Mechanistic Study Of The Regulatory Role Of Human Plasminogen And Xanthohumol In The Immune Response Of Macrophages

Background:The innate immune system is the first line of defense of the host,which can respond quickly to harmful stimuli for the first time.Immune cells respond to these threats by activating non-specific germline-encoded pattern recognition receptors.Pattern recognition receptors trigger downstream inflammation by recognizing PAMPs and DAMPs to eliminate microbial infections and repair damaged tissues.Mononuclear phagocytes are the key instructors of inflammation or immune tolerance in the immune system.These immune cells exist in multiple parts of the human body,indicating their important role in regulating immune function.Monocytes are mononuclear phagocytes that exist in the circulation and have strong activity and functionality.In pathological conditions associated with acute or chronic inflammation,monocytes migrate into the affected tissue and differentiate into tissue macrophages.Therefore,monocytes and macrophages are the key cellular components of the host defense system against pathogens and have important functions in both innate immunity and adaptive immunity.Inflammation and tissue damage can transform the balance between hemostasis and bleeding into a thrombosis and anti-fibrinolytic state,and establish a physical barrier for the body to resist pathogens.However,if the coagulation state cannot be eliminated in time,it will cause disease,such as DIC.Plasminogen is a protein with seven domains.It has more than a dozen cell surface receptors.It can be used as a ligand to bind to other cells to regulate cell behavior and further affect immune and inflammatory processes.In contrast to its activation for plasmin to exert its inflammatory properties,plasminogen itself also exhibits a variety of anti-inflammatory and immunosuppressive reactions.Siglec-7 can specifically recognize the structure of ?-(2,8)disialic acid,and exerts an immunosuppressive function due to the ITIM motif contained in its cytoplasm.Siglec-7 has a high degree of species specificity,only humans and a few primates have it,and it is mainly expressed on mononuclear macrophages and NK cells.NLRP3 inflammasome is a cytoplasmic signal complex that mediates the activation of inflammatory mediators,and is also a key integration point for cellular stress.It can respond to various stimuli,such as viral RNA,lysosomal damage,and extracellular ATP,Glycolysis,reactive oxygen species,mitochondrial damage,and changes in the permeability of cell membranes to potassium ions.The powerful inflammatory potential of NLRP3 and its role in disease make it an attractive drug target.As a traditional Chinese medicine,hops can also be used to treat anxiety,insomnia,mild pain and indigestion.The traditional medical value of hops is attributed to its presence in a variety of Xn.Previous studies have demonstrated several beneficial effects of Xn,including anti-oxidative,anti-inflammatory,anti-cancer,and anti-viral activities.As the main subgroup of innate immune cells,macrophages are one of the important culprits of inflammation,so the regulation of macrophages and their secreted products has become our research focus.In the two parts of our research,we respectively explored the function and mechanism of the human protein(plasminogen)and plant natural extracts(xanthohumol)that negatively regulate the immune response of macrophages.In the first part,we discovered that plasminogen can specifically bind to the macrophage membrane receptor Siglec-7 for the first time,and that plasminogen can mediate the immunosuppressive function of macrophages in a way that does not depend on the activation of plasmin.This biological significance goes beyond its role as a fibrinolytic system to promote thrombolysis,and has a certain effect on the use of plasminogen to achieve therapeutic potential.In the second part,we found that xanthohumol can inhibit the assembly and activation of NLRP3 inflammasomes by preventing the formation of ASC dimer and inhibiting mitochondrial transport.Our results proved that xanthohumol prevents inflammation through inhibition of NLRP3 inflammasome.Part ?:Plasminogen Blunts Innate Immune and Inflammatory Responses through Siglec-7Objective:Using BMDMs and human monocyte-derived macrophages:1.To clarify the inhibitory effect of Xn on NLRP3 inflammasome;2.To investigate the mechanisms by which Xn inhibits the activation of NLRP3 inflammasome.Methods:1.SPR measurements of the binding between Plg and Siglec-7.2.Molecular docking predicts the interaction site of plasminogen and siglec-7.3.Construct overexpression plasmids and mutants to prepare MST samples.4.MST affinity analysis of Plg toward Siglec7.5.Chromogenic substrate method determines plasmin generation from human Plg.6.lentivirus mediated RNA interference expression of Siglec-7 on U-937 cell line.7.qPCR was used to analyze human Siglec-7 and IL-6 mRNA expression.8.ELISA assays were used to analyze human IL-1? and IL-6 in medium supernatants.9.Immunoblotting was used to analyze Siglec-7,SHP-1,Flag and ?-actin expression in cell extracts.Results:1.SPR measurements of the binding between Plg and Siglec-7,The calculated dissociation constants(Kd)between human Plg and Siglec-7 was 1.94×10-7M.2.Molecular docking analysis of the specific binding sites of human Plg and Siglec-7.3.MST affinity analysis of Plg toward Siglec7,there is indeed a strong interaction between Siglec-7 and human Plg,the interaction between Plg and Siglec-7 through the kringle3 domain and V-set domain.The structure of sialic acid may not play a major role in the combination.4.Plasmin proteolytic activity as measured by chromogenic substrate degradation by transfected 293T cells,U-937 cells,or co-incubation of Plg and Siglec-7,Siglec-7 does not affect the activation of Plg to plasmin.5.Plg can inhibit the protein secretion of IL-6 and IL-1?,and the inhibitory effect of disappears when knockdown Sigle-7.6.Plg enhances endogenous SHP-1 association to Siglec-7.Conclusion:1.SPR and MST shows that there is indeed a strong interaction between Siglec-7 and human Plg;2.The interaction between Plg and Siglec-7 through the V-set domain and Kringle3 domain;3.Siglec-7 does not affect the activation of Plg to plasmin;4.Plg engages an inhibitory through Siglec-7 to blunt innate immune and inflammatory responses;5.Plg enhances endogenous SHP-1 association to Siglec-7?Part ?:Xanthohumol Prevents Inflammation through Inhibition of NLRP3 InflammasomeObjective:Using BMDMs and human monocyte-derived macrophages:1.To clarify the inhibitory effect of Xn on NLRP3 inflammasome;2.To investigate the mechanisms by which Xn inhibits the activation of NLRP3 inflammasome.Methods:1.NLRP3 inflammsome activation:LPS-primed BMDMs or PMA-differentiated THP-1 cells were stimulated with nigericin,MSU,Alum,and R837 to activate NLRP3 inflammsome.2.Immunoblotting was used to analyze pro-IL-1?,pro-caspase-1,ASC dimer,ASC monomer,?-actin expression in cell extracts.Medium supernatants were analyzed by immunoblotting for p20.3.ELISA assays were used to analyze IL-1?,IL-18 and TNF-? release in medium supernatants.4.Confocal laser scanning microscopy was used to analyze cell fluorescent protein expression.5.The LDH release kit was used to detect the inhibitory of Xn in the medium supernatants of macrophages on pyroptosis induced by NLRP3 activation.6.Intracellular NAD+levels were measured using an NAD/NADH quantification kit.Results:1.In mouse BMDMs experiments,Xn was treated before LPS primed macrophages.Immunoblotting results showed that Xn inhibited pro-IL-1? expression and caspase-1 activation in a dose-dependent manner;ELISA results showed that Xn dose-dependently inhibited the secretion of IL-1?,IL-18,and TNF-?.2.In mouse BMDMs experiments,LPS-primed macrophages were treated with Xn and then stimulated with nigericin.Immunoblotting results showed that Xn inhibited caspase-1 activation in a dose-dependent manner;ELISA results showed that Xn dose-dependently inhibited the secretion of IL-1? and IL-18,but had no effect on the secretion of TNF-?.3.Different types of stimulis were used to activate NLRP3 inflammasome of mouse BMDMs,immunoblotting and Elisa results respectively showed that Xn down-regulated the activation of caspase-1 and the secretion of IL-1?.Moreover,ELISA results demonstrated that EGCG inhibited IL-1? secretion in human THP-1 differentiated macrophages.4.Increased mitochondrial damage was found in the process of Xn inhibition of NLRP3 inflammasomes activation induced by nigericin or MSU,including the detection of indicators such as ROS,??m,NAD+.Immunofluorescence pretreatment with Xan inhibited mtROS overproduction triggered by nigericin or MSU,but Xn aggravates mitochondrial damage during NLRP3 inflammasome activation.5.Immunofluorescence showed that Xn prevented nigericin-induced translocation of impaired mitochondria to perinuclear area,and this tubulin-driven mitochondrial spatial localization process does not depend on acetylated?-tubulin.6.Immunoblotting showed that Xn reduced the formation of ASC dimers during the assembly process of NLRP3 inflammasome;Immunofluorescence results also showed that ASC speckles were significantly reduced.Conclusion:1.Xn inhibits the priming,assembly and activation of NLRP3 inflammasome;2.Xn inhibits the activation of NLRP3 inflammasome by blocking spatial localization of downstream mitochondria,and the formation of ASC dimers and ASC speckles.