Postoperative Study Of Pattern Recognition Receprtor Expression And Functionality In Children With Congenital Heart Disease

Background:Cardiopulmonary bypass(CPB)is a technique that temporarily takes over the organic functions during surgery,maintaining the circulation of blood of the patient's body.CPB is nowadays widely used in the pediatric heart surgery.However,previous studies have shown that CPB is associated with reduced immunity and increased risk of postoperative infection,and the underlying mechanisms are largely unknown.This thesis aims to study the expression level and function of pattern recognition receptors(PRRs)inside and outside cells after CPB,and to elucidate the effects of CPB on the innate immune in children with congenital heart diseases(CHD),which will provide useful information for the prevention and the treatment of postoperative infection in children with CHD.Part One:Postoperative study of Toll-like Receptors(TLRs)expression and function in children with CHDObjective:To assess the influence of CPB on surface expression and function of TLR2 and TLR4 on monocytes in CHD children.Methods:Peripheral blood samples were collected from 20 children with CHD at the followingtime points:before operation,at the end of CPB,and 1,3,and 7 days after CPB.The levels of CD 14,TLR2 and TLR4 on monocytes at each time point were quantified using flow cytometry.The phosphorylation level of NF-?B p65,MAPK p38,and ERK1/2 upon LPS or BLP stimulation were further measured.Moreover,the inflamnatory cytokines in the culture supermatants were measured by enzyme-linked immunosorbent assay(ELISA).Results:1.The level of CD14,TLR4,and TLR2 were markedly decreased after CPB,which were recovered at day 1 postoperatively.2.The activation of NF-?B p65 and ERK1/2 upon LPS or BLP stimulationin monocytes were substantially inhibited after CPB,which were recovered at day 1 postoperatively.There was no significant differences in phosphorylated p38 expression upon LPS stimulation at different periods of CPB,but the activation of p38 upon BLP stimulation was inhibited after CPB.3.The levels of TNF-?,IL-1?,IL-6,and IL-8 in response to stimulation with LPS were significantly reduced compared with those before CPB.The levels of TNF-?,IL-6,and IL-8 upon BLP stimulation were also decreased,while the level of IL-10 upon LPS or BLP stimulation was increased after CPB.Conclusions:The expression of CD14/TLR4/TLR2,the TLR mediated signaling pathways,and associated cytokines secretion were substantially inhibited after CPB,which correlated with the suppressed innate immune response and may account,at least inpart,for the increased risk of postoperative bacterial infection in CHD children.Part Two:Postoperative study of NOD-Iike receptors(NLRs)expression and function in children with CHDObjective:To assess the influence of CPB on expression and function of NODI,NOD2 in CHD children.Methods:Peripheral blood samples were collected from 50 children with CHD at the following time points:before operation,at the end of CPB,and 1,3,and 7 days after CPB.The levels of NODI,NOD2 at each time point were quantified using Real-time PCR.After ligand stinulation,the levels of TNF-??IL-6 in the culture supernatants were measured by ELISA.Results:1.The level of NODI was decreasedat day 1 postoperatively,which were recovered at day 3 post-CPB.The level of NOD2 may also decreased after CPB,but no significant difference was found.2.The levels of TNF-? and IL-6 upon Tri-DAP stimulation were significantly reduced compared with those before CPB.The level of IL-6 was recovered at day 1 postoperatively,while the level of TNF-? was recovered at day 3 post-CPB.The level of TNF-? upon MDP stimulation vas also decreased after CPB,which was recovered at day 1 postoperatively.There was no significant difference found in the MDP induced expression of IL-6 at different periods of CPB.Conclusions:The expression of NODI were significantly inhibited postoperatively.The levels of TNF-? and IL-6 upon Tri-DAP or MDP stimulation were also reduced,which may imply the inhibition of NOD post-CPB mediated innate immune response.Part three:Postoperative study of RIG-? like receptors(RLRs)expression and function in children with CHDObjective:To assess the influence of CPB on surface expression and function of RIG-??MDA5 on monocytes in CHD children.Methods:Peripheral blood samples were collected from 30 children with CHD at the followingtime points:before operation,at the end of CPB,and 1,3,and 7 days after CPB.The levels of RIG-? and MDA5 at each time point were quantified using Real-time PCR.After ligand stimulation,Western Blot was used to detect the expressions of phospho-IRF3/IRf3 and NF-?B phospho-p65/p65.The levels of IFN-? and IFN-? in the culture supernatants were measured by ELISA.Results:1.The level of RIG-? was decreased at day 1,3,and 7 postoperatively.The level of MDA5 at day 1 post-CPB was also down-regulated compared with that before CPB,which was recovered at day 3 post-CPB.2.The phosphorylation of IRF3 in PBMCs at 1 day CT3)after operation was lower than that before operation,and the production of IFN-? was also decreased.Conclusions:The expression of RIG-?and MDA5 were significantly inhibited postoperatively.The level of IFN-a upon Poly(I:C)stimulation was reduced,which may imply the inhibition of intracellular RLR mediated innate iunune response.As a result,the ability of CHD children to resist postoperative viral infection may be impaired.