Immune Tolerance Activates M2 Microglia To Resist Epilepsy Through Intestinal Flora

Background&objective:Microglia have both epileptic and antiepileptic effects in the epileptic brain.Inducing the transition of microglia from pro-inflammatory M1 phenotype to anti-inflammatory M2 phenotype is a therapeutic strategy to alleviate epileptic seizures.Microglia after immune training or immune tolerance can acquire the ability to differentiate into specific phenotypes.To achieve the purpose of regulating microglia and then controlling epileptic seizures.At present,the brain-intestine axis is an important bridge to explore central nervous system diseases.Intestinal microflora can affect energy extraction,inflammation and neuroendocrine regulation in other distal organs.Therefore,intentional manipulation of intestinal flora may be another strategy to treat nervous system diseases.Methods:The experimental animals were divided into three groups:control group,immune training group and immune tolerance group.Mice in the immune tolerance group were injected with LPS(4×LPS)four times for four consecutive days.In the immunization training group,LPS was injected once on the first day,and then a placebo(1×LPS)was injected three times for three consecutive days.The control group was injected with a placebo(PBS)four times for four consecutive days.Two weeks later,Kainic Acid(KA)was injected into the hippocampus to simulate seizures.The seizure rate,seizure grade and seizure duration of mice were monitored by video behavior.In vivo multi-channel recording of hippocampal field potential and energy density.Feces of epileptic mice were collected 24 hours later and 16S rDNA sequencing was performed.Serum levels of cytokines IL-1β and IL-10 were detected.The contents of Iba1,IL-10,iNOS,CD68,CD86 and Arg-1 in the hippocampus and Claudin-5 in the intestinal tract were detected by Western Blot.FJB staining was used to compare the degeneration and necrosis of hippocampal neurons after epilepsy in three groups.Results:The release of IL-1β in serum was very low,but the release of IL-10 was increased in mice treated with 4×LPS,which indicated the establishment of immune tolerance.The immune tolerance group prolonged the incubation period of acute seizures induced by KA,decreased Racine score,reduced seizure duration,and decreased the average energy density of EEG activity.By 16S rDNA sequencing,it was found that the dominant flora in the immune tolerance group was Ruminococcaceae.Ruminococcaceae tend to consume more resistant starch and dietary fiber in whole grains and produce Short-chain fatty acid(SCFA).These metabolites have anti-inflammatory and intestinal barrier functions.Subsequently,we confirmed that the protein content of intestinal tight junction protein Claudin-5 was more expressed in the immune tolerance group.Western blotting of Iba1,iNOS and Arg-1 showed that microglia were activated during an epileptic seizure.Compared with the immune tolerance and control group,iNOS and CD86,which represent the M1 phenotype,were more expressed in an immune training group.The contents of Arg-1 and CD68,which represent the M2 phenotype,were significantly higher in the immune tolerance group than those in the immune training group and control group.Based on the above results,the number of FJB positive cells in the hippocampus of the immune tolerance group was significantly lower than that of an immune training group.Therefore,immune tolerance can indeed alleviate neuronal degeneration caused by epilepsy.Conclusion:Our study found that Ruminococcus,as the dominant flora in the immune tolerant group,played an anti-inflammatory and intestinal barrier function.It can reduce systemic inflammatory reactions and promote microglia differentiation.This study demonstrated for the first time that immune training or immune tolerance induced microglia to differentiate into different phenotypes.The relationship between intestinal flora and microglia phenotype during immune status changes was evaluated and verified in the epilepsy model.We developed a method for initiating an anti-inflammatory procedure(i.e.,microglia differentiate into M2 phenotype)in the early stage of epilepsy.Because of the close relationship between the brain and intestinal axis,intestinal flora may be a promising additional therapy for epilepsy.