Investigation Of The Underlying Mechanism By Which BLP-induced Tolerance Heightens Bactericidal Activity Through Enhancing Caspase-1/11 Activation In Macrophages

As an important component of organism defence systems,tolerance to bacterial cell wall components bacterial lipoprotein(BLP)represents an essential regulatory mechanism during biological evolution,which characterized by enhanced protection against infection,but the underlying mechanisms is undetermined.Our previous work has shown that,the important intracellular pattern recognition receptors,NOD1 and NOD2 are probably involved in enhanced bactericidal activity in BLP-tolerized macrophages.Similar to TLRs,another PRR,NOD1 and NOD2 are the two main members of the family of NLRs which are involved in host innate immune response to bacterial infection.NLRs present in the cytoplasm of immune cells which are triggered upon detection of pathogen-associated molecular patterns(PAMPs)such as lipopolysaccharide,peptidoglycan,viral RNA,or danger-associated molecular patterns(DAMPs).Upon detection of intracellular pathogen,NLRs associated in multiprotein complexes called inflammasome through canonical inflammasome or non-canonical inflammasome activation that mediate Caspase-1 or Caspase-11 activation,the mature and secretion of IL-1β and IL-18,and pyroptosis.Both TLR and NOD signaling act synergistically which constitute an essential component of the host innate immune system to protect against bacterial infection.It is not clear whether Caspase-1/11 activation and pyroptosis induced by NLR inflammasome activation are involved in protection effects of BLP-induced tolerance.Thus,the article was designed to explore whether BLP-induced tolerance heightens bactericidal activity through enhancing Caspase-1/11 activation in macrophages and its potential mechanism.The study consists of three parts:1.BLP-induced tolerance enhances the activation of Caspase-1 and Caspase-11 in BMDMs.2.BLP-induced tolerance heightens bactericidal activity through enhancing Caspase-1 activation in macrophages.3.BLP-induced tolerance regulates pyroptosis in macrophages and its underlying molecular mechanism.The main results include:1.Comparing with Naive macrophages,the expression of mRNA and protein levels of Caspase-1,Caspase-11 up-regulated in BLP-tolerized macrophages measured by real-time RT-PCR and Western blot.And the expression of mRNA and protein levels of pro-caspase-1,pro-caspase-11 up-regulated significantly after suffered from S.aureus and S.typhi as interval of stimuli was prolonged.And the activation of Caspase-1 and Caspase-11 enhanced in BLP-tolerized macrophages.Simultaneously,the expression of NLRP3、NLRC4 and ASC up-regulated in BLP-tolerized macrophages measured by confocal microscopy.2.BLP-tolerized BMDMs displayed significantly enhanced phagocytosis measured by Flow Cytometer.Z-YVAD-FMK,the specific inhibitor of Caspase-1,does not affect the enhanced phagocytosis ability of BLP-tolerized macrophages.In killing assay,we found that the BLP tolerance-enhanced bactericidal activity can be inhibited with Z-YVAD-FMK in macrophages suffered with S.aureus but not S.typhi.And the inhibitory effect is dose-dependent.The immunofluorescence results showed that phagosomal acidification after ingestion of S.aureus enhanced significantly in BLP-tolerized peritoneal macrophages.And Z-YVAD-FMK impairs the enhanced acidification.3.The Flow Cytometer results showed that the 7-AAD staining positive cells decreased significantly in BLP-tolerized macrophages compared with Na?ve BMDMs.Using Cytotoxicity Detection Kit,we found that the release of LDH reduced significantly in BLP-tolerized BMDMs.And Z-YVAD-FMK inhibit the release of LDH both in Naive and BLP-tolerized macrophages.Similarly,the PI staining positive cells decreased significantly in BLP-tolerized macrophages.And Z-YVAD-FMK reduced the PI staining positive cells both in Na?ve and BLP-tolerized macrophages.ASC specks increased significantly in Naive macrophages suffered with S.aureus and S.typhi.In addition,the secrete of IL-1β increased in BLP-tolerized macrophages.And both in transcription or translation level and cleaved GSDMD,the expression of GSDMD down-regulated in BLP-tolerized macrophages,especially on early infective stage.In conclusion,we demonstrate that:1.BLP tolerance enhances the activation of Caspase-1 and Caspase-11 in macrophages suffered from bacterial infection.And the mechanism may be associated with the expression(or formation)of NLRP3 and NLPC4 inflammasome components.2.The BLP tolerance-enhanced bactericidal activity is associated with phagosome acidification induced by enhanced Caspase-1 activation.3.In addition,BLP tolerance protects against pathogens by regulating cell pyroptosis.And the potential mechanism probably is associated with the decreased ASC specks formation and down-regulated of GSDMD expression in BLP-tolerized macrophages.