| BackgroundTuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis(MTB),a disease that is the largest number of deaths caused by a single pathogen worldwide.The mortality rate has exceeded that of AIDS.It is reported that there are 10.4 million new cases of tuberculosis in 2015 and 1.8 million deaths from tuberculosis.As a result,tuberculosis is still a serious threat to human health in the world,and it is an urgent global public health problem for sustainable development.MTB is a facultative intracellular parasite,which will mainly sojourn in the host M? after the invasion of body.As the first line of defense for tuberculosis infection,macrophages express a variety of pattern recognition receptors,where TLRs are the key molecules that mediate host recognition of MTB and anti-tuberculosis immune response.After recognized by macrophages by TLR,MTB can stimulate macrophages to produce a series of proinflammatory cytokines to build inflammatory microenvironment.On the other hand,to promote the bactericidal inflammatory response is also a mechanism leading to pathological damage.Whether the host is in the latency or active period,excessive dysregulation of the inflammatory response is the core of the tuberculosis showed the pathological features.Therefore,the regulation of macrophage inflammatory response induced by MTB infection is an important step in controlling the development of tuberculosis.It has been confirmed that there is a negative regulatory mechanism of intracellular TLR signalling,one of its important components is ?-arrestin 2.?-arrestin 2 regulates the effect of MAPK,NF-?B and other pathways,and?-arrestin 2 is an important immunoreactive negative regulator of TLR/IL-1R signalling downstream.MAPK pathway is a key signalling pathway in infected Mcp,and one of its members-ERK1/2 is involved in anti-MTB innate immune TLR downstream signalling pathway.ERK1/2 pathway has a wide variety of effects that affect the outcome of MTB infection.Therefore,our study focuses on the regulation of ERK1/2 pathway by ?-arrestin 2.In addition,the ubiquitination modification is critical for the effect of the?-arrestin 2 regulatory signalling pathway.A number of studies have demonstrated that ubiquitination of ?-arrestin 2 is necessary for its scaffold protein activity on ERK1/2.However,the regulation of ERK1/2 pathway in TLR downstream by P-arrestin 2 ubiquitination under MTB infection has not been reported.Our study firstly found that ?-arrestin 2-dependent proinflammatory cytokines expression,which are regulated by ERK1/2 activation,was inhibited after silencing E3 ubiquitin ligase Mdm2.It can be seen that ?-arrestin 2 ubiquitination is related to ERK1/2 activation closely under MTB infection.Our study explored the effect and mechanism of ?-arrestin 2 on the regulation of ERK1/2 pathway the role of ?-arrestin 2 ubiquitination in this process,therefore,regulating the inflammatory response of macrophages infected with MTB.Investigation of the above mechanism will help to develop anti-tuberculosis drugs that accurately regulate MAPK pathway through ?-arrestin 2,which is of great significance to Tuberculosis control.Objective1.To detect the effect of MTB on the expression of ?-arrestin 2 in M? and the effect of p-arrestin 2 on the removal of MTB in M?;2.To detect the effect of ?-arrestin 2 on inflammatory response of M? under MTB infection;3.To explore the molecular mechanism of ?-arrestin 2 in regulating inflammation response and antibacterial effect of M?.Method1.To detect the effect of MTB on the expression of ?-arrestin 2 in M? and the effect of ?-arrestin 2 on the removal of MTB in M?(1)The expression of ?-arrestin 2 in macrophages mRNA and protein was detected by Real-Time Quantitative RT-PCR and Western blot respectively.(2)The antimicrobial activity of MTB-infected macrophages after silencing ?-arrestin2 was detected by CFU assay.2.To detect the effect of ?-arrestin 2 on inflammatory response of Mcp under MTB infectionReal-Time Quantitative RT-PCR and Luminex technique were used to detect the expression of proinflammatory and anti-inflammatory cytokines in MTB-infected macrophages after silencing ?-arrestin 2.3.To explore the molecular mechanism of ?-arrestin 2 in regulating inflammation response and antibacterial effect of M?(1)The cells were treated with ERK1/2 pathway inhibitor U0126.To detect the expression of ?-arrestin 2 expression silencing on the expression of Mcp proinflammatory cytokines in ERK1/2 pathway;(2)Western blot was used to detect the activation of ERK1/2 pathway in MTB-infected macrophages after silencing ?-arrestin 2.(3)Immunofluorescence-laser scanning confocal microscopy was used to observe the localization and translocation of ?-arrestin 2 and p-ERK1/2 in MTB-infected macrophages.(4)Immunoprecipitation was used to detect the binding of ?-arrestin 2 to ERK1/2 pathway protein and the ubiquitination of ?-arrestin 2 in MTB-infected macrophages.(5)Real-Time Quantitative RT-PCR was used to detect the expression of cytokines in Mdm2 expression silencing on MTB-infected macrophages.Results1.The expression of ?-arrestin 2 mRNA was down-regulated in the macrophages after infection.On the contrary,the expression of ?-arrestin 2 protein was gradually increased,and the expression of ?-arrestin 2 mRNA was down-regulated at 72 h after infection;Silencing ?-arrestin 2 expression can promote the antibacterial activity of Mcp.2.After silencing ?-arrestin 2 expression,the expression of proinflammatory cytokine TNF-?,IL-1? and IL-6 in BCG-infected M? was significantly upregulated.However,the changes of anti-inflammatory factors IL-10 and TGF-? in THP-1-M? and primary M? were different.The effect of ?-arrestin 2 on the expression of proinflammatory cytokines is consistent with its effect on the activity of MTB killing in M?,suggesting that it regulates the activity of MTB killing in M? by regulating the expression of proinflammatory cytokines.3.After inhibiting ERK1/2 pathway,the effect of ?-arrestin 2 expression silencing on the expression of proinflammatory cytokines can be reversed.The expression of?-arrestin 2 did not alter the activity of ERK1/2 in BCG-infected THP-1-M?.However,endogenous ?-arrestin 2 can inhibit the nuclear translocation of p-ERK1/2.In the meanwhile,the binding of ?-arrestin 2 to p-ERKl/2 and p-TPL2 increased after BCG infecting,and the level of ubiquitination of ?-arrestin 2 was also upregulated.The effect of Mdm2 expression silence was similar to that after silencing ?-arrestin 2.Conclusions?-arrestin 2 is upregulated in MTB-infected M? and is involved in the regulation of MTK-induced ERK1/2 nuclear translocation in the downstream of TLR,thereby regulating the inflammatory response and antibacterial activity of the infected Mcp.In this process,the level of ?-arrestin 2 ubiquitination is upregulated,and inhibition of ubiquitination enhances the effect of transcriptional regulation of ERK1/2 pathway. |
PDF Full Text Request