Low Dose IL-2 Improves Neuroinflammation In Autism By Affecting Microglia

Objective : Autism Spectrum Disorders(ASD)is a complex neurodevelopmental disorder characterized by deficits in social communication and repetitive and stereotypical behaviors.Because the specific pathogenic factors are not clear,there are no specific therapeutic drugs.Current studies consider that genes,environment and immunity are involved in the pathogenesis of ASD,and there is increasing evidence that Teff /Treg imbalance plays an important role in ASD.Our research group previously used low-dose(30,000IU)IL-2 to selectively up-regulate Treg cells to treat ASD inbred mice--BTBR T+Itpr3tf/J(BTBR)mice,and found that low-dose IL-2 treatment could promote TGF-β secretion by correcting peripheral Teff /Treg imbalance.Down-regulation of TFH,TH17 cells and related cytokines can reduce inflammatory lesions of ASD and thus improve abnormal behaviors of autistic BTBR mice.However,whether low-dose IL-2 can regulate the activation state of brain microglia and neuroinflammation of BTBR mice has not been reported.Therefore,in this study,natural BTBR T+Itpr3tf/J mice without modeling and with good stability were used as the research object,and low-dose IL-2 was used as the intervention method to observe the effects of low-dose IL-2 treatment on the number and polarization type of microglia cells in the prefrontal cortex,striatum and hippocampus of autistic BTBR mice.Microglia cultured in vitro were treated with IL-2 and the possible mechanism of action was investigated.Methods: Male BTBR mice aged 6-8 weeks were injected subcutaneously with low dose of IL-2,and male C57BL/6J(C57)mice and BTBR mice in the control group were injected with equal volume of normal saline,with continuous injection for7 days and one rest day as a course of treatment,for a total of four courses.After treatment,behavioral changes of mice were detected by three-box social experiment,grooming experiment,bead burying experiment and open field experiment.After that,brain tissue samples were collected,and the number and morphology of microglia cells in prefrontal cortex,striatum and hippocampus of each group were detected by immunofluorescence method,and microglia cells were labeled with iba1.Transcriptome sequencing was performed on the cortex and hippocampus of BTBR mice in normal saline group and low-dose IL-2 group.Real-time quantitative PCR was used to detect the expression levels of inflammatory factors in the prefrontal cortex,striatum and hippocampus.The levels of M1-type microglia and M2-type microglia in prefrontal cortex,striatum and hippocampus were detected by flow cytometry.After the primary microglia of autistic BTBR mice were stimulated with IL-2,transcriptome sequencing was performed on the primary microglia and m RNA expression levels of inflammatory cytokines were detected.Results:1.Low dose of IL-2 can reduce the number of microglia,especially activated microglia,in the prefrontal cortex,striatum and hippocampus of autistic BTBR mice.2.Low dose of IL-2 can reduce the expression levels of pro-inflammatory cytokines IL-1β,IL-6 and TNF-α in the prefrontal cortex,striatum and hippocampus of autistic BTBR mice.The expression levels of anti-inflammatory cytokines TGF-βand arg-1 were increased.3.Low dose of IL-2 can reduce the ratio of M1-type microglia to M2-type microglia in the prefrontal cortex and hippocampus of autistic BTBR mice.4.Low-dose IL-2 treatment can reduce the expression of microglia-related genes in the prefrontal cortex and hippocampus of autistic BTBR mice.5.Using IL-2 to stimulate primary microglia of BTBR mice in vitro can change the expression of IL-2-related pathway genes and reduce IL-6 m RNA expression level.Conclusion: Low-dose IL-2 therapy can regulate the activation state of microglia,reduce brain inflammation and improve abnormal behavior of autistic BTBR mice,which is a potential treatment for ASD.Low dose of IL-2 may directly act on microglia cells and change their function,and the specific mechanism needs follow-up research.