Dissertation > Medicine, health > Basic Medical > Medical Immunology

Expression of SOCS-1Proteins through the TLR4/MyD88Direct Signaling Pathway in Group A Streptococcus Infected Macrophages

Author WuJingHua
Tutor WeiLin
School Hebei Medical University
Course Immunology
Keywords GAS Macrophages SOCS-1 Cytokine activation pathway TLR4/MyD88signaling pathway
CLC R392
Type PhD thesis
Year 2014
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Objectives: Macrophages are important to fight to infection, and it wasactivated combined with a variety of pathogenic bacteria through its surfacepattern recognition receptors (PRR),which could identificated of the pathogenassociated molecular patterns (PAMPs). TLR, is important PRR, dimers wassynthesized when combined with PAMPs.MyD88molecules was recruited,andactivated its downstream IRAK which can activate the TRAF6to activate NF-κB.Then NF-κB transposited to the nucleus,and combined with specificprotein on the DNA sequence to inducte the specific gene and proteintranslation.The activity of macrophages is influenced by a variety of negativeregulatory proteins, including suppressor of cytokine signaling (SOCS),whichis adjustable immune cell activation to ensure the moderation of immuneresponse.SOCS proteins play a role in negative feedback regulation of manyimmune cells. SOCS is composed of at least eight kinds of ingredients,SOCS1-7and CIS.And the SOCS-1was produced through cytokinescombined cytokines receptors to activate JAK/STAT pathway.Many studiessuggest that the activation of some cytokines, such as IFN-, IL-10, IL-2and colony stimulating factor, could induce the SOCS-1protein expression.Among these the IFN receptor pathway is the classic way to itsproduction.When interferon combined its receptors, the tyrosine kinase (JAK-1and TyK2) was activated.Then STAT1was phosphorylated and transferedinto the nucleus to activate genes SOCS-1,and produce SOCS-1protein.Recently,these was reported that SOCS-1could induce through theTLR, Dectin-1pathway,and its stimulants is LPS, CpG, ans yeastpolysaccharide and so on. At present, more and more studies suggest that the composition ofmicroorganism and parasite can also induce the SOCS expression.Group A streptococcus (GAS) could lead to pharyngitis, soft tissueinfection, and many other respiratory infections.GAS-infected macrophagescould induce inflammatory cytokine IL-1, IL6and TNF-a and IFN-through activate TLR2and TLR4located in the membrane.GAS has a abilityto evade the host immune attack which is the reason of its persist in the bodyand cause some serious infectious diseases,and another phenomenon is a fewcells in GAS-infection induced necrotic areas.So in order to further understand GAS strategy to evade themacrophages, and explore the generation mechanism of SOCS-1, weconducted the following experiments:1, SOCS-1proteins expression inGAS-infected macrophages were detected.2, In view of the cytokinesactivation pathway is a classic pathways to induce the SOCS-1expression,wemeasure the SOCS-1levels in coincubated with cycloheximide (inhibition ofeukaryotic cell protein synthesis).3, Because of the classic pathway ofSOCS-1induction, we use IFN-neutralizing antibodies to verify the role ofIFN-to induce SOCS-1.4, Many studies have shown that TLR pathway could directly induce theexpression of SOCS-1, but its mechanism is unclear. Therefore, we woulddetect TLR2and TLR4activation in GAS-infected macrophages to confirmthe relationship the TLR signaling pathway and SOCS-1proteins expression.5,The key proteins (TLR4、MYD88、NF-κB) in induction of SOCS-1will bestudy through using of inhibitors, knockout mice.6, NF-κB is a diversityproteins, regulate a variety of genes transcription involved in immuneresponse and immune regulation related molecules.So we decide to researchNF-κB regulation on SOCS1expression.This study will help to enrich the mechanism of the interact immune cellsand bacteria, especially rich the understanding of the mechanism of host cellsrapid, direct expression of negative regulation factor which could helpmicrobial to evade host immune systems.So it will broaden the establishment and development of anti-infection immunity.Methods:1GAS-infected macrophage RAW264.7\BMDMs(MOI100:1) were detectedthe SOCS-1expression.2The relations of cytokines and SOCS-1expression was definited in GASinfected macrophages coincubate cycloheximide.2.1After cycloheximide incubate with macrophages30minutes, themacrophages were infectec with GAS,after1hour remove GAS and continueto develop6hours. SOCS-1was detected by RT-PCR and Western blot, aswell as the activation of the JAK/STAT pathway was test meantime.2.2IFN-was detected in GAS, nonviable GAS infectedmacrophages(RT-PCR).2.3The JAK/STAT pathway activation and SOCS-1expression was detectedafter blocking IFN-with a specific antibody.3The relationship will be confirmed in SOCS-1expression and TLR4activation.3.1TLR2/TLR4expression were detected in GAS and inviable GAS infectedmacrophages (RT-PCR).3.2After blocking TLR4with macrophages30minutes, the macrophages wereinfectec with GAS,after1hour remove GAS and continue to develop6hours.SOCS-1was detected by RT-PCR and Western blot, as well as the activationof the JAK/STAT pathway was test meantime.3.3The relationship between TLR4signaling and SOCS-1expression wasconfirmed by BMDMs from TLR4-/-mice again.4To study the relationship between GAS-induced SOCS-1expression andTLR4/MyD88pathway.4.1MyD88,JAK1, STAT1protein were detected in macrophages byimmunofluorescence and immune coprecipitation.4.2According to the above results, if MyD88and JAK1, STAT1exis ascomplex of in macrophages, we will detect the complex activation byimmunofluorescence and immune co-precipitation. 4.3TLR4/MyD88direct pathway will be comfirmed by mmuneprecipitationwith when cytokines activation pathway was excluded throughcoincubated with cycloheximide.4.4TLR4/MYD88direct pathway was further confirmed in GAS infectionthrough MYD88-/-macrophages.5The impaction of NF-κB activation to GAS-induced SOCS-1expression willbe cofirmed.5.1The relationship between JAK/STAT pathway activation and NF-κBactivation will be confirmed through activation of JAK and STAT proteinsafter GAS infected1h,2h,4h,6h.5.2According to the above results, theregulation was further confirmed byblcoking to NF-κB signaling.Results:1GAS-induced SOCS-1expression in early stage in macrophagesGAS and nonviable GAS (MOI100:1) respectively stimulate RAW264.7macrophages and BMDMs1hour, characteristics of SOCS-1expression asfollows: The SOCS-1gene had significantly increased after4h of GASinfection,peaked at6h, begin to decline after8h.However, although theinviable GAS-induced SOCS1mRNA elevated moderately6-8hours afterinfection,its level increased much smaller than GAS group. In GAS-infectedBMDMs, SOCS-1mRNA expression is the same as in RAW264.7cells, onlygreater than RAW264.7cells. SOCS-1protein begin to express after6hoursin GAS-infected macrophages,but inviable GAS don’t induce expression in10h.2GAS infected macrophages induced SOCS-1expression partly depends onthe IFN-βactivation pathway2.1IFN-βbegan to rise in GAS infection RAW264.7macrophages at2h, andpeaked at6h.2.2The IFN-βactivation pathway is not the only way to induce the expressionof SOCS-1through coincubating anti-IFN-βand GAS.2.3In order to check the SOCS-1expression whether depend on the cytokines activation pathway, Cycloheximide (it could inhibit protein translation ofeukaryotic cells) was used to coincubate with macrophages.The results showthat STAT1proteins were still phosphorylated in the cycloheximide,and itreveals GAS itself could induce SOCS-1expression besides cytokinesactivation dependent pathway.3GAS-infection induced TLR4activationWhen macrophages was infected with GAS, TLR2and TLR4located incells surface were activated, and peaked at4h after infection, was about8times of normal, and begun to decline after6hours. While the macrophagesinfected by inviable GAS,the TLR4has not been activated; TLR2activation has no difference in GAS group and inviable GAS infection within6hours.4TLR4play an important role in GAS-induced SOCS-1expression4.1In order to further determine whether the expression of SOCS-1wasdepend on the TLR4activation, we use the TLR4neutralizing antibody blockdthe TLR4on the macrophages.And the results show that the SOCS-1expression was declined in the present of anti-TLR4.So it could confirm thatTLR4play a role in the GAS-induced SOCS-1expression.4.2It was further confirmed that the SOCS-1expression depend on the TLR4activation in theTLR4-/-BMDMs cells.5SOCS-1early expression depends on the MyD885.1Through immunofluorescence and immune precipitation,we could believethat Jak1, STAT1and MyD88exist in RAW264.7macrophages as a complexin macrophages. It was found that when JAK1associated with MyD88,phosphorylation was rapidly activated in macrophages that were post-infectedwith GAS at1-2hrs post-infection, and STAT1was activated andphosphorylated STAT1levels peaked at3to4hrs post-infection.5.2SOCS-1expression levels were diminished in MyD88-/-BMDMs.To further test whether STAT1was phosphorylated by GAS itself throughthe MyD88-JAK1-STAT1complex, we detected p-STAT1levels combinedwith MyD88in the presence of CHX by immunoprecipitation assay. The cells were infected with GAS, or with GAS plus CHX, and then thep-STAT/MyD88complex was detected by immunoprecipitation analysis.Without cytokine activation signaling, the Without cytokine activationsignaling, the p-STAT1levels combined with MyD88were unaffected. Thus,these findings suggested that in GAS-induced infection, GAS itself promptlyactivated STAT1through the TLR4/MyD88-JAK1/STAT1complex.6NF-κB activation regulated early expression of SOCS-1through reducedSTAT1expression levels6.1GAS induced rapid and potent activation of NF-κB and increased STAT1expression. The expression of STAT1increased following NF-κB activationand peaked at6hrs. Having established that GAS infection induces NF-κBactivation and leads to increased STAT1expression; we think there maybe alink between NF-κB activation and STAT1expression.6.2Through pretreated BMDMs with JSH-23(20uM)30min prior toinfection and incubated JSH-23for the indicated times,we found thatGAS-induced expression of STAT-1was strongly diminished, and that thephosphorylation of STAT-1was almost completely abolished in BMDMstreated with JSH-23.And GAS-induced expression of SOCS-1was completelyabolished in cells treated with JSH-23.Conclusion:1GAS-infected macrophages could express SOCS-1proteins in theearly stage.2GAS-induced expression of SOCS-1partly relies on the IFN-activated pathway.3TLR4/MyD88direct pathway plays an important role in GAS-inducedSOCS-1expression in the early stage.4NF-κB signaling could affect SOCS-1protein levels throughregulation to the STAT1protein expression.

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