The Mechanism Of IL-1? Maturation And Secretion In Mice Neutrophils Infected With Streptococcus Pneumoniae

Streptococcus pneumoniae is a major cause of bacterial pneumonia,otitis media,meningitis and sepsis.The elderly and children under the age of 5 are most susceptible Streptococcus pneumoniae can induce host immune cells secretion a series of cytokines such as IL-1 family cytokines and TNFa,and these cytokines in turn contribute to the protective defense against the bacterial.Streptococcus pneumoniae virulence factor pneumolysin(PLY)play a key role during the infection and colonization of Streptococcus pneumoniae.PLY can also trigger the host innate immune response against Streptococcus pneumoniae.Upon infection of Streptococcus pneumoniae,host immune cells such as neutrophils can secretion a series of cytokines.IL-1? as a potent pro inflammatory cytokine,can lead to immune cell recruitment and subsequent amplification of cytokine and chemokine responses leading to eventual bacterial clearance.We have previously reported the precise mechanism of IL-1? maturation and secretion in Streptococcus pneumoniae-infected macrophages.We found that PLY-dependent IL-1? secretion required the activation of NLRP3 and AIM2 inflammasomes.However,the mechanism of IL-1? maturation and secretion in Streptococcus pneumoniae-infected neutrophils is still unclear.In this study,we explore the mechanism of IL-1? maturation and secretion in the mice neutrophils infected with Streptococcus pneumoniae1.The effect of PLY on the maturation and secretion of mice neutrophil IL-1?Mice peritoneal neutrophils were infected with Streptococcus pneumoniae wild-type strain D39 and ply gene deficient strain ?ply.The secretion of cytokines IL-1?,IL-1?,IL-6 and TNFa in culture supernatant were detected with ELISA.And mature and preform of IL-1? in culture supernatant and cell lysate were tested by Western blot.To further conform the role of PLY in processing IL-1?,different concentrations of rPLY protein were used to infect the neutrophils.And then the secretion of IL-1? in the culture supernatant were tested by ELISA.These results showed that,both D39 and ?ply strains induced comparable levels of IL-1?,IL-6 and TNFa secretion.And the secretion of IL-1? in ?ply-infected neutrophils was significantly lower than the D39 infected ones.Moreover,mature form of IL-1? was only appeared in the D39-infected neutrophils,and the preform of IL-1? were found in both D39-and ?ply-infected neutrophils.Furthermore,the rPLY induced the secretion of IL-1? depend on the concentration of rPLY.Collected,these results indicated that mice neutrophils can secret a bound of cytokines including IL-1?,IL-1?,IL-6 and TNFa,and PLY was involved in the maturation and secretion of IL-1?.2.The involvement of the NLRP3 inflammasome in the processing of mice neutrophil IL-1?To examine the role of neutrophil phagocytosis,mice neutrophils were pretreated with Cyto.B before infected the D39 strain,the secretion of IL-1 ? in culture supernatant was tested by ELISA.The result showed that the secretion of IL-1? was significant decreased after Cyto.B treatment.To assess whether the NLRP3 inflammasome is required for neutrophil IL-1? maturation and secretion upon S.pneumoniae infection.Neutrophil of C57BL/6 WT,Caspase-1-/-ASC-/-NLRP3-/-NLRC4-/-and AIM2-/-mice were infected with S.pneumoniae D39 strain,the secretion of IL-1? in culturethe secretion of IL-1? in Caspase-1-/-,ASC-/-and NLRP3-/-mice were markedly decreased,while there were comparable levels of IL-1? secretion in NLRC4-/-and AIM2-/-mice neutrophils.To further examine the involvement of the NLRP3 inflammasome in mice neutrophils IL-1? mature and secretion,neutrophil of C57BL/6 WT,Caspase-1-/-,ASC-/-and NLRP3-/-mice were infected with the D39 strain,then the protein expression of caspase-land IL-1? in the culture supernatants,and the preform of IL-1 ? and caspase-1 in the cell lysates were tested by Western blot.The results showed that the mature form of IL-1? and the active form of caspase-1 were detected in the neutrophils of WT mice upon D39 infection.To determine whether K efflux is required for neutrophil IL-1?processing during S.pneumoniae infection,high and low concentrations of KC1 were added to neutrophils upon D39 infection,the levels of IL-1? secretion were tested by ELISA,and the activation of caspase-1 was detected by Western blot,it was found that high concentration of KC1 markedly blocked the secretion of IL-1? and caspase-1 activation.To examine the role of JNK/Syk signaling pathway in IL-1? processing upon S.pneumoniae secretion.The neutrophils were pretreated with the JNK/Syk inhibitors SP600125/R406 before D39 infection,ELISA were conducted to test the levels of IL-1?secretion in culture supernatants,and Western blotwere conducted to examine the phosphorylated JNK levels,mature form of IL-1?,activation of caspase-1 in the culture supernatants and the preform of IL-1?,caspase-1 in the cell lysates.And the lysates were cross-linked with DSS to test the oligomeriztion of ASC.It was found that the secretion of IL-1? was significantly decreased,phosphorylated JNK levels were significantly decreased and the the caspase-1 activation were markedly blocked after treatment of SP600125 and ASC was only detected as monomers,but as multimers in other groups.Taken together,these suggested that NLRP3 inflammsome in required for mice neutrophils IL-1? maturation and secretion and the process need K efflux.And the upstream signaling pathway JNK can modulate the processing of neutrophils IL-1?through regulating the oligomerization of ASC and then caspase-1 activation during S.pneumoniae infection.Moreover,phagocytosis of S.pneumoniae by neutrophils plays a key role in the secretion of IL-1?.3.The effect of neutrophil serine proteases on neutrophil IL-1?processing upon S.pneumoniae infection.To assess whether neutrophil serine proteases contribute to neutrophil IL-1?processing upon S.pneumoniae infection.Neutrophils were pretreated with serine proteases inhibitors(PMSF,ONO-5046)and pan-caspase inhibitor Z-VAD-FMK before infected with D39,the secretion of IL-1? in culture supernatant was tested by ELISA.The result showed that the secretion of IL-1? was significant decreased after the inhibitors treatment.Western blotwere conducted to examine the mature form of IL-1?,activation of caspase-1 in the culture supernatants and the preform of IL-1?,caspase-1 in the cell lysates.And the lysates were cross-linked with DSS to test the oligomeriztion of ASC.These results showed that serine proteases inhibitors significantly blocked the activation of caspase-1 and oligomerization of ASC.Collected,indicated that neutrophil serine proteases effect IL-1? procession through modulated ASC oligomeriztion and then caspase-1 activation during S.pneumoniae infection.Taken together,this study demonstrated that neutrophils secrete IL-1? through a mechanism that is dependent on the NLRP3 inflammasome-Caspase-1 axis upon S.pneumoniae infection,and K efflux,JNK activity were indispensable for this process.Further,we highlighted that neutrophil serine proteases,notably NE participate in IL-1?secretion through regulating ASC oligomerization and consequent caspase-1 activation.We also showed the key role of PLY in neutrophil IL-1? maturation and secretion.