The Effect And Activation Mechanism Of AIM2 Inflammasome In Host Defense Against Streptococcus Pneumoniae Infection

Streptococcus pneumoniae is an opportunistic,Gram-positive,extracellular pathogen that often colonizes on the mucosal surfaces of human upper respiratory tract.S.pneumoniae can cause severe invasive diseases worldwide,such as pneumonia,otitis media and meningitis and mainly affects immunocompromised people such as young children and the old people.Pneumolysin?PLY?is one of the most important virulence factors of S.pneumoniae that can induce host innate immunity.Studies have been shown that inflammasome,a multiprotein complexe that typically consists of pattern recognition receptors,apoptosis-associated speck-like protein containing a CARD?ASC?and pro-caspase-1,plays an important role in host innate immunity.Inflammasome is involved in IL-1?maturation and secretion which plays a key role in defense against bacterial infection.Our previous study showed that S.pneumoniae could induce the assembly of the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3?NLRP3?/absent in melanoma 2?AIM2?inflammasome,which mediates the activation of caspase-1 and the subsequent maturation of Interleukin-1??IL-1??;NLRP3 and ASC deficiency significantly impaired host defense against S.pneumoniae.However,the role of AIM2 inflammasom in host defense against S.pneumoniae in vivo is unclear and how S.pneumoniae initiates inflammasomes activation remains to be fully elucidated.In this study,we employed an intranasal infection model of S.pneumoniae in AIM2^-/-and WT mice and using primary mouse macrophages as a cell model to examine the effects of AIM2 inflammasome in host defense against S.pneumoniae infection and the mechanism of inflammasome activation in S.pneumoniae-infected macrophage.1.The role of AIM2 in host defense against S.pneumoniae infectionWT mice and AIM2^-/-mice were intranasally infected with 5×10⁷ CFU of S.pneumoniae D39,and then survival of infected mice were monitored every day until 14days postinfection.Lung tissues were obtained 48 h postinfection for bacterial CFU counting and histology.Lungs were homogenized,serial diluted in PBS and plated onto blood agar to count CFU after 12 h culture.For histology,the lungs were fixed with 10%formaldehyde,embedded in paraffin,stained with H&E and observed histological changes by microscopy.To assess whether AIM2 has an impact on cytokine production upon S.pneumoniae infection in vivo,the levels of IL-1?,IL-12,IL-6 and TNF?in bronchoalveolar lavage fluid?BALF?at 12 h after infection were compared between WT and AIM2^-/-mice by ELISA.The results showed that 18.75%?3/16?of WT mice died within 2 weeks,while AIM2^-/-mice showed a higher mortality rate at 50%?7/14?.The bacterial burdens in the lungs of AIM2^-/-mice were much 10 times higher than those in WT mice.H&E staining showed that there was a more severe pulmonary inflammatory response in the AIM2^-/-group.Lungs from AIM2^-/-mice displayed obvious histopathologic inflammatory reactions,such as infiltration of inflammatory cells and hyperplasia of interstitial tissues.The concentrations of IL-1?in BALF were significantly lower in AIM2^-/-mice than in WT mice after S.pneumoniae infection,while IL-6,IL-12and TNF?levels showed no difference.AIM2^-/-mice displayed increased susceptibility to intranasal infection with S.pneumoniae in comparison to WT mice,which show higher mortality,higher bacterial colonization and aggravated inflammation in the lungs.These results suggest that AIM2 plays a protective role in host defense against S.pneumoniae.2.The effects of AIM2 inflammasome on maturation and secretion of IL-1?in S.pneumoniae-infected macrophageTo assess whether AIM2 inflammasome is required for the secretion of IL-1?upon infection with S.pneumoniae,primary mouse macrophages?PECs?from WT mice were infected with S.pneumoniae D39.Real-time RT-PCR was carried out to determine the expression of AIM2.Results showed that the expression of AIM2 was strongly increased following infected with S.pneumoniae.PECs from WT and AIM2^-/-mice were infected with D39,and the IL-1?secretion levels and caspase-1 activation were detected by ELISA or Western blot.Compared with WT group,S.pneumoniae-induced the secretion of IL-1?and caspase-1 activation decreased in PECs from AIM2^-/-mice.In order to examine the role of cytoplasmic potassium efflux in inflammasome activation upon S.pneumoniae infection,different concentrations of KCl were added to culture medium to block cytoplasmic potassium efflux.Level of IL-1?in cultural supernatant was examined by ELISA.Western blot was carried out to determine the activation of caspase-1.And the oligomerization of ASC was also determined by Western blot after chemical crosslinking.Studies results showed that increasing the potassium concentration in the culture medium almost blocked ASC oligomerization,caspase-1activation and subsequent secretion of IL-1?,with no difference in the level of TNF?.As shown,a significant reduction in S.pneumoniae-induced ASC oligomerization was also observed in AIM2^-/-PECs.Thus,it appeared that the AIM2 inflammasome is involved in IL-1?maturation and secretion in macrophages infected with S.pneumoniae,and cytoplasmic potassium efflux is critical for inflammasome activation upon this process.3.The mechanism of inflammasome activation in S.pneumoniae- infected macrophageTo examine the effects of kinases on inflammasome activation induced by S.pneumoniae infection,PECs were pretreated with a series of common kinase inhibitors and then infected by S.pneumoniae D39.The inflammasome activity was examined by ELISA and Western blot.Results showed that inhibitors of Spleen tyrosine kinase?Syk?and c-Jun N-terminal kinase?JNK?decreased the oligomerization of ASC and subsequent caspase-1 activation and IL-1?secretion,compared to that of the DMSO control group.It suggests Syk and JNK signaling are required for S.pneumoniae-induced activation of inflammasome.To address the relevance of PLY to inflammasome activation,macrophages were infected with D39 or?ply strain and phosphorylation of Syk/JNK was detected by Western blot.Infecting cells with the D39 strain,but not the?ply strain,led to significantly upregulated phosphorylation of Syk and JNK.To further test whether the PLY protein itself is sufficient to confer Syk and JNK activation,macrophages were treated with different concentrations of recombinant PLY?rPLY?.Western blot showed that increasing the concentration of rPLY led to a dose-dependent increase in Syk/JNK phosphorylation.To explore the relationship between JNK and Syk in S.pneumoniae infection,Syk inhibitor R406 or DMSO was used to pretreat PECs before infection.The phosphorylation of JNK induced by D39 infection was almost abolished by inhibiting Syk signaling compared with control group.In conclusion,these results indicated that PLY is involved in S.pneumoniae-induced inflammasome activaition by mediating Syk and JNK activation and Syk may act upstream of JNK in S.pneumoniae-infected macrophages.Taken together,these results suggested that the Syk/JNK signaling pathway may play a vital role in the inflammasome activation and modulate host immune responses against S.pneumoniae.