Distinct Properties Of TLRs Mediated Signal Pathways Exposed In The Secondary Immune Response

Immune response is a biological process that occurs after the body's immune system is stimulated by antigens in order to eliminate antigens.According to the characteristics,acquisition form and effect mechanism of immune response recognition,it can be divided into two categories: innate immunity and adaptive immune response.Among them,innate immunity,also known as inherent immunity or non-specific immunity,is the innate immune defense function formed during the genesis and evolution of the organism.It establishes the first line of defense against pathogenic microbial infection and participates in the initiation and regulation of adaptive immune response.Innate immune cells are an important part of innate immunity,including phagocytes,dendritic cells and NK cells and so on.Monocyte-phagocyte system,which includes pre-bone marrow mononuclear cells,peripheral blood mononuclear cells and tissue macrophages(M?).Macrophages are one of the cell types with the most active biological activities in vivo,which are participated in immune defense and immune self-stabilization of the body,and can process and present antigens,regulate immune response and mediate the inflammatory response.Pattern recognition receptors(PRRs)are a class of recognition molecule mainly expressed on the surface of innate immune cells or the membranes of intracellular organelles,which can recognize one or more pathogen-associated molecular patterns(PAMPs).Through the recognition of PAMPs,PRRs can activate related signaling pathways and lead to a series of immune inflammatory reactions.However,it is often precisely because of the over-activation of these pattern recognition receptors that leads to the occurrence of a series of autoimmune diseases,such as rheumatoid arthritis,psoriasis,and systemic lupus erythematosus and so on.Important PRRs include mannose receptors(MR),NOD-like receptors(NLR)and Toll-like receptors(TLR),etc.Among them,Toll-like receptors(TLRs)are one of the most studied and representative transmembrane proteins,which is mainly expressed in dendritic cells,macrophages and other antigen-presenting cell(APC).It can specifically identify and bind the specific components of viruses,fungi,bacteria and parasites,such as lipopolysaccharides,peptidoglycans,bacterial DNA,double-stranded RNA and so on.TLRs can induce inflammation by mediating activation of MyD88-dependent pathway and TRIF-dependent pathway,thus eliminating foreign pathogens.Except that TLR3 is through TRIF pathway,all the other TLRs can activate the downstream signaling pathway by using MyD88 as the adaptor molecule,leading to the release of a large numbers of inflammatory factors such as TNF-a,IL-6 and interferon.With extensive and in-depth research findings,TLRs play an increasingly important role in activating innate immune response.Although TLR-mediated pre-inflammatory response is a basic form of innate immunity,in fact,the pathogen PAMPs often activate multiple TLRs at the same time,leading to different immune effects such as superposition,synergy or antagonism.Numerous studies have also shown that the interaction of TLRs signaling pathways plays an important role in maintaining the immune homeostasis of the organism,and the excessive immune inflammation induced by TLRs signaling pathways often leads to the disorder of immune system regulation.However,the roles and underlying mechanisms of TLRs in the process of mutual coordination are still unclear.Secondly,TLRs re-response is an immune response that occurs when the organism contacts the pathogen again.Studies have found that when the host responded again,the immune effects are usually sometimes strong or weak.In recent years,more and more viewpoints have been put forward to explain these different innate immune behaviors by immune memory and tolerance,and the underlying mechanism is still unclear.Therefore,this issue has become a research hotspot in recent years.In this paper,we used mouse Raw264.7 macrophages as the research object,starting from the characteristics of macrophage immune re-response,using TLRs(TLR3,TLR4,TLR7/8,TLR9)ligands to pretreat macrophages,and then cross-stimulating with these different TLRs ligands to construct the re-response model.On this basis,the changes of immune response and cell activity of macrophages after TLRs reactivation were observed.Firstly,different concentrations of TLRs agonists,including Poly(I:C)(TLR3 VII agonist),CpG-1826(TLR9 agonist),R848(TLR7/8 agonist)and LPS(TLR4 agonist),were used to treat mouse Raw264.7 cells,respectively.Saturated stimulatory concentrations of these TLRs agonists were found.Subsequently,MTT and flow cytometry were used to detect the effects of these saturated stimulatory concentrations on the vitality and apoptosis of macrophages,and the results showed that they had little effect.Then,because both TLR9 and TLR7/8 depend on the MyD88 signaling pathway to activate downstream signaling,enzyme-linked immunosorbent assay(ELISA)was used to detect the release of inflammatory factors in the supernatant of Raw264.7 cells pretreated with different concentrations of TLR9 agonist CpG-1826 or saturated concentrations of TLR7/8 agonist R848,to study the effect of pre-activation of MyD88 signaling pathway on secondary immune response of macrophages.The results showed that the immune response of the cells decreased with the increase of pretreatment concentration,while the pretreated cells were re-stimulated by TLR3 agonist Poly(I:C),the cellular immune response was not decreased,but enhanced.To further verify this result,we detected the expression level of IRAK protein downstream of TLR7/8 and TLR9-dependent MyD88 signaling pathway by Western blot,and found that the expression of IRAK protein decreased significantly after the pretreatment.We also detected that the cells pretreated with MyD88-dependent TLRs agonists were re-stimulated after a certain time interval,and found that the immune response of macrophages could not be restored to the initial state.Similarly,since TLR3 only relies on TRIF signaling pathway to activate downstream signaling,we also detected the release of inflammatory factors in the supernatant of Raw264.7 cells pretreated with different concentrations of TLR3 agonist Poly(I:C)by ELISA after they were re-cross-stimulated by CpG-1826,R848 and Poly(I:C),in order to investigated the effect of pre-activation of TRIF pathway on the secondary immune response of macrophages.And the results were interesting to note that the pretreatment of cellular immune response ability was enhanced.Secondly,because TLR4 can activate both MyD88 and TRIF signaling pathways at the same time,Raw264.7 cells were pretreated with different concentrations of TLR4 agonist LPS,to study the effect of TLR4 activation on secondary immune response of macrophages.The results showed that when the cells were stimulated by CpG-1826,R848 and LPS,the immune response ability of cells was decreased,while that of cells re-stimulated by Poly(I:C)was significantly enhanced.These results suggest that the pre-activation of MyD88 pathway reduces the secondary response mediated by itself,while the pre-activation of TRIF pathway enhances the subsequent secondary response of macrophages,and whether TRIF pathway itself or MyD88 pathway is re-activated,the immune response of macrophages is enhanced.These results suggest that macrophages activated by different TLRs exhibit different secondary response abilities,which are closely related to the activated pathway.Interestingly,we observed the morphological changes of cells activated by different TLRs under optical microscope,and found a consistent correlation that when MyD88 pathway was activated,Raw264.7 cells became larger,pseudopodia elongated and cytoplasm increased.When pretreated cells received secondary stimulation,the cell morphology did not continue to swelling.However,when TRIF pathway was activated,the morphology of cells was basically the same as that of normal cells.Above all,this study indicates,the responses of different TLRs can be classified into two categories,one is the enhancement of the re-response,the other is the weakening of the re-response.These distinct properties are related to the signal pathways on which they depend.Among them,MyD88 pathway mediated response is rapid,saturation attack makes the cell swelling to the limit,leading to the exhaustion of macrophage resources,and its immune response ability is difficult to return to normal level in a short time,thus showing non-specific immune anergy in re-response.Secondly,although the activation of MyD88 pathway leads to its own anergy,it does not affect the responsiveness of TRIF pathway,which recurs the independency of two pathways.On the contrary,the completed activation of TRIF pathway did not make cells overreacted,and the cell morphology does not swell of saturation attack.Moreover,the activation of TRIF pathway would enhance the ability of itself and MyD88 pathway to re-respond,thus reflecting the non-specific irritation characteristics.This study not only gives us a deeper understanding of the different roles of the two TLRs signaling pathways in innate immunity and the interaction between TLRs,but also provides a new idea for innate immune regulation in the future.