Study On The Mechanism Of IL-38 Promoting Microglial Phagocytosis Through PI3K-RAC1 Signaling Pathwa

Background:Alzheimer’s disease(AD),characterized by extracellular amyloid deposition and intracellular neurofibrillary tangles,is a progressive neurodegenerative disease.Its clinical manifestations include cognitive decline and memory loss,accompanied by a series of psychiatric symptoms and behavioral changes.So far,the etiology of AD is not completely clear yet.There are several hypotheses being put forward,including the cholinergic theory,starch-like(amyloid)peptide hypothesis,Tau protein hypothesis and the involvement of genetic factor.Amyloid peptide theory believes that the metabolic abnormalities of brain give rise to toxic protein fragments deposition in brain and formation of amyloid plaques,which results in neuronal death eventually.Microglia is the resident immune cell and phagocytes in the central nervous system,which can clear microorganisms,cell debris and protein aggregates in the brain through phagocytosis,but the underlying mechanism remains poorly understood.Objectives:To determine a positive regulator of microglial phagocytosis,38 human interleukins were screened.Then the effects of interleukin-38(IL-38)on the phagocytosis of various cargos were investigated in rat primary microglia and the mechanism was explored.Finally,the effects of IL-38 on pathological plaques clearance and behavioral performance were further examined by overexpressing IL-38 in AD mice model APPSWE/PS1△E9 mice.Methods:1)38 human interleukin overexpression vectors were constructed and transfected into BV-2 microglia cell line individually.The effect of each vector on dextran clearance was detected and the most effective interleukin factor was determined to be IL-38.2)Rat primary astrocytes,microglia and neurons were isolated and cultured to detect the mRNA and protein expression of IL-38 and its receptors IL-1R1,IL-1RL2 and IL-1RAPL2 respectively.3)Rat primary microglia and iPSC-induced microglia were treated with human recombinant IL-38 protein for 24 hours with different concentrations(0,5 and 25 ng/ml)and for different durations(0,6,12 and 24 hours)with 25 ng/ml IL-38,and their phagocytosis of different cargos(Microspheres,Aβ,Dextran,Transferrin and Zymosan)were tested.4)The recruitment of IL-38 receptors IL-1R1,IL-1RL2 and IL-1RAPL2 on the microglia membrane were examined after treating the microglia with IL-38 protein for different durations(0,1,5,15,30 and 60 mins).The PI3K-RAC1 signalling pathway and the change upon its inhibition were detected by Western Blot.5)AAV-IL-38 and the control AAV-LUC were constructed and injected stereotactically together with Aβ oligomers into the hippocampus of C57BL/6J mice,after which the Aβ oligomer clearance was examined.AAV-IL-38 and the control AAV-LUC were injected stereotactically into the hippocampus of AD model APPSWE/PS1△E9 mice.Then the clearance of pathological plaques in the brain and the learning and memory abilities of the mice were detected.Results:1)The screen results showed that IL-38 can promote the clearance of dextran in BV2 microglia cells.2)The expression detection results showed that IL-38 is primarily expressed in neurons and its receptor IL-1RL2 receptor is mainly expressed in microglia.3)The intracellular level of phagocytic cargos detected by immunofluorescence and flow cytometry revealed that IL-38 promotes the phagocytosis of Microspheres,Aβ and dextran in rat primary microglia,and promotes the phagocytosis of Zymosan in iPSC-induced microglia cells.This effect shows concentrationdependent and time-dependent manner.4)The results of IL-38 receptors recruitment indicated that IL-1RL2 accumulates on the microglia membrane after 15-minute IL-38 treatment,and its recruitment disappears after 60-minute treatment.IL-38 activates the PI3K-RAC1 pathway,promotes the phosphorylation of PI3K,AKT and VAV,and increases the level of RAC1-GTP.After inhibiting PI3K activity,microglia no longer respond to IL-38 treatment,with unchanged levels of phosphorylation PI3K,AKT and VAV.Meanwhile,microglia no longer promote the phagocytosis of Microspheres.5)Compared with the control AAV-LUC injection group,Aβ oligomers in the hippocampus of C57BL/6J mice were decreased after AAV-IL-38 injection.After AAV-IL-38 injection into the hippocampus of APPSWE/PS1△E9 mice,the plaque area in the hippocampus is reduced while there is no significant change in the cortical area.6)After AAV-IL-38 injection,APPSWE/PS1△E9 mice show normal trajectories in the open field test.And they performed better in the water maze experiment.To be specific,they take less time to find the platform and cross the platform with much more times.Conclusions:IL-38 promotes phagocytosis of microglia through PI3K-RAC1 pathway,facilitates the clearance of pathological plaques in the brain of APPSWE/PS1△E9 mice,and improves their learning and memory abilities,suggesting a potential therapeutic role of IL38 in Alzheimer’s disease.