by flow cytometry(FCM)after DOX treatment.The results showed that DOX treatment significantly increased the proportion of mitochondria damaged cells.DOX 50 ?g/ml(P<0.01)and DOX 100 ?g/ml(P<0.01)increases the level ROS and mitoROS significantly in a manner of dose-dependent.Moreover,the expression level of oxidative stress related genes could be significantly promoted,which indicated that DOX treatment could lead to a certain level of oxidative stress.While,DOX treatment did not affect the mitochondrial membrane potential.These results indicated that DOX did not inhibit the proliferation of IPEC-J2 cells,but it could induce the accumulation of mitochondria in the injured cells and induce oxidative stress.2.DOX induces complete autophagy and mitochondrial autophagy in IPEC-J2 cellsDOX can cause the accumulation of mitochondria in the cells,but it will not cause apoptosis.Autophagy can remove the damaged mitochondria to maintain cell homeostasis.Firstly,to investigate whether DOX induces autophagy in IPEC-J2 cells,the changes of LC3-?/LC3-? were detected by Western-blot,and the distribution of GFP-LC3B was observed by fluorescence microscopy.The results showed that DOX 50 ?g/ml(P<0.01)and DOX ?g/ml(P<0.001)significantly increased the expression of LC3-?/LC3-?.The number of GFP-LC3B puncta in cells also increased in a dose-dependent manner.In order to further verify whether DOX induces mitochondrial autophagy in IPEC-J2 cells to remove damaged mitochondria,the structural changes of mitochondria were observed by transmission electron microscopy after treated with DOX,and the co localization of mitochondria and autophagy in IPEC-J2 cells was observed by laser scanning microscope.The results showed that after DOX treatment with 24h,the morphology of mitochondria showed swelling and deformation,and the cristae disappeared,the obvious localization of mitochondria and autophagy was observed,with the increase of concentration,the co-localization was more obvious.In order to investigate whether mitophagy induced by DOX is bound to lysosomes,the mRFP-GFP-Bcl-xL fusion protein was used to observe the acidification of the autophagy.The results showed that with the increase of time,the degree of autophagy increased.These results suggest that DOX can induce complete mitochondrial autophagy in order to remove damaged mitochondria.3.The effect of DOX on innate immunityMitochondria are innate immune signal transduction platforms,mitophagy can inhibit the conduction of RLR signal and inhibit the production of I type IFN.In order to study the effect of DOX on the production of IFN.We detected the mRNA level of IFN? IFIT1,DDX58 induced by poly(I:C)through the RT-PCR after DOX treatment.The results showed that DOX 50 ?g/ml(P<0.001)and DOX g/ml(P<0.001)significantly inhibited the transcription of IFN beta,IFIT1,DDX5 8 in a dose-dependent manner.At the same time,we detected the effect of DOX on the activation of ISRE by the double luciferase reporter assay system.The results showed that DOX significantly inhibited the activation of ISRE induced by poly(I:C).In order to further verify whether DOX inhibits the IFN-I reaction is related to mitophagy.We interfered the ATG5 with shRNA on IPEC-J2 cells and the results showed that ATG5 interference significantly increased DOX induced mitochondrial damage and eliminated the inhibition of IFN? production.The above results suggest that mitophagy induced by DOX can significantly inhibit the type I interferon response in IPEC-J2 cells4.DOX promotes the replication of TGEV in IPEC-J2 cellsInnate immune response is the first line of defense against viral infection.In order to study if the inhibition of IFN production induced by DOX will influence the replication of the virus in IPEC-J2 cells.We detected the virus titer of TGEV infected IPEC-J2 cells for.24h after DOX treatment by TCID50 assay and plaque assay.The results showed that DOX could significantly promote the replication of TGEV in intestinal epithelial cells.Moreover,DOX could not promote the replication of TGEV in the cells that interfered with ATG5.These results suggest that DOX will promote TGEV replication in IPEC-J2 cells.In this study,we demonstrated that DOX induced mitophagy,inhibited innate immune responses and promoted TGEV replication in porcine small intestinal epithelial cells.We provide theoretical basis for revealing the side effects of antibiotic abuse which suggest the importance of rational use of antibiotics.