RIP3Knockout Correct The Embryonic Dysplasia And T Cell Proliferation Defect In FADD Knockout Mouse

Apoptosis and necrosis play a key role in keep embryos development andhomeostasis of the immune syetem. Deletion of the Fas gene leads todevelopment of autoimmune-lymphoproliferative disease.However,thedeletion of FADD,Caspase8or cFLIP resulted in the embryonic lethalityat the day of10.5th of the pregnency.A kind of cell death was found inthe middle pregnancy death of the fetals that is similar to the necrosis.It was exhibitted by the further studies that FADD can suppress RIP1mediated necrosis in embryo development and T cell function. At the sametime it was confirmed that Caspase8is important in keeping away thenecrosis mediated by RIP3. FADD can prevent epithelial cell fromRIP3-mediated necrosis has been demonstrated in previous intestinalepithelial cell related study.T cells proliferation defect in FADDconditional knockout mice were correctted meanwhile by the deletion ofRIP1. Deletion of RIP3restore the proliferation dysfunction of Caspase8deleted T cells.Some studies have indicated that RIP3can interactionwith RIP1.Since RIP3has the similary function with RIP1and RIP1deletioncan rescue the embryos development defect and T cell proliferationdysfunction in FADD knockout mice,we assume that RIP3deletion can alsorescue the embryos development defect and T cell proliferationdysfunction in FADD knockout mouse.To confirm this hypothesis,we do someexperiments as below: At first,we set up mating of FADD+/-RIP3+/-heterozygous6weeks miceto get FADD RIP3double knockout mice.As expecting, FADD and RIP3doubleknockout mice were generated with predicted Mendelian frequencies in ourlab and the DKO mice can not be distinguished from its littermates inweight,food intake,activity and reproduction at the beginning.Actuallyit shows that RIP3deletion can rescue the embryos development defect inFADD knockout mice. It is different from the RIP1FADD double knockoutmice died right after born,the FADD/RIP3DKO mice can survive as thewild type control mice.It implys that the necrosis caused by RIP3wascontrolled by FADD.It consistent with other research that there is noFADD-/-RIP3+/+and FADD-/-RIP3+/-genetype mouse was born at birth andafter that within the offsprings of the all3differtent genetype mating.No matter what is the FADD genetype,the RIP3-/-mice can be born andsurvival the whole life as the wildtype control.In general,The FADD RIP3double knockout mice is visible and can survival like its littermatecontrol.Secondly, we analysed the T cell subgroup by flowcytometry. Enlargetedlymph nodes and spleen was found in these older mice though it is notdifferent with control at the birth.It is the FADD-/-/RIP3-/-DKO micethat develop age-dependent LPD (lymphoproliferative disease) resemblingthat of Fas-/-mice. There are distinct spleen cell count differencebetween DKO and control mice, A CD3、B220double positive and CD4、CD8double negative cell population was found in lymph node and spleen of theseDKO mice.To confirm whether the cell is activated, CD25, CD28, CD69and CTL-A4were stained as the T cell activation marker and analysed byflowcytometry.It shows that the CD69expression is higer in DKO mice,andother markers are negative.A anti-ANA kit was used to test the anti-ANAantigen in DKO mice serum by immunofluorescent techniques.It indicatedthat the DKO mice have unanimous positive results comparing with the control.So wo got the conclusion that the DKO mice occur the LPD and itcan provide a new target for investigating the therapy of LPD.At last, T cell proliferation and cell death were detected. T cellproliferation induced by TCR is a little bit higer than the control inyoung mice,but there is a contray outcome in old DKO mice.Not only youngDKO mice but also old DKO mice have decreased anti-Fas induced thymocytekilling contrasted with their control respectively.It can not bedistinguished of anti-Fas,TNFR1and anti-CD3relatived activationinduced cell death(AICD) from DKO mice and their wild type control thoughthere is a big difference between DKO mice and FADD-/-mice in AICD.In total,our data indicate that RIP3knockout correct the FADD-/-embryonic dysplasia and the DKO mice gave birth to viable thendevelopment with LPD in adult. The CD69expression is higer in DKO micethan the control though there is no difference in others activationmarker.The DKO mice have unanimous positive results contrasted with thecontrol in serum anti-ANA analysed by immunofluorescent.T cellproliferation defect in FADD knockout mice was restored by RIP3deletion.DKO mice thymocyte was resistent to anti-Fas inducedkilling.There is no differencd in DKO mice and wild type control inanti-Fas,TNFR1and anti-CD3induced AICD.It is supposed the reason leadto LPD is the decreased cell death which is caused by blocking twopathway of apoptosis and necrosis at the same time by double knockout ofFADD and RIP3.