The Mechanism Of Il-1? Maturation And Secretion In Macrophages Infected With Streptococcus Pneumoniae

Streptococcus pneumoniae is a Gram-positive extracellular organism that is often colonized in the human nasopharynx,which can cause bacterial pneumonia,otitis media,meningitis and sepsis.The elderly and children are susceptible.As an important virulence factor of Streptococcus pneumoniae,pneumolysin?PLY?plays an extremely important role in the pathogenesis of Streptococcus pneumoniae.On one hand,PLY can bind to cholesterol on the cell membrane surface,intercalate into the cell membrane lipid bilayer,and form a transmembrane pore to cause cell lysis by oligomerization of proteins.PLY induces extensive cytotoxicity,disrupts airway epithelial integrity,and inhibits epithelial cell ciliary movement.It also can destroy the bactericidal capacity of neutrophils and change complement-mediated opsonization.On the other hand,PLY activates the host intate immune response system to produce proinflammatory cytokines that elicit host immune response.A series of cytokines including IL-1?,IL-1?and IL-18 contribute to the host defense against S.pneumoniae infection.Our previous study found that in the S.pneumoniae-infected macrophages,IL-1?and IL-18 secretion and maturation are relied on Caspase-1 activation,which is induced by the assembly of NLRP3 and AIM2 inflammasomes.However,the precise mechanism of IL-1?secretion in S.pneumoniae-infected macrophages remains unclear.In this study,we explore the mechanism of IL-1?maturation and secretion in the macrophage model of Streptococcus pneumoniae infection.1.The role of PLY on the maturation and secretion of IL-1?Macrophages were infected with Streptococcus pneumoniae wild-type strain D39and ply-deficient strain??ply?.The expression of IL-1?mRNA was determined by Real-time RT-PCR.The levels of IL-1?in the culture supernatant and cell lysate were detected by ELISA.Western blotting were used to detect IL-1?protein expression of in culture supernatant and lysate.To verify the role of PLY in the maturation and secretion of IL-1?,macrophages were infected with?ply and different concentrations of cholesterol-treated rPLY,which the level of IL-1?secretion in the supernatant was dected by ELISA.The results showed that a significant increase in the IL-1?mRNA expression was observed in both D39-and?ply-infected macrophages.However,the?ply strain could not induce the IL-1?maturation and secretion in in S.pneumoniae-infected macrophages.Compared to?ply,mature fragments of IL-1?could be detected in the supernatants of D39 infection stystems.The secretion of IL-1?was decreased in macrophages infected with rPLY treated by cholesterol.These results indicated that PLY can increase IL-1?secretion in macrophages infected with S.pneumoniae.The pore forming toxicity of PLY is a key factor in inducing the maturation and secretion of IL-1?,but PLY is dispensable for IL-1?transcription and is critical for the maturation and secretion of IL-1?in in S.pneumoniae-infected macrophages.2.The effects of inflammasome and phagocytosis of macrophages on the maturation and secretion of IL-1?To study the effects of inflammasome on the on the maturation and secretion of IL-1?in S.pneumoniae-infected macrophages,WT,Caspase-1^-/-and ASC^-/-mice macrophages were infected with D39,and the IL-1?secretion levels were detected by ELISA.The results showed that comparable levels of IL-1?were detected among WT,Caspase-1^-/-and ASC^-/-macrophages infected with D39.Western blotting showed that there was the mature fragments of IL-1?presence in the supernatant of D39-infected WT,Caspase-1^-/-and ASC^-/-macrophages.To verify that whether phagocytosis of macrophages by bacteria is necessary,cytochalasin B was added to inhibit phagocytosis of macrophages,the IL-1?secretion was detected by ELISA,and the protein expression of IL-1?was detected by Western blotting.The results showed that cytochalasin B treatment markedly decreased the IL-1?secretion levels and protein expression levels.These results suggested that in the macrophage model of S.pneumoniae infection,the inflammasome is not involved in the maturation and secretion of IL-1?,but phagocytosis of S.pneumoniae by macrophages plays an important role in the maturation and secretion of IL-1?.3.The effect of calpain on the maturation and secretion of IL-1?in S.pneumoniae-infected macrophagesMacrophages were infected with D39.Cells were treated with various calpain inhibitors?calpain inhibitor III,calpain inhibitor IV and EST?and calcium ion chelator BAPTA-AM 3 h after infection,and the levels of IL-1?were measured by ELISA.The results showed that the IL-1?secretion levels were significantly decreased with the presence of calpain inhibitors and BAPTA-AM.The protein levels of?-fodrin in the supernatant and the?-fodrin degradation fragment in the cell lysates were detected by Western blotting.The results showed that various calpain inhibitors and BAPTA-AM reduced the the?-fodrin degradation.To research into the factors of activating calpain,macrophages were infected with D39 and?ply firstly,Western blotting showed that there was no existence of the?-fodrin degradation in?ply-infected macrophages.EDTA,EGTA+Mg²⁺were added and ELISA was used to detect the secretion level of IL-1?in S.pneumoniae-infected macrophages,which aimed to study whether Ca²⁺participated in the maturation and secretion of IL-1?or not.The results showed that the secretion of IL-1?was significantly reduced after adding EDTA and EGTA+Mg²⁺.Western blotting was used to detect the protein level of?-fodrin in the supernatant and cell lysate.The results showed that the degradation of?-fodrin was markedly decreased after adding EDTA and EGTA+Mg²⁺.Furthermore,our study found that in the calcium-free environment,the secretion level of IL-1?was significantly decreased in D39-infected macrophages.But macrophages were infected with?ply,and calcium ionophore A23187 was added 3 h after infection.The results showed that the secretion of IL-1?in the culture supernatant was significantly increased.Macrophages were infected with D39 or?ply strains,the intracellular calcium ion concentration was measured every 3 hours using the Fluo-4 NW kit.The results showed that compared with?ply,the intracellular calcium ion concentration was significantly increased after D39 infection.These results indicated that PLY contributes to the increased intracellular Ca²⁺levels in macrophages infected by S.pneumoniae,followed by calpain activation and IL-1?secretion.The above results elucidated that Streptococcus pneumoniae is phagocytized by macrophages and PLY is released.PLY contributes to the influx of extracellular calcium,the elevation of intracellular calcium by forming pores on the cell membrane,resulting in calpain activation,and finally maturation and secretion of IL-1?.