Function And Mechanism Of RNA Editing Enzyme ADAR1 In Humoral Immune Response

Humoral immunity is the process of antibody-mediated elimination of extracellular antigens and is an important part of the immune system.During humoral immune response,B cells are activated by antigens and differentiate into memory B cells and plasma cells.Plasma cells have a unique cellular structure and can synthesize and secrete antibodies to mediate the clearance of pathogens.Memory B cells have a long-life span and are rapidly activated by the same antigen and respond quickly and efficiently.However,abnormal B cell activation and differentiation will lead to immune deficiency,autoimmune disease,and lymphoma.Therefore,the activation and differentiation of B cells are strictly regulated during humoral immune responses.Understanding the regulatory mechanism of B cell activation and differentiation will help for the treatment of pathogen infection and autoimmune diseases and the design of vaccines.Adenosine deaminase acting on RNA(ADAR)1is the principal enzyme for adenosine to inosine(A-to-I)editing.Previous studies have demonstrated that ADAR1 is a critical player in innate immunity and the development of B and T cells.However,it remains unknown if ADAR1 plays a role in adaptive immune responses.To assess the role of ADAR1 in B cell activation and immune response,we used the Cre-Lox P system to generate ADAR1 conditionally knockout(KO)mice.ADAR1-deficient mice have defective germinal center response and diminished number of memory B cells and antibody secreting cells.However,the plasmablasts,generated during early T cell-dependent or-independent immune response,were increased in ADAR1 KO mice.Our results suggest that ADAR1 plays a key role in B cell fate decisions during humoral immune response.Previous studies have demonstrated that ADAR1 prevents the hyperactivation of multiple ds RNA sensors,including melanoma differentiation-associated protein 5(MDA5),protein kinase R(PKR)and ribonuclease L(RNase L)by acting on cellular endogenous ds RNAs.To evaluate the roles of MDA5,PKR and RNase L in ADAR1 KO B cell mediated humoral immune response,we generated various double mutant mice concurrently deficient in ADAR1 and different downstream ds RNA sensors,and unveiled that the MDA5 pathway is partially required for the germinal center response.However,MDA5 knockout failed to rescue the reduced defect of memory B cells and plasma cells.Moreover,MDA5 knockout also failed to reduce the increased plasmablasts cell number in ADAR1 KO mice during early T cell independent immune response.In addition,PKR and RNase L pathways are dispensable for ADAR1’s role in humoral immune response.These results indicated that ADAR1 regulates humoral immune response independent of PKR and RNase L,and partly depends on MDA5 in germinal center response.Two ADAR1 isoforms exist in mammals,a longer mainly cytoplasm-localized p150 isoform and a shorter primarily nucleus-localized p110 isoform.Studies have showed the two isoforms play different roles in mouse organ development.To explore the function of ADAR1 p150 and ADAR1 p110 isoforms during humoral immune response.We used the Cre-Lox P system to generate Adar1 knock-out and Adar1 p150 or p110 knock-in mice.The results showed that ADAR1 p150 isoform but not the ADAR1 p110 isoform could replace the ADAR1’s role in humoral immune response.These results suggest that the p150 isoform exclusively accounts for the role of ADAR1 in humoral immune response.To further explore the mechanism of ADAR1 p150 regulating germinal center response,we performed mixed bone marrow chimera reconstitution experiments by transferring ADAR1 KO mice bone marrow cells transduced retrovirally with the genes encoding the Zα,ds RBD and catalytic domain mutated ADAR1 p150 proteins and NES-ADAR1p110 fusion protein,which can be distributed in cell cytoplasm.The results showed that the mutation of Zα and ds RBD domains completely abolished the function of ADAR1p150 in germinal center response.The overexpression of NES-ADAR1p110 failed to rescue the defect of germinal center response.These results revealed that ADAR1p150 Zα and ds RBD domains are essential for germinal center response.The Zα and ds RBD domains of ADAR1p150 may have synergistic effect in germinal center response.Gene expression profile analysis by RNA-seq revealed that the differentially expressed genes in activated ADAR1 KO B cells are mainly involved in B cell differentiation,including Blimp-1,IRF4,Pax5,Bach2 and Bcl6,which play crucial roles in the regulation of activated B cell differentiation.Further analysis showed that ADAR1 deletion leads to the hyperactivation of Syk and BLNK,the key molecules of B cell receptor signaling,as well as the hyperactivation of MEK and ERK,the downstream of B cell receptor signaling.These results indicated that ADAR1 plays an important role in regulating B cell receptor signaling.In summary,for the first time,we report that ADAR1 is essential for humoral immune response,and ADAR1 p150 isoform exclusively accounts for its role in this process.Moreover,we reveal that ADAR1 plays a key role in B cell fate decisions by regulating the B cell receptor signaling during B cell activation.