The Effection Of The Activation And Regulatory Of The M Protein On The Macrophages

Objective: GAS is a human-specific pathogen that will lead to a seriousresult without treatment in time. Our labortory has worked a lot about themechanism of inflammatory and immune escape. The M protein is the seriousvirulent factor that coat at the surface of the GAS. Previous work showed thatM protein could trigger serious inflrection and was able to inhibitphagocotosis. At the basic work of our laboratory, our aim is to make a deepresearch to the activation and regulation of Macrophage induced by the Mprotein.Macrophage is one of much more efficient cells at immune. Activatedmacrophageis specifically secret an enormous variety of proinflammatorycytokines such as IL-1β, IL-6, TNF-α, which play essential roles in inducinginflammatory responses. At the same time we are anxious of the function ofthe suppressor of the cytokine. TNF-α induced protein3(A20), an inhibitior ofinflammatory signal transduction factor NF-κB, play an important regulationrole in the innate and adoptive immunity. Here we are eager to know how it isinduced by M protein of GAS. IL-10is a cytokine of violent inhibit thesecretion of proinflammatory cytokines. What is its character of expressionwhen macrophages were stimulated by M protein? Suppressor of cytokinesignaling (socs) is a negative regulator of JAK/STAT pathway, how does it toexpress in the inflammatory process of M protein induced? Upon on thesequestions, we will have a basic research. We expressed the M protein that wassaved by our lab.Then we constructed and expressed protein SpeB, one of asecretion protein by GAS. Different ability of induction cytokines in theRAW264.7cells by two proteins were compared. We hope that the studywould set a foundation to further research on negative feedback regulation ofmacrophage induced by the M protein of GAS. The research aim to a deep realization of GAS, provide the experimentalevidence to the clinical for targe treatment.Methods:1Purification of protein: extract plasmid of expression M protein in thestored bacterial and clone into express bacterial E.coli BL21, and induced byIPTG.The expression produce was detected by SDS-PAGE.Structure theplasmid of expression SpeB protein and express it.2The cytotoxicity of the fusion protein were detected by MTT essay，LPS level in protein are detrimined using a Limulus amebocyte lysate LPSdetection kit and ensure of the protein concentration stimulated on cell. first,M protein and SpeB protein was detected by MTT cell toxicity, then theexpression of A20is reference when stimulate the macrophage byconcentrations of0.1、1、5、10μg/ml of the M protein, in order to determinethe appropriate stimulus concentration.3The M protein and SpeB protein on RAW264.7cell activation andregulation: according to the above experiment, the10μg/ml concentration ofM protein and SpeB protein respectively, stimulation of RAW264.7cells,2,4,8,12hours and execute Realtime-PCR for detect the mRNA expression ofIL-1β, IL-6, TNF-α and negative regulatory factor A20, IL-10, SOCS1andSOCS3.4Detection of A20expression in RAW264.7cells stimulated by Mprotein:10μg/ml concentration of M protein stimulate RAW264.7cells for4,8,12,24hours and then perform the WB to detect the expression of P65, A20and its downstream molecules TRAF6.5Detection of A20expression in BMDM cells of C57mouse stimulatedby M protein:10μg/ml concentration of M protein stimulate mouse BMDMcells4,8,12,24hours and perform the WB to detect the expression of P65,A20and its downstream molecules TRAF6.Results:1M protein concentration was160μg/ml, SpeB protein concentration was75μg/ml.2The MTT essay proved that the M protein had no cytotoxicity when theconcentration≤20μg/ml while the SpeB protein concentration≤16μg/ml.The concentration of LPS in M protein is1.015ng/μl and that inSpeB protein is0.786ng/μl.3Taking PBS as negative control, LPS was used as positive control, Mprotein stimulated RAW264.7cells2,4,8,12hours.Then detectedinflammatory factor TNF-α, IL-1, IL-6and negative regulatory factor of A20,IL-10, SCOS1, SOCS3by the Realtime-PCR. Results showed thepro-inflmmatory cytokines were increased. Among them, TNF-α didn’tincrease significantly compared with IL-1β and IL-6, the expression decreasedgradually after reaching the peak in8hours. Negative regulatory factor A20expression increased, reached the peak in4hours, and IL-10has a weakincrease. SOCS1and SOCS3had little increased. At the same time withprotein SpeB in parallel experiments, results showed that TNF-α, IL-1β, IL-6cytokines were increased, but lower than those of M protein, and theexpression of negative regulatory factor of A20, SOCS1and SOCS3wassignificantly lower than that of M protein after stimulation, the expression ofIL-10is than that in the normal control.4To detect the expression of A20by WB after The M protein stimulatedRAW264.7cells4,8,12,24hours.Result showed that the expression of A20in12hours was gradually increased, the expression of A20after24hours waslower compare with after12hours. M protein has stimulated RAW264.7cells for4,8,12,24hours, performed WB to detect expression of P65andTRAF6showed increased expression of P65obviously and increasedcontinuously in24hours, showed that the NF-κB pathway is activated, and theexpression of TRAF6in a wave way.5The M protein stimulated BMDM cells for the same conditions4,8,12,24hours of WB for detection of A20showed that A20expression increasedgradually in12hours,24hours after the expression level of less than12hours;the expression of P65in24hours is gradually increased and the expression of TRAF6was same to RAW264.7cell.Conclusions:1The M protein and SpeB protein can promote macrophageproinflammatory cytokine secretion of IL-1β, IL-6and TNF-α, but negativefeedback factor expression of the two are different in stimulation, M protein ishigher than that of SpeB protein.2The M protein may activate cells through the NF-κB pathway, it alsowas a negative regulator of A20activation by a certain way.3Negative regulatory factor A20inhibites inflammation through thesubstrate molecule TRAF6in the inflammatory pathway.