Regulatory Effects Of Fn14 Signaling On TLR4-mediated Inflammation Occurred In Small Intestinal Epithelial Cells Of Piglets

TLR4 in intestinal epithelial cells has been shown paly an important role both in inflammatory reaction and homeostatic maintaining following binding of its cognate ligand lipopolysaccharide(LPS).TWEAK-Fn14 axis plays a key role in pathologies caused by excessive or abnormal inflammation responses.However,the cross-talk between the TLR4 and TWEAK/Fn14 signaling pathway in the gastrointestinal tract are limited.TWEAK is constitutively expressed by leukocytes,including monocytes/macrophages,T cells,B cells,natural killer cells,dendritic cells and neutrophils.Fn14 is expressed by many tissue progenitor cells,including epithelial,mesenchymal,and endothelial cells.This study aimed to evaluate potential cross-talk between the TLR4 and TWEAK/Fn14 signaling pathway in LPS stimulated porcine small intential epithelial cell lines ZYM-SIEC02.The obtained results are as follows:1.We collected the small intestine issues of healthy piglets on 1-day-old(neonatal),7-day-old(suckling),33-day-old(weaned),and 55-day-old,and the LPS-treated small intestine issues of 55-day-old piglet.Immunofluorescence detection showed slight labeling of TWEAK was mainly observed in the central lacteals in intestinal villus of the normal piglet on 0-,7-,and 33-day-old.Interestingly,immunostaining of TWEAK was visibly stronger in lamina propria of 55-day-old piglet small intestine.For Fn14,weak staining was observed in the luminal epithelium in the small intestine of normal piglets on 0,7,33,and 55 days of old.Conversely,very weak TWEAK staining in the central lacteals and strong Fn14 staining in the intestinal epithelial cells was observed in the LPS stimulated piglets as compared with age matched control group.Real-time quantitative PCR and Western Blot detection revealed that both the m RNA and protein expression levels of TWEAK and Fn14 were almost consistent with the the results of Immunofluorescence detection,the expression level of TLR4 was in accord with the expression of Fn14.In vitro,porcine small intential epithelial cell lines ZYM-SIEC02 was treated with LPS.QRT-PCR and Western Blot analysis revealed that the expression levels of Fn14 and TLR4 was increased in a dose-dependent manner.Furthermore,Porcine small intential epithelial cell lines ZYM-SIEC02 treated with LPS,anti-Fn14 or anti-TLR4 showed that the expression levels of TLR4 and Fn14 were positive correlation in cells by Western Blot analysis,and the expression levels of Fn14 depended on the expression level of TLR4.2.In order to further study the cross-talk mechanism between TWEAK-Fn14 pathway and TLR4 related pathway in LPS-induced porcine small epithelial cells inflammation,we blocked the LPS-induced TNF-? using an anti-TNF-? antibody.Western blot analysis showed decreased Fn14 levels while unvaried TLR4 levels in LPS stimulated cells treated with blocking antibody compared to those treated with control antibody.This results indicated that the LPS-induced TNF-? had positive regulation for Fn14 expression.Moreover,the LPS-induced TNF-? in ZYM-SIEC02 was significantly reduced by inhibiting Fn14 expression using anti-Fn14 blocking antibody or si RNA,which was companied with the down regulation of My D88 and NF-?B p65 expression.This results revealed that Fn14 augment TLR4-mediated inflammatory responses via downstream My D88-NF-?B p65 pathways.In order to study the impact of TWEAK on inflammation in ZYM-SIEC02,we used recombinant TWEAK protein with different concentration to treat the LPS-stimulated ZYM-SIEC02.Western Blot analysis showed TWEAK suppressed the expression levels of Fn14,My D88,NF-?B p65 and TNF-? in a dose-dependent manner in LPS stimulated ZYM-SIEC02.In addition,Western Blot analysis and immunofluorescence assay aslo found that Occludin staining appeared to be reduced after stimulated with LPS,whereas the treatment of TWEAK inhibited the reduction of Occludin staining in cells in a dose-dependent manner.Collectively,this study demonstrates that the production of TLR4-mediated TNF-? had positive regulation for Fn14 expression levels in LPS stimulated porcine small intestinal epithelial cells,and the activation of Fn14 augments TLR4-mediated inflammatory responses via activation of My D88-NF-?B p65 pathway,which exhibit a positive feedback circulation.Furthermore,TWEAK play a crucial role in suppressing Fn14 expression,regulating immunity levels in porcine small epithelial cells under the context of inflammation,and modulating Occludin expression in LPS-stimulated intestinal epithelial cells.