Role Of M2 Macrophage Exosome-derived LncRNA AK083884 In Regulating Macrophage Polarization During CVB3-induced Viral Myocarditis And Its Mechanism

Objective: This study aims to elucidate the role of M2-type macrophage exosome(M2-Exo)derived lncRNA AK083884 in regulating macrophage polarization in viral myocarditis(VM)and its molecular mechanism to provide a new theoretical basis and experimental basis for designing targeted immunotherapeutic strategies for VM based on the regulation of macrophage polarization.Methods: M2-Exo was isolated using differential centrifugation.Transmission electron microscopy(TEM),nanometer-scale tracer analyzer(NTA),and flow cytometry were used to analyze the size of the M2-Exo particles.Western blot(WB)analysis detected M2-Exo markers.The prediction of the lncRNA most abundantly expressed in M2-Exo was utilized by exosomal lncRNA sequencing combined with bioinformatic analysis and then validated by q RT-PCR.AK083884 small interfering RNA(si RNA)was designed and synthesized to observe the effect of inhibition of M2-Exo-derived AK083884 on the phenotype and function of macrophages.AAV9-AK083884 sh RNA was designed,constructed,and intramyocardial injected into CVB3-infected mice to examine the effects of AK083884 inhibition on cardiac infiltrating macrophage phenotype and function in CVB3-infected mice and its role in VM pathogenesis.AK083884 intracellular localization was determined by RNA FISH.RNA Pull-down and mass spectrometry analysis were performed to identify AK083884 interacting proteins.RIP further confirmed the specific binding of AK083884 to the interacting protein.Results: In this study,we found that M2-Exo can effectively attenuate VM,and the protective effect of M2-Exo disappears after macrophage depletion in vivo.The lncRNA AK083884 in M2-Exo was found to be involved in the regulation of macrophage polarization by exosomal lncRNA sequencing combined with in vitro functional assays.Functional assays indicated that M2-Exo-derived lncRNA AK083884 promotes M2 polarization of macrophage and protects mice from CVB3-induced VM.The study of the proximate gene SOCS2 revealed that AK083884 is involved in M2-Exo-mediated macrophage polarization by targeting the SOCS2/STAT6 signaling pathways.Furthermore,we identified PKM2 as a protein target binding to AK083884 by RNA pull-down and mass spectrometry analysis and found that inhibition of PKM2 could promote the polarization of BMDMs to M2.Intriguingly,the detection of glycolytic capacity in macrophages confirmed that the downregulation of AK083884 promoted glycolysis and attenuated oxidative phosphorylation(OXPHOS).Additionally,co-Immunoprecipitation(co-IP)was performed to reveal that AK083884 could simultaneously interact with PKM2 and HIF-1α,and inhibiting AK083884 can facilitate PKM2 and HIF-1α binding.Conclusion: Collectively,our findings uncover a critical role of M2-Exo-derived lncRNA AK083884 in regulating macrophage polarization in vitro and in vivo,identifying a new player in the development of VM and providing a potential clinically important therapeutic target.