NOD2 Regulates Microglial Inflammation Through The TAK1-NF-?B Pathway And Autophagy Activation In Murine Pneumococcal Meningitis

Background and ObjectivePneumococcal meningitis is a common infection of the central nervous system(CNS)in children,with a high mortality rate of 12-30%in developing country.Although antibiotic therapy has reduced the mortality of pneumococcal meningitis,about 30%of surviving infants still suffer from permanent neurological sequelae,including mental retardation,hearing impairment and seizures.A more complete understanding of the mechanisms underlying the induction of pneumococcal meningitis may have a significant effect on development of new therapeutics.Streptococcus pneumonia is a Gram-positive,extracellular bacterium that is responsible for high mortality and morbidity worldwide.While immune cells likely contribute to the inflammatory responses within the CNS,it has become increasingly apparent that resident microglia play an important role in the inflammation following,pathogen invasion.To accomplish this role,microglia constitutively express nucleotide-binding oligomerization domain-2(NOD2),a member of the NOD-like receptor(NLR)family,that functions as an intracellular receptor for a conserved component of bacterial peptidoglycan(PGN).Upon stimulation by bacteria,NOD2 oligomerizes and recruits the receptor-interacting protein 2 kinase(RIP2/RICK/CARDIAK).Activation of RIP2 stimulates protein kinase cascades,resulting in the activation of NF-?B and the mitogen-activated protein kinases(MAPKs)p38.The dual activation of both NF-?B and MAPK signaling by NOD2 is responsible for the rapid induction of pro-inflammatory responses during pathogen infection.As a serine/threonine kinase,transforming growth factor activated kinase-1(TAK1)is a critical regulator of the NF-?B and MAPK pathways.Studies have shown that TAK1 is required for NOD2-induced NF-?B signaling in mouse embryonic fibroblasts(MEFs).However,our understanding of the accurate molecular mechanisms of NOD2-mediated NF-?activation in pneumococcal meningitis remains limited.Recent studies have shown that autophagy plays a vital role in inflammation,immunity,cancer,and cell death in mammals.Autophagy is an evolutionarily conserved degradation process by which cells digest damaged,aggregated,or aged ingredients.Emerging evidence suggests that autophagy can be induced by several pathogens,such as Shigella flexneri,Listeria monocytogenes and Group A streptococci(GAS)and serves as a potent host defense mechanism against intracellular bacteria.Cargo is engulfed into a double-membrane vesicle known as the autophagosome,which is then fused with lysosomes,ultimately resulting in degradation.Autophagy begins with the interactions between multiple autophagy proteins.ATG5,ATG12 and microtubule-associated protein light chain 3(LC3)are critical for the formation of initial autophagic vacuoles and the maturation of autophagosomes.A critical role for NOD2 in the regulation of autophagy in intestinal epithelial cells(IECs)has been identified,which is associated with the pathogenesis of intestinal bowel diseases(IBD).Recent findings demonstrated a requirement for RIP2 tyrosine kinase activity in NOD2-mediated autophagy.However,the impact of NOD2 interactions with autophagy on S.pneumonia-driven inflammation during pneumococcal meningitis has not been elucidatedOur study aims to explore the role and intrinsic mechanism of NOD2 in pneumococcal meningitis.The first part of the study analyzed the effect of NOD2 on microglial inflammation and autophagy during pneumococcal infection,as well as the role of TAK1-NF-?B signaling pathway in the process.In the second part,the role of NOD2 in pneumococcal meningitis was further verified by detecting neurological functional scores,brain edema,pathological changes,expression of inflammatory mediators and autophagy proteins of pneumococcal meningitis mice.Methods and Results1.Part 1(experiment in vitro):The study of the effect of NOD2 on microglial inflammation and autophagy during pneumococcal infection,as well as the role of TAK1-NF-?B signaling pathway.The main experimental techniques of this study:Cell culture and treatment,Lentiviral transfection,ELISA,RNA extraction and reverse transcription,Real-time PCR,Western blot.Immunofluorescence,et al.(1)Microglial inflammation and expression of NOD2 during pneumococcal infection:BV-2 microglial cells were infected with various MOI(0,25,50,100)of S.pneumonia for different time points(0 h,4 h,8 h,24 h).The levels of inflammatory mediators TNF-?,IL-6,IL-10 and IL-18 in the culture supernatant were determined by ELISA,and LDH was measured by the LDH Cytotoxicity Detection Kit.The results demonstrated that the production of TNF-?,IL-6,IL-10,IL-18 and LDH were increased significantly in a time-and concentration-dependent manner.A significant increase of TNF-?,IL-6,IL-10,IL-18 and LDH was observed at 4 h,8 h or 24 h following infection with S.pneumonia(MOI=50).Furthermore,significant levels of inflammatory cytokines were released into the culture supernatant upon various S.pneumonia MOI of 25,50 or 100 in a concentration-dependent manner.Meanwhile,we detected NOD2 levels of microglia after S.pneumonia infection.The results demonstrated that S.pneumonia induced the expression of NOD2 in aconcentration-and time-dependent manner.These findings suggest that NOD2 is involved in microglial inflammation induced by S.pneumonia.(2)Analysis of NOD2 silencing efficiency after lentiviral transfection with shRNA:Lentiviral transfection with NOD2-shRNA was used to knockout NOD2.GFP in BV-2 microglia was observed by fluorescence microscopy to analyse the transfection efficiency.After lentiviral transfection with NOD2-shRNA for 72 h,the transfection efficiency was(75.241±2.46)%·The silencing efficiency of NOD2 was detected by real-time PCR.After lentiviral transfection with NOD2-shRNA,the expression of NOD2 mRNA in BV-2 microglia was significantly decreased compared with that in the control group.(3)The effect of NOD2 on microglial inflammation and the role of TAK1-NF-?B signaling pathway during pneumococcal infection:To investigate the contribution of NOD2 to BV-2 microglial activation induced by S.pneumonia,we performed lentiviral transfection of shRNA to knockdown NOD2.The results showed that the expression of TNF-?,IL-6,IL-10,IL-18 and LDH increased significantly after S.pneumonia infection,as well as expression of p-TAKI and NF-?B p65.Knockdown of NOD2 resulted in a significant decrease in inflammatory mediators as well as expression of p-TAK1 and NF-?B p65 in S.pneumonia-infected microglial cells.To further assess the role of the TAK1-NF-?B pathway in NOD2-mediated microglial activation,5Z-7-oxozeaenol,an inhibitor of TAK1,was utilized to reduce the generation of p-TAK1,We determined the expression of inflammatory mediators as well as NOD2,p-TAK1,TAK1 and NF-?B p65 after treatment with NOD2 shRNA and 5Z-7-oxozeaenol.The results demonstrated that the expression of p-TAK1 and NF-?B p65 was significantly decreased when NOD2 shRNA and 5Z-7-oxozeaenol were applied alone or in combination during S.pneumonia infection,as well as decreased production of inflammatory mediators.Taken together,these results suggest that NOD2 mediates microglial activation by activating the TAK1-NF-?B pathway during S.pneumonia infection.(4)The regulation of NOD2 to microglial autophagy during pneumococcal infection:To examine whether autophagy is stimulated by S.pneumonia and the role of NOD2 in the process,the expression of autophagy-related proteins was examined by Western blot.S.pneumonia induced a gradual increase of LC3 ?,Beclin1 and Atg5,and the expression of autophagy-related proteins decreased after NOD2 silencing by lentivirus transfection of shRNA.To further confirm that NOD2 promoted microglial autophagy during S.pneumonia infection,the autophagosome-like dots were visualized by staining with anti-LC3 antibody.Upon the initiation of autophagy,cytosolic LC3 ? is converted to LC3 ?,which is linked to autophagosomal membranes.Infection of BV-2 microglia with S.pneumonia for 8 h led to the redistribution of LC3 from a diffuse pattern to the typical punctate pattern.In addition,treatment with NOD2 shRNA reduced LC3 autophagosome formation.Taken together,these results indicate that NOD2 promotes microglial autophagy during S.pneumonia infection.(5)The effect of autophagy to microglial inflammation during pneumococcal infection:To understand the role of autophagy in microglial inflammation,BV-2 microglial cells were treated with an autophagy inducer(rapamycin,Rapa)or inhibitor(3-methyladenine,3-MA).The results showed that the production of TNF-?,IL-6,IL-10,IL-18 and LDH was significantly elevated by S.pneumonia infection,whereas activation of the autophagy pathway by rapamycin suppressed the levels of inflammatory mediators.Furthermore,the levels of S,pneumonia-induced inflammatory mediators were increased when autophagy was inhibited by 3-MA.These results suggest that the activation of autophagy mitigates the inflammatory cellular responses of S.pneumonia-stimulated microglia.2.Part 2(animal experiment):The effect of NOD2 on inflammation and autophagy in brain tissue of pneumococcal meningitis mice.Forty-eight male C57BL/6 mice(6-8 weeks,20-24g)were randomly divided into 4 groups(control group,S.pneumonia group,NOD2 shRNA group,and NOD2 shRNA+S.pneumonia group).To investigate whether NOD2 mediates inflammation and autophagy in the mouse brain during pneumococcal meningitis,C57BL/6 mice were lentivirally transfected with shRNA via intracerebral ventricular injection to knockdown NOD2,and the silencing efficiency of NOD2 was detected by real-time PCR 14 days later.Pneumococcal meningitis was induced by intracerebral injection of a bacterial suspension containing 1.0×107 CFU/ml of S.pneumonia into the lateral ventricle.The neurological function of mice was evaluated at 8 h and 24 h after bacterial infection.The brain edema index was calculated using the following formula:(wet weight-dry weight)/wet weight × 100%.The levels of inflammatory mediators TNF-?,IL-6,IL-10 and IL-18 in brain were determined by ELISA,and LDH was measured by the LDH Cytotoxicity Detection Kit.HE staining was used to observe the meningeal pathological changes.To investigate the relationship between NOD2 and autophagy in pneumococcal meningitis mice,we detected the expression of NOD2 and autophagy-related proteins LC3?,Atg5 and Beclinl in brain by Western blot.(1)Analysis of NOD2 silencing efficiency after lentiviral transfection with shRNA:Lentiviral transfection with NOD2-shRNA to lateral ventricle was used to knockout NOD2.The silencing efficiency of NOD2 was detected by real-time PCR.The expression of NOD2 mRNA in brain was significantly decreased compared with that in the control group.(2)Establishment of the mouse model of pneumococcal meningitis:After infection of S.pneumonia for 8h,the cerebrospinal fluid of mice was retained for bacterial culture.The same strain of S.pneumonia with a high bacterial concentration was cultured,while no bacteria found in the mice of control group,which confirmed the establishment of mouse model of pneumococcal meningitis.(3)Clinical manifestation and neurological functional test:Neurological disorders occurred to various degrees in mice after infection with S.pneumonia for 8h and 24h,including weariness,reduced feeding and spontaneous activity,drowsiness,paralysis,ataxia,seizures and even death,while the control group showed no obvious neurological disorders.The Loeffler scoring method was used to evaluate the neurological function of mice.The results showed that the scores decreased significantly at 8 h and 24 h after S.pneumonia infection compared with the control group.The scores were significantly increased with administration of NOD2 shRNA compared with the S.pneumonia group at 24 h.(4)Detection of brain edema index:The index of brain edema was enhanced in the S.pneumonia group at 24 h after injection compared with that in the control group and was decreased by NOD2 silencing.(5)Detection of inflammatory mediators in brain:Expression of the inflammatory mediators TNF-?,IL-6,IL-10,IL-18 and LDH was elevated in the brain of S.pneumonia-infected mice.However,NOD2-shRNA treatment reduced the level of cytokines.(6)HE staining of brain tissue:The results demonstrated that neutrophil infiltration was increased after S.pneumonia infection,along with tissue hyperemia,edema and vascular destruction,but knockdown of NOD2 attenuated the inflammatory response.(7)Expression of autophagy-related proteins in brain:The expression of LC3?,Beclinl and Atg5 in the brain was significantly increased after S.pneumonia infection,but decreased after NOD2-shRNA treatment.Conclusion1.NOD2 promotes microglial activation to release inflammatory mediators through the TAK1-NF-?B signaling pathway during S.pneumonia infection.2.Autophagy can be induced by S.pneumonia infection,and NOD2 mediates the activation of autophagy.Autophagy contributes to the clearance of inflammatory mediators.3.In the mouse model of pneumococcal meningitis,NOD2 mediates the inflammation of brain,causing neurological disorders,brain edema,meningeal inflammatory infiltration,etc.Meanwhile,NOD2 promotes the activation of autophagy in brain.