Study On The Role Of CD317 In Macrophage-mediated Innate Immune Response

Innate immune response is the first line of defense against pathogen invasion,it plays an important role in the early stage of pathogen invasion,and complements adaptive immunity.Pattern recognition receptors(PRRs),which recognize pathogens by \ specific pathogen-associated molecular patterns(PAMPs),and activate immune response signal transductions,in early host defense against microbial infection,which serve as a bridge between innate immunity and adaptive immunity.Toll-like Receptors(TLRs)are an important pattern recognition receptor family in innate immunity and play an important role in the innate immune response to various infections.TLRs signal transduction can be classified into My D88-dependent and My D88-independent according to the different adaptor proteins recruited.In classic My D88-dependent pathway,the combination of TLR and My D88 can recruit IL-1receptor associated kinase 4(IRAK4)and activate other family members of IRAK.IRAKs then dissociates from the complex and activates tumor necrosis factor receptorassociated factor-6(TRAF6).By activating TAK1,TRAF6 further phosphorylates the IκKs complex inducing IκB phosphorylation and degradation.Subsequently,the transcription factor NF-κB is activated and translocated into the nucleus to induce the expression of various pro-inflammatory cytokines,such as IL-1,IL-6,and TNF-α.In most pathogenic microorganism infections,TLRs plays a positive role in the outcome of the disease.However,going too far is as bad as not going far enough,its abnormal activation often leads to inflammation or autoimmune damage.Therefore,TLR signaling needs to be strictly regulated to maintain immune homeostasis.CD317 also called Bst2 is a type Ⅱ transmembrane glycoprotein,which has been previously reported to have antiviral role by binding HIV-1,SIV,other viruses and display anti-tumor effect.However,in recent years,several studies have shown that CD317 can activate NF-κB,and thus further regulate antiviral immune response in the body.For all this,we still lack experimental evidence and in-depth studies on whether CD317 has a regulatory effect on TLRS-mediated signal transduction currently.Therefore,this study explore the regulatory effect of CD317 on My D88-dependent TLRs signal transduction using primary peritoneal macrophages from CD317 knockout mice and THP-1-induced macrophages as models.The main research contents include the following five aspects:(1)The effect of TLR4/7 signal activation on CD317 expression: Peritoneal primary macrophages(PM)derived from mouse and THP-1-induced macrophages(Td M)were selected as the research objects.After LPS or IMQ stimulation,fluorescent quantitative PCR was used to detect the expression level of CD317 m RNA,while Western blot or FACS was used to verify the changes in protein level.The results showed that CD317 can be induced by TLR4/7 signal,and may be involved in TLR4/7signal transduction or functional development process.(2)Creation of Bst2 knockout mouse: Bst2 knockout mice were constructed by CRISPR/Cas9 Bst2 targeted gene editing.The genotypes of the progeny mouse were identified by PCR amplification.After CD317 knockout mice were successfully obtained,the peritoneal macrophages of wild-type(WT)and knockout mice(KO)were further isolated and CD317 expression was detected by flow cytometry to verify the knockout effect.(3)Impacts of CD317 knockdown/knockout on the expression of TLR4/7 induced pro-inflammatory cytokines: WT and CD317 KO PM cells and CD317 si RNA knockdown Td M cells were collected at different time points after stimulated with LPS or IMQ.The expression levels of CD317,TNF-α,IL-1β,IL-6 other pro-inflammatory cytokines m RNA were detected using real-time fluorescence quantitative PCR.The results showed that CD317 knockdown/knockout significantly inhibited the expression of TLR4/7 induced pro-inflammatory cytokines.(4)The effect of CD317 knockdown/knockout on the activation of NF-κB signaling pathway: Western blot was used to analyze the activation of MAPK and NF-κB key pathways which is downstream of My D88-dependent TLR4,to study the mechanism by which CD317 affects the expression of TLR4-mediated proinflammatory cytokines.CD317 deficiency significantly inhibited the activation of NF-κB,but had little effect on the p38 signal in the MAPK signaling pathway downstream of TLRs signaling.(5)Study on the interaction between CD317 and My D88/TRAF6: CD317 and My D88 or TRAF6 expression plasmid were co-expressed in HEK293 T.Immunoprecipitation was used to investigate whether CD317 could interact with My D88 and TRAF6,(downstream proteins of My D88-dependent TLRs),to explore the potential mechanism of CD317 involvment in TLRs signal regulation.The results showed that CD317 could interact with My D88 and TRAF6,possibly enhance the activation of NF-κB through this interaction.In conclusion,this study confirmed that CD317 promotes My D8-dependent TLRs signal transduction by the production of related pro-inflammatory cytokines,and CD317 interacts with key proteins such as My D88 and TRAF6 downstream of TLR,which points out the direction for molecular mechanism exploration.This study not only enriches our understanding of the function of CD317,especially its role in innate immune regulation,but also provides new targets and theoretical guidance for the development of therapeutic strategies for related diseases.