| The innate immune system is the most effective line of host defense and plays a critical role in defense against a majority of microbial infections.When bacterial and other microorganisms invade the body,its highly conserved structures such as lipopolysaccharide(LPS),bacterial lipoproteins(BLP)and peptidoglycan(PGN)are recognized by the corresponding sensors such as Toll-like receptors(TLRs)and NOD-like receptors(NLRs)on the macrophages,mast cells,dendritic cells,neutrophils and other innate immune cells.At the same time of this recognition the host produces an immune response against bacterial infection.These conserved microbial components are termed as pathogen associated molecular patterns(PAMPs),and the innate receptors or sensors that present on the host cell surface or in the cytosolic compartment are known as pattern-recognition receptors(PRRs).After recognizing PAMPs,the PRRs can activate various signal cascades that generally converge to transcription factors including nuclear transcription factor-κB,activator protein(AP)-1,and interferon regulatory factors(IRFs),which then induce the release of inflammatory mediators such as pro-inflammatory and anti-inflammatory cytokines and chemokines.Meanwhile,pathogenic microorganisms are phagocytized and killed.All the above processes participate in the clearance of pathogenic microorganisms.The NF-κB family is a key player in controlling both innate and adaptive immunity.In mammals,this protein family includes five members:p50,p52,p65,c-Rel and RelB.Under resting conditions,NF-κB dimers are bound to their inhibitory proteins such as IκBa,which sequester NF-κB complexes in the cytoplasm;when the cells are stimulated by bacteria and other inducers,the IκB proteins become phosphorylated,then ubiquitylated,and subsequently degraded by the proteasome.Following degradation of IκB proteins,NF-κB dimers translocate to the nucleus,where they bind to the κB binding site of their target genes and participate in regulating the transcription of a large number of genes,such as cytokines,chemokines,antimicrobial peptides.It is known that a heterodimer of the p65 subunit associated with either a p50 or p52 subunit is the main active form of NF-κB and widely expressed in a variety of cell types.BLP as the most abundant protein in the outer membrane of both Gram-positive and Gram-negative bacteria is mainly released by the growing or cracked bacteria.As a member of PAMPs,BLP can be recognized by its specific PRR known as Toll-like receptor 2(TLR2)and at the same time it can trigger the immune responses,and induce the release of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and other pro-inflammatory cytokines by monocytes and macrophages.These pro-inflammatory cytokines play a critical role in the defensive immune response to eliminate bacterial pathogens from the host.However,overproduction of pro-inflammatory is harmful to the body and can lead to septic shock and even death.The phenomenon of the body’s(or cell’s)tolerance to bacterial cell wall components including by LPS and BLP was found in the studying of reasonable regulation of inflammation.Initially researchers found that when pretreated with a sub-lethal dose of LPS,laboratory animal or monocytes/macrophages would be irresponsive or hypo-responsive to a subsequent LPS stimulation with a diminished production of pro-inflammatory cytokines,a reduced inflammatory damage and a significant survival advantage.This phenomenon is well established and termed LPS tolerance.Just as the LPS tolerance described above,pretreatment of laboratory animal or monocytes/macrophages with BLP can also induced tolerance to a second BLP challenge,which is called BLP tolerance.Moreover,except for self-tolerance,BLP can also induce cross-tolerance to bacteria and LPS.So BLP tolerance is considered as both a protective regulatory mechanism forming in the long term process of biological evolution and an important part of the host defense response,but its mechanism remains not fully understood.One typical features of BLP tolerance is the down-regulation of TLRs signaling pathway,showing as the suppressed expression of TLR2 and interleukin-1 receptor-associated kinase(IRAK-1)and the reduced release of inflammatory cytokines such as TNF-α、IL-6.Enhanced bacterial clearance of macrophages is another feature of BLP tolerance,which may be an important way of the protective effects of BLP tolerance.Currently,the signal transduction mechanisms involving in regulating the enhanced bacterial clearance of macrophages,in the case of down-regulated of TLRs pathway,have rarely been reported.Phagocytosis is essential in host defense against microbial pathogens,which is mainly performed by specialized phagocytes,such as macrophages.At the very beginning of phagocytosis,the plasmalemma of phagocytic cells invaginates inwardly to engulf microbial pathogens and to form the nascent phagosome,which lacks the microbicidal and degradative capacity required for pathogen elimination.These capabilities are acquired subsequently,through a process termed as phagosome maturation,which requires a fusion of phagosome with lysosomes and also needs the acquisition and activation of a series of oxidative,acidifying and hydrolytic enzymes.Phagosome maturation is an essential process for successful clearance of bacteria and needs a great number of proteins to work together.At present,it is known that lysosomal enzymes and membrane transport regulators play an important role in the phagosome maturation process.Since the enhanced bacterial clearance effect of BLP-tolerized body(or cell)is closely associated with phagosome maturation,it will be helpful to explore the change of phagosome maturation related protein,to know whether they are involved in the enhanced bacterial clearance,to clarify the relevant signaling molecules and pathways that control these changes,and most importantly to let us understand the underlying mechanisms of the protective regulatory role of BLP tolerance.Based on the above understandings,our previous study chose bone marrow-derived macrophages(BMM)as our cell model,and used RT2 PCR Array to compare the expression differences of phagosome maturation-related genes between the BLP-tolerized(100 ng/ml BLP for 24 h)and Naive BMM infected by Gram-negative bacteria.Results showed that the expression of acp2,acp5,rab10,rab20,rabgef1,camp,ctsc,nos2 were significantly up-regulated in BLP-tolerized BMM.The acp2 and acp5 encode the lysosomal acid phosphatase 2/5(Acp2/5),rab10 and rab20 encode two members(Rab10 and Rab20)of the Rab GTPases family that are implicated in controlling the trafficking of phagosomes and other membrane-bound organelles,rabgef1 encodes RAB guanine nucleotide exchange factor 1(Rabgef1),camp encodes the cathelicidin antimicrobial peptide(Camp),ctsc encodes cathepsin C(Ctsc),nos2 encods inducible nitric oxide synthase(iNOS).In addition,we also found that when stimulated with bacteria,NF-κB pathway in the BLP-tolerized BMM would be reactivated,but the mitogen-activated protein kinase,MAPK)signaling pathway still at a low level.As we know TLRs related pathways are down-regulated in BLP-tolerized BMM.But,it is still unknown that whether the activation of NF-κB pathway is mediated by the cytoplasmic receptors NOD 1/2,whether this activated NF-κB pathway is involved in the enhanced phagosome maturation.To address all these issues will help us to understand the protective mechanisms of BLP tolerance and develop a new way to prevent and treat septic shock.Based on the above knowledge and our previous work,the purposes of this study are as follows:1.To compare the bacterial engulfment and intracellular killing between BLP-tolerized and Naive BMM.Firstly,FITC-E.coli were used to infect BMM,then the bacterial engulfment was observed under Zeiss fluorescence microscope,or objectively the mean fluorescence of cell lysate was detected by Spectra Max M5.Next,using the bactericidal experiments,we compare the intracellular bacterial killing capacity for live E.coli between BLP-tolerized and Naive BMM.2.To verify the phagosome maturation related genes that were screened by RT2 PCR Array.Firstly,the mRNA levels of Acp2,Acp5,Rab10,Rab20,Rabgefl,Camp,Ctsc,iNOS in macrophages pre-treated with 100 ng/ml BLP for 24 h were detected by real-time PCR.Then the protein levels of Acp5 and Rab10 were further confirmed by Western blot.3.To observe the influence of bacterial infections on the p65 activity in the BLP-tolerized and Naive BMM.The location and nuclear translocation of p65 were shown by immunofluorescence in BLP-tolerized and Naive BMM infected with heat-killed Salmonella typhi.4.To investigate whether the activated p65 involved in the regulation of phagosome maturation related molecules.Firstly,BAY-11-7082,a specific inhibitor of NF-κB,was chosen to inhibit the activation of NF-κB signaling pathway.Next,Acp2,Acp5,iNOS mRNA expression differences between the BAY-11-7082 inhibited group and DMSO control group were detected by real-time PCR.5.To explore whether cytoplasmic receptors NOD 1/2 involved in the expression of phagosome maturation related molecules through mediating the p65 activation in BMM infected by heat-killed Salmonella typhi.Firstly,specific siRNA mixture were transfected by electroporation method into BMM in order to down-regulate the expression of NOD1 and(or)NOD2.Then,the p65 nuclear translocation was observed by immunofluorescence in both the double interfered group and NC control group.Finally,the effects of NOD1 or NOD2 down-regulation on the mRNA expression of Acp5,Rab10 and iNOS in the BLP-tolerized BMM infected by bacteria were measured by real-time PCR.6.To clarify the role of phagosome maturation related molecules Rab10 in the enhanced bactericidal capacity of BLP-tolerized BMM.The expression of RablO was down-regulated by transfecting specific siRNA into the BLP-tolerized BMM firstly.Then the phagocytic and bactericidal capacity between the interfered group and NC control group were compared.The results were as follows:1.After 30 minutes’ incubation with FITC-E.coli,stronger green fluorescence was observed by Zeiss microscope in the BLP-tolerized BMM than Naive.The mean fluorescence of its cell lysate detected by Spectra Max M5 was significantly higher than Naive BMM as well(*P<0.05,t test).Live E.coli were incubated with BMM for 30 minutes and then discarded.After this incubation cells were cultured at 37 C for another 30 or 60 minutes.The intracellular bacterial killing capacity for live E.coli between BLP-tolerized and Naive BMM had no significant differences at the point of 30 minutes,but was significantly different at the point of 60 mintues.2.Acp2,Acp5,Rab10,Rab20,Camp,iNOS mRNA levels in the BLP-tolerized BMM were significantly higher than Naive BMM(*P<0.05,t test);but Rabgefl and Ctsc had no significant differences.The protein levels of Acp5 and Rab10 significantly increased in BLP-tolerised macrophages;followingbacteria stimulation,Acp5 and Rab10 increased in Naive BMM,and stayed at a high level in BLP-tolerized BMM.3.Under resting conditions,p65 of Naive BMM almost located in the cytoplasm;except for its main cytoplasm distribution p65 of BLP-tolerized BMM also had a small number distribution in the nucleus.When attacked by the Salmonella typhi for 30 and 60 minutes,p65 mainly located in the nucleus in both the Naive and BLP-tolerized BMM,indicating that p65 rapidly shifted from the cytoplasm to the nuclear after Salmonella typhi infected.The nuclear translocation of p65 revealed the activation of NF-κB signaling pathway.4.After inhibiting the activation of pathway in infected macrophages by IκBa phosphorylation inhibitor(BAY-11-7082),the changes of Acp5,Rab10,iNOS mRNA expression in bacteria infected macrophages were detected.Results showed that:in Naive BMM infected with bacteria,Acp5 and iNOS mRNA levels were significantly lower in NF-κB inhibited group(BAY-11-7082)compared with control group(DMSO)at the point of 60 minutes,but Rab10 had no significant difference.In BLP-tolerized BMM infected with bacteria,Acp5,Rab10 and iNOS mRNA levels all had significant differences between the two groups at the point of 60 minutes.These results demonstrated that p65 activation was involved in the expression regulation of Acp5,Rab10 and iNOS in macrophages infected with bacteria.5.Under resting state,p65 was mainly distributed in the cytoplasm both in the NC or NOD 1/2 double inferred Naive BMM;in BLP-tolerized BMM,p65 located mainly in cytoplasm with a small number distribution in the nucleus in both groups.When attacked by the Salmonella typhi for 30 and 60 minutes,p65 mainly located in the nucleus in Naive-NC group;the nuclear translocation of p65 were delayed in Naive NOD 1/2 inferred group,where p65 were distributed both in the cytoplasm and nucleus.The same phenomenon could also be observed in the BLP-tolerized BMM,but the differences between the NC and NOD1/2 double inferred group were less obvious comparing to Naive BMM.6.Following down regulation of NOD1,the Rab10 and iNOS mRNA expression in BLP-tolerized BMM were significantly lower compared with NC group at the point of 60 minutes;but Acp5 mRNA had no significant difference between two groups.Following down regulation of NOD2,the Acp5,the Rab10 and iNOS mRNA expression in BLP-tolerized BMM were significantly lower compared with NC group at the point of 30 minutes;but there were no significant differences at the point of 60 minutes.7.Down-regulating of Rab10 using RNA interference(RNAi)in BLP-tolerized BMM,there was no significant differences between NC and inferred group in the phagocytosis experiment;bactericidal experiments shown that the intracellular bacterial killing was significantly influenced at both 30 and 60 time point.These results revealed that Rab10 down-regulating had no effect on engulfment of bacteria,but could weaken the bactericidal effect significantly.According to the above results,we could get conclusions as follows:1.Compared with Naive BMM,the bacterial engulfment and intracellular killing were both enhanced in BLP-tolerized BMM;2.The expression of phagosome maturation related molecules such as Acp2,Acp5,Rab10,Rab20,Camp and iNOS were up-regulated in BLP-tolerized BMM;3.p65 could be activated and translocated into nucleus in the BLP-tolerized BMM infected by bacteria;4.p65 activation involved in the expression regulation of Acp5,Rab10 and iNOS in the BLP-tolerized BMM infected by bacteria;5.Cytoplasmic receptors NODI and NOD2 mediated p65 activation and the expression regulation of Acp5,Rab10,iNOS in the BLP-tolerized BMM infected by bacteria;6.Rab10 involved in the enhanced bactericidal effect of BLP-tolerized BMM. |
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