Protective Effect And Mechanism Of Protein Vaccine And PGRN On Pneumococcal Meningitis Infection

Objective:Bacterial meningitis is a common infectious diseases leading death worldwide.Streptococcus pneumoniae is a Gram-positive extracellular pathogen and is one of the most common pathogens of bacterial meningitis.It mainly affects children and the elderly.Although Streptococcus pneumoniae is planting on the nasopharynx usually,in addition to causing mild infections such as otitis media and sinusitis,it can break through the blood-brain barrier to form pneumococcal meningitis when suffering life-threatening infections.Pneumococcal meningitis has a mortality rate of 20%to 30%,and most of the survivors have different degrees of neurological sequelae.At present,the prevention of pneumococcal meningitis mainly depends on vaccines.The polysaccharide-conjugated vaccines are very effective in reducing the morbidity of invasive pneumococcal diseases caused by vaccine-type strains.However,there is a decrease in the effectiveness of these vaccines caused by the serotype substitution.Therefore,it is important to develop new vaccines that do not rely on serotypes.Previous research showed that the fusion protein DnaJ-?A146Ply can resist a variety of lethal infectious diseases caused by Streptococcus pneumoniae and it is worth developing,but whether it has a protective effect on meningitis is unknown.PGRN is a neurotrophic factor,previous studies showed a more severe symptom in meningitis mice lacking PGRN,suggesting that PGRN has protective effects and therapeutic potential,but the mechanism of how PGRN protects pneumococcal meningitis mice is unknown.Therefore,our study will explore whether the DnaJ-?A146Ply has a protective effect on pneumococcal meningitis,and the mechanism by which PGRN protects pneumococcal meningitis mice,and provide new strategies of prevention and treatment for meningitis.Method:Construction of meningitis models:Using the stereotaxic apparatus by the intraventricular injection method,15?L of Streptococcus pneumoniae serotype III suspension was slowly injected into the lateral ventricle of the mouse,and the bacteria volume was 5×10~6 CFU per mouse.After active immunization for three times,100?L of Tigr4 was injected through the tail vein,and the bacteria volume was 5×10~8 CFU per mouse.Passive immune antiserum:Concentrated anti-DnaJ-?A146Ply serum or WT control serum was injected into the brains of experimental or control mice via the lateral ventricle.The volume of the concentrated serum was5?L,and then the Streptococcus pneumoniae serotype III or Tigr4 were injected immediately through the lateral ventricle.The amount of serotype III was 5×10~6 CFU per mouse,and Tigr4 was 1×10~8 CFU per mouse.Protective effect of DnaJ-?A146Ply on pneumococcal meningitis:After active or passive immunization,mice were infected with Streptococcus pneumoniae,and the brain bacterial load,inflammatory factor expression,and inflammation infiltration were observed.Protective mechanism of PGRN on mice suffered pneumococcal meningitis:Mouse microglioma BV-2 cells were used to establish cell model and pyroptosis was detected by Western blot.The same age and weight of C57/BL6 WT and pgrn-/-mice were selected to establish meningitis models via lateral ventricle injection with Streptococcus pneumoniae serotype III.Western blot was used to detect the expression of NLRP3,ASC,Caspase-1,GSDMD,IL-1?,IL-18 in the brain of meningitis mice.MCC950 was used for the reverse validation.Results:After the active immunization of DnaJ-?A146Ply for 3 times,the antibody titer reached to 10~6,which could be used to collect serum and subsequent active immune experiments.Anti-DnaJ-?A146Ply serum was injected in the lateral ventricle of mice for the passive immunization and then constructed meningitis models with Streptococcus pneumoniae serotype III or Tigr4.The brain bacterial load showed that compared with the control group,anti-DnaJ-?A146Ply serum group was lower(p<0.05 in serotype?group and p<0.01 in Tigr4group).The inflammatory factors of brain homogenate were detected by ELISA,the expression of TNF-?and IFN-?in the brain of meningitis mice injected with anti-DnaJ-?A146Ply serum were down-regulated(p<0.05).Brain sections HE staining results showed that compared with the WT serum group,the infiltration of inflammatory cells in the lateral ventricle and pia mater of anti-DnaJ-?A146Ply serum meningitis group were less.C57/BL6 mice actively immunized with DnaJ-?A146Ply three times were injected with 100?L 5×10~8 CFU of Streptococcus pneumoniae serotype Tigr4 via the tail vein.The brain bacterial load showed that compared with the PBS group,DnaJ-?A146Ply group was significantly reduced(p<0.05).After 6 hours of treatment with Streptococcus pneumoniae serotype III,BV-2 cells models swelled and deformed,and LDH expression in the supernatant was increased,western blot results showed that the expression of NLRP3,ASC,Caspase-1,IL-1?,IL-18 in the brain of meningitis mice were increased(p<0.05).After injection with 5×10~6CFU of Streptococcus pneumoniae serotype III through the lateral ventricle for 24 hours,LDH release of the brain homogenate was increased in pgrn-/-mice.Western blot results showed that the expression of NLRP3,ASC,Caspase-1,GSDMD,IL-1?,IL-18 in the brain of meningitis mice were increased(p<0.05),and the expression of pgrn-/-meningitis mice was higher than that of WT meningitis mice(p<0.05).Pgrn-/-mice were injected with MCC950 24 hours before(i.p.),then Streptococcus pneumoniae serotype?was injected via lateral ventricle.The results of the 24-hour brain bacteria load showed that MCC950pretreated mice had a downward trend.Conclusion:The fusion protein DnaJ-?A146Ply has protective effect on meningitis in mice caused by various serotypes of Streptococcus pneumoniae,and can reduce bacterial invasion and inflammatory infiltration in the brain.Pyroptosis occurred in the brain of mice suffered pneumococcal meningitis,and pgrn-/-mice suffered more severe,indicating that PGRN plays a protective role in combating pyroptosis in pneumococcal meningitis.