Study On Coordination Between TLR And Nod Signaling In Innate Immunity Against Sepsis

Background:Microbial infection-associated sepsis and septic shock are the major cause of mortality in hospitalized children worldwide.The innate immune system plays a key role by forming the first line of host defense against microbial infection and sepsis via activation of both TLR signaling and Nod signaling through PRRs.Previous studies have focused on impact of either TLR signaling or Nod signaling on innate immunity-associated responses to microbial infection;however,the synergistic effect of activation of both TLR and Nod signaling on initiating and augmenting the inflammatory response and antimicrobial activity is largely unexplored.In our research,we will investigate the synergistic effect of TLR and Nod signaling in sepsis.Moreover,we will further investigate whether activation of both TLR and Nod signaling is essential to induce a maximal inflammatory response and an efficient antimicrobial response,and the underlying mechanisms involved.We hope our work will provide new data that will add new information on immune targeting and medicines on sepsis.Part Ⅰ The expression and clinical values of TLR and Nod signaling in microbial infectionObjective:To explore the expression and clinical values of TLR and Nod signaling and their synergistic effects in microbial infection-associated sepsis.Methods:Peripheral blood samples of sepsis children were collected and plasma was separated to examine the production of inflammatory cytokines.The expression of TLR2,TLR4,Nod1 and Nod2 were analyzed through Fluorescent Quantatitive PCR.We stimulated PBMC from healthy volunteers with TLR and NLR agonist alone and their combination to analyze the production of inflammatory cytokines.We also extracted proteins to assess the activation of NF-κB and MAPK p38 pathways.Results:Compared with the control group,the levels of IL-6 and IL-10 were significantly higher(P<0.05,P<0.01)in sepsis group,but the levels of IL-8,IL-1β and TNFa had no obvious differences.The expression of TLR4 increased(P<0.05),while Nod1 decreased(P<0.01)significantly in sepsis group comparing with the control group.However,the expression of TLR2 and Nod2 had no differences.After being stimulated with LPS or BLP,the release of IL-8,IL-6 and TNFα increased significantly(P<0.01)in PBMC from healthy volunteers,but the release of IL-1β had no changes.Stimulated with both LPS and MDP in PBMC lead to an amplified release of cytokines and activation of the NF-κB pathway with increased phosphorylation of NF-κB p65 at Ser586 when compared to PBMC stimulated with LPS or MDP alone,but no augmentative effects appear on phosphorylation of MAPK p38 at Th180/Try182.Conclusion:Sepsis in children leads to dysfunctions of TLR and Nod signaling and abnormal release of inflammatory cytokines.Stimulated with both TLR and Nod agonists in PBMC lead to an amplified release of cytokines and activation of the NF-κB pathway,but not the MAPK p38 pathway.Part Ⅱ The crosstalk of TLR and Nod signaling in inflammatory responseObjective:To study the release of cytokines and chemokines and the activation of inflammatory signaling pathways of BMMs after being activated of both TLR and Nod signaling.Methods:We stimulated BMMs with TLR and NLR agonist alone and their combination to analyze the production of inflammatory cytokines and chemokines through CBA or ELISA technologies.We assessed TLR and Nod mediated upstream and downstream pathways in BMMs stimulated with TLR and Nod agonists at different time pointsResults:Stimulation of BMMs with LPS resulted in an increased release of inflammatory cytokines TNF-α,IL-6,and IL-12p70,and chemokine CXCL2(P<0.01 versus PBS-treated macrophages),whereas Tri-DAP or MDP stimulation caused slighter but significant increases in TNF-α,IL-6,IL-12p70,and CXCL2 release(P<0.05 versus PBS-treated macrophages).Of note,a combined stimulation of LPS with Tri-DAP or MDP maximized the inflammatory response with substantially augmented release of TNF-α,IL-6,IL-12p70,and CXCL2 when compared to the response observed with LPS,Tri-DAP,or MDP alone(P<0.05,P<0.01).Consistent with the finding from the TLR4 agonist LPS stimulation,stimulation of macrophages with the TLR2 agonist BLP also has the same augmented inflammatory response.Stimulation of BMMs with LPS,Tri-DAP,MDP,or their combinations did not affect TLR-and Nod-mediated upstream pathways including the expression of TLR4,Nod1,Nod2,MyD88,IRAK1,RIP2,and CARD9.However,stimulation of BMMs with a combination of LPS plus Tri-DAP or MDP resulted in a strong activation in the downstream NF-κB pathway with substantially increased phosphorylation of NF-κB p65,but had no augmentative effect on phosphorylation of MAPK p38.We further stimulated BMMs with BLP,Tri-DAP,MDP,or their combinations,and observed a substantially augmented activation of NF-κB,but not the MAPK p38 pathway.Conclusion:Activation of both TLR and Nod signaling results in an optimal inflammatory response with enhanced inflammatory cytokines and chemokines release,which is associated with an amplified downstream activation of the NF-κB pathway,but not the MAPK p38 pathway.PartⅢ The crosstalk of TLR and Nod signaling in innate phagocyte-associated bactericidal activityObjective:To prove that the synergistic effects of TLR and Nod signaling can lead to an enhanced antimicrobial response.Methods:To examine bacterial uptake,phagocytosis and intracellular killing functions of macrophages after being activated by TLR and Nod signaling.The expression of phagocytic receptors and maturation of phagosome are also examinated.Results:Stimulation with LPS,Tri-DAP,or MDP alone did not affect macrophage-associated bactericidal activity;however,a combined stimulation of LPS with Tri-DAP or MDP significantly enhanced uptake and phagocytosis of S.typhimurium,with substantially increased intracellular killing of the ingested S.typhimurium(P<0.05 versus macrophages stimulated with LPS,Tri-DAP,or MDP alone).BLP stimulation in combination with Tri-DAP or MDP also resulted in an augmented bactericidal activity against gram-positive S.aureus.Stimulation with LPS or BLP,but not Tri-DAP or MDP,increased surface expression of CR3 and Fc y R on macrophages(P<0.05 versus PBS-treated macrophages),and a combined stimulation of LPS/BLP with Tri-DAP or MDP resulted in a further upregulation of CR3 and Fc y R expression(P<0.05 versus macrophages stimulated with LPS or BLP alone).A combined stimulation of LPS/BLP with Tri-DAP or MDP substantially accelerated phagosomal acidification after ingestion of bacterial(P<0.01 versus macrophages stimulated singly).Consistent with an accelerated phagosomal acidification,macrophages stimulated by a combination of LPS or BLP plus either Tri-DAP or MDP displayed significantly increased phagosome fusion at 30,60,and 90 min after ingestion of S.typhimurium(P<0.05 versus macrophages stimulated with LPS,Tri-DAP,or MDP alone)or S.aureus(P<0.05 versus macrophages stimulated with BLP,Tri-DAP,or MDP alone)as determined in a cell-free organelle system.Conclusion:Activation of both TLR and Nod signaling leads to an augmented antimicrobial response with increased bactericidal activity,which is correlated with up-regulated phagocytic receptors expression and accelerated phagosome maturation.