Toll-like Receptor 2-mediated Autophagy Regulates Microglia Cell Survival By Modulating The M1/M2 Phenotype

Backgrounds and ObjectiveAutophagy is still a hot research topic at present.Autophagy is an extremely conservative physiological process in cells that depends on lysosomes and is an effective mechanism for recycling intracellular substances.Autophagy is a multi-step dynamic physiological process,including five parts:induction,extension,encapsulation,maturation and degradation.Autophagy can maintain intracellular stability by degrading intracellular dysfunction or unnecessary cytoplasmic components(such as endoplasmic reticulum,mitochondria,etc.)to promote cell survival in unfavorable environments(such as hunger,hypoxia,infection).Moderate autophagy can protect cells in adverse environment,but excessive autophagy will cause cell death,which is called autophagic death.Studies have found that a large number of autophagy lysosomes can be found in dead cells,which wrap cytoplasm and organelles.Most substances in cytoplasm are degraded,but the nucleus remains intact.Studies have found that many important proteins or cytokines have regulatory effects on autophagy.For example,P53 upregulates apoptosis regulator(PUMA),which belongs to an important member of BH3 protein family,plays an important role in inducing cell apoptosis.Studies have found that the loss of PUMA significantly reduces the number of microglial cells.Experiments have found that PUMA can inhibit the autophagy of microglial cells and inhibit the activation of ERK,thus reducing the apoptosis of microglial cells.Meanwhile,experiments have found that the increased expression of PUMA can also enhance the autophagy of microglial cells.At present,many researches have found that signal pathway can regulate autophagy.TOLL-like receptor pathway and NOD pathway have been studied extensively,especially TLR2 and TLR4 pathway,in which TLR2 is expressed on glial cell membrane,belonging to Toll-like family and can activate microglia.Studies have found that peptidoglycan(PGN),as a ligand of TLR2,can stimulate microglia activation and induce cell death by activating autophagy.The study found that PGN treatment significantly increased BV-2 microglial cell death.At the same time,the experiment found that the concentration change of pro-inflammatory factors such as TNF-a and Fas ligand has little relation with cell death,but 3-MA can inhibit cell death,proving that apoptosis is not necrotic death but autophagic death.In addition,there are experiments to establish TLR2 deficient mice model.The expression of LC3Ⅱ in mice treated with PGN did not increase and could not induce apoptosis of brain cells in these mice.The results show that PGN activates microglial autophagy and promotes cell death through TLR2 pathway.Studies have proved that autophagy is closely related to inflammatory reaction or immune regulation.It can regulate excessive inflammatory action or immune reaction,thus preventing autoimmune diseases and inflammatory diseases.Microglia are important neuroimmune cells,which play an important role in innate immune response.Activated microglia can release various cytokines and neurotrophic factors,induce tissue repair,and play a neuroprotective role.Overactivated microglia induce inflammatory reaction and release a large number of cytokines and neuroinflammatory factors such as NO,IL-6,IL-1B,TNF-a,IL-10,Arg-1,etc.The accumulation of inflammatory factors leads to neurotoxicity of the central nervous system due to nitrogen and oxygen imbalance.Studies have found that activated microglia express a variety of functional states,mainly including pro-inflammatory Ml and anti-inflammatory M2 states.In M1 state,the cell surface mostly expresses CD16/32,CD86 and CD40 markers,in M2 state,the cell surface expresses marker CD206,and microglia release different inflammatory chemical factors under different phenotypes.Much research has been done on the regulation of autophagy by receptor pathways,but little has been done in-depth research,especially on the role of autophagy in regulating the phenotypic transformation of microglia.Based on the role of TLR2 in identifying microbial infection and activating inflammation,this subject studies the relationship between autophagy and TLR2 pathway in microglial activation.In the experiment,TLR2 agonists and antagonists were used to explore the relationship between TLR2 and microglial autophagy,to study the effect of autophagy on microglial M1/M2 phenotype.The experiment further observed the effect of different phenotypic changes on cells and explored new strategies for brain cell protection.Some studies have found that autophagy-induced drugs may be beneficial in clinical treatment of nervous system infections in adult.Whether such drugs benefit children with neurological infections has not been studied.This study is based on the effect of regulating autophagy level on cell survival under the condition of intracranial infection in mice.To some extent,it can provide theoretical support for this conjecture and provide a prospect for the treatment of streptococcal meningitis in children.Research content1.Study on the relationship between activation of microglia TLR2 pathway and autophagy.2.TLR2-mediated autophagy on microglial M1/M2 phenotypic transformation and its effect on microglial cells3.Study on the survival effect of autophagy on rat nerve cells under intracranial infection.Research methodsIn vitro1.Microglia cells were co-cultured with different concentrations of PGN,and the expression levels of autophagy-related proteins LC3I/II and Beclin-1 were detected by western blot at different time points.The experimental concentration and detection time points of PGN were screened.2.The interference sequence of TLR2-siRNA was screened to knock out genes,and the expression level of TLR2 receptor protein after treatment was evaluated by western blot.3.TLR2-siRNA treatment group and PGN stimulation group were established,and the expression levels of autophagy-related proteins LC3Ⅰ/Ⅱ and Beclin-1 after TLR2-KO were detected by western blot.4.Immunofluorescence was used to observe the autophagic protein LC3B in TLR2-siRNA treatment group and PGN treatment group.Confocal microscope was used to observe and evaluate the expression level of autophagic protein,which proved the relationship between TLR2 pathway and autophagy.5.The control group,experimental group,Pam3CSK4 treatment group and CU-CPT22 treatment group were set up.The expression levels of autophagy-related proteins LC3Ⅰ/Ⅱ and Beclin-1 were determined by western blot,and the effect of TLR2 receptor modulator on autophagy was observed.6.Immunofluorescence was used to observe LC3B and LAMP-1 proteins in each group.Autophagy-related proteins were observed under confocal microscope.The number of Autophagous plaque was observed and calculated.7.ELISA was used to detect the expression levels of M1-related factors TNF-a,IL-6 and specific marker CD86.The expression levels of M2-related factors IL-10,Arg-1 and specific marker D206 were also detected.To evaluate the effect of autophagy intensity on cell phenotype transformation.8.Apoptosis kit Annexin V-PE/7AAD was used to label microglial cells,and flow cytometry was used to detect the level of apoptosis.In vivo1.TLR2-KO mouse model was established and TLR2 gene was knocked out by TLR2-lentivirus transfection.2.Control group,experimental group,TLR2-KO group and 3-MA treatment group were set up.Paraffin sections were made from brain.The autophagy number of microglial cells was observed and counted according to CDllb and LC3B immune markers.3.Nissl staining was used to observe and count neuronal apoptosis.4.Detection of water content in brain tissue of mouse brain tissue infection model.5.Neural function score was performed in the mouse brain tissue infection model.ResultsPart 1The relationship between activation of TLR2 pathway in microglia and autophagy1.1 Determination of optimal PGN concentration for infecting microglia and time point for observing autophagy level in microgliaWe tested three PGN concentrations(5,15,and 30 μg/ml)to determine the optimal PGN concentration and time point of autophagy detection.Autophagy-associated proteins extracted from infected BV2 cells in vitro were analyzed using western blotting at 12 h and 24 h culture time points.Autophagy was significantly increased at the 24 h time point compared to the 12 h time point.In addition,15 μg/ml PGN concentration gave rise to highest autophagy expression and showed comparable autophagy level to that of 30 μg/ml PGN concentration.Thus,15μg/ml PGN concentration and detection at the 24 h time point were selected for use in subsequent experiments.1.2 Selection of siRNA sequence to produce TLR2-KOThe siRNA sequence for TLR2-KO was selected from three different siRNA sequences which were each transfected into BV2 cells.TLR2 protein level in BV2 cells after siRNA treatment was assessed using western blotting after 48 h of culture with GADPH as the loading control.The third siRNA sequence(si-TLR2-3)was selected for subsequent experiments as it showed the highest TLR2 inhibition with an approximate 4-fold lower TLR2 expression.1.3 TLR2-KO inhibit autophagy expression of microglia under PGN infectionTo study the relationship between autophagy level and the TLR2 pathway,the siRNA-TLR2-KO group and a control group were established.Western blotting was performed to detect the expression of autophagy-associated proteins LC3I/II and Beclin-1 at the 24 h culture time point and immunofluorescence enabled observation of GFP-LC3 autophagic plaques.Higher autophagy(increased number of autophagic plaques)was observed in the PGN group but not in the TLR2-KO group compared to the control group.This indicates that PGN enhances autophagy by activating the TLR2 pathway.Part 2Studies on the phenotypic transformation and effects of TLR2-mediated autophagy on microglia M1/M22.1 TLR2 agonists/antagonists regulate autophagy expression in vitroTLR2 regulator Pam3CSK4 and cu-cpt22 were added,Western blotting was performed to detect the expression of autophagy-associated proteins LC3Ⅰ/Ⅱ and Beclin-1 at the 24 h culture time point.The results showed that Pam3CSK4 enhanced the expression of TLR2 receptor and the expression of autophagy related proteins LC3Ⅰ/Ⅱ and beclin-1.Through LC3B(green)/LAMP-1(red)immunofluorescence experiment,increased number of autophagosomes was observed in the PGN and PGN+Pam3CSK4 groups.2.2 TLR2-mediated autophagy regulates the M1/M2 phenotype transformation of microgliaWe determined the effect of autophagy on microglia phenotype transition and cell survival status.The expression levels of M1-specific marker CD86 and M1-associated TNF-α and IL-6 cytokines,and the M2-specific marker CD206 and M2-associated IL-10 and Arg-1 cytokines,were assessed using ELISA at the 24 h culture time point.we found increased autophagy and pro-inflammatory marker expression with obvious transition to the M1 subtype and inhibition of M2 signatures in the PGN+Pam3CSK4 group.The reverse was observed in the PGN+CU-CPT22 group wherein microglia switched to the M2 phenotype with concomitant decrease in M1-related protein expression.2.3 Autophagy regulates microglial phenotype transformation and affects cell survival,and M1 promotes apoptosisTo observe the effect of phenotypic transformation on cell survival.Flow cytometry showed that autophagy was enhanced with increased apoptosis and the extent of apoptosis was consistent with the switching to the pro-inflammation M1 subtype.Part 3Study on the effect of microglia autophagy on the survival of nerve cells in mice under intracranial infection3.1 The effects of TLR2-KO and autophagy inhibitors on autophagy expression in vivoMouse TLR2 receptor was knocked out by lentivirus transfection,and the experiment was designed into 4 groups.All the experimental groups were injected with drugs(PGN,PGN+3MA,placebo)by lateral ventricle localization injection.The number of autophagosomes in the microglia of the PGN group was the highest and autophagy was significantly reduced in the TLR2-KO and 3-MA groups.3.2 The effects of autophagy on nerve cell injuryThe survival of nerve cells was observed by nissl staining.Damaged or apoptotic microglia was most significant in the PGN group unlike in the TLR2-KO and 3-MA groups which showed more intense Nissl staining,indicating that TLR2 inhibition or autophagy inhibition confers cytoprotective effects to microglia.3.3 The water content of brain tissue in mouse model of brain tissue infectionIt was found that there was no significant difference in water content between TLR2-KO and autophagy inhibitor groups,In PGN group,the water content of brain tissue increased significantly,Therefore TLR2-KO and autophagy inhibitor groups can effectively reduce the water content of brain tissue.3.4 The detection of nerve function in the mouse model of brain tissue infection.Studies have shown that there was no significant difference in neurological function between the tlr2-ko and autophagy inhibitor groups,while in the PGN group,the neurological function of mice was impaired,and both tlr2-ko and autophagy inhibitor could effectively protect the neurological function of mice.Conclussion1.PGN can activate autophagy in microglia in vitro or in vivo.2.Activation of the TLR2 pathway can activate autophagy,and the level of autophagy is inhibited after interference with TLR2 gene expression3.Autophagy can regulate the phenotypic transformation of microglia,Induction of autophagy can promote the transformation of microglia to M1 type,and increase cell death,but Inhibition of autophagy promotes the transformation of microglia to M2 type and reduce cell death.4.Inhibition of autophagy or knockout of TLR2 gene can reduce the damage of nerve cells,facilitate to the survival of neuronal cells in vitro and in vivo,but Induction of autophagy can promote cell death.