The Role And Mechanism Of The Inhibitor NMS-873 In Dectin-1 Antifungal Signaling Pathway

Fungi are ubiquitous;we inhale several hundreds of Aspergillus sp.spores each day,most of us are colonized with Candida sp.and other fungal species,and in our lifetime,we will be exposed to hundreds of potentially infective fungal species.With the prevalence of these microorganisms and relatively low incidence of pathogenicity,it is easy to overlook their threat to public health.Despite being a commensal microbe for healthy individuals,C.albicans can be a mortal pathogen to AIDS,immunocompromised patients.Since currently available therapy can be ineffective and toxic,better preventive and therapeutic strategies are urgent need for fungal infections,which require best understanding the host defense mechanisms.Up to now,four families of PRRs have been shown to recognize fungal pathogens and to be capable of inducing cellular responses.Such as Toll-like receptor(TLR),NOD-like receptor(NLR),RIG-I-like receptor(RLR)and C-type lectin receptor(CLR)families CLR and their signaling pathways are essential for antifungal immunity.A prototypic CLR is Dectin-1,which is crucial for defense against fungi.Ligand binding by Dectin-1 and subsequent receptor clustering triggers phosphorylation of the immunoreceptor tyrosine-based activation motif(ITAM)-like sequence within the cytoplasmic domains of the Dectin-1 receptors,leading to the consecutive recruitment of SHP2 and the tyrosine kinase SYK,followed promoting the formation of the CARD9-BCL10-MALT1 scaffold complex.This scaffold initiates a signaling cascade that leads to activation of both the canonical NF-?B pathway and the non-canonical NF-?B pathway.Tyrosine phosphorylation occurred in pivotal molecules during Dectin-1 activation and mediated antifungal innate immunity,including SYK,SHP2,PLC-y2 and PKC?.However,it is not clear whether exit other key molecular that regulate Dectin-1 pathway activation,it is worth for us to further elucidate.Therefore,this study will explore whether there are other key regulators that can mediate the Dectin-1 signaling pathway.First,we confirm that the cytokine IL-4 can significantly up-regulate the activation of Dectin-1 signaling pathway.There is also exist tyrosine phosphorylation of other key molecules independent and dependent of SYK.After preliminary explore,we found that small molecule inhibitor NMS-873 can effectively inhibit the activation of Dectin-1 signaling pathway and affect the expression of antifungal related genes.In terms of mechanism,the regulation of NMS-873 on the signal activation of Dectin-1 pathway is likely to affect the recruitment of kinase SYK and phosphatase SHP2 by Dectin-1,and then impairs the downstream signal transduction to regulate the antifungal genes' productionObjectives:Exploring the specific mechanism of antifungal innate immune response from the point of view of signaling pathway.To study whether there are other key mediators that can regulate the activation of Dectin-1 signaling pathway.Methods:1.Confirm the cytokine IL-4 that can up-regulate the Dectin-1 signaling pathway.The effect of IL-4 on the signal activation of Dectin-1 pathway was detected by Western Blot,and the tyrosine phosphorylation modified bands in WT cells at different time points were detected by specific tyrosine phosphorylation antibody p-Tyr(4G10).2.To explore the role of kinase SYK on Dectin-1 signaling pathway and to observe whether exist other key molecules are modified by tyrosine phosphorylation during the engagement of Dectin-1 independent of SYK.BMDMs and BMDCs were used to detect the effect of kinase SYK on the activation of Dectin-1 pathway in WT and SYK deficient cells.Investigate whether there is tyrosine phosphorylation of other key molecules independent of SYK.3.Investigate whether the key molecule of necrosis RIPK3 affects the activation of Dectin-1 signaling pathway.WB were used to check whether RIPK3 directly affects the Dectin-1 signaling pathway's activation.The expression of inflammatory cytokines and chemokines induced by Zymosan was detected by Real-time PCR.4.Investigate the effect of NMS-873 on Zymosan-induced Dectin-1 pathway's activation.WB to check the effect of NMS-873 and Celastrol on Zymosan induced the activation of dectin-1 signaling pathway in differentiated BMDMs,and then comparing the two inhibitors' effect.Then we focus on the NMS-873 and elucidating its role in dectin-1 signaling pathway.The changes of Zymosan induced inflammatory cytokines and chemokine gene's expression were detected by Real Time PCR.The Zymosan and Zymosan D induced expressions of Cxcl1?Il-1? and Il-6 were detected by using ELISA5.Investigate the effect of NMS-873 on Zymosan induced Dectin-1 activation with TLRs blockedWB to check the effect of NMS-873 on Zymosan induced Dectin-1 activation in differentiated Myd88 knock out BMDMs.The changes of Dectin-1 ligands induced inflammatory cytokines and chemokine gene's expression were detected by Real Time PCR.6.Investigate the specific mechanism of the effect of NMS-873 on the activation of Dectin-1 pathway and whether it affected the recruitment of key molecules including SYK and SHP2 to the cell membraneThe cells were divided into different components,including P1,P25,P100 and S100,by using different centrifugation speeds.WB to check the phosphorylation of Dectin-1 mediated molecular in different cell part to evaluate its function.Results:1.IL-4 upregulates Dectin-1 signaling pathway's activation2.Spleen tyrosine kinase(SYK)mediates the activation of Dectin-1 signaling3.RIPK3 has no effect on the Dectin-1 signal transduction pathways upstream of the activation of CARD94.NMS-873 attenuates the Dectin-1 signaling pathway's activation induced by Zymosan5.NMS-873 attenuates Dectin-1 signaling pathway independent of TLRs6.NMS-873 impairs the phosphorylation of SYK and SHP2 in P25 and P100 componentsSummary:We found that small molecular inhibitors NMS-873 and Celastrol regulate Dectin-1 signal's transduction.Specifically,the NMS-873 can impair the antifungal genes'production.In mechanism,NMS-873 may affect the recruitment of kinase SYK and phosphatase SHP2 to the cell membrane,and then attenuates the downstream signal's transduction.