| BackgroundBacterial meningitis is a common and serious infectious disease of the central nervous system worldwide.Streptococcus pneumoniae is the most common pathogen of bacterial meningitis in children.With the popularization of vaccines and application of effective antibiotics,the incidence rate of pneumococcal meningitis has decreased significantly.Nevertheless,the neurological sequelae are still common and the mortality is still as high as 15%-51%worldwide.Meanwhile,about half of the survivors suffered from various long-term neurological diseases,including movement disorders,seizures,mental retardation,hearing impairment and cognitive decline.Therefore,in addition to effective anti-infection treatment,in-depth understanding of the pathogenesis of pneumococcal meningitis may promote the exploring of new treatments.CD93(cluster of differentiation)is a type Ⅰ transmembrane glycoprotein composed of 652 amino acids.It belongs to the C-type lectin XIV family and can be expressed on a variety of cell surfaces.Previous studies have identified that CD93 plays an important role in the pathology of systemic lupus erythematosus,rheumatoid arthritis,cancer,cerebral ischemia-reperfusion,coronary artery disease,etc.Under the stimulation of inflammation,CD93 can release from the cell surface and form sCD93(soluble CD93),suggesting the important role of CD93 in immune regulation and inflammatory reaction.Meanwhile,CD93 is up-regulated in mouse models of stroke and autoimmune cerebrospinal meningitis,suggesting that CD93 have a neuromodulation effect.CD93 was also called complement C1q receptor,which plays an important role in complement activation and innate immunity,and participates in the interaction between the immune system and microorganisms.Complement C1q related regulatory proteins are crucial in the regulation of the immune system,in which CD93 may be one of the important regulatory proteins.Meanwhile,the intracellular segment of CD93 may interact with the PDZ domain of GIPC(GAIP(G alpha interacting protein)-interacting protein COOH)protein and regulate cytoskeleton remodeling,which maybe a signal pathway of CD93 mediated inflammatory response.In bacterial meningitis,neurotoxicity induced by systemic inflammatory response,microglia activation and direct toxicity of bacterial compounds can lead to long-term sequelae or even death.Microglia are immune sentinels of the brain and play a significant role as a main important macrophage of the central nervous system.Besides,there are also meningeal macrophages,perivascular macrophages and recruited monocytes in the central nervous system,which also play important roles.After activation,microglia can differentiate into M1 or M2 phenotypes.The activated M1 phenotype can promote the release of inflammatory factors,which leads to the increase of vascular endothelial permeability,releasing a large number of reactive oxygen species and nitrogen substances,which leads to the further destruction of the blood-brain barrier,and eventually leads to respiratory burst and neuronal death.Activated M2 phenotype microglia is characterized by releasing of anti-inflammatory factors and neurotrophic factors,which plays a neuroprotective role by promoting neuronal recovery and remodeling.Therefore,microglia play a dual role in the development of disease.Regulating the differentiation of microglia into M2 phenotype may enhance neuroprotection,but the mechanism is not completely clear.Integrin family is expressed on the surface of all types of cells and plays an important role in mediating cell adhesion and migration in the inflammatory process.Many different types of integrin are expressed on the surface of microglia.Integrin β1 can mediate signal pathways,which plays an important role in cell proliferation,migration,differentiation and other biological reaction process and pathogenic process.Integrin β1 plays a key role in monocyte/macrophage recruitment and migration to inflammatory sites,and previous studies have showed that CD93 can modulate integrin β1 signaling.Currently,the mechanism of CD93 in infection and inflammatory responses is unclear,and integrin β1 may play an important role in its signaling pathway.Therefore,this study first detected the level of sCD93 in children with bacterial meningitis,and further explored the expression of CD93 and its related proteins and their effects on microglia differentiation using rat S.pneumoniae meningitis model and HAPI rat microglia.Meanwhile,we tried to explore its related mechanism through Clq and integrin β1,which made us deeply understand the inflammatory reaction of S.pneumoniae,and provide a new clinical view for the diagnosis and treatment of pneumococcal meningitis.Objective1.To investigate the level of sCD93 in cerebrospinal fluid of children with bacterial meningitis.2.Specify the expression and effect of CD93,C1q,GIPC and integrin β1 and other related proteins in the rat model of pneumococcal meningitis.3.Further explore the effect of CD93 and integrin β1 on regulation of microglia differentiation in S.pneumoniae infection.MethodPart I:Expression of sCD93 in cerebrospinal fluid of children with bacterial meningitis1.Clinical data analysisThe clinical data of 32 children in the Department of Pediatrics of Qilu Hospital of Shandong University from July 1,2018 to October 31,2022 were collected and analyzed.Among them,22 children were set as the observation group(8 cases of acute bacterial meningitis and 14 cases of viral encephalitis),and 10 children with febrile convulsion were set as the control group.We analyzed the children’s sex,age,inpatient days,fever days,convulsions,use of antiepileptic drugs,cerebrospinal fluid examination results,peripheral blood examination results,brain imaging,etc.2.Detection of the level of sCD93 in cerebrospinal fluid in different groupsCollect cerebrospinal fluid samples of each group,and detect the level of sCD93 in cerebrospinal fluid of different groups by ELISA.Part II:Investigation of the mechanism of CD93 in rat pneumococcal meningitis1.Establishment and grouping of rat pneumococcal meningitis modelMale SD rats were injected with S.pneumoniae(1.5×108cfu/ml)into the cerebellomedullary cistern to establish the rat S.pneumoniae meningitis model.Rats were divided into control group,sham operation group,normal saline group and S.pneumoniae group(2h,6h,12h and 24h after injection).Neurobehavioral assessment was performed before the rats were killed.HE staining was used to observe brain tissue damage.2.Detection of sCD93,IL(Interleukin,IL)-6,TNF(tumor necrosis factor,TNF)-α,iNOS(inducible Nitric Oxide Synthase,iNOS),CD93,Clq and GIPC levels in different groupsCerebrospinal fluid was extracted and sCD93,IL-6 and TNF-α level were detected by ELISA.Brain tissue was extracted,and the levels of iNOS,CD93,C1q and GIPC in brain tissue were detected by Western blotting.3.Detection of the interaction between CD93 with Clq and GIPC by immunoprecipitationThe rat brain tissue was extracted,and the immunoprecipitation complex was obtained by reacted with anti-CD93 antibody.The levels of CD93,C1q and GIPC in the immune complex were detected by Western blotting.4.Detection of the interaction between CD93 and GIPC by immunofluorescence co-localizationExtract rat brain tissue,make brain tissue sections,add anti-CD93 antibody,anti-GIPC antibody and fluorescent labeled secondary antibody,and observe the expression of CD93 and GIPC in cerebral cortex by fluorescence microscope.5.Detection the phenotype of inflammatory cell by flow cytometryMononuclear cells of rat brain were extracted.M1(CD11b+/CD45-/CD86+,CD11b+/CD45+/CD86+)and M2(CD11b+/CD45-/CD206+,CD11b+/CD45+/CD206+)phenotype of microglial cells and other macrophages in central nervous system were detected by flow cytometry.Meanwhile,the cerebral cortex was extracted and the cytokines IL-6,TNF-α,IL-10,TGF-β level were detected by ELISA.Integrin β1 level of brain tissue was detected by Western blotting.Part III:Effect of CD93 on microglia differentiation in S.pneumoniae infection1.Construction the model of S.pneumoniae infecting HAPI rat microglia in vitroHAPI rat microglia were infected in vitro with S.pneumoniae in different concentrations(MOI=25/50/100)and until different time points(1h,2h,4h,8h and 24h).LPS was set as positive control group,and the level of sCD93 in cell culture medium was detected by ELISA to determine the optimal concentration and time.The rate of infected cells was determined by Wright staining.Collect cell precipitation,lyse and dilute,then inoculate and cultivate,and calculate the number of living bacteria.The cell survival rate was detected by Calcein AM/PI fluorescence staining and the level of C1q in microglia was detected by Western blotting.2.Changes of differentiation phenotype and inflammatory factors of microglia infected by S.pneumoniae under the intervention of Clq and PDBu(Phorbol 12,13-Dibutyrate,PDBu)HAPI microglia were infected with S.pneumoniae.The cell culture medium sCD93,IL-6,TNF-α,IL-10 and TGF-β were detected by ELISA;NO level was detected by biochemical test kit;integrin β1 was detected by Western blot;and phenotype of microglia was detected by Flow cytometry.All the levels above were compared between control group,C1q group,PDBu group and C1q+PDBu group.3.Changes of differentiation phenotype and inflammatory factors of microglia infectedby S.pneumoniae under the down-regulation of CD93 expression by siRNA Interfere the expression of CD93 in microglia by three types of siCD93,verify the bestdown-regulated siCD93 by PCR and Western blot for subsequent experiments.Infect HAPI microglia with S.pneumoniae.The cell culture medium IL-6,TNF-α,IL-10 and TGF-β were detected by ELISA;integrin β1 was detected by Western blot;and phenotype of microglia was detected by flow cytometry.All the levels above were compared between control group,C1q group,siCD93 group,siNS group,and siCD93+C1q group.HAPI microglia treated with siCD93 was coated by recombinant CD93(1 μg/ml,2.5 μg/ml,5 μg/ml,10 μg/ml,20 μg/ml),which was infected by S.pneumoniae to the target time.The M1 and M2 differentiation phenotype of microglia was detected by flow cytometry.4.Expression level of CD93 and changes of differentiation phenotype,inflammatoryfactors and cell migration ability of microglia infected by S.pneumoniae under the down-regulation of integrin β1 by siRNA Interfere the expression of integrin β1 in microglia by three types of siIntegrin β1,verifythe best down-regulated siIntegrin β1 by PCR and Western blot for subsequent experiments.Infect microglia with S.pneumoniae.The cell culture medium IL-6,TNF-α,IL-10 and TGFβ were detected by ELISA;level of CD93 was detected by Western blot;phenotype of microglia was detected by flow cytometry;and the ability of cell migration was detected by cell migration test.All the levels above were compared between control group,C1q group.PDBu group,siNS group,siIntegrin β1 group,siIntegrin β1+C1q group,siIntegrin β1+PDBu group and siIntegrin β1+C1q+PDBu group.HAPI microglia treated with siIntegrinβ1 was coated by recombinant CD93(1 μg/ml,2.5 μg/ml,5 μg/ml,10 μg/ml,20μg/ml),which were infected by S.pneumoniae to the target time.The M1 and M2 differentiation phenotype of microglia was detected by flow cytometry.Statistical analysisThe datas were expressed as means±SD,and the enumeration datas were expressed as percentage.We used one-way analysis of variance(ANOVA)followed by the LSD and Tamhane’s T2 tests,and independent-sample t-test,χ2 test and rank sum test to analyze the differences between groups.Statistical analysis was performed using the SPSS 28.0 software.The p value less than 0.05 was considered statistically significant.ResultsPart Ⅰ:1.General data and clinical analysisThere were 8 children with acute bacterial meningitis,including 5 males and 3 females,aged from 2 months to 6 years and 8 months,3 cases of cerebrospinal fluid culture were positive,7 cases of cerebrospinal fluid pneumococcal antigen were positive,S.pneumoniae was detected in 5 cases of cerebrospinal fluid with mNGS(metagenomic Next-Generation Sequencing,mNGS),and Haemophilus influenzae was detected in 1 case of cerebrospinal fluid with mNGS.There were 14 children with viral encephalitis,including 6 males and 8 females,aged from 7 months and 22 days to 13 years and 4 months,Enterovirus RNA was detected in 2 cases of cerebrospinal fluid,and EB virus DNA was detected in 6 cases of cerebrospinal fluid.There are 10 children with febrile convulsion,including 4 males and 6 females,aged from 1 year and 5 months to 5 years and 6 months.The mean hospital stay in the acute bacterial meningitis group was 25.88±8.87 days,and the days of fever was 17.63±21.78 days,which was significantly higher than that in the febrile convulsion group and viral encephalitis group(p<0.01).87.5%of the children in the acute bacterial meningitis group had abnormal brain MRI or CT,which was significantly higher than that in the febrile convulsion group and viral encephalitis group(p<0.01),and compared with viral encephalitis group and febrile convulsion group,only 62.5%of patients with acute bacterial meningitis got improved(p<0.01).The number of cerebrospinal-fluid cells of children in acute bacterial meningitis group was(534.50±682.69)×106/L,and that in viral group was(69.93±89.89)×106/L,which were significantly increased compared with the febrile convulsion group(p<0.01).Meanwhile,the proportion of neutrophils in cerebrospinal fluid with acute bacterial meningitis was significantly increased,with an average of 52.88 ± 30.80%(p<0.01),accompanied by a significant increase in protein,with an average of 4.01±6.49g/L(p<0.01),and a significant decrease in sugar content,with an average of 1.81±1.49 mmol/L(p<0.05).The level of peripheral blood leukocyte in the acute bacterial meningitis group((12.11±9.64)×109/L)was higher than that of the viral encephalitis group((9.90±5.25)×109/L)and the febrile convulsion group((8.39±4.98)×109/L),but there was no significant difference between the three groups(p>0.05).The average CRP in the acute bacterial meningitis group was 135.12±127.18 mg/L,which was significantly higher than that in the febrile convulsion group(21.01±44.27mg/L)and the viral encephalitis group(16.52±26.71 mg/L)(p<0.01).The average PCT in the acute bacterial meningitis group was 10.26±13.65 ng/ml,which was significantly higher than that in the febrile convulsion group(0.28±0.62 ng/ml)and the viral encephalitis group(0.24±0.41 ng/ml)(p<0.01).2.The level of sCD93 in cerebrospinal fluid of children with bacterial meningitis increasedThe level of sCD93 in cerebrospinal fluid of viral encephalitis group and acute bacterial meningitis group was higher than that of the febrile convulsion group.Compared with the febrile convulsion group,the level of sCD93 in acute bacterial meningitis group was significantly higher(p<0.05),but there was no statistical difference in viral encephalitis group(p>0.05).Part II:1.The neurobehavioral ability decreased and brain tissue injury in rat pneumococcal meningitisSix hours after S.pneumoniae infection,the neurobehavioral ability of rats began to decrease significantly.With the extension of infection time,the reduction of behavioral ability became worse(p<0.05,p<0.01),with 4.29±0.52 points in sp-6h group,4.00 ± 0.63 points in SP-12h group and 3.14 ± 1.55 points in SP-24h group respectively.Two hours after S.pneumoniae infection,the number of inflammatory cells infiltration and neuronal cell pyknosis in the brain tissue increased significantly.With the prolongation of infection time,the brain tissue damage was gradually aggravated(p<0.05,p<0.01).2.S.pneumoniae can induce the secretion increase of sCD93 and cytokines IL-6 and TNF-α in cerebrospinal fluid and up-regulation of CD93,Clq and GIPC in brain tissueTwo hours after S.pneumoniae infection,the expression of sCD93 in cerebrospinal fluid was first significantly up-regulated(p<0.01).The level of TNF-α began to increase significantly at 6 hours after infection(p<0.01),and the level of IL-6 began to increase significantly at 12 hours after infection(p<0.01).Inducible NOS in brain tissue began to increase significantly at 2 hours after infection(p<0.01),the expression of CD93 and C1q in brain tissue began to increase significantly at 6 hours after infection(p<0.01),and GIPC began to increase at 12 hours after infection(p<0.05).3.Immunocoprecipitation showed the interaction between CD93 and ClqIn CD93 immune complex,C1q protein was detected by Western blot,but GIPC protein was not detected.At 12 hours and 24 hours after infection,the expression of C1q in the immune complex began to increase significantly(p<0.05,p<0.01).4.Immunofluorescence co-localization showed that CD93 and GIPC were co-located on cerebral cortexImmunofluorescence staining showed that the expression of CD93 and GIPC on cerebral cortex increased significantly 24 hours after infection,and they were co-located on the cell membrane of cerebral cortex(p<0.05).5.Rat pneumococcal meningitis is dominated by proinflammatory M1 phenotype cellsIn rat pneumococcal meningitis,M1 phenotype microglia(CD11b+CD45-CD86+)and M1 phenotype in other type of central macrophage(CD11b+CD45+CD86+)began to increase significantly 2 hours after infection(p<0.01),and M2 phenotype microglia(CD11b+CD45-CD206+)began to increase significantly 12 hours after infection and M2 phenotype in other type of central macrophage(CD11b+CD45+CD206+)began to increase significantly 2 hours after infection(p<0.01,p<0.05),but M1 phenotype was significantly dominant,and about 80%of M1 phenotype cells were microglia.The levels of IL-6,TNF-αand IL-10,TGF-β up-regulated accompanied with inflammatory cells differentiation(p<0.05,p<0.01).Integrin β1 expression began to significantly increase 2 hours after infection(p<0.01).Part Ⅲ:1.The model of S.pneumoniae infecting HAPI microglia in vitro was ConstructedThe level of sCD93 significantly increased with the MOI in a time-dependent manner,reaching a peak at 8 h after infection.At 8 h after S.pneumoniae infection(MOI=100),sCD93 levels was significantly higher than those of other groups(p<0.01).After S.pneumoniae(MOI=100)infected HAPI microglia cells for 8 h,we observed that about 6.788%HAPI microglia cells died and about 35.9%of microglia cells contained intracellular bacteria We also observed about 44000 CFU/mL of cell precipitate,accompanied with C1q levels elevation(p<0.01).2.Clq and PDBu promote the releasing of sCD93,the level of integrin β1 and differentiation of M1 phenotype microglia in S.pneumoniae infected microgliaEight hours after S.pneumoniae infected microglia,the levels of sCD93 in cell culture medium and intracellular NO in C1p group,PDBu group and C1q+PDBu group increased significantly(p<0.01).Meanwhile,integrin β1 was significantly increased(p<0.01),especially in C1q+PDBu group(p<0.01).M1 and M2 microglia increased significantly in C1q group,PDBu group and C1q+PDBu group(p<0.05,p<0.01),especially for M1 microglia,and the corresponding cytokine IL-6,TNF-α and IL-10,TGF-β also significantly increased(p<0.01).3.Down regulation of CD93 reduces integrin β1 expression and differentiation to M1 phenotype of microglia infected by S.pneumoniaePCR and Western blot showed that siCD93-2 was more effective in reducing the transcription and protein expression of CD93(p<0.01).The expression of microglia integrinβ1 was down-regulated after siCD93-2 down regulated CD93 expression(p<0.01),accompanied with down-regulation of the proportion of M1 type microglia(p<0.01)and up-regulation of the proportion of M2 phenotype(p<0.01).With the change of the proportion of microglia in M1 phenotype and M2 phenotype,the corresponding level of cytokine IL-6,TNF-α and IL-10,TGF-β also changed significantly(p<0.05,p<0.01).When the microglia treated with siCD93 was coated with different concentrations recombinant CD93,and then infected by S.pneumoniae,the proportion of M1 microglia significantly increased,and the proportion of M2 microglia significantly decreased.With the increase of recombinant CD93 concentration,the proportion of M1 microglia gradually increased,while the proportion of M2 microglia gradually decreased(p<0.01,p<0.05).4.Down-regulation of integrin β1 reduces the expression of CD93,the migration ability and differentiation to M1 phenotype of microglia infected by S.pneumoniaePCR and Western blot validation showed siIntegrin β1-2 significantly can effectively reduce the transcription and protein expression of integrin β1(p<0.01).The expression of CD93 in microglia was down regulated after siIntegrin β1-2 down regulated integrin β1 expression(p<0.01);the migration ability of microglia was reduced(p<0.01),and the proportion of M1 microglia was significantly reduced(p<0.01),while the proportion of M2 phenotype was significantly increased(p<0.01).With the change of the proportion of microglia M1 phenotype and M2 phenotype,the corresponding level of cytokine IL-6,TNFa and IL-10,TGF-β also changed significantly(p<0.05,p<0.01).When the microglia treated with siIntegrinβ1 was coated with different concentrations recombinant CD93,and then infected by S.pneumoniae,the proportion of M1 microglia significantly increased,and the proportion of M2 microglia significantly decreased.With the increase of recombinant CD93 concentration,the proportion of M1 microglia gradually increased,while the proportion of M2 microglia gradually decreased(p<0.01).Conclusion1.The level of sCD93 in cerebrospinal fluid of children with acute bacterial meningitis increased.Soluble CD93 may be a new inflammatory factor in S.pneumoniae meningitis infection.C1q and PDBu promoted the release of sCD93 from microglia.2.The expression of CD93,GIPC,C1q,integrinβ1 increased in brain tissue infected by S.pneumoniae.Meanwhile,inflammatory cells activated and differentiated,mainly composed of M1 phenotype microglia.CD93 and integrin β1 can regulate the differentiation of microglia into M1 phenotype.3.CD93 and GIPC were co-located on the cell membrane of cerebral cortex,which may activate downstream signal pathways and regulate cell phagocytosis and inflammatory response.4.Down regulation of CD93/sCD93 reduced the differentiation of microglia into M1 phenotype and induced microglia to differentiate into M2 phenotype. |
PDF Full Text Request